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EMILIN-3 is a glycoprotein of the extracellular matrix belonging to a family that contains a characteristic N-terminal cysteine-rich EMI domain. Currently, EMILIN-3 is the least characterized member of the EMILIN/Multimerin family. Using RNA, immunohistochemical and protein chemistry approaches, we carried out a detailed characterization of the expression and of the biochemical properties of EMILIN-3 in mouse. During embryonic and postnatal development, EMILIN-3 showed a peculiar and dynamic pattern of gene expression and protein distribution. EMILIN-3 mRNA was first detected at E8.5-E9.5 in the tail bud and in the primitive gut, and at later stages it became abundant in the developing gonads and in the osteogenic mesenchyme. Interestingly, and in contrast to other EMILIN/Multimerin genes, EMILIN-3 was not found in the cardiovascular system. Despite the absence of the globular C1q domain, immunoprecipitation and western blot analyses demonstrated that EMILIN-3 forms disulfide-bonded homotrimers and higher-order oligomers. Circular dichroism spectroscopy indicated that the most C-terminal part of EMILIN-3 has a substantial ?-helical content and forms coiled-coil structures, involved in EMILIN-3 homo-oligomerization. Transfection experiments with recombinant constructs showed that the EMI domain contributes to the higher-order self-assembly but was dispensable for homotrimer formation. EMILIN-3 was found to bind heparin with high affinity, a property mediated by the EMI domain, thus revealing a new function for this domain that may contribute to the interaction of EMILIN-3 with other ECM and/or cell surface molecules. Finally, in vitro experiments showed that EMILIN-3 is able to function as an extracellular regulator of the activity of TGF-? ligands.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/NUW-g0loq1k" height="1" width="1"/>
All thermophilic and hyperthermophilic archaea encode homologs of dimeric Alba (Sac10b) proteins that bind cooperatively at high density to DNA. Here, we report the 2.0 Å resolution crystal structure of an Alba2 (Ape10b2)-dsDNA complex from Aeropyrum pernix K1. A rectangular tube-like structure encompassing duplex DNA reveales the positively charged residues in the monomer-monomer interface of each dimer packing on either side of the bound dsDNA in successive minor grooves. The extended hairpin loop connecting the strands ?3-?4 undergoes significant conformational changes upon DNA-binding to accommodate the other Alba2-dimer during oligomerization. Mutational analysis of key interacting residues confirmed the specificity of Alba2-dsDNA interactions.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/cSeZ8yyECOs" height="1" width="1"/>
Eukaryotic linker or H1 histones modulate DNA compaction and gene expression in vivo. In mammals, these proteins exist as multiple isotypes with distinct properties suggesting a functional significance to the heterogeneity. Linker histones typically have a tripartite structure, composed of a conserved central globular domain flanked by a highly variable short amino-terminal domain and a longer highly basic carboxyl-terminal domain. We hypothesized that the variable terminal domains of individual subtypes contribute to their functional heterogeneity by influencing chromatin-binding interactions. We developed a novel dual-color fluorescence recovery after photobleaching (FRAP) assay system in which two H1 proteins fused to spectrally separable fluorescent proteins can be co-expressed and their independent binding kinetics simultaneously monitored in a single cell. This approach was combined with domain swap and point mutagenesis to determine the roles of the terminal domains in the differential binding characteristics of the linker histone isotypes, H10 and H1c. Exchanging the N-terminal domains between H10 and H1c changed their overall binding affinity to that of the other variant. In contrast, switching the C-terminal domains altered the chromatin interaction surface of the globular domain. These results indicate that linker histone subtypes bind to chromatin in an intrinsically specific manner and that the highly variable terminal domains contribute to differences between subtypes. The methods developed in this study will have broad applications in studying dynamic properties of additional histone subtypes and other mobile proteins.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/gYZFvSq2niM" height="1" width="1"/>
Chromatin structure organization is crucial for regulating many fundamental cellular processes. However, the molecular mechanism that regulates the assembly of higher-order chromatin structure remains poorly understood. In this study, we demonstrate that the Brd4 (bromodomain-containing protein 4) protein participates in the maintenance of higher-order chromatin structure. Brd4, a member of the BET family of proteins, has been shown to play important roles in cellular growth control, cell cycle progression, and cancer development. We apply in situ single cell chromatin imaging and micrococcal nuclease (MNase) assay to show that Brd4 depletion leads to a large scale chromatin unfolding. A dominant negative inhibitor encoding the double bromodomains (BDI/II) of Brd4 can competitively dissociate endogenous Brd4 from chromatin to trigger severely fragmented chromatin morphology. Mechanistic studies using Brd4 truncation mutants reveal that the Brd4 C-terminal domain (CTD) is crucial for maintaining normal chromatin structure. Using the bimolecular fluorescence complementation (BiFC) technology, we demonstrate that Brd4 molecules interact intermolecularly on chromatin and that replacing Brd4 molecules by BDI/II causes abnormal nucleosome aggregation and chromatin fragmentation. These studies establish a novel structural role of Brd4 in supporting the higher chromatin architecture.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/9_IpTLC7A1I" height="1" width="1"/>
Microenvironmental acidosis is a common feature of inflammatory loci, in which clearance of apoptotic cells is necessary for the resolution of inflammation. Although it is known that a low pH environment affects immune function, its effect on apoptotic cell clearance by macrophages has not been fully investigated. Here, we show that treatment of macrophages with low pH medium resulted in increased expression of stabilin-1 out of several receptors, which are known to be involved in PS-dependent removal of apoptotic cells. Reporter assays showed that the -120/-1 region of the mouse stabilin-1 promoter was a low pH-responsive region and provided evidence that extracellular low pH mediated transcriptional activation of stabilin-1 via Ets-2. Furthermore, extracellular low pH activated c-Jun N-terminal kinase (JNK), thereby inducing translocation of Ets-2 into the nucleus. When macrophages were preincubated with low pH medium, phagocytosis of PS-exposed RBC and PS-coated beads by macrophages was enhanced. Blockade of stabilin-1 in macrophages abolished the enhancement of phagocytic activity by low pH. Thus, our results demonstrate that a low pH microenvironment upregulates stabilin-1 expression in macrophages, thereby modulating the phagocytic capacity of macrophages and suggest roles for stabilin-1 and Ets-2 in the maintenance of tissue homeostasis by the immune system.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/KnoFn-SJbpU" height="1" width="1"/>
Integrins are heterodimeric type I membrane cell adhesion molecules that are involved in many biological processes. Integrins are bi-directional signal transducers because their cytoplasmic tails are docking sites for cytoskeletal and signaling molecules. Kindlins are cytoplasmic molecules that mediate inside-out signaling and activation of the integrins. The three kindlin paralogs in humans are kindlin-1, -2, and -3. Each of these contains a 4.1-ezrin-radixin-moesin (FERM)-domain and a pleckstrin homology (PH) domain. Kindlin-3 is expressed in platelets, hematopoietic cells and endothelial cells. Here we show that kindlin-3 is involved in integrin ?L?2 outside-in signaling. It also promotes micro-clustering of integrin ?L?2. We provide evidence that kindlin-3 interacts with the receptor for activated-C kinase 1 (RACK1), a scaffold protein that folds into a seven-blade propeller. This interaction involves the PH domain of kindlin-3 and blades 5-7 of RACK1. Using the SKW3 human T lymphoma cells, we show that integrin ?L?2 engagement by its ligand ICAM-1 promotes the association of kindlin-3 with RACK1. We also show that kindlin-3 co-localizes with RACK1 in polarized SKW3 cells and human T lymphoblasts. Our findings suggest that kindlin-3 plays an important role in integrin ?L?2 outside-in signaling.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/WJspnov2ZXA" height="1" width="1"/>
UDP-galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). As in prokaryotic UGMs, the flavin needs to be reduced for the enzyme to be active. Here we present the first eukaryotic UGM structures from Aspergillus fumigatus (AfUGM). The structures are of UGM alone, with the substrate UDP-Galp and with the inhibitor UDP. Additionally, we report the structures of AfUGM bound to substrate with oxidized and educed flavin. These structures provide insight into substrate recognition and structural changes observed upon substrate binding involving the mobile loops and the critical arginine residues R182 and R327. Comparison with prokaryotic UGM reveals that despite low sequence identity with known prokaryotic UGMs the overall fold is largely conserved. Structural differences between prokaryotic UGM and AfUGM result from inserts in AfUGM. A notable difference from prokaryotic UGMs is that AfUGM contains a third flexible loop (loop III) above the si-face of the isoalloxazine ring that changes position depending on the redox state of the flavin cofactor. This loop flipping has not been observed in prokaryotic UGMs. In addition we have determined the crystals structures and steady-state kinetic constants of the reaction catalyzed by mutants R182K, R327K, R182A and R327A. These results support our hypothesis that R182 and R327 play important roles in stabilizing the diphosphates of the nucleotide sugar and help to facilitate the positioning of the galactose moiety for catalysis.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/RUKR-oAp1ZQ" height="1" width="1"/>
How nucleotide excision repair (NER) machinery gains access to damaged chromatinized DNA templates and how the chromatin structure is modified to promote efficient repair of the non-transcribed genome remain poorly understood. The UV-damaged DNA-binding protein complex (UV-DDB, consisting of DDB1 and DDB2, the latter of which is mutated in xeroderma pigmentosum group E patients, XP-E) is a substrate-recruiting module of the cullin 4B-based E3 ligase complex, DDB1-CUL4BDDB2. We previously reported that the deficiency of UV-DDB E3 ligases in ubiquitinating histone H2A at UV-damaged DNA sites in the XP-E cells contributes to the faulty NER in these skin cancer-prone patients. Here, we reveal the mechanism by which monoubiquitination of specific H2A lysine residues alters nucleosomal dynamics and subsequently initiates NER. We show that DDB1-CUL4BDDB2 E3 ligase specifically binds to mononucleosomes assembled with human recombinant histone octamers and nucleosome-positioning DNA containing CPD or 6-4PP photolesions. We demonstrate functionally that ubiquitination of H2A Lys119/Lys120 is necessary for destabilization of nucleosomes and concomitant release of DDB1-CUL4BDDB2 from photolesion-containing DNA. Nucleosomes in which these lysines are replaced with arginines are resistant to such structural changes, and arginine mutants prevent the eviction of H2A and dissociation of polyubiquitinated DDB2 from UV-damaged nucleosomes. The partial eviction of H3 from the nucleosomes is dependent on ubiquitinated H2A Lys119/Lys120. Our results provide mechanistic insight into how post-translational modification of H2A at the site of a photolesion initiates the repair process and directly affects the stability of the human genome.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/k43oX6OWnmo" height="1" width="1"/>
The catalytic subunit of cAMP-dependent protein kinase (PKA) is a member of the AGC subfamily of protein kinases. While PKA has served as a structural model for the protein kinase superfamily, all previous structures of the catalytic subunit contain a phosphorylated activation loop. To understand the structural effects of activation loop phosphorylation at Thr197 we used a PKA mutant which does not autophosphorylate at Thr197. The enzyme crystallized in the apo-state and the structure was solved to 3.0 angstroms. The N-lobe is rotated by 18° relative to the wild type apoenzyme which illustrates that the enzyme likely exists in a wide range of conformations in solution due to the uncoupling of the N- and C-lobes. Several regions of the protein including the activation loop are disordered in the structure and there are alternate main chain conformations for the magnesium positioning loop and catalytic loop causing a complete loss of hydrogen bonding between these two active site structural elements. These alterations are reflected in a 20-fold decrease in the apparent phosphoryl transfer rate as measured by pre-steady-state kinetic methods.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/So_XPze8PaM" height="1" width="1"/>
Hyperphosphorylated tau is the major component of neurofibrillary tangles in Alzheimer's disease (AD), and the tangle distribution is largely overlapped with zinc containing glutamatergic neurons, suggesting that zinc released in synaptic terminals may play a role in tau phosphorylation. To explore this possibility, we treated the cultured hippocampal slices or primary neurons with glutamate or Bic/4-AP to increase the synaptic activity with or without pre-treatment of zinc chelators, and then detected the phosphorylation levels of tau. We found that glutamate or Bic/4-AP treatment caused tau hyperphosphorylation at multiple AD-related sites, including Ser396, Ser404, Thr231, and Thr205, while application of intracellular or extracellular zinc chelators, or blockade of zinc release by extracellular calcium omission almost abolished the synaptic activity-associated tau hyperphosphorylation. The zinc release and translocation of excitatory synapses in the hippocampus was detected, and zinc-induced tau hyperphosphorylation was also observed in cultured brain slices incubated with exogenously supplemented zinc. Tau hyperphosphorylation induced by synaptic activity was strongly associated with inactivation of protein phosphatase 2A (PP2A), and this inactivation can be reversed by pre-treatment of zinc chelator. Together, these results suggest that synaptically released zinc promotes tau hyperphosphorylation through PP2A inhibition.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/ptlDfHMyoP4" height="1" width="1"/>
Hematopoietic progenitor kinase 1 (HPK1) is a Ste20-like serine/threonine kinase that suppresses immune responses and autoimmunity. B-cell receptor (BCR) signaling activates HPK1 by inducing BLNK/HPK1 interaction. Whether HPK1 can reciprocally regulate BLNK during BCR signaling is unknown. Here we show that HPK1-deficient B cells display hyper-proliferation and hyper-activation of IKK and MAPKs (ERK, p38, and JNK) upon the ligation of BCR. HPK1 attenuates BCR-induced cell activation via inducing BLNK threonine152 phosphorylation, which mediates BLNK/14-3-3 binding. Furthermore, threonine152-phosphorylated BLNK is ubiquitinated at lysine37, 38 and 42 residues, leading to attenuation of MAPK and IKK activation in B cells during BCR signaling. These results reveal a novel negative feedback regulation of BCR signaling by HPK1-mediated phosphorylation, ubiquitination, and subsequent degradation of the activated BLNK.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/ytsFqOC-sIc" height="1" width="1"/>
? See referenced article, J. Biol. Chem. 2012, 287, 4451–4461
In 1996, hydrogen sulfide, best known for its rotten egg smell, was proposed to be a neurotransmitter. The suggestion that hydrogen sulfide had a biological role was controversial until 2008, when researchers showed that deletion of an enzyme that produced hydrogen sulfide in mice caused the animals to develop hypertension. More studies revealed that hydrogen sulfide affected macrophage infiltration, apoptosis, and mitochondrial respiration in the heart. In this Paper of the Week, a team led by Balakuntalam S. Kasinath at the University of Texas Health Science Center and the South Texas Veterans Health Care System delved into how hydrogen sulfide influences renal physiology and disease. They showed that hydrogen sulfide mitigated some of the damage caused by high glucose, which stimulates aberrant matrix protein synthesis in glomerular epithelial cells, by activating AMP kinase. AMP kinase is a sensor of decreased ATP energy in cells and blocks anabolic processes required for matrix protein synthesis. Kasinath's team also demonstrated that diabetic mice suffered from lower expression levels of key enzymes involved in hydrogen sulfide production. The investigators concluded, “Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.”
jbc;287/7/5180/FU1F1FU1
Immunoperoxidase staining of the kidney showed a reduction in the expression of an enzyme involved in hydrogen sulfide production, cystathionine ?-synthase, in type 1 diabetic mice (OVE26) compared with control mice.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/0YwR7dDAu7A" height="1" width="1"/>
We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked ???? core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of Tm = 52 °C, enhanced the protein thermostability by 36 °C (Tm = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ?10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000–25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500–3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/Syxj3HCLOq0" height="1" width="1"/>
We have solved the x-ray structure of the N-terminal half of the yeast kinetochore protein Ndc10 at 1.9 Å resolution. This essential protein is a key constituent of the budding yeast centromere and is essential for the recruitment of the centromeric nucleosome and establishment of the kinetochore. The fold of the protein shows unexpected similarities to the tyrosine recombinase/?-integrase family of proteins, most notably Cre, with some variation in the relative position of the subdomains. This finding offers new insights into kinetochore evolution and the adaptation of a well studied protein fold to a novel role. By comparison with tyrosine recombinases and mutagenesis studies, we have been able to define some of the key DNA-binding motifs.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/D_l3w7GbpGY" height="1" width="1"/>
The ATP-binding cassette transporter ABCB4 is a phosphatidylcholine translocator specifically expressed at the bile canalicular membrane in hepatocytes, highly homologous to the multidrug transporter ABCB1. Variations in the ABCB4 gene sequence cause progressive familial intrahepatic cholestasis type 3. We have shown previously that the I541F mutation, when reproduced either in ABCB1 or in ABCB4, led to retention in the endoplasmic reticulum (ER)/Golgi. Here, Madin-Darby canine kidney cells expressing ABCB1-GFP were used as a model to investigate this mutant. We show that ABCB1-I541F is not properly folded and is more susceptible to in situ protease degradation. It colocalizes and coprecipitates with the ER chaperone calnexin and coprecipitates with the cytosolic chaperone Hsc/Hsp70. Silencing of calnexin or overexpression of Hsp70 have no effect on maturation of the mutant. We also tested potential rescue by chemical and pharmacological chaperones. Thapsigargin and sodium 4-phenyl butyrate were inefficient. Glycerol improved maturation and exit of the mutant from the ER. Cyclosporin A, a competitive substrate for ABCB1, restored maturation, plasma membrane expression, and activity of ABCB1-I541F. Cyclosporin A also improved maturation of ABCB4-I541F in Madin-Darby canine kidney cells. In HepG2 cells transfected with ABCB4-I541F cDNA, cyclosporin A allowed a significant amount of the mutant protein to reach the membrane of bile canaliculi. These results show that the best strategy to rescue conformation-defective ABCB4 mutants is provided by pharmacological chaperones that specifically target the protein. They identify cyclosporin A as a potential novel therapeutic tool for progressive familial intrahepatic cholestasis type 3 patients.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/7_wY6PxWbZk" height="1" width="1"/>
Saframycin A (SFM-A) is a potent antitumor antibiotic that belongs to the tetrahydroisoquinoline family. Biosynthetic studies have revealed that its unique pentacyclic core structure is derived from alanine, glycine, and non-proteinogenic amino acid 3-hydroxy-5-methyl-O-methyltyrosine (3-OH-5-Me-OMe-Tyr). SfmD, a hypothetical protein in the biosynthetic pathway of SFM-A, was hypothesized to be responsible for the generation of the 3-hydroxy group of 3-OH-5-Me-OMe-Tyr based on previously heterologous expression results. We now report the in vitro characterization of SfmD as a novel heme-containing peroxidase that catalyzes the hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine using hydrogen peroxide as the oxidant. In addition, we elucidated the biosynthetic pathway of 3-OH-5-Me-OMe-Tyr by kinetic studies of SfmD in combination with biochemical assays of SfmM2, a methyltransferase within the same pathway. Furthermore, SacD, a counterpart of SfmD involved in safracin B biosynthesis, was also characterized as a heme-containing peroxidase, suggesting that SfmD-like heme-containing peroxidases may be commonly involved in the biosynthesis of SFM-A and its analogs. Finally, we found that the conserved motif HXXXC is crucial for heme binding using comparative UV-Vis and Magnetic Circular Dichroism (MCD) spectra studies of SfmD wild-type and mutants. Together, these findings expand the category of heme-containing peroxidases and set the stage for further mechanistic studies. In addition, this study has critical implications for delineating the biosynthetic pathway of other related tetrahydroisoquinoline family members.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/yt4v25w989g" height="1" width="1"/>
Epithelial to mesenchymal transition (EMT) and pulmonary fibrogenesis require epithelial integrin ?3?1-mediated cross-talk between TGF?1 and Wnt signaling pathways. One hallmark of this cross-talk is pY654-?-catenin accumulation, but whether pY654-?-catenin is a biomarker of fibrogenesis or functionally important is unknown. To clarify further the role of ?-catenin in fibrosis, we explored pY654-?-catenin generation and function. ?3?1 was required for TGF?1-mediated activation of Src family kinases, and Src inhibition blocked both pY654 and EMT in primary alveolar epithelial cells (AECs). TGF?1 stimulated ?-catenin/Lef1-dependent promoter activity comparably in immortalized AECs stably expressing WT ?-catenin as well as Y654E or Y654F ?-catenin point mutants. But EMT was abrogated in the Tyr to Phe mutant. pY654-?-catenin was sensitive to the axin ?-catenin turnover pathway as inhibition of tankyrase 1 led to high AEC axin levels, loss of pY654-?-catenin, and inhibition of EMT ex vivo. Mice given a tankyrase inhibitor (50 mg/kg orally) daily for 7 days beginning 10 days after intratracheal bleomycin had improved survival over controls. Treated mice developed raised axin levels in the lung that abrogated pY654-?-catenin and attenuated lung Snail1, Twist1, ?-smooth muscle actin, and type I collagen accumulation. Total ?-catenin levels were unaltered. These findings identify Src kinase(s) as a mediator of TGF?1-induced pY654-?-catenin, provide evidence that pY654-?-catenin levels are a critical determinant of EMT and fibrogenesis, and suggest regulation of axin levels as a novel therapeutic approach to fibrotic disorders.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/J5NU9-8mNec" height="1" width="1"/>
Regulated intramembrane proteolysis is a widely accepted concept describing the processing of various transmembrane proteins via ectodomain shedding followed by an intramembrane cleavage. The resulting cleavage products can be involved in reverse signaling. Presenilins, which constitute the active center of the ?-secretase complex, signal peptide peptidase (SPP), and its homologues, the SPP-like (SPPL) proteases are members of the family of intramembrane-cleaving aspartyl proteases of the GXGD-type. We recently demonstrated that Bri2 (itm2b) is a substrate for regulated intramembrane proteolysis by SPPL2a and SPPL2b. Intramembrane cleavage of Bri2 is triggered by an initial shedding event catalyzed by A Disintegrin and Metalloprotease 10 (ADAM10). Additionally primary sequence determinants within the intracellular domain, the transmembrane domain and the luminal juxtamembrane domain are required for efficient cleavage of Bri2 by SPPL2b. Using mutagenesis and circular dichroism spectroscopy we now demonstrate that a high ?-helical content of the Bri2 transmembrane domain (TMD) reduces cleavage efficiency of Bri2 by SPPL2b, while the presence of a GXXXG dimerization motif influences the intramembrane cleavage only to a minor extent. Surprisingly, only one of the four conserved intramembrane glycine residues significantly affects the secondary structure of the Bri2 TMD and thereby its intramembrane cleavage. Other glycine residues do not influence the ?-helical content of the transmembrane domain nor its intramembrane processing.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/5ib65WQcRAM" height="1" width="1"/>
The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule that plays a central role in the etiology of diabetes complications, inflammation, and neurodegeneration. The cytoplasmic domain of RAGE (C-terminal RAGE; ctRAGE) is critical for RAGE-dependent signal transduction. As the most membrane-proximal event, mDia1 binds to ctRAGE, and it is essential for RAGE ligand-stimulated phosphorylation of AKT and cell proliferation/migration. We show that ctRAGE contains an unusual ?-turn that mediates the mDia1-ctRAGE interaction and is required for RAGE-dependent signaling. The results establish a novel mechanism through which an extracellular signal initiated by RAGE ligands regulates RAGE signaling in a manner requiring mDia1.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/mXnjTJ29Yqk" height="1" width="1"/>
Slc26a2 is a ubiquitously expressed SO42? transporter with high expression levels in cartilage and several epithelia. Mutations in SLC26A2 are associated with diastrophic dysplasia. The mechanism by which Slc26a2 transports SO42? and the ion gradients that mediate SO42? uptake are poorly understood. We report here that Slc26a2 functions as an SO42?/2OH?, SO42?/2Cl?, and SO42?/OH?/Cl? exchanger, depending on the Cl? and OH? gradients. At inward Cl? and outward pH gradients (high Cl?o and low pHo) Slc26a2 functions primarily as an SO42?o/2OH?i exchanger. At low Cl?o and high pHo Slc26a2 functions increasingly as an SO42?o/2Cl?i exchanger. The reverse is observed for SO42?i/2OH?o and SO42?i/2Cl?o exchange. Slc26a2 also exchanges Cl? for I?, Br?, and NO3? and Cl?o competes with SO42? on the transport site. Interestingly, Slc26a2 is regulated by an extracellular anion site, required to activate SO42?i/2OH?o exchange. Slc26a2 can transport oxalate in exchange for OH? and/or Cl? with properties similar to SO42? transport. Modeling of the Slc26a2 transmembrane domain (TMD) structure identified a conserved extracellular sequence 367GFXXP371 between TMD7 and TMD8 close to the conserved Glu417 in the permeation pathway. Mutation of Glu417 eliminated transport by Slc26a2, whereas mutation of Phe368 increased the affinity for SO42?o 8-fold while reducing the affinity for Cl?o 2 fold, but without affecting regulation by Cl?o. These findings clarify the mechanism of net SO42? transport and describe a novel regulation of Slc26a2 by an extracellular anion binding site and should help in further understanding aberrant SLC26A2 function in diastrophic dysplasia.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/d-Erd_Ix7Og" height="1" width="1"/>
Inactivating mutations in the breast cancer susceptibility gene BRCA2 cause gross chromosomal rearrangements. Chromosome structural instability in the absence of BRCA2 is thought to result from defective homology-directed DNA repair. Here, we show that BRCA2 links the fidelity of telomere maintenance with genetic integrity. Absence of BRCA2 resulted in signs of dysfunctional telomeres, such as telomere shortening, erosions, and end fusions in proliferating mouse fibroblasts. BRCA2 localized to the telomeres in S phase in an ATR-dependent manner, and its absence resulted in the accumulation of common fragile sites, particularly at the G-rich lagging strand, and increased the telomere sister chromatid exchange in unchallenged cells. The incidence of common fragile sites and telomere sister chromatid exchange increased markedly after treatment with replication inhibitors. Congruently, telomere-induced foci were frequently observed in the absence of Brca2, denoting activation of the DNA damage response and abnormal chromosome end joining. These telomere end fusions constituted a significant portion of chromosome aberrations in Brca2-deficient cells. Our results suggest that BRCA2 is required for telomere homeostasis and may be particularly important for the replication of G-rich telomeric lagging strands.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/EmzahpL78ZA" height="1" width="1"/>
It is well accepted that the Mdm2 ubiquitin ligase acts as a major factor in controlling p53 stability and activity in vivo. Although several E3 ligases have been reported to be involved in Mdm2-independent p53 degradation, the roles of these ligases in p53 regulation in vivo remain largely unknown. To elucidate the physiological role of the ubiquitin ligase ARF-BP1, we generated arf-bp1 mutant mice. We found that inactivation of arf-bp1 during embryonic development in mice resulted in p53 activation and embryonic lethality, but the mice with arf-bp1 deletion specifically in the pancreatic ?-cells (arf-bp1FL/Y/RIP-cre) were viable and displayed no obvious abnormality after birth. Interestingly, these mice showed dramatic loss of ?-cells as mice aged, and >50% of these mice died of severe diabetic symptoms before reaching 1 year of age. Notably, the diabetic phenotype of these mice was largely reversed by concomitant deletion of p53, and the life span of the mice was significantly extended (p53LFL/FL/arf-bp1FL/Y/RIP-cre). These findings underscore an important role of ARF-BP1 in maintaining ?-cell homeostasis in aging mice and reveal that the stability of p53 is critically regulated by ARF-BP1 in vivo.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/fkd4j-qF6Jg" height="1" width="1"/>
Phagocytosis occurs primarily through two main processes in macrophages: the Fc? receptor- and the integrin ?M?2-mediated processes. Complement C3bi-opsonized particles are known to be engulfed through integrin ?M?2-mediated process, which is regulated by RhoA GTPase. C3 toxin fused with Tat-peptide (Tat-C3 toxin), an inhibitor of the Rho GTPases, was shown to markedly inhibit the phagocytosis of serum (C3bi)-opsonized zymosans (SOZs). However, 8CPT-2Me-cAMP, an activator of exchange protein directly activated by cAMP (Epac, Rap1 guanine nucleotide exchange factor), restored the phagocytosis of the SOZs that was previously inhibited by the Tat-C3 toxin. In addition, a constitutively active form of Rap1 GTPase (CA-Rap1) also restored the phagocytosis that was previously reduced by a dominant negative form of RhoA GTPase (DN-RhoA). This suggests that Rap1 can replace the function of RhoA in the phagocytosis. Inversely, CA-RhoA rescued the phagocytosis that was suppressed by DN-Rap1. These findings suggest that both RhoA and Rap1 GTPases collectively regulate the phagocytosis of SOZs. In addition, filamentous actin was reduced by the Tat-C3 toxin, which was again restored by 8CPT-2Me-cAMP. Small interfering profilin suppressed the phagocytosis, suggesting that profilin is essential for the phagocytosis of SOZs. Furthermore, 8CPT-2Me-cAMP increased the co-immunoprecipitation of profilin with Rap1, whereas Tat-C3 toxin decreased that of profilin with RhoA. Co-immunoprecipitations of profilin with actin, Rap1, and RhoA GTPases were augmented in the presence of GTP?S rather than GDP. Therefore, we propose that both Rap1 and RhoA GTPases regulate the formation of filamentous actin through the interaction between actin and profilin, thereby collectively inducing the phagocytosis of SOZs in macrophages.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/LFjE46-nrXc" height="1" width="1"/>
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The wrong images were inserted into the last row of Fig. 1C. The corrected figure is shown below.
jbc;287/7/5181/FU1F1FU1<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/slZSPimGnB4" height="1" width="1"/>
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The source of IFIT2?/? mouse embryo fibroblasts (MEFs) was inadvertently left out in the original manuscript. IFIT2?/? MEFs were generated by the laboratory of Dr. Ganes Sen (Cleveland Clinic) and were a gift to Dr. Michael Diamond.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/6H_t_aIkzj0" height="1" width="1"/>
Expression of the ISG15 specific protease USP18 is highly induced by type I interferons. The two main functions of USP18, i.e. its enzymatic activity and down-regulation of type I interferon signaling, are well characterized. However, to date all functional studies focused on full-length USP18. Here, we report that translation of human USP18 is initiated by a rare start codon (CUG). Usage of this non-canonical initiation site with its weak translation initiation efficiency promotes expression of an N-terminal truncated isoform (USP18-sf). In addition, an internal ribosome entry site (IRES) located in the 5?-coding region of USP18 also contributes to translation of USP18-sf. Functionally, both isoforms exhibit enzymatic activity and interfere with type I interferon signaling. However, USP18-sf shows different subcellular distribution compared with the full-length protein and enhanced deISGylation activity in the nucleus. Taken together, we report the existence of an N-terminal truncated isoform of USP18, whose expression is controlled on translational level by two independent mechanisms providing translational flexibility as well as cell type-specific resistance to inhibition of cap-dependent translation.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/dS_7FBJkMjc" height="1" width="1"/>
Hypercholesterolemia is a well-known risk factor for cardiovascular disease. In the heart, activation of KACh mediates the vagal (parasympathetic) negative chronotropic effect on heart rate. Yet, the effect of cholesterol on KACh is unknown. Here we show that cholesterol plays a critical role in modulating KACh currents (IK,ACh) in atrial cardiomyocytes. Specifically, cholesterol enrichment of rabbit atrial cardiomyocytes led to enhanced channel activity while cholesterol depletion suppressed IK,ACh. Moreover, a high-cholesterol diet resulted in up to 3-fold increase in IK,ACh in rodents. In accordance, elevated currents were observed in Xenopus oocytes expressing the Kir3.1/Kir3.4 heteromer that underlies IK,ACh. Furthermore, our data suggest that cholesterol affects IK,ACh via a mechanism which is independent of both PI(4,5)P2 and G??. Interestingly, the effect of cholesterol on IK,ACh is opposite to its effect on IK1 in atrial myocytes. The latter are suppressed by cholesterol enrichment and by high-cholesterol diet, and facilitated following cholesterol depletion. These findings establish that cholesterol plays a critical role in modulating IK,ACh in atrial cardiomyocytes via a mechanism independent of the channel's major modulators.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/R7Rgmm1t2dM" height="1" width="1"/>
The biosynthesis of chlorophyll, an essential cofactor for photosynthesis, requires the ATP-dependent insertion of Mg2+ into protoporphyrin IX catalyzed by the multisubunit enzyme magnesium chelatase. This enzyme complex consists of the I subunit, an ATPase that forms a complex with the D subunit, and an H subunit that binds both the protoporphyrin substrate and the magnesium protoporphyrin product. In this study we used electron microscopy and small-angle x-ray scattering to investigate the structure of the magnesium chelatase H subunit, ChlH, from the thermophilic cyanobacterium Thermosynechococcus elongatus. Single particle reconstruction of negatively stained apo-ChlH and Chl-porphyrin proteins was used to reconstitute three-dimensional structures to a resolution of ?30 Å. ChlH is a large, 148-kDa protein of 1326 residues, forming a cage-like assembly comprising the majority of the structure, attached to a globular N-terminal domain of ?16 kDa by a narrow linker region. This N-terminal domain is adjacent to a 5 nm-diameter opening in the structure that allows access to a cavity. Small-angle x-ray scattering analysis of ChlH, performed on soluble, catalytically active ChlH, verifies the presence of two domains and their relative sizes. Our results provide a basis for the multiple regulatory and catalytic functions of ChlH of oxygenic photosynthetic organisms and for a chaperoning function that sequesters the enzyme-bound magnesium protoporphyrin product prior to its delivery to the next enzyme in the chlorophyll biosynthetic pathway, magnesium protoporphyrin methyltransferase.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/CU2OuOVUF7A" height="1" width="1"/>
In physiological conditions, both ?-amyloid precursor protein (?APP) and cellular prion (PrPc) undergo similar disintegrin-mediated ?-secretase cleavage yielding N-terminal secreted products referred to as soluble amyloid precursor protein-? (sAPP?) and N1, respectively. We recently demonstrated that N1 displays neuroprotective properties by reducing p53-dependent cell death both in vitro and in vivo. In this study, we examined the potential of N1 as a neuroprotector against amyloid ? (A?)-mediated toxicity. We first show that both recombinant sAPP? and N1, but not its inactive parent fragment N2, reduce staurosporine-stimulated caspase-3 activation and TUNEL-positive cell death by lowering p53 promoter transactivation and activity in human cells. We demonstrate that N1 also lowers toxicity, cell death, and p53 pathway exacerbation triggered by Swedish mutated ?APP overexpression in human cells. We designed a CHO cell line overexpressing the London mutated ?APP (APPLDN) that yields A? oligomers. N1 protected primary cultured neurons against toxicity and cell death triggered by oligomer-enriched APPLDN-derived conditioned medium. Finally, we establish that N1 also protects neurons against oligomers extracted from Alzheimer disease-affected brain tissues. Overall, our data indicate that a cellular prion catabolite could interfere with A?-associated toxicity and that its production could be seen as a cellular protective mechanism aimed at compensating for an sAPP? deficit taking place at the early asymptomatic phase of Alzheimer disease.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/46nTnbOztkw" height="1" width="1"/>
Phosphorylation is an important posttranslational modification of proteins in living cells and primarily serves regulatory purposes. Several methods were employed for isolating phosphopeptides from proteolytically digested plasma membranes of Arabidopsis thaliana. After a mass spectrometric analysis of the resulting peptides we could identify 10 different phosphorylation sites in plasma membrane H+-ATPases AHA1, AHA2, AHA3, and AHA4/11, five of which have not been reported before, bringing the total number of phosphosites up to 11, which is substantially higher than reported so far for any other P-type ATPase. Phosphosites were almost exclusively (9 of 10) in the terminal regulatory domains of the pumps. The AHA2 isoform was subsequently expressed in the yeast Saccharomyces cerevisiae. The plant protein was phosphorylated at multiple sites in yeast, and surprisingly, seven of nine of the phosphosites identified in AHA2 were identical in the plant and fungal systems even though none of the target sequences in AHA2 show homology to proteins of the fungal host. These findings suggest an unexpected accessibility of the terminal regulatory domain of plasma membrane H+-ATPase to protein kinase action.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/XZdQkThNbkc" height="1" width="1"/>
Bile acid-like molecules named dafachronic acids (DAs) control the dauer formation program in Caenorhabditis elegans through the nuclear receptor DAF-12. This mechanism is conserved in parasitic nematodes to regulate their dauer-like infective larval stage, and as such, the DAF-12 ligand binding domain has been identified as an important therapeutic target in human parasitic hookworm species that infect more than 600 million people worldwide. Here, we report two x-ray crystal structures of the hookworm Ancylostoma ceylanicum DAF-12 ligand binding domain in complex with DA and cholestenoic acid (a bile acid-like metabolite), respectively. Structure analysis and functional studies reveal key residues responsible for species-specific ligand responses of DAF-12. Furthermore, DA binds to DAF-12 mechanistically and is structurally similar to bile acids binding to the mammalian bile acid receptor farnesoid X receptor. Activation of DAF-12 by cholestenoic acid and the cholestenoic acid complex structure suggest that bile acid-like signaling pathways have been conserved in nematodes and mammals. Together, these results reveal the molecular mechanism for the interplay between parasite and host, provide a structural framework for DAF-12 as a promising target in treating nematode parasitism, and provide insight into the evolution of gut parasite hormone-signaling pathways.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/vjwJdfj8xCg" height="1" width="1"/>
DNA double strand breaks (DSB) are repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Recent genetic data in yeast shows that the choice between these two pathways for the repair of DSBs is via competition between the NHEJ protein, Ku, and the HR protein, Mre11/Rad50/Xrs2 (MRX) complex. To study the interrelationship between human Ku and Mre11 or Mre11/Rad50 (MR), we established an in vitro DNA end resection system using a forked model dsDNA substrate and purified human Ku70/80, Mre11, Mre11/Rad50, and exonuclease 1 (Exo1). Our study shows that the addition of Ku70/80 blocks Exo1-mediated DNA end resection of the forked dsDNA substrate. Although human Mre11 and MR bind to the forked double strand DNA, they could not compete with Ku for DNA ends or actively mediate the displacement of Ku from the DNA end either physically or via its exonuclease or endonuclease activity. Our in vitro studies show that Ku can block DNA resection and suggest that Ku must be actively displaced for DNA end processing to occur and is more complicated than the competition model established in yeast.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/YY_JnqYUr1M" height="1" width="1"/>
Apicomplexan parasites are responsible for high impact human diseases such as malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular pathogens are dependent on both de novo lipid biosynthesis as well as the uptake of host lipids for biogenesis of parasite membranes. Genome annotations and biochemical studies indicate that apicomplexan parasites can synthesize fatty acids via a number of different biosynthetic pathways that are differentially compartmentalized. However, the relative contribution of each of these biosynthetic pathways to total fatty acid composition of intracellular parasite stages remains poorly defined. Here, we use a combination of genetic, biochemical, and metabolomic approaches to delineate the contribution of fatty acid biosynthetic pathways in Toxoplasma gondii. Metabolic labeling studies with [13C]glucose showed that intracellular tachyzoites synthesized a range of long and very long chain fatty acids (C14:0–26:1). Genetic disruption of the apicoplast-localized type II fatty-acid synthase resulted in greatly reduced synthesis of saturated fatty acids up to 18 carbons long. Ablation of type II fatty-acid synthase activity resulted in reduced intracellular growth that was partially restored by addition of long chain fatty acids. In contrast, synthesis of very long chain fatty acids was primarily dependent on a fatty acid elongation system comprising three elongases, two reductases, and a dehydratase that were localized to the endoplasmic reticulum. The function of these enzymes was confirmed by heterologous expression in yeast. This elongase pathway appears to have a unique role in generating very long unsaturated fatty acids (C26:1) that cannot be salvaged from the host.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/isugQdqLwbo" height="1" width="1"/>
Compromised clearance of all-trans-retinal (atRAL), a component of the retinoid cycle, increases the susceptibility of mouse retina to acute light-induced photoreceptor degeneration. Abca4?/?Rdh8?/? mice featuring defective atRAL clearance were used to examine the one or more underlying molecular mechanisms, because exposure to intense light causes severe photoreceptor degeneration in these animals. Here we report that bright light exposure of Abca4?/?Rdh8?/? mice increased atRAL levels in the retina that induced rapid NADPH oxidase-mediated overproduction of intracellular reactive oxygen species (ROS). Moreover, such ROS generation was inhibited by blocking phospholipase C and inositol 1,4,5-trisphosphate-induced Ca2+ release, indicating that activation occurs upstream of NADPH oxidase-mediated ROS generation. Because multiple upstream G protein-coupled receptors can activate phospholipase C, we then tested the effects of antagonists of serotonin 2A (5-HT2AR) and M3-muscarinic (M3R) receptors and found they both protected Abca4?/?Rdh8?/? mouse retinas from light-induced degeneration. Thus, a cascade of signaling events appears to mediate the toxicity of atRAL in light-induced photoreceptor degeneration of Abca4?/?Rdh8?/? mice. A similar mechanism may be operative in human Stargardt disease and age-related macular degeneration.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/BMVgfUIs5F0" height="1" width="1"/>
Caenorhabditis elegans and human HRG-1-related proteins are conserved, membrane-bound permeases that bind and translocate heme in metazoan cells via a currently uncharacterized mechanism. Here, we show that cellular import of heme by HRG-1-related proteins from worms and humans requires strategically located amino acids that are topologically conserved across species. We exploit a heme synthesis-defective Saccharomyces cerevisiae mutant to model the heme auxotrophy of C. elegans and demonstrate that, under heme-deplete conditions, the endosomal CeHRG-1 requires both a specific histidine in the predicted second transmembrane domain (TMD2) and the FARKY motif in the C terminus tail for heme transport. By contrast, the plasma membrane CeHRG-4 transports heme by utilizing a histidine in the exoplasmic (E2) loop and the FARKY motif. Optimal activity under heme-limiting conditions, however, requires histidine in the E2 loop of CeHRG-1 and tyrosine in TMD2 of CeHRG-4. An analogous system exists in humans, because mutation of the synonymous histidine in TMD2 of hHRG-1 eliminates heme transport activity, implying an evolutionary conserved heme transport mechanism that predates vertebrate origins. Our results support a model in which heme is translocated across membranes facilitated by conserved amino acids positioned on the exoplasmic, cytoplasmic, and transmembrane regions of HRG-1-related proteins. These findings may provide a framework for understanding the structural basis of heme transport in eukaryotes and human parasites, which rely on host heme for survival.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/e30KfJsQ808" height="1" width="1"/>
The proton-coupled folate transporter (PCFT; SLC46A1) is a proton-folate symporter that is abundantly expressed in solid tumors and normal tissues, such as duodenum. The acidic pH optimum for PCFT is relevant to intestinal absorption of folates and could afford a means of selectively targeting tumors with novel cytotoxic antifolates. PCFT is a member of the major facilitator superfamily of transporters. Because major facilitator superfamily members exist as homo-oligomers, we tested this for PCFT because such structures could be significant to PCFT mechanism and regulation. By transiently expressing PCFT in reduced folate carrier- and PCFT-null HeLa (R1-11) cells and chemical cross-linking with 1,1-methanediyl bismethanethiosulfonate and Western blotting, PCFT species with molecular masses approximating those of the PCFT dimer and higher order oligomers were detected. Blue native polyacrylamide gel electrophoresis identified PCFT dimer, trimer, and tetramer forms. PCFT monomers with hemagglutinin and His10 epitope tags were co-expressed in R1-11 cells, solubilized, and bound to nickel affinity columns, establishing their physical associations. Co-expressing YPet and ECFP*-tagged PCFT monomers enabled transport and fluorescence resonance energy transfer in plasma membranes of R1-11 cells. Combined wild-type (WT) and inactive mutant P425R PCFTs were targeted to the cell surface by surface biotinylation/Western blots and confocal microscopy and functionally exhibited a “dominant-positive” phenotype, implying positive cooperativity between PCFT monomers and functional rescue of mutant by WT PCFT. Our results demonstrate the existence of PCFT homo-oligomers and imply their functional and regulatory impact. Better understanding of these higher order PCFT structures may lead to therapeutic applications related to folate uptake in hereditary folate malabsorption, and delivery of PCFT-targeted chemotherapy drugs for cancer.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/P0PnShXHEqI" height="1" width="1"/>
Myocardial ischemia is characterized by reduced blood flow to cardiomyocytes, which can lead to acidosis. Acidosis decreases the calcium sensitivity and contractile efficiency of cardiac muscle. By contrast, skeletal and neonatal muscles are much less sensitive to changes in pH. The pH sensitivity of cardiac muscle can be reduced by replacing cardiac troponin I with its skeletal or neonatal counterparts. The isoform-specific response of troponin I is dictated by a single histidine, which is replaced by an alanine in cardiac troponin I. The decreased pH sensitivity may stem from the protonation of this histidine at low pH, which would promote the formation of electrostatic interactions with negatively charged residues on troponin C. In this study, we measured acid dissociation constants of glutamate residues on troponin C and of histidine on skeletal troponin I (His-130). The results indicate that Glu-19 comes in close contact with an ionizable group that has a pKa of ?6.7 when it is in complex with skeletal troponin I but not when it is bound to cardiac troponin I. The pKa of Glu-19 is decreased when troponin C is bound to skeletal troponin I and the pKa of His-130 is shifted upward. These results strongly suggest that these residues form an electrostatic interaction. Furthermore, we found that skeletal troponin I bound to troponin C tighter at pH 6.1 than at pH 7.5. The data presented here provide insights into the molecular mechanism for the pH sensitivity of different muscle types.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/DzOmw3sYEz4" height="1" width="1"/>
Unfolding of the gene expression program that converts precursor cells to their terminally differentiated counterparts is critically dependent on the nucleosome-remodeling activity of the mammalian SWI/SNF complex. The complex can be powered by either of two alternative ATPases, BRM or BRG1. BRG1 is critical for development and the activation of tissue specific genes and is found in two major stable configurations. The complex of BRG1-associated factors termed BAF is the originally characterized form of mammalian SWI/SNF. A more recently recognized configuration shares many of the same subunits but is termed PBAF in recognition of a unique subunit, the polybromo protein (PBRM1). Two other unique subunits, BRD7 and ARID2, are also diagnostic of PBAF. PBAF plays an essential role in development, apparent from the embryonic lethality of Pbmr1-null mice, but very little is known about the role of PBAF, or its signature subunits, in tissue-specific gene expression in individual differentiation programs. Osteoblast differentiation is an attractive model for tissue-specific gene expression because the process is highly regulated and remains tightly synchronized over a period of several weeks. This model was used here, with a stable shRNA-mediated depletion approach, to examine the role of the signature PBAF subunit, ARID2, during differentiation. This analysis identifies a critical role for ARID2-containing complexes in promoting osteoblast differentiation and supports a view that the PBAF subset of SWI/SNF contributes importantly to maintaining cellular identity and activating tissue-specific gene expression.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/Rwc999X3h00" height="1" width="1"/>
Initiation, a major rate-limiting step of host protein translation, is a critical target in many viral infections. Chronic hepatitis C virus (HCV) infection results in hepatocellular carcinoma. Translation initiation, up-regulated in many cancers, plays a critical role in tumorigenesis. mTOR is a major regulator of host protein translation. Even though activation of PI3K-AKT-mTOR by HCV non-structural protein 5A (NS5A) is known, not much is understood about the regulation of host translation initiation by this virus. Here for the first time we show that HCV up-regulates host cap-dependent translation machinery in Huh7.5 cells through simultaneous activation of mTORC1 and eukaryotic translation initiation factor 4E (eIF4E) by NS5A. NS5A, interestingly, overexpressed and subsequently hyperphosphorylated 4EBP1. NS5A phosphorylated eIF4E through the p38 MAPK-MNK pathway. Both HCV infection and NS5A expression augmented eIF4F complex assembly, an indicator of cap-dependent translation efficiency. Global translation, however, was not altered by HCV NS5A. 4EBP1 phosphorylation, but not that of S6K1, was uniquely resistant to rapamycin in NS5A-Huh7.5 cells, indicative of an alternate phosphorylation mechanism of 4EBP1. Resistance of Ser-473, but not Thr-308, phosphorylation of AKT to PI3K inhibitors suggested an activation of mTORC2 by NS5A. NS5A associated with eIF4F complex and polysomes, suggesting its active involvement in host translation. This is the first report that implicates an HCV protein in the up-regulation of host translation initiation apparatus through concomitant regulation of multiple pathways. Because both mTORC1 activation and eIF4E phosphorylation are involved in tumorigenesis, we propose that their simultaneous activation by NS5A might contribute significantly to the development of hepatocellular carcinoma.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/CfVtd0YKiiU" height="1" width="1"/>
The important role of G protein-coupled receptor homo/heteromerization in receptor folding, maturation, trafficking, and cell surface expression has become increasingly evident. Here we investigated whether the human ?-opioid receptor (h?OR) Cys-27 variant that shows inherent compromised maturation has an effect on the behavior of the more common Phe-27 variant in the early secretory pathway. We demonstrate that h?OR-Cys-27 acts in a dominant negative manner and impairs cell surface delivery of the co-expressed h?OR-Phe-27 and impairs conversion of precursors to the mature form. This was demonstrated by metabolic labeling, Western blotting, flow cytometry, and confocal microscopy in HEK293 and human SH-SY5Y neuroblastoma cells using differentially epitope-tagged variants. The h?OR-Phe-27 precursors that were redirected to the endoplasmic reticulum-associated degradation were, however, rescued by a pharmacological chaperone, the opioid antagonist naltrexone. Co-immunoprecipitation of metabolically labeled variants revealed that both endoplasmic reticulum-localized precursors and mature receptors exist as homo/heteromers. The existence of homo/heteromers was confirmed in living cells by bioluminescence resonance energy transfer measurements, showing that the variants have a similar propensity to form homo/heteromers. By forming both homomers and heteromers, the h?OR-Cys-27 variant may thus regulate the levels of receptors at the cell surface, possibly leading to altered responsiveness to opioid ligands in individuals carrying the Cys-27 variant.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/HjDWG9xvqqY" height="1" width="1"/>
?-Aminobutyric acid (GABA) release from inhibitory interneurons located within the cerebellar cortex limits the extent of neuronal excitation in part through activation of metabotropic GABAB receptors. Stimulation of these receptors triggers a number of downstream signaling events, including activation of GIRK channels by the G?? dimer resulting in membrane hyperpolarization and inhibition of neurotransmitter release from presynaptic sites. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key regulator of GABABR signaling in cerebellum. RGS6 is enriched in the granule cell layer of the cerebellum along with neuronal GIRK channel subunits 1 and 2 where RGS6 forms a complex with known binding partners G?5 and R7BP. Mice lacking RGS6 exhibit abnormal gait and ataxia characterized by impaired rotarod performance improved by treatment with a GABABR antagonist. RGS6?/? mice administered baclofen also showed exaggerated motor coordination deficits compared with their wild-type counterparts. Isolated cerebellar neurons natively expressed RGS6, GABABR, and GIRK channel subunits, and cerebellar granule neurons from RGS6?/? mice showed a significant delay in the deactivation kinetics of baclofen-induced GIRK channel currents. These results establish RGS6 as a key component of GABABR signaling and represent the first demonstration of an essential role for modulatory actions of RGS proteins in adult cerebellum. Dysregulation of RGS6 expression in human patients could potentially contribute to loss of motor coordination and, thus, pharmacological manipulation of RGS6 levels might represent a viable means to treat patients with ataxias of cerebellar origin.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/XzOXBIuXfMs" height="1" width="1"/>
Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (QD) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the QD site (MSQD) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with 1H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon ? carrying the methyl group, which is ascribed to a strong asymmetry of the MSQD binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ?1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the N? of the His-66 residue, an axial ligand of the distal heme bD. Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the QD site are discussed, in light of the unusually high thermodynamic stability of MSQD.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/pTZHXj6itxM" height="1" width="1"/>
Serine hydroxymethyltransferase 1 (SHMT1) expression limits rates of de novo dTMP synthesis in the nucleus. Here we report that SHMT1 is ubiquitinated at the small ubiquitin-like modifier (SUMO) consensus motif and that ubiquitination at that site is required for SHMT1 degradation. SHMT1 protein levels are cell cycle-regulated, and Ub-SHMT1 levels are lowest at S phase when SHMT1 undergoes SUMO modification and nuclear transport. Mutation of the SUMO consensus motif increases SHMT1 stability. SHMT1 interacts with components of the proteasome in both the nucleus and cytoplasm, indicating that degradation occurs in both compartments. Ubc13-mediated ubiquitination is required for SHMT1 nuclear export and increases stability of SHMT1 within the nucleus, whereas Ubc9-mediated modification with Sumo2/3 is involved in nuclear degradation. These data demonstrate that SUMO and ubiquitin modification of SHMT1 occurs on the same lysine residue and determine the localization and accumulation of SHMT1 in the nucleus.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/nWwT9w5VzgU" height="1" width="1"/>
BubR1 functions as a crucial component that monitors proper chromosome congression and mitotic timing during cell division. We investigated molecular regulation of BubR1 and found that BubR1 was modified by an unknown post-translation mechanism during the cell cycle, resulting in a significant mobility shift on denaturing gels. We termed it BubR1-M as the nature of modification was not characterized. Extended (>24 h) treatment of HeLa cells with a microtubule disrupting agent including nocodazole and taxol or release of mitotic shake-off cells into fresh medium induced BubR1-M. BubR1-M was derived from neither phosphorylation nor acetylation. Ectopic expression coupled with pulling down analyses showed that BubR1-M was derived from SUMO modification. Mutation analysis revealed that lysine 250 was a crucial site for sumoylation. Significantly, compared with the wild-type control, ectopic expression of a sumoylation-deficient mutant of BubR1 induced chromosomal missegregation and mitotic delay. Combined, our study identifies a new type of post-translational modification that is essential for BubR1 function during mitosis.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/lPs3SAutfSo" height="1" width="1"/>
As extracellular proteins age, they undergo and accumulate nonenzymatic post-translational modifications that cannot be repaired. We hypothesized that these could be used to systemically monitor loss of extracellular matrix due to chronic arthritic diseases such as osteoarthritis (OA). To test this, we predicted sites of deamidation in cartilage oligomeric matrix protein (COMP) and confirmed, by mass spectroscopy, the presence of deamidated (Asp64) and native (Asn64) COMP epitopes (mean 0.95% deamidated COMP (D-COMP) relative to native COMP) in cartilage. An Asp64, D-COMP-specific ELISA was developed using a newly created monoclonal antibody 6-1A12. In a joint replacement study, serum D-COMP (p = 0.017), but not total COMP (p = 0.5), declined significantly after replacement demonstrating a joint tissue source for D-COMP. In analyses of 450 participants from the Johnston County Osteoarthritis Project controlled for age, gender, and race, D-COMP was associated with radiographic hip (p < 0.0001) but not knee (p = 0.95) OA severity. In contrast, total COMP was associated with radiographic knee (p < 0.0001) but not hip (p = 0.47) OA severity. D-COMP was higher in soluble proteins extracted from hip cartilage proximal to OA lesions compared with remote from lesions (p = 0.007) or lesional and remote OA knee (p < 0.01) cartilage. Total COMP in cartilage did not vary by joint site or proximity to the lesion. This study demonstrates the presence of D-COMP in articular cartilage and the systemic circulation, and to our knowledge, it is the first biomarker to show specificity for a particular joint site. We believe that enrichment of deamidated epitope in hip OA cartilage indicates a lesser repair response of hip OA compared with knee OA cartilage.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/tBDkaP1BEfw" height="1" width="1"/>
Several lines of evidence suggest that HAb18G/CD147 interacts with the integrin variants ?3?1 and ?6?1. However, the mechanism of the interaction remains largely unknown. In this study, mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, was used to study the CD147-integrin ?1 subunit interaction. CD147 in human hepatocellular carcinoma (HCC) cells was interfered with by small hairpin RNA. Nude mouse xenograft model and metastatic model of HCC were used to detect the role of CD147 in carcinogenesis and metastasis. We found that the extracellular membrane-proximal domain of HAb18G/CD147 (I-type domain) binds at the metal ion-dependent adhesion site in the ?A domain of the integrin ?1 subunit, and Asp179 in the I-type domain of HAb18G/CD147 plays an important role in the interaction. The levels of the proteins that act downstream of integrin, including focal adhesion kinase (FAK) and phospho-FAK, were decreased, and the cytoskeletal structures of HCC cells were rearranged bearing the HAb18G/CD147 deletion. Simultaneously, the migration and invasion capacities, secretion of matrix metalloproteinases, colony formation rate in vitro, and tumor growth and metastatic potential in vivo were decreased. These results indicate that the interaction of HAb18G/CD147 extracellular I-type domain with the integrin ?1 metal ion-dependent adhesion site motif activates the downstream FAK signaling pathway, subsequently enhancing the malignant properties of HCC cells.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/SlD4_F8B9CU" height="1" width="1"/>
Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/PPdAexYKHcs" height="1" width="1"/>
LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca2+. Recent crystal structures have been obtained for the protein in the apo- and Ca2+-bound forms. In this work, we produced three LipL32 mutants (D163–168A, Q67A, and S247A) and evaluated their ability to interact with Ca2+ and with ECM glycoproteins and human plasminogen. The D163–168A mutant modifies aspartate residues involved in Ca2+ binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca2+ affinity as the wild-type protein. We then evaluated if Ca2+ binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163–168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca2+ ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/allkOY_zaRA" height="1" width="1"/>
Soluble proteins are enriched in the endoplasmic reticulum (ER) by retrograde transport from the Golgi that is mediated by the KDEL receptors. In addition to the classic carboxyl-terminal KDEL motif, a variety of sequence variants are also capable of receptor binding that result in ER localization. Although different ER localization signals that exhibit varying affinities for the KDEL receptors exist, whether there are functional implications was unknown. The present study determines whether AGR2 requires a specific ER localization signal to be functionally active. AGR2 is expressed in most human adenocarcinomas and serves a role in promoting growth and the transformed phenotype. Using two different cell lines in which AGR2 induces expression of either the EGFR ligand amphiregulin or the transcription factor CDX2, only the highly conserved wild-type carboxyl-terminal KTEL motif results in the appropriate outcome. Deletion of the KTEL motif results in AGR2 secretion and loss of AGR2 function. AGR2 function is also lost when ER residence is achieved with a carboxyl-terminal KDEL or KSEL instead of a KTEL motif. Thus variations in ER localization sequences may serve a specific functional role, and in the case of AGR2, this role is served specifically by KTEL.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/224_Qke6Wno" height="1" width="1"/>
Regulation of the assembly and function of G-protein heterotrimers (G?·GDP/G??) is a complex process involving the participation of many accessory proteins. One of these regulators, GPSM3, is a member of a family of proteins containing one or more copies of a small regulatory motif known as the GoLoco (or GPR) motif. Although GPSM3 is known to bind G?i·GDP subunits via its GoLoco motifs, here we report that GPSM3 also interacts with the G? subunits G?1 to G?4, independent of G? or G?·GDP subunit interactions. Bimolecular fluorescence complementation studies suggest that the G?-GPSM3 complex is formed at, and transits through, the Golgi apparatus and also exists as a soluble complex in the cytoplasm. GPSM3 and G? co-localize endogenously in THP-1 cells at the plasma membrane and in a juxtanuclear compartment. We provide evidence that GPSM3 increases G? stability until formation of the G?? dimer, including association of the G?-GPSM3 complex with phosducin-like protein PhLP and T-complex protein 1 subunit eta (CCT7), two known chaperones of neosynthesized G? subunits. The G? interaction site within GPSM3 was mapped to a leucine-rich region proximal to the N-terminal side of its first GoLoco motif. Both G? and G?i·GDP binding events are required for GPSM3 activity in inhibiting phospholipase-C? activation. GPSM3 is also shown in THP-1 cells to be important for Akt activation, a known G??-dependent pathway. Discovery of a G?/GPSM3 interaction, independent of G?·GDP and G? involvement, adds to the combinatorial complexity of the role of GPSM3 in heterotrimeric G-protein regulation.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/sg-6zh1Eii8" height="1" width="1"/>
Fibrin (Fn) enhances plasminogen (Pg) activation by tissue-type plasminogen activator (tPA) by serving as a template onto which Pg and tPA assemble. To explore the contribution of the Pg/Fn interaction to Fn cofactor activity, Pg variants were generated and their affinities for Fn were determined using surface plasmon resonance (SPR). Glu-Pg, Lys-Pg (des(1–77)), and Mini-Pg (lacking kringles 1–4) bound Fn with Kd values of 3.1, 0.21, and 24.5 ?m, respectively, whereas Micro-Pg (lacking all kringles) did not bind. The kinetics of activation of the Pg variants by tPA were then examined in the absence or presence of Fn. Whereas Fn had no effect on Micro-Pg activation, the catalytic efficiencies of Glu-Pg, Lys-Pg, and Mini-Pg activation in the presence of Fn were 300- to 600-fold higher than in its absence. The retention of Fn cofactor activity with Mini-Pg, which has low affinity for Fn, suggests that Mini-Pg binds the tPA-Fn complex more tightly than tPA alone. To explore this possibility, SPR was used to examine the interaction of Mini-Pg with Fn in the absence or presence of tPA. There was 50% more Mini-Pg binding to Fn in the presence of tPA than in its absence, suggesting that formation of the tPA-Fn complex exposes a cryptic site that binds Mini-Pg. Thus, our data (a) indicate that high affinity binding of Pg to Fn is not essential for Fn cofactor activity, and (b) suggest that kringle 5 localizes and stabilizes Pg within the tPA-Fn complex and contributes to its efficient activation.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/lhG4EHM6cr8" height="1" width="1"/>
The RanBP2 nucleoporin contains an internal repeat domain (IR1-M-IR2) that catalyzes E3 ligase activity and forms a stable complex with SUMO-modified RanGAP1 and UBC9 at the nuclear pore complex. RanBP2 exhibits specificity for SUMO1 as RanGAP1-SUMO1/UBC9 forms a more stable complex with RanBP2 compared with RanGAP1-SUMO2 that results in greater protection of RanGAP-SUMO1 from proteases. The IR1-M-IR2 SUMO E3 ligase activity also shows a similar preference for SUMO1. We utilized deletions and domain swap constructs in protease protection assays and automodification assays to define RanBP2 domains responsible for RanGAP1-SUMO1 protection and SUMO1-specific E3 ligase activity. Our data suggest that elements in both IR1 and IR2 exhibit specificity for SUMO1. IR1 protects RanGAP1-SUMO1/UBC9 and functions as the primary E3 ligase of RanBP2, whereas IR2 retains the ability to interact with SUMO1 to promote SUMO1-specific E3 ligase activity. To determine the structural basis for SUMO1 specificity, a hybrid IR1 construct and IR1 were used to determine three new structures for complexes containing UBC9 with RanGAP1-SUMO1/2. These structures show more extensive contacts among SUMO, UBC9, and RanBP2 in complexes containing SUMO1 compared with SUMO2 and suggest that differences in SUMO specificity may be achieved through these subtle conformational differences.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/eR0Z53Q92IE" height="1" width="1"/>
Filopodia are dynamic actin-rich cell surface protrusions involved in cell migration, axon guidance, and wound healing. The RhoGTPase Cdc42 generates filopodia via IRSp53, a multidomain protein that links the processes of plasma membrane deformation and actin dynamics required for their formation in mammalian cells. The Src homology 3 domain of IRSp53 binds to the actin regulators Mena, Eps8, WAVE1, WAVE2, mDia1, and mDia2. We show that mDia1 and WAVE2 synergize with IRSp53 to form filopodia. IRSp53 also interacts directly with these two proteins within filopodia, as observed in acceptor photobleaching FRET studies. Measurement of filopodium formation by time-lapse imaging of live cells also revealed that depleting neuronal cells of either mDia1 or WAVE2 protein decreases the ability of IRSp53 to induce filopodia. In contrast, IRSp53 does not appear to partner WAVE1 or mDia2 to give rise to these structures. In addition, although all three isoforms of mDia are capable of inducing filopodia, IRSp53 requires only mDia1 to do so. These findings suggest that mDia1 and WAVE2 are important Src homology 3 domain partners of IRSp53 in forming filopodia.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/jQpKvtor6IQ" height="1" width="1"/>
The proinflammatory cytokines IL-17A and IL-17F are primarily produced by Th17 lymphocytes. Both are involved in host defense mechanisms against bacterial and fungal pathogens and contribute to the development of various autoimmune diseases. T lymphocytes from patients with systemic lupus erythematosus (SLE) display increased expression of transcription factor cAMP-responsive element modulator ? (CREM?), which has been documented to account for aberrant T cell function and contributes to the pathogenesis of SLE. Here, we provide evidence that IL-17F expression is reduced in SLE T cells. We demonstrate that CREM? binds to a yet unidentified CRE site within the proximal promoter. This results in reduced IL-17F expression in SLE T lymphocytes and is independent of activating epigenetic patterns (increased histone H3 Lys-18 acetylation, reduced histone H3 Lys-27 trimethylation, and CpG-DNA demethylation). Forced CREM? expression in human T lymphocytes results in reduced IL-17F expression. Our findings demonstrate extended involvement of CREM? in cytokine dysregulation in SLE by contributing to a disrupted balance between IL-17A and IL-17F. An increased IL-17A/IL-17F ratio may aggravate the proinflammatory phenotype of SLE.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/6qvygnvPFyE" height="1" width="1"/>
Regenerating islet-derived 1? (Reg-1?)/lithostathine, a member of a family of secreted proteins containing a C-type lectin domain, is expressed in various organs and plays a role in proliferation, differentiation, inflammation, and carcinogenesis of cells of the digestive system. We previously reported that Reg-1? is overexpressed during the very early stages of Alzheimer disease, and Reg-1? deposits were detected in the brain of patients with Alzheimer disease. However, the physiological function of Reg-1? in neural cells remains unknown. Here, we show that Reg-1? is expressed in neuronal cell lines (PC12 and Neuro-2a) and in rat primary hippocampal neurons (E17.5). Reg-1? is mainly localized around the nucleus and at the membrane of cell bodies and neurites. Transient overexpression of Reg-1? or addition of recombinant Reg-1? significantly increases the number of cells with longer neurites by stimulating neurite outgrowth. These effects are abolished upon down-regulation of Reg-1? by siRNA and following inhibition of secreted Reg-1? by antibodies. Moreover, Reg-1? colocalizes with exostosin tumor-like 3 (EXTL3), its putative receptor, at the membrane of these cells. Overexpression of EXTL3 increases the effect of recombinant Reg-1? on neurite outgrowth, and Reg-1? is not effective when EXTL3 overexpression is down-regulated by shRNA. Our findings indicate that Reg-1? regulates neurite outgrowth and suggest that this effect is mediated by its receptor EXTL3.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/eImjDMuKshE" height="1" width="1"/>
Channelrhodopsin-2 (ChR2) is a light-gated cation channel widely used as a biotechnological tool to control membrane depolarization in various cell types and tissues. Although several ChR2 variants with modified properties have been generated, the structural determinants of the protein function are largely unresolved. We used bioinformatic modeling of the ChR2 structure to identify the putative cationic pathway within the channel, which is formed by a system of inner cavities that are uniquely present in this microbial rhodopsin. Site-directed mutagenesis combined with patch clamp analysis in HeLa cells was used to determine key residues involved in ChR2 conductance and selectivity. Among them, Gln-56 is important for ion conductance, whereas Ser-63, Thr-250, and Asn-258 are previously unrecognized residues involved in ion selectivity and photocurrent kinetics. This study widens the current structural information on ChR2 and can assist in the design of new improved variants for specific biological applications.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/R9RhoPG8nd4" height="1" width="1"/>
l-Cysteine desulfurases provide sulfur to several metabolic pathways in the form of persulfides on specific cysteine residues of an acceptor protein for the eventual incorporation of sulfur into an end product. IscS is one of the three Escherichia coli l-cysteine desulfurases. It interacts with FdhD, a protein essential for the activity of formate dehydrogenases (FDHs), which are iron/molybdenum/selenium-containing enzymes. Here, we address the role played by this interaction in the activity of FDH-H (FdhF) in E. coli. The interaction of IscS with FdhD results in a sulfur transfer between IscS and FdhD in the form of persulfides. Substitution of the strictly conserved residue Cys-121 of FdhD impairs both sulfur transfer from IscS to FdhD and FdhF activity. Furthermore, inactive FdhF produced in the absence of FdhD contains both metal centers, albeit the molybdenum cofactor is at a reduced level. Finally, FdhF activity is sulfur-dependent, as it shows reversible sensitivity to cyanide treatment. Conclusively, FdhD is a sulfurtransferase between IscS and FdhF and is thereby essential to yield FDH activity.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/D2TSrKcG3oo" height="1" width="1"/>
Notch1 receptor functions as a critical controller of cell fate decisions and also as a key regulator of cell growth, differentiation, and proliferation in invertebrates and vertebrates. In this study, we have demonstrated that the adaptor protein Fe65 attenuates Notch1 signaling via the accelerated degradation of the membrane-tethered Notch1 in the cytoplasm. Fe65 also suppresses Notch1 transcriptional activity via the dissociation of the Notch1-IC-recombining binding protein suppressor of hairless (RBP)-Jk complex within the nucleus. Fe65 is capable of forming a trimeric complex with Itch and membrane-tethered Notch1, and Fe65 enhances the protein degradation of membrane-tethered Notch1 via an Itch-dependent proteasomal pathway. Collectively, our results demonstrate that Fe65 carries out different functions depending on its location in the regulation of Notch1 signaling.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/AD708HdivNQ" height="1" width="1"/>
PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with ?4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Ac? can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Ac?2. Higher levels of the PP2Ac?2 mRNA, equivalent to the level of the longer PP2Ac? mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Ac?2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Ac?2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the ?4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, ?4 out-competes PME-1 and LCMT-1 for binding to both PP2Ac? isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit. Our findings add important new insights into the complex mechanisms of PP2A regulation.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/EdGnKhpX9qg" height="1" width="1"/>
Most commensal and food bacteria lack heme biosynthesis genes. For several of these, the capture of environmental heme is a means of activating aerobic respiration metabolism. Our previous studies in the Gram-positive bacterium Lactococcus lactis showed that heme exposure strongly induced expression of a single operon, called here hrtRBA, encoding an ortholog of the conserved membrane hrt (heme-regulated transporter) and a unique transcriptional regulator that we named HrtR. We show that HrtR expressed as a fusion protein is a heme-binding protein. Heme iron interaction with HrtR is non-covalent, hexacoordinated, and involves two histidines, His-72 and His-149. HrtR specifically binds a 15-nt palindromic sequence in the hrtRBA promoter region, which is needed for hrtRBA repression. HrtR-DNA binding is abolished by heme addition, which activates expression of the HrtB-HrtA (HrtBA) transporter in vitro and in vivo. The use of HrtR as an intracellular heme sensor appears to be conserved among numerous commensal bacteria, in contrast with numerous Gram-positive pathogens that use an extracellular heme-sensing system, HssRS, to regulate hrt. Finally, we show for the first time that HrtBA permease controls heme toxicity by its direct and specific efflux. The use of an intracellular heme sensor to control heme efflux constitutes a novel paradigm for bacterial heme homeostasis.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/lat3TXoZn7I" height="1" width="1"/>
Our objective was to determine whether lipocalin-2 (Lcn2) regulates cardiomyocyte apoptosis, the mechanisms involved, and the functional significance. Emerging evidence suggests that Lcn2 is a proinflammatory adipokine associated with insulin resistance and obesity-related complications, such as heart failure. Here, we used both primary neonatal rat cardiomyocytes and H9c2 cells and demonstrated for the first time that Lcn2 directly induced cardiomyocyte apoptosis, an important component of cardiac remodeling leading to heart failure. This was shown by detection of DNA fragmentation using TUNEL assay, phosphatidylserine exposure using flow cytometry to detect annexin V-positive cells, caspase-3 activity using enzymatic assay and immunofluorescence, and Western blotting for the detection of cleaved caspase-3. We also observed that Lcn2 caused translocation of the proapoptotic protein Bax to mitochondria and disruption of mitochondrial membrane potential. Using transient transfection of GFP-Bax, we confirmed that Lcn2 induced co-localization of Bax with MitoTracker® dye. Importantly, we used the fluorescent probe Phen Green SK to demonstrate an increase in intracellular iron in response to Lcn2, and depleting intracellular iron using an iron chelator prevented Lcn2-induced cardiomyocyte apoptosis. Administration of recombinant Lcn2 to mice for 14 days increased cardiomyocyte apoptosis as well as an acute inflammatory response with compensatory changes in cardiac functional parameters. In conclusion, Lcn2-induced cardiomyocyte apoptosis is of physiological significance and occurs via a mechanism involving elevated intracellular iron levels and Bax translocation.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/ZMV9pAPNFp0" height="1" width="1"/>
Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b558) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91phox and CeCl3 cytochemistry showed the presence of gp91phox and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b558 is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b558-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5?-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b558 under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNF?, and CD40L as physiological inducers of cyt b558 exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b558, which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b558 did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b558 depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/Jy_6lLNI2Io" height="1" width="1"/>
The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ?60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ?30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5?-flap or 5?-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772–1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/b_pluilt5eM" height="1" width="1"/>
Recent work has uncovered the “GET system,” which is responsible for endoplasmic reticulum targeting of tail-anchored proteins. Although structural information and the individual roles of most components of this system have been defined, the interactions and interplay between them remain to be elucidated. Here, we investigated the interactions between Get3 and the Get4-Get5 complex from Saccharomyces cerevisiae. We show that Get3 interacts with Get4-Get5 via an interface dominated by electrostatic forces. Using isothermal titration calorimetry and small-angle x-ray scattering, we further demonstrate that the Get3 homodimer interacts with two copies of the Get4-Get5 complex to form an extended conformation in solution.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/kZtapBKVYas" height="1" width="1"/>
Immune complexes composed of IgG-opsonized pathogens, particles, or proteins are phagocytosed by macrophages through Fc? receptors (Fc?Rs). Macrophages primed with IFN? or other pro-inflammatory mediators respond to Fc?R engagement by secreting high levels of cytokines and nitric oxide (NO). We found that unprimed macrophages produced lower levels of NO, which required efficient calcium (Ca2+) flux as demonstrated by using macrophages lacking selenoprotein K, which is required for Fc?R-induced Ca2+ flux. Thus, we further investigated the signaling pathways involved in low output NO and its functional significance. Evaluation of inducible, endothelial, and neuronal nitric-oxide synthases (iNOS, eNOS, and nNOS) revealed that Fc?R stimulation in unprimed macrophages caused a marked Ca2+-dependent increase in both total and phosphorylated nNOS and slightly elevated levels of phosphorylated eNOS. Also activated were three MAP kinases, ERK, JNK, and p38, of which ERK activation was highly dependent on Ca2+ flux. Inhibition of ERK reduced both nNOS activation and NO secretion. Finally, Transwell experiments showed that Fc?R-induced NO functioned to increase the phagocytic capacity of other macrophages and required both NOS and ERK activity. The production of NO by macrophages is conventionally attributed to iNOS, but we have revealed an iNOS-independent receptor/enzyme system in unprimed macrophages that produces low output NO. Under these conditions, Fc?R engagement relies on Ca2+-dependent ERK phosphorylation, which in turn increases nNOS and, to a lesser extent, eNOS, both of which produce low levels of NO that function to promote phagocytosis.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/10i429SS2XY" height="1" width="1"/>
Pathogenic bacteria can resist their microenvironment by changing the expression of virulence genes. In Salmonella typhimurium, some of these genes are controlled by the two-component system PhoP-PhoQ. Studies have shown that activation of the system by cationic antimicrobial peptides (AMPs) results, among other changes, in outer membrane remodeling. However, it is not fully clear what characteristics of AMPs are required to activate the PhoP-PhoQ system and whether activation can induce resistance to the various AMPs. For that purpose, we investigated the ability of a broad repertoire of AMPs to traverse the inner membrane, to activate the PhoP-PhoQ system, and to induce bacterial resistance. The AMPs differ in length, composition, and net positive charge, and the tested bacteria include two wild-type (WT) Salmonella strains and their corresponding PhoP-PhoQ knock-out mutants. A lacZ-reporting system was adapted to follow PhoP-PhoQ activation. The data revealed that: (i) a good correlation exists among the extent of the positive charge, hydrophobicity, and amphipathicity of an AMP and its potency to activate PhoP-PhoQ; (ii) a +1 charged peptide containing histidines was highly potent, suggesting the existence of an additional mechanism independent of the peptide charge; (iii) the WT bacteria are more resistant to AMPs that are potent activators of PhoP-PhoQ; (iv) only a subset of AMPs, independent of their potency to activate the system, is more toxic to the mutated bacteria compared with the WT strains; and (v) short term exposure of WT bacteria to these AMPs does not enhance resistance. Overall, this study advances our understanding of the molecular mechanism by which AMPs activate PhoP-PhoQ and induce bacterial resistance. It also reveals that some AMPs can overcome such a resistance mechanism.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/V57gRWtuisg" height="1" width="1"/>
NF-E2-related factor 2 (NRF2; also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares several domains with the original Nrf2 (now Nrf2a) but lacks the Neh4 transactivation domain. Zebrafish-human comparisons demonstrate conserved synteny involving nrf2 and hox genes, indicating that nrf2a and nrf2b are co-orthologs of human NRF2. nrf2a and nrf2b displayed distinct patterns of expression during embryonic development; nrf2b was more highly expressed at all stages. Embryos in which Nrf2a expression had been knocked down with morpholino oligonucleotides were more sensitive to tert-butylhydroperoxide but not tert-butylhydroquinone, whereas knockdown of Nrf2b did not affect sensitivity of embryos to either chemical. Gene expression profiling by microarray identified a specific role for Nrf2b as a negative regulator of several genes, including p53, cyclin G1, and heme oxygenase 1, in embryos. Nrf2a and Nrf2b exhibited different mechanisms of cross-talk with the Ahr2 signaling pathway. Together, these results demonstrate distinct roles for nrf2a and nrf2b, consistent with subfunction partitioning, and identify a novel negative regulatory role for Nrf2b during development. The identification of zebrafish nrf2 co-orthologs will facilitate new understanding of the multiple roles of NRF2 in protecting vertebrate embryos from oxidative damage.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/beg4YJlkDSY" height="1" width="1"/>
The regulation of endothelial function by insulin is consistently abnormal in insulin-resistant states and diabetes. Protein kinase C (PKC) activation has been reported to inhibit insulin signaling selectively in endothelial cells via the insulin receptor substrate/PI3K/Akt pathway to reduce the activation of endothelial nitric-oxide synthase (eNOS). In this study, it was observed that PKC activation differentially inhibited insulin receptor substrate 1/2 (IRS1/2) signaling of insulin's activation of PI3K/eNOS by decreasing only tyrosine phosphorylation of IRS2. In addition, PKC activation, by general activator and specifically by angiotensin II, increased the phosphorylation of p85/PI3K, which decreases its association with IRS1 and activation. Thr-86 of p85/PI3K was identified to be phosphorylated by PKC activation and confirmed to affect IRS1-mediated activation of Akt/eNOS by insulin and VEGF using a deletion mutant of the Thr-86 region of p85/PI3K. Thus, PKC and angiotensin-induced phosphorylation of Thr-86 of p85/PI3K may partially inhibit the activation of PI3K/eNOS by multiple cytokines and contribute to endothelial dysfunction in metabolic disorders.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/_HA2e8-pDqI" height="1" width="1"/>
14-3-3 proteins regulate key processes in eukaryotic cells including nitrogen assimilation in plants by tuning the activity of nitrate reductase (NR), the first and rate-limiting enzyme in this pathway. The homodimeric NR harbors three cofactors, each of which is bound to separate domains, thus forming an electron transfer chain. 14-3-3 proteins inhibit NR by binding to a conserved phosphorylation site localized in the linker between the heme and molybdenum cofactor-containing domains. Here, we have investigated the molecular mechanism of 14-3-3-mediated NR inhibition using a fragment of the enzyme lacking the third domain, allowing us to analyze electron transfer from the heme cofactor via the molybdenum center to nitrate. The kinetic behavior of the inhibited Mo-heme fragment indicates that the principal point at which 14-3-3 acts is the electron transfer from the heme to the molybdenum cofactor. We demonstrate that this is not due to a perturbation of the reduction potentials of either the heme or the molybdenum center and conclude that 14-3-3 most likely inhibits nitrate reductase by inducing a conformational change that significantly increases the distance between the two redox-active sites.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/UElpbjujY3A" height="1" width="1"/>
Prion diseases or transmissible spongiform encephalopathy diseases are typically characterized by deposition of abnormally folded partially protease-resistant host-derived prion protein (PrPres), which is associated with activated glia and increased release of cytokines. This neuroinflammatory response may play a role in transmissible spongiform encephalopathy pathogenesis. We previously reported that brain homogenates from prion-infected mice induced cytokine protein release in primary astroglial and microglial cell cultures. Here we measured cytokine release by cultured glial cells to determine what factors in infected brain contributed to activation of microglia and astroglia. In assays analyzing IL-12p40 and CCL2 (MCP-1), glial cells were not stimulated in vitro by either PrPres purified from infected mouse brains or prion protein amyloid fibrils produced in vitro. However, significant glial stimulation was induced by clarified scrapie brain homogenates lacking PrPres. This stimulation was greatly reduced both by antibody to cyclophilin A (CyPA), a known mediator of inflammation in peripheral tissues, and by cyclosporine A, a CyPA inhibitor. In biochemical studies, purified truncated CyPA fragments stimulated a pattern of cytokine release by microglia and astroglia similar to that induced by scrapie-infected brain homogenates, whereas purified full-length CyPA was a poor stimulator. This requirement for CyPA truncation was not reported in previous studies of stimulation of peripheral macrophages, endothelial cell cardiomyocytes, and vascular smooth muscle cells. Therefore, truncated CyPA detected in brain following prion infection may have an important role in the activation of brain-derived primary astroglia and microglia in prion disease and perhaps other neurodegenerative or neuroinflammatory diseases.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/WrcrolLSN4Y" height="1" width="1"/>
IL-22 is an immunoregulatory cytokine displaying pathological functions in models of autoimmunity like experimental psoriasis. Understanding molecular mechanisms driving IL-22, together with knowledge on the capacity of current immunosuppressive drugs to target this process, may open an avenue to novel therapeutic options. Here, we sought to characterize regulation of human IL22 gene expression with focus on the established model of Jurkat T cells. Moreover, effects of the prototypic immunosuppressant cyclosporin A (CsA) were investigated. We report that IL-22 induction by TPA/A23187 (T/A) or ?CD3 is inhibited by CsA or related FK506. Similar data were obtained with peripheral blood mononuclear cells or purified CD3+ T cells. IL22 promoter analysis (?1074 to +156 bp) revealed a role of an NF-AT (?95/?91 nt) and a CREB (?194/?190 nt) binding site for gene induction. Indeed, binding of CREB and NF-ATc2, but not c-Rel, under the influence of T/A to those elements could be proven by ChIP. Because CsA has the capability to impair I?B kinase (IKK) complex activation, the IKK?/? inhibitor IKKVII was evaluated. IKKVII likewise reduced IL-22 induction in Jurkat cells and peripheral blood mononuclear cells. Interestingly, transfection of Jurkat cells with siRNA directed against IKK? impaired IL22 gene expression. Data presented suggest that NF-AT, CREB, and IKK? contribute to rapid IL22 gene induction. In particular the crucial role of NF-AT detected herein may form the basis of direct action of CsA on IL-22 expression by T cells, which may contribute to therapeutic efficacy of the drug in autoimmunity.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/XpEgYj7fOJM" height="1" width="1"/>
UDP-galactopyranose mutase (UGM) requires reduced FAD (FADred) to catalyze the reversible interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). Recent structural and mechanistic studies of UGM have provided evidence for the existence of an FAD-Galf/p adduct as an intermediate in the catalytic cycle. These findings are consistent with Lewis acid/base chemistry involving nucleophilic attack by N5 of FADred at C1 of UDP-Galf/p. In this study, we employed a variety of FAD analogues to characterize the role of FADred in the UGM catalytic cycle using positional isotope exchange (PIX) and linear free energy relationship studies. PIX studies indicated that UGM reconstituted with 5-deaza-FADred is unable to catalyze PIX of the bridging C1–OP? oxygen of UDP-Galp, suggesting a direct role for the FADred N5 atom in this process. In addition, analysis of kinetic linear free energy relationships of kcat versus the nucleophilicity of N5 of FADred gave a slope of ? = ?2.4 ± 0.4. Together, these findings are most consistent with a chemical mechanism for UGM involving an SN2-type displacement of UDP from UDP-Galf/p by N5 of FADred.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/nQ7kqF5P1U8" height="1" width="1"/>
Neuronostatin, a recently discovered peptide encoded by somatostatin gene, is involved in regulation of neuronal function, blood pressure, food intake, and drinking behavior. However, the biological effects of neuronostatin on cardiac myocytes are not known, and the intracellular signaling mechanisms induced by neuronostatin remain unidentified. We analyzed the effect of neuronostatin in isolated perfused rat hearts and in cultured primary cardiomyocytes. Neuronostatin infusion alone had no effect on left ventricular (LV) contractile function or on isoprenaline- or preload-induced increase in cardiac contractility. However, infusion of neuronostatin significantly decreased the positive inotropic response to endothelin-1 (ET-1). This was associated with an increase in phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK). Treatment of both neonatal and adult cardiomyocytes with neuronostatin resulted in reduced cardiomyocyte viability. Inhibition of JNK further increased the neuronostatin-induced cell death. We conclude that neuronostatin regulates cardiac contractile function and cardiomyocyte survival. Receptors for neuronostatin need to be identified to further characterize the biological functions of the peptide.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/RXIa3JO80Uw" height="1" width="1"/>
Runt-related transcription factors (RUNX1, RUNX2, and RUNX3) are key lineage-specific regulators of progenitor cell growth and differentiation but also function pathologically as cancer genes that contribute to tumorigenesis. RUNX2 attenuates growth and stimulates maturation of osteoblasts during bone formation but is also robustly expressed in a subset of osteosarcomas, as well as in metastatic breast and prostate tumors. To assess the biological function of RUNX2 in osteosarcoma cells, we examined human genomic promoter interactions for RUNX2 using chromatin immunoprecipitation (ChIP)-microarray analysis in SAOS-2 cells. Promoter binding of both RUNX2 and RNA polymerase II was compared with gene expression profiles of cells in which RUNX2 was depleted by RNA interference. Many RUNX2-bound loci (1550 of 2339 total) exhibit promoter occupancy by RNA polymerase II and contain the RUNX consensus motif 5?-((T/A/C)G(T/A/C)GG(T/G). Gene ontology analysis indicates that RUNX2 controls components of multiple signaling pathways (e.g. WNT, TGF?, TNF?, and interleukins), as well as genes linked to cell motility and adhesion (e.g. the focal adhesion-related genes FAK/PTK2 and TLN1). Our results reveal that siRNA depletion of RUNX2, PTK2, or TLN1 diminishes motility of U2OS osteosarcoma cells. Thus, RUNX2 binding to diverse gene loci may support the biological properties of osteosarcoma cells.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/cjPWkeYkiEA" height="1" width="1"/>
Glutathione (GSH) has several important functions in eukaryotic cells, and its intracellular concentration is tightly controlled. Combining mathematical models and 35S labeling, we analyzed Saccharomyces cerevisiae sulfur metabolism. This led us to the observation that GSH recycling is markedly faster than previously estimated. We set up additional in vivo assays and concluded that under standard conditions, GSH half-life is around 90 min. Sulfur starvation and growth with GSH as the sole sulfur source strongly increase GSH degradation, whereas cadmium (Cd2+) treatment inhibits GSH degradation. Whatever the condition tested, GSH is degraded by the cytosolic Dug complex (composed of the three subunits Dug1, Dug2, and Dug3) but not by the ?-glutamyl-transpeptidase, raising the question of the role of this enzyme. In vivo, both DUG2/3 mRNA levels and Dug activity are quickly induced by sulfur deprivation in a Met4-dependent manner. This suggests that Dug activity is mainly regulated at the transcriptional level. Finally, analysis of dug2? and dug3? mutant cells shows that GSH degradation activity strongly impacts on GSH intracellular concentration and that GSH intracellular concentration does not affect GSH synthesis rate. Altogether, our data led us to reconsider important aspects of GSH metabolism, challenging notions on GSH synthesis and GSH degradation that were considered as established.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/VxuBPZA0wxk" height="1" width="1"/>
Macrophage exiting from inflammatory sites is critical to limit the local innate immune response. With tissue insult, resident tissue macrophages rapidly efflux to lymph nodes where they modulate the adaptive immune response, and inflammatory macrophages attracted to the site of injury then exit during the resolution phase. However, the mechanisms that regulate macrophage efflux are poorly understood. This study has investigated soluble forms of integrin ?2 whose levels are elevated in experimental peritonitis at times when macrophages are exiting the peritoneum, suggesting that its proteolytic shedding may be involved in macrophage efflux. Both constitutive and inducible metalloproteinase-dependent shedding of integrin ?2 from mouse macrophages are demonstrated. Soluble integrin ?2 is primarily released as a heterodimeric complex with ?M that retains its ability to bind its ligands intracellular adhesion molecule-1, fibrin, and collagen and thus may serve as a soluble antagonist. In a model of accelerated exiting, administration of a metalloproteinase inhibitor prevents macrophage efflux by 50% and impedes loss of macrophage integrin ?2 from the cell surface. Exiting of peritoneal macrophages in mice lacking integrin ?2 is accelerated, and antibody disruption of integrin ?2-substrate interactions can reverse 50% of the metalloprotease inhibitor blockade of macrophage exiting. Thus, our study demonstrates the ability of metalloproteinase-mediated shedding of integrin ?2 to promote macrophage efflux from inflammatory sites, and the release of soluble integrin heterodimers may also limit local inflammation.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/15cxr2jGhBE" height="1" width="1"/>
Cardiolipin (CL) is a major membrane phospholipid specifically localized in mitochondria. At the cellular level, CL has been shown to have a role in mitochondrial energy production, mitochondrial membrane dynamics, and the triggering of apoptosis. However, the in vivo role of CL in multicellular organisms is largely unknown. In this study, by analyzing deletion mutants of a CL synthase gene (crls-1) in Caenorhabditis elegans, we demonstrated that CL depletion selectively caused abnormal mitochondrial function and morphology in germ cells but not in somatic cell types such as muscle cells. crls-1 mutants reached adulthood but were sterile with reduced germ cell proliferation and impaired oogenesis. In the gonad of crls-1 mutants, mitochondrial membrane potential was significantly decreased, and the structure of the mitochondrial cristae was disrupted. Contrary to the abnormalities in the gonad, somatic tissues in crls-1 mutants appeared normal with respect to cell proliferation, mitochondrial function, and mitochondrial morphology. Increased susceptibility to CL depletion in germ cells was also observed in mutants of phosphatidylglycerophosphate synthase, an enzyme responsible for producing phosphatidylglycerol, a precursor phospholipid of CL. We propose that the contribution of CL to mitochondrial function and morphology is different among the cell types in C. elegans.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/nc_HSHLKqgk" height="1" width="1"/>
Protein post-translational modification by S-nitrosylation conveys a ubiquitous influence of nitric oxide on signal transduction in eukaryotic cells. The wide functional purview of S-nitrosylation reflects in part the regulation by S-nitrosylation of the principal protein post-translational modifications that play a role in cell signaling, including phosphorylation, acetylation, ubiquitylation and related modifications, palmitoylation, and alternative Cys-based redox modifications. In this minireview, we discuss the mechanisms through which S-nitrosylation exerts its broad pleiotropic influence on protein post-translational modification.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/GgUoNHfErIc" height="1" width="1"/>
Introduction The cache of coupled reduction-oxidation (i.e. redox) reactions in mammals is ultimately enabled by the cellular nicotinamide currency, i.e. the NAD+/NADH and NADP+/NADPH redox couples. Oxidative metabolism, which results in the capture of reducing equivalents primarily as NADH, represents capital investment, whereas biosynthetic reactions, which utilize NADPH, represent expenditures. From the central nicotinamide warehouse, conversion to alternative currencies diversifies the cellular energy portfolio. The mitochondrial membrane is the site of currency conversion to ATP, which occurs via the electron transfer chain and involves a four-electron reduction of oxygen to water. The oxidative phosphorylation process is, however, leaky, and reactive oxygen species (ROS),2 representing incomplete oxidation products of this pathway, are side products. ROS are also generated at other loci, such as NADPH oxidase, by metabolic enzymes, such as lipoxygenase, and in response to extracellular signals, such as growth hormones and cytokines. Long viewed as noxious, ROS are produced in a controlled manner that is now recognized as critical for signaling within and between cells. ROS are crucial for regulating such processes as cell division, circadian rhythms, and the immune response. On the other hand, uncontrolled ROS levels spell death. The intracellular redox “climate” is varied and influenced by multiple regulators. In the cytoplasm, protein regulators, such as thioredoxin, and small molecule antioxidants, such as glutathione and cysteine, maintain distinct redox potentials, revealing that the various redox nodes are not in equilibrium with each other (1). Within organelles, the potentials of the very same redox couples vary; the redox...<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/ZGO3fuPej_s" height="1" width="1"/>
Many therapeutic proteins possessing a small size are rapidly cleared from circulation. Half-life extension strategies have therefore become increasingly important to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Here, we performed a comparative analysis of the half-life extension properties of various bacterial immunoglobulin-binding domains (IgBDs) derived from Staphylococcus protein A (SpA), Streptococcus protein G (SpG), and Finegoldia (formerly Peptostreptococcus) protein L (PpL). These domains, composed of 50–60 amino acid residues, were fused to the C terminus of a single-chain Fv and a bispecific single-chain diabody, respectively. All fusion proteins were produced in mammalian cells and retained their antigen-binding properties. The half-lives of the antibody molecules were prolonged to varying extents for the different IgBDs. The strongest effects in mice were observed for domain C3 of SpG (SpGC3) followed by domains B and D of SpA, suggesting that SpGC3 is particularly useful to extend the plasma half-life of small proteins.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/ABeMt5j963Q" height="1" width="1"/>
Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H2S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase ?, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase ? inhibitor, had the same effect. Renal cortical content of cystathionine ?-synthase and cystathionine ?-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/QIxoy76TzOM" height="1" width="1"/>
99% of all mitochondrial proteins are synthesized in the cytosol, from where they are imported into mitochondria. In contrast to matrix proteins, many proteins of the intermembrane space (IMS) lack presequences and are imported in an oxidation-driven reaction by the mitochondrial disulfide relay. Incoming polypeptides are recognized and oxidized by the IMS-located receptor Mia40. Reoxidation of Mia40 is facilitated by the sulfhydryl oxidase Erv1 and the respiratory chain. Although structurally unrelated, the mitochondrial disulfide relay functionally resembles the Dsb (disufide bond) system of the bacterial periplasm, the compartment from which the IMS was derived 2 billion years ago.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/0xb74hPtmDs" height="1" width="1"/>
Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17?-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/ENlZYFZvbb8" height="1" width="1"/>
The production of mitochondrial reactive oxygen species occurs as a consequence of aerobic metabolism. Mitochondrial oxidants are increasingly viewed less as byproducts of metabolism and more as important signaling molecules. Here, I review several notable examples, including the cellular response to hypoxia, aspects of innate immunity, the regulation of autophagy, and stem cell self-renewal capacity, where evidence suggests an important regulatory role for mitochondrial oxidants.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/x3KVeA3bAOg" height="1" width="1"/>
Cys is much different from other common amino acids in proteins. Being one of the least abundant residues, Cys is often observed in functional sites in proteins. This residue is reactive, polarizable, and redox-active; has high affinity for metals; and is particularly responsive to the local environment. A better understanding of the basic properties of Cys is essential for interpretation of high-throughput data sets and for prediction and classification of functional Cys residues. We provide an overview of approaches used to study Cys residues, from methods for investigation of their basic properties, such as exposure and pKa, to algorithms for functional prediction of different types of Cys in proteins.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/jtXWO50SYwM" height="1" width="1"/>
Synaptic adhesion-like molecules (SALMs) are a family of cell adhesion molecules involved in neurite outgrowth and synapse formation. Of the five family members, only SALM1, -2, and -3 contain a cytoplasmic C-terminal PDZ-binding motif. We have found that SALM1 is unique among the SALMs because deletion of its PDZ-binding motif (SALM1?PDZ) blocks its surface expression in heterologous cells. When expressed in hippocampal neurons, SALM1?PDZ had decreased surface expression in dendrites and the cell soma but not in axons, suggesting that the PDZ-binding domain may influence cellular trafficking of SALMs to specific neuronal locations. Endoglycosidase H digestion assays indicated that SALM1?PDZ is retained in the endoplasmic reticulum (ER) in heterologous cells. However, when the entire C-terminal tail of SALM1 was deleted, SALM1 was detected on the cell surface. Using serial deletions, we identified a region of SALM1 that contains a putative dileucine ER retention motif, which is not present in the other SALMs. Mutation of this DXXXLL motif allowed SALM1 to leave the ER and enhanced its surface expression in heterologous cells and neurons. An increase in the number of protrusions at the dendrites and cell body was observed when this SALM1 mutant was expressed in hippocampal neurons. With electron microscopy, these protrusions appeared to be irregular, enlarged spines and filopodia. Thus, enrichment of SALM1 on the cell surface affects dendritic arborization, and intracellular motifs regulate its dendritic versus axonal localization.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/QWnrrPXqyOY" height="1" width="1"/>
Overexpression of metadherin (MTDH) has been documented in many solid tumors and is implicated in metastasis and chemoresistance. MTDH has been detected at the plasma membrane as well as in the cytoplasm and nucleus, and the function of MTDH in these locales remains under investigation. In the nucleus, MTDH acts as a transcription co-factor to induce expression of chemoresistance-associated genes. However, MTDH is predominantly cytoplasmic in prostate tumors, and this localization correlates with poor prognosis. Herein, we used endometrial cancer cells as a model system to define a new role for MTDH in the cytoplasm. First, MTDH was primarily localized to the cytoplasm in endometrial cancer cells, and the N-terminal region of MTDH was required to maintain cytoplasmic localization. Next, we identified novel binding partners for cytoplasmic MTDH, including RNA-binding proteins and components of the RNA-induced silencing complex. Nucleic acids were required for the association of MTDH with these cytoplasmic proteins. Furthermore, MTDH interacted with and regulated protein expression of multiple mRNAs, such as PDCD10 and KDM6A. Depletion of cytoplasmic MTDH was associated with increased stress granule formation, reduced survival in response to chemotherapy and the tyrosine kinase inhibitor BIBF1120, Rad51 nuclear accumulation, and cell cycle arrest at G2/M. Finally, in vivo tumor formation was abrogated with knockdown of cytoplasmic MTDH. Taken together, our data identify a novel function for cytoplasmic MTDH as an RNA-binding protein. Our findings implicate cytoplasmic MTDH in cell survival and broad drug resistance via association with RNA and RNA-binding proteins.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/0XvKsyHENwM" height="1" width="1"/>
Aerobic organisms generate reactive oxygen species as metabolic side products and must achieve a delicate balance between using them for signaling cellular functions and protecting against collateral damage. Small molecule (e.g. glutathione and cysteine)- and protein (e.g. thioredoxin)-based buffers regulate the ambient redox potentials in the various intracellular compartments, influence the status of redox-sensitive macromolecules, and protect against oxidative stress. Less well appreciated is the fact that the redox potential of the extracellular compartment is also carefully regulated and is dynamic. Changes in intracellular metabolism alter the redox poise in the extracellular compartment, and these are correlated with cellular processes such as proliferation, differentiation, and death. In this minireview, the mechanism of extracellular redox remodeling due to intracellular sulfur metabolism is discussed in the context of various cell-cell communication paradigms.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/TPBElgXXbeU" height="1" width="1"/>
Peroxiredoxins (Prxs) contain an active site cysteine that is sensitive to oxidation by H2O2. Mammalian cells express six Prx isoforms that are localized to various cellular compartments. The oxidized active site cysteine of Prx can be reduced by a cellular thiol, thus enabling Prx to function as a locally constrained peroxidase. Regulation of Prx via phosphorylation in response to extracellular signals allows the local accumulation of H2O2 and thereby enables its messenger function. The fact that the oxidation state of the active site cysteine of Prx can be transferred to other proteins that are less intrinsically susceptible to H2O2 also allows Prx to function as an H2O2 sensor.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/IMgI3-rkVZ8" height="1" width="1"/>
The major physiological inhibitor of plasminogen activator, type I plasminogen activator inhibitor (PAI-1) controls blood clotting and tissue remodeling events that involve cell migration. Transforming growth factor type ? (TGF?) and epidermal growth factor (EGF) interact synergistically to increase PAI-1 mRNA and protein levels in human HepG2 and mink Mv1Lu cells. Other growth factors that activate tyrosine kinase receptors can substitute for EGF. EGF and TGF? regulate PAI-1 by synergistically activating transcription, which is further amplified by a decrease in the rate of mRNA degradation, the latter being regulated only by EGF. The combined effect of transcriptional activation and mRNA stabilization results in a rapid two-order of magnitude increase in the level of PAI-1. TGF? also increases the sensitivity of the cells to EGF, thereby recruiting the cooperation of EGF at lower than normally effective concentrations. The contribution of EGF to the regulation of PAI-1 involves the MAPK pathway and the synergistic interface with the TGF? pathway is downstream of Mek1/2 and involves neither phosphorylation of Erk1/2 nor Smad2/3. Synergism requires the presence of both Smad and AP-1 recognition sites in the promoter. This work demonstrates the existence of a multidimensional cellular mechanism by which EGF and TGF? are able to promote large and rapid changes in PAI-1 expression.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/2UMa0YHCLIo" height="1" width="1"/>
The lysin motif receptor like kinase, NFP, is a key protein in the legume Medicago truncatula for the perception of lipochitooligosaccharidic Nod Factors, which are secreted bacterial signals essential for establishing the nitrogen-fixing legume-rhizobia symbiosis. Predicted structural and genetic analyses strongly suggest that NFP is at least part of a Nod factor receptor, but few data are available about this protein. Characterization of a variant encoded by the mutant allele nfp-2 revealed the sensitivity of this protein to the endoplasmic reticulum quality control mechanisms, affecting its trafficking to the plasma membrane. Further analysis revealed that the extensive N-glycosylation of the protein is not essential for biological activity. In the NFP extracellular region, two CXC motifs and two other Cys residues were found to be involved in disulphide bridges and these are necessary for correct folding and localization of the protein. Analysis of the intracellular region revealed its importance for biological activity but suggests that it does not rely on kinase activity. This work shows that NFP trafficking to the plasma membrane is highly sensitive to regulation in the endoplasmic reticulum and has identified structural features of the protein, particularly disulphide bridges involving CXC motifs in the extracellular region that are required for its biological function.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/dnbp4NRFsXY" height="1" width="1"/>
Unbiased proteomic screens provide a powerful tool for defining protein-protein interaction networks. Previous studies employed Multidimensional Protein Identification Technology (MudPIT) to identify the Sox2-interactome in embryonic stem cells (ESC) undergoing differentiation in response to a small increase in the expression of epitope-tagged Sox2. Thus far, the Sox2-interactome in ESC has not been determined. To identify the Sox2-interactome in ESC, we engineered ESC for inducible expression of different combinations of epitope-tagged Sox2 along with Oct4, Klf4 and c-Myc. Epitope-tagged Sox2 was used to circumvent the lack of suitable Sox2 antibodies needed to perform an unbiased proteomic screen of Sox2-associated proteins. Although i-OS-ESC differentiate when both Oct4 and Sox2 are elevated, i-OSKM-ESC do not differentiate, even when the levels of the four transcription factors are coordinately elevated ~2- to 3-fold. Our findings with i-OS-ESC and i-OSKM-ESC provide new insights into the reasons why ESC undergo differentiation when Sox2 and Oct4 are elevated in ESC. Importantly, the use of i-OSKM-ESC enabled us to identify the Sox2-interactome in undifferentiated ESC. Using MudPIT, we identified >70 proteins that associate with Sox2 in ESC. We extended these findings by testing the function of the Sox2-assoicated protein Smarcd1, and demonstrate that knockdown of Smarcd1 disrupts the self-renewal of ESC and induces their differentiation. Together, our work provides the first description of the Sox2-interactome in ESC and indicates that Sox2, along with other master regulators, is part of a highly integrated protein-protein interaction landscape in ESC.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/XWEHnhZY31s" height="1" width="1"/>
The protein ING4 binds to histone H3 trimethylated at Lys4 (H3K4me3) through its C-terminal PlantHomeoDomain (PHD), thus recruiting the HBO1 histone acetyltransferase complex to target promoters. The structure of the PHD finger bound to an H3K4me3 peptide has been described, as well as the disorder and flexibility in the ING4 central region. We report the crystal structure of the ING4 N-terminal domain, which shows an antiparallel coiled-coil homodimer with each protomer folded into a helix-loop-helix structure. This arrangement suggests that ING4 can bind simultaneously two histone tails, on the same or different nucleosomes. Dimerization has a direct impact on ING4 tumor suppressor activity since monomeric mutants lose the ability of inducing apoptosis after genotoxic stress. Homology modeling based on the ING4 structure suggests that other ING dimers may also exist.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/dbRSJ3GwVGY" height="1" width="1"/>
The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The family's structural features and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this has only been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations, through transient receptor potential channels, that trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly-changing local cellular water availability. Moreover, since calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/U_Qj1aosX4M" height="1" width="1"/>
Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins however in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP) which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen-receptor (ER) positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts (MEFs) that lack mhdac6 localize survivin predominantly to the nuclear compartment while wild-type MEFs localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/7oj23v_YvSo" height="1" width="1"/>
Discrimination of metabolic models based on high-throughput metabolomics data, reflecting various internal and external perturbations, is essential for identifying the components that contribute to the emerging behavior of metabolic processes. Here, we investigate twelve different models of the mitochondrial electron transport chain (ETC) in Arabidopsis thaliana during dark-induced senescence in order to elucidate the alternative substrates to this metabolic pathway. Our findings demonstrate that the coupling of the proposed computational approach, based on dynamic flux balance analysis, with time-resolved metabolomics data results in model-based confirmations of the hypotheses that during dark-induced senescence in Arabidopsis : (i) under conditions where the main substrate for the ETC are not fully available, isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase are able to donate electrons to the ETC, (ii) phytanoyl-CoA does not act even as an indirect substrate of the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex, and (iii) the mitochondrial ?-aminobutyric acid transporter has functional significance in maintaining mitochondrial metabolism. Our study provides a basic framework for future in silico studies of alternative pathways in mitochondrial metabolism under extended darkness whereby the role of its components can be computationally discriminated based on available molecular profile data.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/sDIAk0_qRQk" height="1" width="1"/>
The Smc5/6 complex belongs to the Structural Maintenance of Chromosomes (SMC) family that also includes cohesin and condensin. In Saccharomyces cerevisiae the Smc5/6 complex contains six essential Non-Smc-Elements (Nse1-6). Very little is known about how these additional elements contribute to complex function except for Nse2/Mms21, which is an E3 SUMO ligase important for Smc5 sumoylation. Characterization of two temperature sensitive mutants, nse5-ts1 and nse5-ts2, demonstrates the importance of Nse5 within the Smc5/6 complex for its stability and functionality at forks during hydroxyurea (HU)-induced replication stress. Both NSE5 alleles showed a marked reduction in Smc5 sumoylation to levels lower than those observed with mms21-11, a mutant of Mms21 that is deficient in SUMO ligase activity. However, a phenotypic comparison of nse5-ts1 and nse5-ts2 revealed a separation of importance between Smc5 sumoylation and the function of the Smc5/6 complex during replication. Only cells carrying the nse5-ts1 allele exhibited defects such as dissociation of the replisome from stalled forks, the formation of fork-associated homologous recombination (HR) intermediates, and HU sensitivity that is additive with mms21-11. These defects are attributed to a failure in Smc5/6 localization to forks in nse5-ts1 cells. Overall, these data support the premise that Nse5 is important for vital interactions between components within the Smc5/6 complex, which is a prerequisite for its functionality during replication stress<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/j82JPfJQhXM" height="1" width="1"/>
Filamin A (FLNa) is a cross-linker of actin filaments and serves as a scaffold protein mostly involved in the regulation of actin polymerisation. It is ubiquitously distributed and null-mutations have strong consequences on embryonic development in humans, with organ defects which suggest deficiencies in cell migration. We have previously reported that macrophages, the archetypal migratory cells, use the protease and podosome-dependent mesenchymal migration mode in dense 3D environments, whereas they use the protease- and podosome-independent amoeboid mode in more porous matrices. Since FLNa has been shown to localise to podosomes, we hypothesized that the defects seen in patients carrying FLNa mutations could be related to the capacity of certain cell types to form podosomes. Using strategies based on FLNa knock-out, knock-down, and rescue, we show that FLNa: (i) is involved in podosome stability and their organization as rosettes and 3D-podosomes, (ii) regulates the proteolysis of the matrix mediated by podosomes in macrophages, (iii) is required for podosome rosette formation triggered by Hck and (iv) is necessary for mesenchymal migration but dispensable for amoeboid migration. These new functions assigned to FLNa, particularly its role in mesenchymal migration, could be directly related to the defects in cell migration described during the embryonic development in FLNa-defective patients.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/GuE95RViuKQ" height="1" width="1"/>
The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family, which allows cross talk between mitochondria, where cell energy is mainly produced, and cytosol, where cell energy is mainly consumed. The members of this family share numerous structural and functional characteristics. Resolution of the atomic structure of the bovine Ancp, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in the movements of the protein to perform translocation of nucleotides from one side of the membrane to the other. They correspond to three prolines located in the odd-numbered transmembrane helices (TMH), Pro27, Pro132 and Pro229. The corresponding residues of the yeast Ancp (Pro43, Ser147 and Pro247) were mutated into alanine or leucine, one at a time and analysis of the various mutants evidenced a crucial role of Pro43 and Pro247 during nucleotide transport. Beside, replacement of Ser147 does not inactivate Ancp and this is discussed in view of the conservation of the three prolines at equivalent positions in the Ancp sequences. These prolines belong to the signature sequences of the mitochondrial carriers and we propose they play a dual role in the mitochondrial ADP/ATP carrier function and biogenesis. Unexpectedly their mutations cause more general effects on the mitochondrial biogenesis and morphology, as evidenced by measurements of respiratory rates, cytochrome contents and also clearly highlighted by fluorescence microscopy.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/1WQnMn3XemQ" height="1" width="1"/>
Recent findings have suggested that reactive oxygen species (ROS) are important signaling molecules for regulating plant responses to abiotic and biotic stress, and that there exist source- and kind-specific pathways for ROS signaling. In plant cells, a major source of ROS are chloroplasts, in which thylakoid membrane-bound ascorbate peroxidase (tAPX) plays a role in the regulation of H2O2 levels. Here, to clarify the signaling function of H2O2 derived from the chloroplast, we created a conditional system for producing H2O2 in the organelle by chemical-dependent tAPX silencing using estrogen-inducible RNAi. When the expression of tAPX was silenced in leaves, levels of oxidized protein in chloroplasts increased in the absence of stress. Microarray analysis revealed that tAPX silencing affects the expression of a large set of genes, some of which are involved in the response to chilling and pathogens. In response to tAPX silencing, the transcript levels of C-REPEAT/DRE BINDING FACTOR (CBF1), a central regulator for cold acclimation, was suppressed, resulting in a high sensitivity of tAPX-silenced plants to cold. Furthermore, tAPX silencing enhanced the levels of salycilic acid (SA) and the response to SA. Interestingly, we found that tAPX silencing-responsive genes were up- or down-regulated by high light (HL), and that tAPX silencing had a negative effect on expression of ROS-responsive genes under HL, suggesting synergistic and antagonistic roles of chloroplastic H2O2 in HL response. The present findings provide a new insight into the role of H2O2-triggered retrograde signaling from chloroplasts in the response to stress in planta.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/IZmvMS1shoM" height="1" width="1"/>
The pneumococcal autolysin LytA is a virulence factor involved in autolysis, as well as fratricidal- and penicillin-induced lysis. In this study, we used biochemical and molecular biological approaches to elucidate which factors control the cytoplasmic translocation and lytic activation of LytA. We show that LytA is mainly localized intracellularly as only a small fraction was found attached to the extracellular cell wall. By manipulating the extracellular concentration of LytA, we found that the cells were protected from lysis during exponential growth, but not in the stationary phase, and that a defined threshold concentration of extracellular LytA dictates the onset of autolysis. Stalling growth through nutrient depletion or the specific arrest of cell wall synthesis sensitized cells for LytA-mediated lysis. Inhibition of cell wall association via the choline binding domain of an exogenously added enzymatically inactive form of LytA revealed a potential substrate for the amidase domain within the cell wall where the formation of nascent peptidoglycan occurs.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/9xIRPrum1Ok" height="1" width="1"/>
?-Synuclein (AS) is associated with both sporadic and familial forms of Parkinson's disease (PD). In sporadic disease, wild-type AS fibrillates and accumulates as Lewy bodies within dopaminergic neurons of the substantia nigra. The accumulation of misfolded AS is associated with the death of these neurons, which underlies many of the clinical features of PD. In addition, a rare missense mutation in AS, A30P, is associated with highly penetrant, autosomal dominant PD, although the pathogenic mechanism is unclear. A30P AS fibrillates more slowly than the wild-type protein in vitro, and has been reported to preferentially adopt a soluble, protofibrillar conformation. This has led to speculation that A30P forms aggregates that are distinct in structure as compared to wild-type AS. Here we perform a detailed comparison of the chemical shifts and secondary structures of these fibrillar species, based upon our recent characterization of full-length wild-type fibrils. We have assigned A30P AS fibril chemical shifts de novo and used them to empirically determine its secondary structure. Our results illustrate that although A30P forms fibrils more slowly than WT in vitro, the chemical shifts and secondary structure of the resultant fibrils are in high agreement, demonstrating a conserved ?-sheet core.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/_IDjKyTLegQ" height="1" width="1"/>
Transcriptional up-regulation of the plasminogen activator inhibitor type-2 (PAI-2) gene is a major response to cellular stress. The expression of PAI-2 is induced by a variety of cytokines and growth factors which act in a cell type- and differentiation stage-dependent manner. We previously reported that the human PAI-2 gene promoter is controlled by three major transcription regulatory domains - an inducible proximal promoter, an upstream silencer (PAUSE-1) and a distal transactivator region between -5100 and -3300, that appears to overcome inhibition mediated by the silencer. The distal transactivator region is inducible by the phorbol ester PMA, a potent activator of the protein kinase C (PKC) pathway that is a powerful inducer of PAI-2 gene expression in monocytes, macrophages and myelomonocytic cells as well as in epidermal keratinocytes. Here we show that a 21 bp region (-4952/-4932) containing an AP-1 element, is both necessary and sufficient for PMA-induced transactivator activity in PAI-2 expressing U937 cells. This site specifically binds FosB in PAI-2 expressing U937 cells, but not in PAI-2 non-expressing HeLa cells; and overexpression of FosB, c-Fos, or c-Jun in HeLa cells is sufficient to cause derepression of transcription from the PAI-2 promoter. While FosB is likely to be involved in transactivator-mediated derepression of PAI-2 transcription in macrophage-like cells, as exemplified by the U937 cell line, c-Jun may be functional in other cell types. These data suggest a model for the transcriptional control of the human PAI-2 gene and further our understanding of the molecular basis for its tissue-specific expression.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/Lm56qhDKnQo" height="1" width="1"/>
The yeast Pah1p phosphatidate phosphatase, which catalyzes the penultimate step in the synthesis of triacylglycerol and plays a role in the transcriptional regulation of phospholipid synthesis genes, is a cytosolic enzyme that associates with the nuclear/endoplasmic reticulum membrane to catalyze the dephosphorylation of phosphatidate to yield diacylglycerol. Pah1p is phosphorylated on seven (Ser(110), Ser(114), Ser(168), Ser(602), Thr(723), Ser(744), and Ser(748)) sites that are targets for proline-directed proteins kinases. In this work, we showed that the seven sites are phosphorylated by Pho85p-Pho80p, a protein kinase-cyclin complex known to regulate a variety of cellular processes. The phosphorylation of recombinant Pah1p was time- and dose-dependent, and dependent on the concentrations of ATP (3.7 ?M) and Pah1p (0.25 ?M). Phosphorylation reduced (6-fold) the catalytic efficiency (Vmax/Km) of Pah1p and reduced (3-fold) its interaction (Kd) with liposomes. Alanine mutations of the seven sites ablated the inhibitory effect that Pho85p-Pho80p had on Pah1p activity and on the interaction with liposomes. Analysis of pho85? mutant cells, phosphate-starved wild type cells, and cells expressing phosphorylation-deficient forms of Pah1p indicated that loss of Pho85p-Pho80p phosphorylation reduced Pah1p abundance. In contrast, lack of Nem1p-Spo7p, the phosphatase complex that dephosphorylates Pah1p at the nuclear/endoplasmic reticulum membrane, stabilized Pah1p abundance. While loss of phosphorylation caused a decrease in abundance, a greater amount of Pah1p was associated with membranes when compared with phosphorylated enzyme, and the loss of phosphorylation allowed bypass of the Nem1p-Spo7p requirement for Pah1p function in the synthesis of triacylglycerol.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/q9xX3dPXwXU" height="1" width="1"/>
Since the discovery and isolation of ?-synuclein (?-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that ?-syn produced in E. Coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in ?-helical structure (1,2). However, it remains unknown whether or not this putative tetramer is the main physiological form of ?-syn in the brain. In this study, we investigated the oligomeric state of ?-syn in mouse, rat and human brains. To assess the conformational and oligomeric state of native ?-syn in complex mixtures, we generated ?-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed ?-syn to these standards using native and denaturing gel electrophoresis techniques, size exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent ?-syn expressed in the nervous system exist predominantly as an unfolded monomer. Similar results were observed when human ?-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, SH-SY5Y). Furthermore, we show that ?-syn expressed in E. Coli and purified under denaturing or non-denaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that ?-syn becomes structured upon interaction with other proteins and/or biological membranes.<img src="http://feeds.feedburner.com/~r/JBC_PapersInPress/~4/tBhiFO7crtc" height="1" width="1"/>
Posted on 9 February 2012 | 1:16 pm
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