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PC (pyruvate carboxylase) is a biotin-containing enzyme that catalyses the HCO3{Minus Sign}- and MgATP-dependent carboxylation of pyruvate to form oxaloacetate. This is a very important anaplerotic reaction, replenishing oxaloacetate withdrawn from the tricarboxylic acid cycle for various pivotal biochemical pathways. PC is therefore considered as an enzyme that is crucial for intermediary metabolism, controlling fuel partitioning toward gluconeogenesis or lipogenesis and in insulin secretion. The enzyme was discovered in 1959 and over the last decade there has been much progress in understanding its structure and function. PC from most organisms is a tetrameric protein that is allosterically regulated by acetyl-CoA and aspartate. High-resolution crystal structures of the holoenzyme with various ligands bound have recently been determined, and have revealed details of the binding sites and the relative positions of the biotin carboxylase, carboxyltransferase and biotin carboxyl carrier domains, and also a unique allosteric effector domain. In the presence of the allosteric effector, acetyl-CoA, the biotin moiety transfers the carboxy group between the biotin carboxylase domain active site on one polypeptide chain and the carboxyltransferase active site on the adjacent antiparallel polypeptide chain. In addition, the bona fide role of PC in the non-gluconeogenic tissues has been studied using a combination of classical biochemistry and genetic approaches. The first cloning of the promoter of the PC gene in mammals and subsequent transcriptional studies reveal some key cognate transcription factors regulating tissue-specific expression. The present review summarizes these advances and also offers some prospects in terms of future directions for the study of this important enzyme.
The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp266 in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis–trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis–trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3–E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.
The ‘stalk’ is a large ribosomal subunit domain that regulates translation. In the present study the role of the ribosomal stalk P proteins in modulating ribosomal activity has been investigated in human cells using RNA interference. A strong down-regulation of P2 mRNA and a drastic decrease in P2 protein in a stable human cell line was achieved using a doxycycline-inducible system. Interestingly, the amount of P1 protein was similarly decreased in these cells, in contrast with the expression of P1 mRNA. The loss of P1/P2 proteins produced a decrease in the growth rate of these cells, as well as an altered polysome pattern with reduced translation efficiency, but without affecting the free 40 S/60 S subunit ratio. A decrease in the ribosomal-subunit joining capacity was also observed. These data indicate that P1/P2 proteins modulate cytoplasmic translation by influencing the interaction between subunits, thereby regulating the rate of cell proliferation.
hBVR (human biliverdin reductase) is an enzyme that reduces biliverdin (the product of haem oxygenases HO-1 and HO-2 activity) to the antioxidant bilirubin. It also functions as a kinase and as a transcription factor in the MAPK (mitogen-activated protein kinase) signalling cascade. Fluorescence correlation spectroscopy was used to investigate the mobility of hBVR in living cells and its function in the nuclear transport of haematin for induction of HO-1. In transiently transfected HeLa cells only kinase-competent hBVR translocates to the nucleus. A reduced mobility in the nucleus of haematin-treated cells suggests formation of an hBVR–haematin complex and its further association with large nuclear components. The binding of haematin is specific, with the formation of a 1:1 molar complex, and the C-terminal 7-residue fragment KYCCSRK296 of hBVR contributes to the binding. The following data suggest formation of dynamic complexes of hBVR–haematin with chromatin: (i) the reduction of hBVR mobility in the presence of haematin is greater in heterochromatic regions than in euchromatic domains and (ii) hBVR mobility is not retarded by haematin in nuclear lysates that contain only soluble factors. Moreover, hBVR kinase activity is stimulated in the presence of double-stranded DNA fragments corresponding to HO-1 antioxidant and HREs (hypoxia response elements), as well as by haematin. Experiments with nuclear localization, export signal mutants and si-hBVR [siRNA (small interfering RNA) specific to hBVR] indicate that nuclear localization of hBVR is required for induction of HO-1 by haematin. Because gene regulation is energy-dependent and haematin regulates gene expression, our data suggest that hBVR functions as an essential component of the regulatory mechanisms for haem-responsive transcriptional activation.
The hTRPC [human TRPC (canonical transient receptor potential)] family of non-selective cation channels is proposed to mediate calcium influx across the plasma membrane via PLC (phospholipase C)-coupled receptors. Heterologously expressed hTRPC3 and hTRPC7 have been localized at the cell surface; however, a large intracellular component has also been noted but not characterized. In the present study, we have investigated the intracellular pool in COS-7 cells and have shown co-localization with markers for both the TGN (trans-Golgi network) and the cis-Golgi cisternae by immunofluorescence microscopy. Addition of BFA (Brefeldin A) to cells expressing hTRPC3 or hTRPC7 resulted in the redistribution of the Golgi component to the endoplasmic reticulum, indicating that this pool is present in both the Golgi stack and the TGN. Expression of either TRPC3 or TRPC7, but not TRPC1 or the cell surface marker CD8, resulted in a 2–4-fold increase in secreted alkaline phosphatase in the extracellular medium. Based on these results, we propose that an additional function of these members of the hTRPC family may be to enhance secretion either by affecting transport through the Golgi stack or by increasing fusion at the plasma membrane.
Whereas ligation of the CD95 death receptor in the plasma membrane of so-called type I cells leads to a direct caspase 8-dependent activation of downstream effector caspases, mitochondrial amplification of caspase 8-derived signals is required in so-called type II cells in order to execute apoptotic cell death. In type I cells CD95L (CD95 ligand) binding to CD95 results in a ceramide-dependent formation of the DISC (death-inducing signalling complex) and caspase 8-dependent CD95 clustering in the plasma membrane, followed by an internalization of these multimeric-receptor–DISC complexes. In contrast, in the hepatocyte, a type II cell, the bulk of CD95 is stored intracellularly under resting conditions and only a few ‘sentinel’ CD95 receptors are present in the plasma membrane. However, their activation by CD95L is sufficient to trigger a caspase 8-dependent endosomal acidification and a ceramide-dependent trafficking of intracellularly stored CD95 to the plasma membrane, thereby amplifying CD95 activation. Thus, in both type I and type II cells, ceramide and CD95 receptor endo- and exo-cytosis are involved in CD95-mediated apoptosis, but apparently in different ways. This, however, is not the only effect of CD95 ligation on intracellular membrane flow in type II cells, and evidence has been presented that soon after CD95 ligation Golgi elements intermix caspase-dependently with mitochondria. In this issue of the Biochemical Journal, Matarrese et al. report another aspect on endocytosis in response to CD95 ligation in type II cells, namely a caspase-independent endocytosis with vesicle translocation to the mitochondrial compartment, suggestive of an interplay between both organelles in the sense of an ‘organelle scrambling’. Thus early effects of CD95 activation on intracellular membrane flow may be much more complex than previously thought, but much has still to be learned about signalling mechanisms and the role they play in apoptosis.
Ago (Argonaute) proteins are essential effectors of RNA-mediated gene silencing. To explore potential regulatory mechanisms for Ago proteins, we examined the phosphorylation of human Ago2. We identified serine-387 as the major Ago2 phosphorylation site in vivo. Phosphorylation of Ago2 at serine-387 was significantly induced by treatment with sodium arsenite or anisomycin, and arsenite-induced phosphorylation was inhibited by a p38 MAPK (mitogen-activated protein kinase) inhibitor, but not by inhibitors of JNK (c-Jun N-terminal kinase) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]. MAPKAPK2 (MAPK-activated protein kinase-2) phosphorylated bacterially expressed full-length human Ago2 at serine-387 in vitro, but not the S387A mutant. Finally, mutation of serine-387 to an alanine residue or treatment of cells with a p38 MAPK inhibitor reduced the localization of Ago2 to processing bodies. These results suggest a potential regulatory mechanism for RNA silencing acting through Ago2 serine-387 phosphorylation mediated by the p38 MAPK pathway.
GSNO (S-nitrosoglutathione) is emerging as a key regulator in NO signalling as it is in equilibrium with S-nitrosated proteins. Accordingly, it is of great interest to investigate GSNO metabolism in terms of competitive pathways and redox state. The present study explored ADH3 (alcohol dehydrogenase 3) in its dual function as GSNOR (GSNO reductase) and glutathione-dependent formaldehyde dehydrogenase. The glutathione adduct of formaldehyde, HMGSH (S-hydroxymethylglutathione), was oxidized with a kcat/Km value approx. 10 times the kcat/Km value of GSNO reduction, as determined by fluorescence spectroscopy. HMGSH oxidation in vitro was greatly accelerated in the presence of GSNO, which was concurrently reduced under cofactor recycling. Hence, considering the high cytosolic NAD+/NADH ratio, formaldehyde probably triggers ADH3-mediated GSNO reduction by enzyme-bound cofactor recycling and might result in a decrease in cellular S-NO (S-nitrosothiol) content in vivo. Formaldehyde exposure affected S-NO content in cultured cells with a trend towards decreased levels at concentrations of 1–5 mM, in agreement with the proposed mechanism. Product formation after GSNO reduction to the intermediate semimercaptal responded to GSH/GSNO ratios; ratios up to 2-fold allowed the spontaneous rearrangement to glutathione sulfinamide, whereas 5-fold excess of GSH favoured the interception of the intermediate to form glutathione disulfide. The sulfinamide and its hydrolysis product, glutathione sulfinic acid, inhibited GST (glutathione transferase) activity. Taken together, the findings of the present study provide indirect evidence for formaldehyde as a physiological trigger of GSNO depletion and show that GSNO reduction can result in the formation of GST inhibitors, which, however, is prevented under normal cellular redox conditions.
Achieving an understanding of how apoptosis/PCD (programmed cell death) is integrated within cellular responses to environmental and intracellular signals is a daunting task. From the sensation of a stimulus to the point of no return, a programme of cell death must engage specific pro-death components, whose effects can in turn be enhanced or repressed by downstream regulatory factors. In recent years, considerable progress has been made in our understanding of how components involved in these processes function. We now know that some of the factors involved in PCD networks have ancient origins that pre-date multicellularity and, indeed, eukaryotes themselves. A subject attracting much attention is the role that the actin cytoskeleton, itself a cellular component with ancient origins, plays in cell death regulation. Actin, a key cellular component, has an established role as a cellular sensor, with reorganization and alterations in actin dynamics being a well known consequence of signalling. A range of studies have revealed that actin also plays a key role in apoptosis/PCD regulation. Evidence implicating actin as a regulator of eukaryotic cell death has emerged from studies from the Animal, Plant and Fungal Kingdoms. Here we review recent data that provide evidence for an active, functional role for actin in determining whether PCD is triggered and executed, and discuss these findings within the context of regulation of actin dynamics.
The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC 3.5.1.88], a plant specific deformylase, has been determined at a resolution of 2.4 Å (1 Å=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substrate binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr178 as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr2 in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr178 to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr178 can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.
Exocytosis is regulated by NO in many cell types, including neurons. In the present study we show that syntaxin 1a is a substrate for S-nitrosylation and that NO disrupts the binding of Munc18-1 to the closed conformation of syntaxin 1a in vitro. In contrast, NO does not inhibit SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein] receptor} complex formation or binding of Munc18-1 to the SNARE complex. Cys145 of syntaxin 1a is the target of NO, as a non-nitrosylatable C145S mutant is resistant to NO and novel nitrosomimetic Cys145 mutants mimic the effect of NO on Munc18-1 binding in vitro. Furthermore, expression of nitrosomimetic syntaxin 1a in living cells affects Munc18-1 localization and alters exocytosis release kinetics and quantal size. Molecular dynamic simulations suggest that NO regulates the syntaxin–Munc18 interaction by local rearrangement of the syntaxin linker and H3c regions. Thus S-nitrosylation of Cys145 may be a molecular switch to disrupt Munc18-1 binding to the closed conformation of syntaxin 1a, thereby facilitating its engagement with the membrane fusion machinery.
Thermoanaerobacterium thermosulfurigenes cyclodextrin glucanotransferase primarily catalyses the formation of cyclic {alpha}-(1,4)-linked oligosaccharides (cyclodextrins) from starch. This enzyme also possesses unusually high hydrolytic activity as a side reaction, thought to be due to partial retention of ancestral enzyme function. This side reaction is undesirable, since it produces short saccharides that are responsible for the breakdown of the cyclodextrins formed, thus limiting the yield of cyclodextrins produced. To reduce the competing hydrolysis reaction, while maintaining the cyclization activity, we applied directed evolution, introducing random mutations throughout the cgt gene by error-prone PCR. Mutations in two residues, Ser-77 and Trp-239, on the outer region of the active site, lowered the hydrolytic activity up to 15-fold with retention of cyclization activity. In contrast, mutations within the active site could not lower hydrolytic rates, indicating an evolutionary optimized role for cyclodextrin formation by residues within this region. The crystal structure of the most effective mutant, S77P, showed no alterations to the peptide backbone. However, subtle conformational changes to the side chains of active-site residues had occurred, which may explain the increased cyclization/hydrolysis ratio. This indicates that secondary effects of mutations located on the outer regions of the catalytic site are required to lower the rates of competing side reactions, while maintaining the primary catalytic function. Subsequent functional analysis of various glucanotransferases from the superfamily of glycoside hydrolases also suggests a gradual evolutionary progression of these enzymes from a common ‘intermediate-like’ ancestor towards specific transglycosylation activity.
Following its identification as a liver-expressed antimicrobial peptide, the hepcidin peptide was later shown to be a key player in iron homoeostasis. It is now proposed to be the ‘iron hormone’ which, by interacting with the iron transporter ferroportin, prevents further iron import into the circulatory system. This conclusion was reached using the corresponding synthetic peptide, emphasizing the functional importance of the mature 25-mer peptide, but omitting the possible functionality of its maturation. From urine-purified native hepcidin, we recently demonstrated that a proportion of the purified hepcidin had formed iron–hepcidin complexes. This interaction was investigated further by computer modelling and, based on the sequence similarity of hepcidin with metallothionein, a three-dimensional model of hepcidin, containing one atom of iron, was constructed. To characterize these complexes further, the interaction with iron was analysed using different spectroscopic methods. Monoferric hepcidin was identified by MS, as were possibly other complexes containing two and three atoms of iron respectively, although these were present only in minor amounts. UV/visible absorbance and CD studies identified the iron-binding events which were facilitated at a physiological pH. EPR spectroscopy identified the ferric state of the bound metal, and indicated that the iron–hepcidin complex shares some similarities with the rubredoxin iron–sulfur complex, suggesting the presence of Fe3+ in a tetrahedral sulfur co-ordination. The potential roles of iron binding for hepcidin are discussed, and we propose either a regulatory function in the maturation of pro-hepcidin into active hepcidin or as the necessary link in the interaction between hepcidin and ferroportin.
MUC5B is the predominant polymeric mucin in human saliva [Thornton, Khan, Mehrotra, Howard, Veerman, Packer and Sheehan (1999) Glycobiology 9, 293–302], where it contributes to oral cavity hydration and protection. More recently, the gene for another putative polymeric mucin, MUC19, has been shown to be expressed in human salivary glands [Chen, Zhao, Kalaslavadi, Hamati, Nehrke, Le, Ann and Wu (2004) Am. J. Respir. Cell Mol. Biol. 30, 155–165]. However, to date, the MUC19 mucin has not been isolated from human saliva. Our aim was therefore to purify and characterize the MUC19 glycoprotein from human saliva. Saliva was solubilized in 4 M guanidinium chloride and the high-density mucins were purified by density-gradient centrifugation. The presence of MUC19 was investigated using tandem MS of tryptic peptides derived from this mucin preparation. Using this approach, we found multiple MUC5B-derived tryptic peptides, but were unable to detect any putative MUC19 peptides. These results suggest that MUC19 is not a major component in human saliva. In contrast, using the same experimental approach, we identified Muc19 and Muc5b glycoproteins in horse saliva. Moreover, we also identified Muc19 from pig, cow and rat saliva; the saliva of cow and rat also contained Muc5b; however, due to the lack of pig Muc5b genomic sequence data, we were unable to identify Muc5b in pig saliva. Our results suggest that unlike human saliva, which contains MUC5B, cow, horse and rat saliva are a heterogeneous mixture of Muc5b and Muc19. The functional consequence of these species differences remains to be elucidated.
Participation of diverse organelles in the intracellular signalling that follows CD95/Fas receptor ligation encompasses a series of subcellular changes that are mandatory for, or even bolster, the apoptotic cascade. In the present study, we analysed the role of endocytosis in the propagation of cell death signalling after CD95/Fas engagement in type II cells (CEM cells). We show that this receptor–ligand interaction triggers endocytosis independently of any caspase activation. This FasL (Fas ligand)-induced endocytosis also leads to an early and directional ‘movement’ of endocytic vesicles towards the mitochondrial compartment. In turn, this cross-talk between endosomal and mitochondrial compartments was followed by the loss of the mitochondrial membrane potential and apoptosis execution. This cell remodelling was absent in receptor-independent cell death, such as that induced by the mitochondriotropic drug staurosporine, and in a CEM cell line selected for its multidrug resistance (CEM VBL100). In these cells a reduced FasL (Fas ligand)-induced endocytosis and a reduced organelle cross-talk corresponded to a reduced apoptosis. Altogether, these findings suggest a key role of endocytosis in the propagation and amplification of the CD95/Fas-activated signalling leading to type II cell demise.
Although RecQ5{beta} is a ssDNA (single-stranded DNA)-stimulated ATPase and an ATP-dependent DNA helicase with strand-annealing activities, its cellular function remains to be explored. In the present paper, we used immunopurification and MS-based analyses to show that human DNA helicase RecQ5{beta} is associated with at least four RNAP II (RNA polymerase II) subunits. RecQ5{beta} was also present in complexes immunoprecipitated using three different antibodies against the large subunit of RNAP II, or in complexes immunoprecipitated using an anti-FLAG antibody against either FLAG–RNAP II 33 kDa subunit or FLAG–Pin1. Different regions of the non-helicase domain of the RecQ5{beta} molecule were associated with hypophosphorylated and hyperphosphorylated forms of the RNAP II large subunit independently of DNA and RNA. RecQ5{beta} was also found in nuclear chromatin fractions and associated with the coding regions of the LDL (low-density lipoprotein) receptor and {beta}-actin genes. Knockdown of the RecQ5{beta} transcript increased the transcription of those genes. The results of the present study suggest that RecQ5{beta} has suppressive roles in events associated with RNAP II-dependent transcription.
In eukaryotes, ORC (origin recognition complex), a six-protein complex, is the most likely initiator of chromosomal DNA replication. ORC belongs to the AAA+ (ATPases associated with a variety of cellular activities) family of proteins and has intrinsic ATPase activity derived from Orc1p, one of its subunits. To reveal the role of this ATPase activity in Saccharomyces cerevisiae (baker's yeast) ORC, we mutated the Orc1p sensor 1 and sensor 2 regions, which are important for ATPase activity in AAA+ proteins. Plasmid-shuffling analysis revealed that Asn600, Arg694 and Arg704 are essential for the function of Orc1p. In yeast cells, overexpression of Orc1R694Ep inhibited growth, caused inefficient loading of MCM (mini-chromosome maintenance complex of proteins) and slowed the progression of S phase. In vitro, purified ORC-1R [ORC with Orc1R694Ep (Orc1p Arg694{Rightwards Arrow}Glu mutant)] has decreased ATPase activity in the presence or absence of origin DNA. However, other activities (ATP binding and origin DNA binding) were indistinguishable from those of wild-type ORC. The present study showed that Arg694 of the Orc1p subunit is important for the ATPase activity of ORC and suggests that this ATPase activity is required for efficient MCM loading on to origin DNA and for progression of S phase.
Members of the Ago (Argonaute) protein family are the mediators of small RNA-guided gene-silencing pathways including RNAi (RNA interference), translational regulation by miRNAs (microRNAs) and transcriptional silencing. Recent findings by Zeng et al. in this issue of the Biochemical Journal demonstrate that Ago proteins are post-translationally modified by phosphorylation of Ser387. Mutating Ser387 to alanine leads to reduced localization of human Ago2 to cytoplasmic P-bodies (processing bodies), cellular sites where RNA turnover and, at least in part, miRNA-guided gene regulation occurs. Zeng et al. further show that a member of the MAPK (mitogen-activated protein kinase) signalling pathway phosphorylates Ago2 at Ser387, suggesting that Ago2-mediated gene silencing might be linked to distinct signalling pathways.
CopZ, an Atx1-like copper chaperone from the bacterium Bacillus subtilis, functions as part of a complex cellular machinery for Cu(I) trafficking and detoxification, in which it interacts specifically with the transmembrane Cu(I)-transporter CopA. Here we demonstrate that the cysteine residues of the MXCXXC Cu(I)-binding motif of CopZ have low proton affinities, with both exhibiting pKa values of 6 or below. Chelator competition experiments demonstrated that the protein binds Cu(I) with extremely high affinity, with a small but significant pH-dependence over the range pH 6.5–8.0. From these data, a pH-corrected formation constant, {beta}2={Tilde Operator}6×1022 M{Minus Sign}2, was determined. Rapid exchange of Cu(I) between CopZ and the Cu(I)-chelator BCS (bathocuproine disulfonate) indicated that the mechanism of exchange does not involve simple dissociation of Cu(I) from CopZ (or BCS), but instead proceeds via the formation of a transient Cu(I)-mediated protein–chelator complex. Such a mechanism has similarities to the Cu(I)-exchange pathway that occurs between components of copper-trafficking pathways.
SHP (small heterodimer partner; NR0B2) is an atypical orphan NR (nuclear receptor) that functions as a transcriptional co-repressor by interacting with a diverse set of NRs and transcriptional factors. HNF-6 (hepatocyte nuclear factor-6) is a key regulatory factor in pancreatic development, endocrine differentiation and the formation of the biliary tract, as well as glucose metabolism. In this study, we have investigated the function of SHP as a putative repressor of HNF-6. Using transient transfection assays, we have shown that SHP represses the transcriptional activity of HNF-6. Confocal microscopy revealed that both SHP and HNF-6 co-localize in the nuclei of cells. SHP physically interacted with HNF-6 in protein–protein association assays in vitro. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated that SHP inhibits the DNA-binding activity of HNF-6 to an HNF-6-response element consensus sequence, and the HNF-6 target region of the endogenous G6Pase (glucose 6-phosphatase) promoter respectively. Northern blot analysis of HNF-6 target genes in cells infected with adenoviral vectors for SHP and SHP siRNAs (small inhibitory RNAs) indicated that SHP represses the expression of endogenous G6Pase and PEPCK (phosphoenolpyruvate carboxykinase). Our results suggest that HNF-6 is a novel target of SHP in the regulation of gluconeogenesis.
by Laurie A. Graham, Stephen C. Lougheed, K. Vanya Ewart, Peter L. Davies
Fishes living in icy seawater are usually protected from freezing by endogenous antifreeze proteins (AFPs) that bind to ice crystals and stop them from growing. The scattered distribution of five highly diverse AFP types across phylogenetically disparate fish species is puzzling. The appearance of radically different AFPs in closely related species has been attributed to the rapid, independent evolution of these proteins in response to natural selection caused by sea level glaciations within the last 20 million years. In at least one instance the same type of simple repetitive AFP has independently originated in two distant species by convergent evolution. But, the isolated occurrence of three very similar type II AFPs in three distantly related species (herring, smelt and sea raven) cannot be explained by this mechanism. These globular, lectin-like AFPs have a unique disulfide-bonding pattern, and share up to 85% identity in their amino acid sequences, with regions of even higher identity in their genes. A thorough search of current databases failed to find a homolog in any other species with greater than 40% amino acid sequence identity. Consistent with this result, genomic Southern blots showed the lectin-like AFP gene was absent from all other fish species tested. The remarkable conservation of both intron and exon sequences, the lack of correlation between evolutionary distance and mutation rate, and the pattern of silent vs non-silent codon changes make it unlikely that the gene for this AFP pre-existed but was lost from most branches of the teleost radiation. We propose instead that lateral gene transfer has resulted in the occurrence of the type II AFPs in herring, smelt and sea raven and allowed these species to survive in an otherwise lethal niche.
by Alberto Valbuena, Sandra Blanco, Francisco M. Vega, Pedro A. Lazo
Background
The vaccinia-related kinase 1 (VRK1) protein, an activator of p53, can be proteolytically downregulated by an indirect mechanism, which requires p53-dependent transcription.
Principal Findings
In this work we have biochemically characterized the contribution of several p53 transcriptional cofactors with acetyl transferase activity to the induction of VRK1 downregulation that was used as a functional assay. Downregulation of VRK1 induced by p53 is prevented in a dose dependent manner by either p300 or CBP, but not by PCAF, used as transcriptional co-activators, suggesting that p53 has a different specificity depending on the relative level of these transcriptional cofactors. This inhibition does not require p53 acetylation, since a p53 acetylation mutant also induces VRK1 downregulation. PCAF can not revert the VRK1 protection effect of p300, indicating that these two proteins do not compete for a common factor needed to induce VRK1 downregulation. The protective effect is also induced by the C/H3 domain of p300, a region implicated in binding to several transcription factors and SV40 large T antigen; but the protective effect is lost when a mutant C/H3Del33 is used. The protective effect is a consequence of direct binding of the C/H3 domain to the transactivation domain of p53. A similar downregulatory effect can also be detected with VRK2 protein.
Conclusions/Significance
Specific p53-dependent effects are determined by the availability and ratios of its transcriptional cofactors. Specifically, the downregulation of VRK1/VRK2 protein levels, as a consequence of p53 accumulation, is thus dependent on the levels of the p300/CBP protein available for transcriptional complexes, since in this context this cofactor functions as a repressor of the effect. These observations point to the relevance of knowing the cofactor levels in order to determine one effect or another.
by Ursula Schwab, Tuulikki Seppänen-Laakso, Laxman Yetukuri, Jyrki Ågren, Marjukka Kolehmainen, David E. Laaksonen, Anna-Liisa Ruskeepää, Helena Gylling, Matti Uusitupa, Matej Orešič
Background
The effect of weight loss on different plasma lipid subclasses at the molecular level is unknown. The aim of this study was to examine whether a diet-induced weight reduction result in changes in the extended plasma lipid profiles (lipidome) in subjects with features of metabolic syndrome in a 33-week intervention.
Methodology/Principal Findings
Plasma samples of 9 subjects in the weight reduction group and 10 subjects in the control group were analyzed using mass spectrometry based lipidomic and fatty acid analyses. Body weight decreased in the weight reduction group by 7.8±2.9% (p<0.01). Most of the serum triacylglycerols and phosphatidylcholines were reduced. The decrease in triacylglycerols affected predominantly the saturated short chain fatty acids. This decrease of saturated short chain fatty acid containing triacylglycerols correlated with the increase of insulin sensitivity. However, levels of several longer chain fatty acids, including arachidonic and docosahexanoic acid, were not affected by weight loss. Levels of other lipids known to be associated with obesity such as sphingolipids and lysophosphatidylcholines were not altered by weight reduction.
Conclusions/Significance
Diet-induced weight loss caused significant changes in global lipid profiles in subjects with abnormal glucose metabolism. The observed changes may affect insulin sensitivity and glucose metabolism in these subjects.
by Kathleen H. Rubins, Lisa E. Hensley, George W. Bell, Chunlin Wang, Elliot J. Lefkowitz, Patrick O. Brown, David A. Relman
Background
Poxviruses engage in a complex and intricate dialogue with host cells as part of their strategy for replication. However, relatively little molecular detail is available with which to understand the mechanisms behind this dialogue.
Methodology/Principal Findings
We designed a specialized microarray that contains probes specific to all predicted ORFs in the Monkeypox Zaire (MPXV) and Vaccinia Western Reserve (VACV) genomes, as well as >18,000 human genes, and used this tool to characterize MPXV and VACV gene expression responses in vitro during the course of primary infection of human monocytes, primary human fibroblasts and HeLa cells. The two viral transcriptomes show distinct features of temporal regulation and species-specific gene expression, and provide an early foundation for understanding global gene expression responses during poxvirus infection.
Conclusions/Significance
The results provide a temporal map of the transcriptome of each virus during infection, enabling us to compare viral gene expression across species, and classify expression patterns of previously uncharacterized ORFs.
It is becoming increasingly clear that the soluble protofibrillar species that proceed amyloid fibril formation are associated with a range of neurodegenerative disorders such as Alzheimer's and Parkinson diseases. Computer simulations of the processes that lead to the formation of these oligomeric species are starting to make significant contributions to our understanding of the determinants of protein aggregation. We simulate different systems at constant concentration but with a different number of peptides and we study the how the finite number of proteins affects the underlying free energy of the system and therefore the relative stability of the species involved in the process. If not taken into account, this finite size effect can undermine the validity of theoretical predictions regarding the relative stability of the species involved and the rates of conversion from one to the other. We discuss the reasons that give rise to this finite size effect form both a probabilistic and energy fluctuations point of view and also how this problem can be dealt by a finite size scaling analysis.
4C8 is a new mouse anti-human CD34 monoclonal antibody (mAb), which recognizes class II CD34 epitopes and can be used for clinical hematopoietic stem/progenitor cell selection. In an attempt to improve its safety profiles, we have developed a humanized antibody of 4C8 by complementarity-determining region (CDR) grafting method in this study. Using a molecular model of 4C8 built by computer-assisted homology modelling, framework region (FR) residues of potential importance to the antigen binding were identified. A humanized version of 4C8, denoted as h4C8, was generated by transferring these key murine FR residues onto a human antibody framework that was selected based on homology to the mouse antibody framework, together with the mouse CDR residues. The resultant humanized antibody was shown to possess antigen-binding affinity and specificity similar to that of the original murine antibody, suggesting that it might be an alternative to mouse anti-CD34 antibodies routinely used clinically.
A bacterial semicarbazide-sensitive amine oxidase (SSAO) was purified and characterized from Mycobacterium sp. strain JC1 DSM 3803 grown on benzylamine. During the purification procedures, the enzyme was tending to aggregate and exhibited heterogeneity in native PAGE. The heterogeneous forms having amine oxidase (AO) activity could be separated by their native molecular weights using gel-filtration chromatography. Most of the AOs behaved as dimers (Mr 150,000) composed of a 75-kDa subunit, but some aggregated to form tetramers (Mr 300,000). Besides their native molecular weight, subunit composition and Vmax value, both forms (dimer and tetramer) have almost identical biochemical properties (e.g. subunit size, optimum pH and temperature, activation energy, Km value on benzylamine, substrate and inhibitor specificities). When AO activity was observed by activity staining, the best-oxidized substrate was benzylamine, although the AO also oxidized tyramine and histamine. The AO was strongly inhibited by semicarbazide and isoniazid, but KCN did not affect its activity. The purified enzyme was shown to contain 2.39 mol of copper per mole of subunit, but there were no evidences of topaquinone co-factor involvement, when tested by absorption spectrum analysis and redox-cycling staining for quinoprotein detection.
Bacillus subtilis (Bs) DivIVA comprises coiled-coil structures and self-associates forming a 10–12 mer complex in vitro. Using bioinformatic approaches, we determined that Enterococcus faecalis (Ef) DivIVA comprises four coiled-coil domains, one at the N-terminus, the second and the third in the central region of the protein and the fourth at the C-terminus. We determined that DivIVAEf self-interacts and forms a 10–12 multimeric complex. Point mutations or deletions of the central regions predicted bioinformatically to disrupt the coiled-coil structures either eliminated or weakened DivIVAEf self-interaction and reduced oligomerization. Mutations disrupting the N- and C-terminal coiled-coils of DivIVAEf did not affect DivIVAEf oligomerization. The introduction of DivIVAEf mutations to both the N-terminal and the central coiled-coil domains were lethal unless rescued by expressing wild-type DivIVAEfin trans. E. faecalis cells expressing these mutations displayed aberrant cell morphology, indicating disruption of the normal cell division phenotype. The results in E. faecalis also indicate that both the N-terminal and the central coiled-coil structures of DivIVAEf are indispensable for proper biological function. Overexpression of wild-type DivIVAEf in both rod-shaped and round Escherichia coli cells resulted in morphological changes, while the overexpression of DivIVAEf mutations failed to induce such alterations.
The modification of proteins by SUMO (small ubiquitin-like modifier) regulates various cellular processes. Sumoylation often occurs on a specific lysine residue within the consensus motif KxE/D. However, little is known about the specificity and selectivity of SUMO target sites. We describe here a SUMO assay with peptide array on solid support for the simultaneous characterization of hundreds of different SUMO target sites. This approach was used to characterize known SUMO substrates. The position of the motif within the peptide and the amino acids flanking the acceptor site affected the efficiency of SUMO modification. Interestingly, a sequence of only four amino acids, corresponding to the SUMO consensus motif without flanking amino acids, was a bona fide target site. Analysis of a peptide library for all variants of the KxE/D consensus motif revealed that the first and third positions in the tetrapeptide preferably contain aromatic amino acid residues. Furthermore, by adding the SUMO E3 ligase PIAS1 to the reaction mixture, we show specific enhancement of the modification of a PIAS1-dependent SUMO substrate in this system. Overall, our results demonstrate that the sumoylation assay with peptide array on solid support can be used for the high-throughput characterization of SUMO target sites, and provide new insights into the composition, selectivity and specificity of SUMO target sites.
Fibrin deposition was universal in the lungs of SARS patients and fgl2 prothrombinase gene, a novel procoagulant, was demonstrated to express highly in a clinically relevant SARS model. To investigate whether and which structural protein of SARS-CoV induced transcription of hfgl2 prothrombinase gene, three eukaryotic expression plasmids expressing nucleocapsid protein (N), membrane protein (M) and spike protein 2 (S2) of SARS-CoV were co-transfected with hfgl2 promoter luciferase-reporter plasmids and β-galactosidase plasmid in CHO cells, respectively. M, N and S2 protein of SARS-CoV were detected by western blotting and immunohistochemistry analysis. Further assays demonstrated that expression of hfgl2 gene was related with N protein, but not with M or S2 protein in THP-1 cells and Vero cells. N protein significantly induced functional procoagulant activity in comparison with control group. Luciferase assay showed that N protein of SARS-CoV could activate the transcription of hfgl2 promoter compared with the pcDNA3.1 empty vector. Site-directed mutagenesis and EMSA assay further demonstrated that transcription factor C/EBP alpha band with its cognate cis-element in hfgl2 promoter. The results showed that N protein of SARS-CoV induced hfgl2 gene transcription dependent on the transcription factor C/EBP alpha, which maybe contribute to the development of thrombosis in SARS.
Proteoglycans carrying heparan sulphate (HS) chains are ubiquitously expressed at cell surfaces and in extra-cellular matrices, and HS chains interact with numerous proteins, including growth factors, morphogens and extra-cellular-matrix proteins. These interactions form the basis of HS-related biological phenomena. Thus, the biosynthesis of HS regulates key events in embryonic development and homeostasis, and deranged HS biosynthesis could cause diseases. EXT1 and EXT2 genes encoding the polymerase responsible for HS biosynthesis are known as causative genes of hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by the formation of multiple cartilaginous tumours. In this review, we will summarize HS biosynthesis in several model animals, the effects on cellular functions by alteration of HS biosynthesis, and HS-associated diseases. This review suggests that HS biosynthetic enzymes would be potential candidates for drug targets in various diseases.
A-crystallin is abundant in the lens of the eye and acts as a molecular chaperone by preventing aggregation of denaturing proteins. We previously found that chemical modification of the guanidino group of selected arginine residues by a metabolic -dicarbonyl compound, methylglyoxal (MGO), makes human A-crystallin a better chaperone. Here, we examined how the introduction of additional guanidino groups and modification by MGO influence the structure and chaperone function of A-crystallin. A-crystallin lysine residues were converted to homoarginine by guanidination with o-methylisourea (OMIU) and then modified with MGO. LC-ESI-mass spectrometry identified homoargpyrimidine and homohydroimidazolone adducts after OMIU and MGO treatment. Treatment with 0.25 M OMIU abolished most of the chaperone function. However, subsequent treatment with 1.0 mM MGO not only restored the chaperone function but increased it by ~40% and ~60% beyond that of unmodified A-crystallin, as measured with citrate synthase and insulin aggregation assays, respectively. OMIU treatment reduced the surface hydrophobicity but after MGO treatment, it was ~39% higher than control. FRET analysis revealed that A-crystallin subunit exchange rate was markedly retarded by OMIU modification, but was enhanced after MGO modification. These results indicate a pattern of loss and gain of chaperone function within the same protein that is associated with introduction of guanidino groups and their neutralization. These findings support our hypothesis that positively charged guanidino group on arginine residues keeps the chaperone function of A-crystallin in check and that a metabolic -dicarbonyl compound neutralizes this charge to restore and enhance chaperone function.
Rare types of glycosylation often occur in a domain-specific manner and are involved in specific biological processes. Well-known examples of such modification are O-linked fucose (O-fucose) and O-linked glucose (O-glucose) glycans on epidermal growth factor (EGF) domains. In particular, O-fucose glycans are reported to regulate the functions of EGF domain-containing proteins such as urinary-type plasminogen activator and Notch receptors. Two glycosyltransferases catalyze the initiation and elongation of O-fucose glycans. The initiation process is catalyzed by O-fucosyltransferase 1, which is essential for Notch signalling in both Drosophila and mice. O-fucosyltransferase 1 can affect the folding, ligand interaction and endocytosis of Notch receptors, and both the glycosyltransferase and non-catalytic activities of O-fucosyltransferase 1 have been reported. The elongation of O-fucose monosaccharide is catalyzed by Fringe-related genes, which differentially modulate the interaction between Notch and two classes of ligands, namely, Delta and Serrate/Jagged. In this article, we have reviewed the recent reports addressing the distinctive features of the glycosyltransferases and O-glycans present on the EGF domains.
One feature of the 3-peptide, which has the amino acid sequence of (Leu-Glu-Thr-Leu-Ala-Lys-Ala)3, that distinguishes it from many other -helix-forming peptides is its ability to form fibrous assemblies that can be observed by transmission electron microscopy. In this study, the effects of Ala->Gln substitution at the e (5th) or g (7th) position in the above heptad sequence of the 3-peptide on the formation of -helix and fibrous assemblies were investigated by circular dichroism spectral measurement and atomic force microscopy. The 5Q3-peptide obtained by Ala->Gln substitution at the e position of the 3-peptide was found to form very short fibrils with long-elliptical shape, whereas the 7Q3-peptide with Gln residues at the g position lost its ability to form such assemblies, in spite of -helix formation in both peptides; the stabilities of both peptides decreased. These results indicate that Ala residues at the g position in the heptad sequence of the 3-peptide are key residues for the formation of fibrous assemblies, which may be due to hydrophobic interactions between -helical bundle surfaces.
Oxidative regeneration pathways of bovine pancreatic ribonuclease A (RNase A), which has four SS linkages, were studied at 25°C and pH 8.0 by using trans-3,4-dihydroxy-1-selenolane oxide (DHSox), a new selenoxide reagent with strong oxidation power. The short-term folding study using a quench-flow instrument (~1 min) revealed that early intermediates (1S, 2S, 3S and 4S) are formed stochastically and irreversibly from the reduced protein (R) and do not have any stable structures. In the long-term folding study (~300 min), on the other hand, slow generation of the key intermediates (des[65–72] and des[40–95]) through SS rearrangement from the 3S intermediate ensemble was observed, followed by slight formation of native RNase A (N). The parallel UV and CD measurements demonstrated that formation of the key intermediates is accompanied with the formation of the native-like structures. Thus, DHSox allowed facile identification of the conformational folding steps coupled with SS rearrangement on the major oxidative folding pathways.
Based on our first structural data of L-threonine dehydrogenase (TDH) of Pyrococcus horikoshii (PhTDH), we examined its catalytic mechanism. The structural analysis indicated that a catalytic zinc atom at the active centre of PhTDH is coordinated by four residues (Cys42, His67, Glu68 and Glu152) with low affinity. These residues are highly conserved in alcohol dehydrogenases (ADHs) and TDHs. Several PhTDH mutants were prepared with respect to Glu152 and other residues, relating to the proton relay system that is substantially a rate-limiting step in ADH. It was found that the E152D mutant showed 3-fold higher turnover rate and reduced affinities toward L-threonine and NAD+, compared to wild-type PhTDH. The kinetic analysis of Glu152 mutants indicated that the carboxyl group of Glu152 is important for expressing the catalytic activity. The results obtained from pH dependency of kinetic parameters suggested that Glu152 to Asp substitution causes the enhancement of deprotonation of His47 or ionization of zinc-bound water and threonine in the enzyme-NAD+ complex. Furthermore, it was predicted that the access of threonine substrate to the enzyme-NAD+ complex induces a large conformational change in the active domain of PhTDH. From these results, we propose here that the proton relay system works as a catalytic mechanism of PhTDH.
The applicability of employing a carbon fibre mesh as an electrochemical sensing substructure for assessing urate transformations within wound exudates is evaluated. Prototype sensor assemblies have been designed and their response characteristics towards uric acid and other common physiological components are detailed. Modification of the carbon fibre sensor through surface anodization and the application of cellulose acetate permselective barriers have been shown to lead to optimized responses and much greater sensitivity (1440% increase) and specificity. These could enable the accurate periodic monitoring of uric acid in wound fluid. The performance characteristics of the composite sensors in whole blood, serum and blister fluid have been investigated.
Urea and guanidine hydrochloride (GdnHCl) denaturation of bovine serum albumin (BSA) were investigated using bromophenol blue (BPB) binding as a probe. Addition of BPB to BSA produced an absorption difference spectrum in the wavelength range, 525–675 nm with a minimum at 587 nm and a maximum at 619 nm. The magnitude of absorption difference (Abs.) at 619 nm decreased on increasing urea/GdnHCl concentration and followed the denaturation curve. The denaturation was found to be a two-state, single-step transition. The transitions started at 1.75 and 0.875 M and completed at 6.5 and 3.25 M with the mid point occurring around 4.0 and 1.5 M urea and GdnHCl concentrations, respectively. The value of free energy of stabilization, $$\Delta {G}_{D}^{{H}_{2}O}$$ as determined from urea and GdnHCl denaturation curves was found to be 4041 and 4602 cal/mol, respectively. Taken together, these results suggest that BPB binding can be used as a probe to study urea and GdnHCl denaturation of BSA.
We analysed four types of free ceramides (Cer 1, Cer 2, Cer 3 and Cer 4) from equine kidneys by electrospray ionization mass spectrometry. Cer 1 was composed of dihydroxy long-chain bases (dLCBs) of (4E)-sphingenine (d18:1), sphinganine and non-hydroxy fatty acids (NFAs); Cer 2 was composed of trihydroxy LCBs (tLCBs) of 4-hydroxysphinganine, t16:0, t18:0, t19:0 and t20:0, and NFAs; Cer 3 was composed of dLCBs, d16:1, d17:1, d18:1, d19:1 and d20:1, and hydroxy FAs (HFAs); and Cer 4 was composed of tLCBs, t16:0, t17:0, t18:0, t19:0 and t20:0, and HFAs. The results indicate all ceramide species containing LCBs with non-octadeca lengths (NOD-LCBs) can be classified into hydroxy-ceramides since these species always consist of tLCBs, and/or HFAs. Furthermore, such species tend to contain FAs with longer acyl chains but contain neither palmitate (C16:0) nor its hydroxylated form (C16:0h). The apoptosis-inducing activities of these hydroxyl-ceramides towards tumour cell lines were compared with that of non-hydroxy-ceramides, dLCB-NFA (Cer 1). Monohydroxy-ceramides, tLCB-NFA (Cer 2) and dLCB-HFA (Cer 3), exhibited stronger activities, whereas dihydroxy-ceramides, tLCB-HFA (Cer 4), exhibited similar or weaker activity than dLCB-NFA (Cer 1), depending on cell lines.
by Edward C. Hsiao, Yuko Yoshinaga, Trieu D. Nguyen, Stacy L. Musone, Judy E. Kim, Paul Swinton, Isidro Espineda, Carlota Manalac, Pieter J. deJong, Bruce R. Conklin
Background
Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.
Methodology
Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.
Conclusions
Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library.
by Silvia Ortega-Gutiérrez, Donmienne Leung, Scott Ficarro, Eric C. Peters, Benjamin F. Cravatt
Background
Phosphoprotein phosphatase 2A (PP2A), a major serine-threonine protein phosphatase in eukaryotes, is an oligomeric protein comprised of structural (A) and catalytic (C) subunits to which a variable regulatory subunit (B) can associate. The C subunit contains a methyl ester post-translational modification on its C-terminal leucine residue, which is removed by a specific methylesterase (PME-1). Methylesterification is thought to control the binding of different B subunits to AC dimers, but little is known about its physiological significance in vivo.
Methodology/Principal Findings
Here, we show that targeted disruption of the PME-1 gene causes perinatal lethality in mice, a phenotype that correlates with a virtually complete loss of the demethylated form of PP2A in the nervous system and peripheral tissues. Interestingly, PP2A catalytic activity over a peptide substrate was dramatically reduced in PME-1(−/−) tissues, which also displayed alterations in phosphoproteome content.
Conclusions
These findings suggest a role for the demethylated form of PP2A in maintenance of enzyme function and phosphorylation networks in vivo.
by Tina M. Bunnell, Michele A. Jaeger, Daniel P. Fitzsimons, Kurt W. Prins, James M. Ervasti
α-Dystrobrevin is a component of the dystrophin-glycoprotein complex (DGC) and is thought to have both structural and signaling roles in skeletal muscle. Mice deficient for α-dystrobrevin (adbn−/−) exhibit extensive myofiber degeneration and neuromuscular junction abnormalities. However, the biochemical stability of the DGC and the functional performance of adbn−/− muscle have not been characterized. Here we show that the biochemical association between dystrophin and β-dystroglycan is compromised in adbn−/− skeletal muscle, suggesting that α-dystrobrevin plays a structural role in stabilizing the DGC. However, despite muscle cell death and DGC destabilization, costamere organization and physiological performance is normal in adbn−/− skeletal muscle. Our results demonstrate that myofiber degeneration alone does not cause functional deficits and suggests that more complex pathological factors contribute to the development of muscle weakness in muscular dystrophy.
by Yan Wei, Mei-Hua Qu, Xing-Sheng Wang, Lan Chen, Dong-Liang Wang, Ying Liu, Qian Hua, Rong-Qiao He
Tau, an important microtubule associated protein, has been found to bind to DNA, and to be localized in the nuclei of both neurons and some non-neuronal cells. Here, using electrophoretic mobility shifting assay (EMSA) in the presence of DNA with different chain-lengths, we observed that tau protein favored binding to a 13 bp or a longer polynucleotide. The results from atomic force microscopy also showed that tau protein preferred a 13 bp polynucleotide to a 12 bp or shorter polynucleotide. In a competitive assay, a minor groove binder distamycin A was able to replace the bound tau from the DNA double helix, indicating that tau protein binds to the minor groove. Tau protein was able to protect the double-strand from digestion in the presence of DNase I that was bound to the minor groove. On the other hand, a major groove binder methyl green as a negative competitor exhibited little effect on the retardation of tau-DNA complex in EMSA. This further indicates the DNA minor groove as the binding site for tau protein. EMSA with truncated tau proteins showed that both the proline-rich domain (PRD) and the microtubule-binding domain (MTBD) contributed to the interaction with DNA; that is to say, both PRD and MTBD bound to the minor groove of DNA and bent the double-strand, as observed by electron microscopy. To investigate whether tau protein is able to prevent DNA from the impairment by hydroxyl free radical, the chemiluminescence emitted by the phen-Cu/H2O2/ascorbate was measured. The emission intensity of the luminescence was markedly decreased when tau protein was present, suggesting a significant protection of DNA from the damage in the presence of hydroxyl free radical.
Biochemistry and structural biology are undergoing a dramatic revolution. Until now, mostly in vitro techniques have been used to study subtle and complex biological processes under conditions usually remote from those existing in the cell. We developed a novel in-cell methodology to post-translationally modify interactor proteins and identify the amino acids that comprise the interaction surface of a target protein when bound to the post-translationally modified interactors. Modifying the interactor proteins causes structural changes that manifest themselves on the interacting surface of the target protein and these changes are monitored using in-cell NMR. We show how Ubiquitin interacts with phosphorylated and non-phosphorylated components of the receptor tyrosine kinase (RTK) endocytic sorting machinery: STAM2 (Signal-transducing adaptor molecule), Hrs (Hepatocyte growth factor regulated substrate) and the STAM2-Hrs heterodimer. Ubiquitin binding mediates the processivity of a large network of interactions required for proper functioning of the RTK sorting machinery. The results are consistent with a weakening of the network of interactions when the interactor proteins are phosphorylated. The methodology can be applied to any stable target molecule and may be extended to include other post-translational modifications such as ubiquitination or sumoylation, thus providing a long-awaited leap to high resolution in cell biochemistry.
by Gyula Timinszky, Miriam Bortfeld, Andreas G. Ladurner
Background
Germline progenitors resist signals that promote differentiation into somatic cells. This occurs through the transient repression in primordial germ cells of RNA polymerase II, specifically by disrupting Ser2 phosphorylation on its C-terminal domain.
Methodology/Principal Findings
Here we show that contrary to expectation the Drosophila polar granule component (pgc) gene functions as a protein rather than a non-coding RNA. Surprisingly, pgc encodes a 71-residue, dimeric, alpha-helical oligopeptide repressor. In vivo data show that Pgc ablates Ser2 phosphorylation of the RNA polymerase II C-terminal domain and completely suppresses early zygotic transcription in the soma.
Conclusions/Significance
We thus identify pgc as a novel oligopeptide that readily inhibits gene expression. Germ cell repression of transcription in Drosophila is thus catalyzed by a small inhibitor protein.
