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Aspergillus fumigatus is the causative agent of aspergillosis, a frequently invasive colonization of the lungs of immunocompromised patients. GNA1 (D-glucosamine-6-phosphate N-acetyltransferase) catalyses the acetylation of GlcN-6P (glucosamine-6-phosphate) to GlcNAc-6P (N-acetylglucosamine-6-phosphate), a key intermediate in the UDP-GlcNAc biosynthetic pathway. Gene disruption of gna1 in yeast and Candida albicans has provided genetic validation of the enzyme as a potential target. An understanding of potential active site differences between the human and A. fumigatus enzymes is required to enable further work aimed at identifying selective inhibitors for the fungal enzyme. In the present study, we describe crystal structures of both human and A. fumigatus GNA1, as well as their kinetic characterization. The structures show significant differences in the sugar-binding site with, in particular, several non-conservative substitutions near the phosphate-binding pocket. Mutagenesis targeting these differences revealed drastic effects on steady-state kinetics, suggesting that the differences could be exploitable with small-molecule inhibitors.
Dysregulation of life and death at the cellular level leads to a variety of diseases. In the nervous system, aberrant neuronal death is an outstanding feature of neurodegenerative diseases. Since the discovery of the caspase family of proteases, much effort has been made to determine how caspases function in disease, including neurodegenerative diseases. Although many papers have been published examining caspases in neuronal death and disease, the pathways have not been fully clarified. In the present review, we examine the potential players in the death pathways, the current tools for examining these players and the models for studying neurological disease. Alzheimer's disease, the most common neurodegenerative disorder, and cerebral ischaemia, the most common cause of neurological death, are used to illustrate our current understanding of death signalling in neurodegenerative diseases. A better understanding of the neuronal death pathways would provide targets for the development of therapeutic interventions for these diseases.
A necessary feature of the immune system, TCR (T-cell receptor) cross-reactivity has been implicated in numerous autoimmune pathologies and is an underlying cause of transplant rejection. Early studies of the interactions of {alpha}{beta} TCRs (T-cell receptors) with their peptide–MHC ligands suggested that conformational plasticity in the TCR CDR (complementarity determining region) loops is a dominant contributor to T-cell cross-reactivity. Since these initial studies, the database of TCRs whose structures have been solved both bound and free is now large enough to permit general conclusions to be drawn about the extent of TCR plasticity and the types and locations of motion that occur. In the present paper, we review the conformational differences between free and bound TCRs, quantifying the structural changes that occur and discussing their possible roles in specificity and cross-reactivity. We show that, rather than undergoing major structural alterations or ‘folding’ upon binding, the majority of TCR CDR loops shift by relatively small amounts. The structural changes that do occur are dominated by hinge-bending motions, with loop remodelling usually occurring near loop apexes. As predicted from previous studies, the largest changes are in the hypervariable CDR3{alpha} and CDR3{beta} loops, although in some cases the germline-encoded CDR1{alpha} and CDR2{alpha} loops shift in magnitudes that approximate those of the CDR3 loops. Intriguingly, the smallest shifts are in the germline-encoded loops of the {beta}-chain, consistent with recent suggestions that the TCR {beta} domain may drive ligand recognition.
The early intermediates in the protein aggregation pathway, the elusive soluble aggregates, play a pivotal role in growth and maturation of ordered aggregates such as amyloid fibrils. Blocking the growth of soluble oligomers is an effective strategy to inhibit aggregation. To decipher the molecular mechanisms and develop better strategies to arrest aggregation, it is imperative to understand how the size, molecular dynamics, activity and growth kinetics of soluble aggregates are affected when aggregation is inhibited. With this objective, in the present study we have investigated the influence of additives such as SDS, CTAB (cetyltrimethylammonium bromide) and DTT (dithiothreitol) on the slow aggregation of HEWL (hen eggwhite lysozyme) at pH 12.2. For this purpose, techniques such as steady-state and time-resolved fluorescence anisotropy of covalently labelled dansyl probe, gel-filtration chromatography, estimation of free thiol groups, thioflavin T and ANS (8-anilinonaphthalene-1-sulfonic acid) fluorescence, CD and atomic-force microscopy were employed to monitor the soluble oligomers over a period spanning 30 days. The results of the present study reveal that: (i) the spontaneous formation of soluble aggregates is irreversible and abolishes activity; (ii) the initial growth of aggregates (0–24 h) is promoted by a gradual increase in the exposure of hydrophobic surfaces; (iii) subsequently intermolecular disulfide bonds are critical for the assembly and stability of aggregates; (iv) the tight molecular packing inside large aggregates which contributed to slow ({Tilde Operator}5 ns) and restricted segmental motion of dansyl probe was clearly loosened up in the presence of additives, enabling fast (1–2 ns) and free motion (unlike DTT, the size of lysozyme complexes with surfactants, was large, due to a conglomeration of proteins and surfactants); (v) the aggregates show reduced helical content compared with native lysozyme, except in the presence of SDS; and (vi) DTT was more potent than SDS/CTAB in arresting the growth of aggregates.
AA (amyloid protein A) amyloidosis in mice is markedly accelerated when the animals are given, in addition to an inflammatory stimulus, an intravenous injection of protein extracted from AA-laden mouse tissue. Previous findings affirm that AA fibrils can enhance the in vivo amyloidogenic process by a nucleation seeding mechanism. Accumulating evidence suggests that globular aggregates rather than fibrils are the toxic entities responsible for cell death. In the present study we report on structural and morphological features of AEF (amyloid-enhancing factor), a compound extracted and partially purified from amyloid-laden spleen. Surprisingly, the chief amyloidogenic material identified in the active AEF was diffusible globular oligomers. This partially purified active extract triggered amyloid deposition in vital organs when injected intravenously into mice. This implies that such a phenomenon could have been inflicted through the nucleation seeding potential of toxic oligomers in association with altered cytokine induction. In the present study we report an apparent relationship between altered cytokine expression and AA accumulation in systemically inflamed tissues. The prevalence of serum AA monomers and proteolytic oligomers in spleen AEF is consistent to suggest that extrahepatic serum AA processing might lead to local accumulation of amyloidogenic proteins at the serum AA production site.
Protein N{alpha}-terminal acetylation is a conserved and widespread protein modification in eukaryotes. Several studies have linked it to normal cell function and cancer development, but nevertheless, little is known about its biological function. In yeast, protein N{alpha}-terminal acetylation is performed by the N-acetyltransferase complexes NatA, NatB and NatC. In humans, only the NatA complex has been identified and characterized. In the present study we present the components of hNatB (human NatB complex). It consists of the Nat3p homologue hNAT3 (human N-acetyltransferase 3) and the Mdm20p homologue hMDM20 (human mitochondrial distribution and morphology 20). They form a stable complex and in vitro display sequence-specific N{alpha}-acetyltransferase activity on a peptide with the N-terminus Met-Asp-. hNAT3 and hMDM20 co-sediment with ribosomal pellets, thus supporting a model where hNatB acts co-translationally on nascent polypeptides. Specific knockdown of hNAT3 and hMDM20 disrupts normal cell-cycle progression, and induces growth inhibition in HeLa cells and the thyroid cancer cell line CAL-62. hNAT3 knockdown results in an increase in G0/G1-phase cells, whereas hMDM20 knockdown decreased the fraction of cells in G0/G1-phase and increased the fraction of cells in the sub-G0/G1-phase. In summary, we show for the first time a vertebrate NatB protein N{alpha}-acetyltransferase complex essential for normal cell proliferation.
LINCL (late-infantile neuronal ceroid lipofuscinosis) is a fatal neurodegenerative disease resulting from mutations in the gene encoding the lysosomal protease TPPI (tripeptidyl-peptidase I). TPPI is expressed ubiquitously throughout the body but disease appears restricted to the brain. One explanation for the absence of peripheral pathology is that in tissues other than brain, other proteases may compensate for the loss of TPPI. One such candidate is another lysosomal aminopeptidase, DPPI (dipeptidyl-peptidase I), which appears to have overlapping substrate specificity with TPPI and is expressed at relatively low levels in brain. Compensation for the loss of TPPI by DPPI may have therapeutic implications for LINCL and, in the present study, we have investigated this possibility using mouse genetic models. Our rationale was that if DPPI could compensate for the loss of TPPI in peripheral tissues, then its absence should exacerbate disease in an LINCL mouse model but, conversely, increased CNS (central nervous system) expression of DPPI should ameliorate disease. By comparing TPPI and DPPI single mutants with a double mutant lacking both proteases, we found that the loss of DPPI had no effect on accumulation of storage material, disease severity or lifespan of the LINCL mouse. Transgenic expression of DPPI resulted in a {Tilde Operator}2-fold increase in DPPI activity in the brain, but this had no significant effect on survival of the LINCL mouse. These results together indicate that DPPI cannot functionally compensate for the loss of TPPI. Therapeutic approaches to increase neuronal expression of DPPI are therefore unlikely to be effective for treatment of LINCL.
Light signal transduction in plants involves an intricate series of pathways which is finely regulated by interactions between specific signalling proteins, as well as by protein modifications such as phosphorylation and ubiquitination. The identification of novel phytochrome-interacting proteins and the precise signalling mechanisms that they mediate is still ongoing. In our present study, we show that the newly identified putative phytochrome-associated protein, PAPP2C (phytochrome-associated protein phosphatase type 2C), interacts in the nucleus with phyA (phytochrome A) and phyB, both in vitro and in vivo. Moreover, the phosphatase activity of PAPP2C and its association with phytochromes were found to be enhanced by red light, indicating that it plays a role in mediating phytochrome signalling. In particular, PAPP2C specifically binds to the N-terminal PHY domain of the phytochromes. We thus speculate that this interaction reflects a unique regulatory function of this phosphatase toward established phytochrome-associated proteins. We also show that PAPP2C effectively dephosphorylates phytochromes in vitro. Interestingly, PAPP2C indirectly mediates the dephosphorylation of PIF3 (phytochrome-interacting factor 3) in vitro. Taken together, we suggest that PAPP2C functions as a regulator of PIF3 by dephosphorylating phytochromes in the nucleus.
Recent clinical data indicates that the emergence of mutant drug-resistant kinase alleles may be particularly relevant for targeted kinase inhibitors. In order to explore how different classes of targeted therapies impact upon resistance mutations, we performed EGFR (epidermal-growth-factor receptor) resistance mutation screens with erlotinib, lapatinib and CI-1033. Distinct mutation spectra were generated with each inhibitor and were reflective of their respective mechanisms of action. Lapatinib yielded the widest variety of mutations, whereas mutational variability was lower in the erlotinib and CI-1033 screens. Lapatinib was uniquely sensitive to mutations of residues located deep within the selectivity pocket, whereas mutation of either Gly796 or Cys797 resulted in a dramatic loss of CI-1033 potency. The clinically observed T790M mutation was common to all inhibitors, but occurred with varying frequencies. Importantly, the presence of C797S with T790M in the same EGFR allele conferred complete resistance to erlotinib, lapatinib and CI-1033. The combination of erlotinib and CI-1033 effectively reduced the number of drug-resistant clones, suggesting a possible clinical strategy to overcome drug resistance. Interestingly, our results also indicate that co-expression of ErbB2 (v-erb-b2 erythroblastic leukaemia viral oncogene homologue 2) has an impact upon the EGFR resistance mutations obtained, suggesting that ErbB2 may play an active role in the acquisition of drug-resistant mutations.
No studies have been performed on the mitochondria of malaria vector mosquitoes. This information would be valuable in understanding mosquito aging and detoxification of insecticides, two parameters that have a significant impact on malaria parasite transmission in endemic regions. In the present study, we report the analyses of respiration and oxidative phosphorylation in mitochondria of cultured cells [ASE (Anopheles stephensi Mos. 43) cell line] from A. stephensi, a major vector of malaria in India, South-East Asia and parts of the Middle East. ASE cell mitochondria share many features in common with mammalian muscle mitochondria, despite the fact that these cells are of larval origin. However, two major differences with mammalian mitochondria were apparent. One, the glycerol–phosphate shuttle plays as major a role in NADH oxidation in ASE cell mitochondria as it does in insect muscle mitochondria. In contrast, mammalian white muscle mitochondria depend primarily on lactate dehydrogenase, whereas red muscle mitochondria depend on the malate–oxaloacetate shuttle. Two, ASE mitochondria were able to oxidize proline at a rate comparable with that of {alpha}-glycerophosphate. However, the proline pathway appeared to differ from the currently accepted pathway, in that oxoglutarate could be catabolized completely by the tricarboxylic acid cycle or via transamination, depending on the ATP need.
Deregulated apoptosis of MCs (mesangial cells) is associated with a number of kidney diseases including end-stage diabetic nephropathy. Cell death by apoptosis is a tightly orchestrated event, whose mechanisms are not completely defined. In the present study we show that the uPA (urokinase-type plasminogen activator)/uPAR (uPA receptor) system can initiate both cell survival and pro-apoptotic signals in human MCs in response to different apoptotic stimuli. uPA abrogated MC apoptosis induced by serum withdrawal conditions and enhanced apoptosis initiated in MCs by high glucose. Effects of uPA were independent of its proteolytic activity and required uPAR for both pro- and anti-apoptotic effects. Studies on the uPAR interactome provide evidence that the opposing effects of uPA were directed via different uPAR-interacting transmembrane partners. Exposure of MCs to RGD (Arg-Gly-Asp) peptide led to abrogation of the anti-apoptotic effect of uPA, which implies involvement of integrins in this process. A pro-apoptotic effect of uPA under high-glucose conditions was mediated via association of uPAR and the cation-independent M6P (mannose-6-phosphate)/IGF2R (insulin-like growth factor 2 receptor). Both receptors were co-precipitated and co-localized in MCs. Studies on the underlying signalling indicate that the ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt and BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) protein were involved in regulation of apoptosis by uPA in MCs. M6P/IGF2R mediated BAD perinuclear localization during apoptosis initiated by uPA and high glucose. In conclusion, we provide evidence that, in MCs, the uPA/uPAR system regulates survival/apoptosis processes in a stimulus-specific fashion via a mitochondria-dependent mechanism and that BAD protein serves as a downstream molecule.
The adaptor protein Lnk is expressed in haemopoietic cells and plays a critical role in haemopoiesis. Animal model studies demonstrated that Lnk acts as a broad inhibitor of signalling pathways in haemopoietic lineages. Lnk belongs to a family of proteins sharing several structural motifs, including an SH2 (Src homology 2) domain which binds phosphotyrosine residues in various signal-transducing proteins. The SH2 domain is essential for Lnk-mediated negative regulation of several cytokine receptors [e.g. Mpl, EpoR (erythropoietin receptor), c-Kit]. Therefore inhibition of the binding of Lnk to cytokine receptors might lead to enhanced downstream signalling of the receptor and thereby to improved haemopoiesis in response to exposure to cytokines (e.g. erythropoietin in anaemic patients). This hypothesis led us to define the exact binding site of Lnk to the stem cell factor receptor c-Kit. Pull-down experiments using GST (glutathione transferase)-fusion proteins of the different domains of c-Kit showed that Lnk almost exclusively binds to the phosphorylated juxtamembrane domain. Binding of Lnk to the juxtamembrane domain was abolished by point mutation of Tyr568 and was competed by peptides with a phosphotyrosine residue at position 568. Co-immunoprecipitation with full-length wild-type or Y568F mutant c-Kit and Lnk confirmed these results, thus showing the importance of this phosphorylated tyrosine residue. Lnk bound directly to c-Kit without requiring other interacting partners. The identification of the binding site of Lnk to c-Kit will be useful to discover inhibitory molecules that prevent the binding of these two proteins, thus making haemopoietic cells more sensitive to growth factors.
ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related) and DNA-PK (DNA-dependent protein kinase), important regulators of genome stability, belong to the PIKK (phosphoinositide 3-kinase-like kinase) family of protein kinases. In the present study, DNA-affinity chromatography was used to identify DNA-binding proteins phosphorylated by these kinases. This resulted in the identification of FUS (fused in sarcoma)/TLS (translocated in liposarcoma) as an in vitro target of the PIKKs. FUS is a member of the Ewing's sarcoma family of proteins that appears to play a role in regulating genome stability, since mice lacking FUS show chromosomal instability and defects in meiosis. The residues in FUS that are phosphorylated in vitro and in vivo were identified, and phospho-specific antibodies were generated to demonstrate that FUS becomes phosphorylated at Ser42in vivo, primarily in response to agents that cause DSBs (double-strand breaks). DSB-induced FUS phosphorylation in vivo at Ser42 requires ATM and not DNA-PK. Although Ser42 is retained in the oncogenic FUS–CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein 10] fusion generated by a t(12;16)(q13;p11) chromosomal translocation, Ser42 in FUS–CHOP is not phosphorylated after DNA damage. These results identify FUS as a new target of the ATM-signalling pathway and strengthen the notion that FUS regulates genome stability.
HNF-4{alpha} (hepatocyte nuclear factor-4{alpha}) is a key regulator of liver-specific gene expression. To understand the mechanisms governing the regulation of HNF-4{alpha} function during the APR (acute-phase response), the effects of transcription co-activators, including p300, PGC-1{alpha} (peroxisome-proliferator-activated receptor-{gamma} co-activator-1{alpha}) and SRC (steroid receptor co-activator)-1{alpha} were investigated in an injury cell model. We have shown previously that the HNF-4{alpha}-sensitive APR genes ApoB (apolipoprotein B), TTR (transthyretin) and {alpha}1-AT ({alpha}1-antitrypsin) were regulated at the DNA binding and transcriptional levels after cytokine stimulation. We now show that co-activators have a differential impact on the transactivation of HNF-4{alpha}-sensitive genes via HNF-4{alpha}-binding sites in ApoB, TTR or {alpha}1-AT promoters. PGC-1{alpha} strongly enhances the transactivation of ApoB and {alpha}1-AT and, to a lesser extent, of TTR, whereas SRC-1{alpha} and p300 only have a weak or no effect on these three genes. More importantly, it was found that PGC-1{alpha} has a novel role in the modulation of the binding ability of HNF-4{alpha} in response to cytokine treatment. Using in vitro and in vivo approaches, electrophoretic mobility-shift and chromatin immunoprecipitation assays, we demonstrate that the reduced HNF-4{alpha}–DNA binding ability induced by cytokines is eliminated by overexpression of PGC-1{alpha}. Cytokine treatment does not significantly alter the protein levels of HNF-4{alpha} and PGC-1{alpha}, but it does reduce the recruitment of PGC-1{alpha} to HNF-4{alpha}-binding sites and thereby decreases transcriptional activity. These results establish the importance of PGC-1{alpha} for HNF-4{alpha} function and describe a new HNF-4{alpha}-dependent regulatory mechanism that is involved in the response to injury.
In Bacillus megaterium, the synthesis of vitamin B12 (cobalamin) and sirohaem diverges at sirohydrochlorin along the branched modified tetrapyrrole biosynthetic pathway. This key intermediate is made by the action of SirC, a precorrin-2 dehydrogenase that requires NAD+ as a cofactor. The structure of SirC has now been solved by X-ray crystallography to 2.8 Å (1 Å = 0.1 nm) resolution. The protein is shown to consist of three domains and has a similar topology to the multifunctional sirohaem synthases Met8p and the N-terminal region of CysG, both of which catalyse not only the dehydrogenation of precorrin-2 but also the ferrochelation of sirohydrochlorin to give sirohaem. Guided by the structure, in the present study a number of active-site residues within SirC were investigated by site-directed mutagenesis. No active-site general base was identified, although surprisingly some of the resulting protein variants were found to have significantly enhanced catalytic activity. Unexpectedly, SirC was found to bind metal ions such as cobalt and copper, and to bind them in an identical fashion with that observed in Met8p. It is suggested that SirC may have evolved from a Met8p-like protein by loss of its chelatase activity. It is proposed that the ability of SirC to act as a single monofunctional enzyme, in conjunction with an independent chelatase, may provide greater control over the intermediate at this branchpoint in the synthesis of sirohaem and cobalamin.
Human GK{beta} (pancreatic {beta}-cell glucokinase) is the main glucose-phosphorylating enzyme in pancreatic {beta}-cells. It shares several structural, catalytic and regulatory properties with Hxk2 (hexokinase 2) from Saccharomyces cerevisiae. In fact, it has been previously described that expression of GK{beta} in yeast could replace Hxk2 in the glucose signalling pathway of S. cerevisiae. In the present study we report that GK{beta} exerts its regulatory role by association with the yeast transcriptional repressor Mig1 (multicopy inhibitor of GAL gene expression 1); the presence of Mig1 allows GK{beta} to bind to the SUC2 (sucrose fermentation 2) promoter, helping in this way in the maintenance of the repression of the SUC2 gene under high-glucose conditions. Since a similar mechanism has been described for the yeast Hxk2, the findings of the present study suggest that the function of the regulatory domain present in these two proteins has been conserved throughout evolution. In addition, we report that GK{beta} is enriched in the yeast nucleus of high-glucose growing cells, whereas it shows a mitochondrial localization upon removal of the sugar. However, GK{beta} does not exit the nucleus in the absence of Mig1, suggesting that Mig1 regulates the nuclear exit of GK{beta} under low-glucose conditions. We also report that binding of GK{beta} to Mig1 allows the latter protein to be located at the mitochondrial network under low-glucose conditions.
NRPSs (non-ribosomal peptide synthetases) and PKSs (polyketide synthases) require post-translational phosphopantetheinylation to become active. This reaction is catalysed by a PPTase (4′-phosphopantetheinyl transferase). The ppt gene of Penicillium chrysogenum, encoding a protein that shares 50% similarity with the stand-alone large PPTases, has been cloned. This gene is present as a single copy in the genome of the wild-type and high-penicillin-producing strains (containing multiple copies of the penicillin gene cluster). Amplification of the ppt gene produced increases in isopenicillin N and benzylpenicillin biosynthesis. A PPTase-defective mutant (Wis54-PPT{Minus Sign}) was obtained. It required lysine and lacked pigment and penicillin production, but it still synthesized normal levels of roquefortine. The biosynthesis of roquefortine does not appear to involve PPTase-mediated modification of the synthesizing enzymes. The PPT{Minus Sign} mutant did not require fatty acids, which indicates that activation of the fatty acid synthase is performed by a different PPTase. Complementation of Wis54-PPT{Minus Sign} with the ppt gene restored lysine biosynthesis, pigmentation and penicillin production, which demonstrates the wide range of processes controlled by this gene.
by Jana Sponarova, Sofia N. Nyström, Gunilla T. Westermark
Spongiform encephalopathies have been reported to be transmitted by blood transfusion even prior to the clinical onset. Experimental AA-amyloidosis shows similarities with prion disease and amyloid-containing organ-extracts can prime a recipient for the disease. In this systemic form of amyloidosis N-terminal fragments of the acute-phase reactant apolipoprotein serum amyloid A are the main amyloid protein. Initial amyloid deposits appear in the perifollicular region of the spleen, followed by deposits in the liver. We used the established murine model and induced AA-amyloidosis in NMRI mice by intravenous injections of purified amyloid fibrils (‘amyloid enhancing factor’) combined with inflammatory challenge (silver nitrate subcutaneously). Blood plasma and peripheral blood monocytes were isolated, sonicated and re-injected into new recipients followed by an inflammatory challenge during a three week period. When the animals were sacrificed presence of amyloid was analyzed in spleen sections after Congo red staining. Our result shows that some of the peripheral blood monocytes, isolated from animals with detectable amyloid, contained amyloid-seed that primed for AA-amyloid. The seeding material seems to have been phagocytosed by the cells since the AA-precursor (SAA1) was found not be expressed by the monocytes. Plasma recovered from mice with AA amyloidosis lacked seeding capacity. Amyloid enhancing activity can reside in monocytes recovered from mice with AA-amyloidosis and in a prion-like way trigger amyloid formation in conjunction with an inflammatory disorder. Human AA-amyloidosis resembles the murine form and every individual is expected to be exposed to conditions that initiate production of the acute-phase reactant. The monocyte-transfer mechanism should be eligible for the human disease and we point out blood transfusion as a putative route for transfer of amyloidosis.
PCaP1, a hydrophilic cation-binding protein, is bound to the plasma membrane in Arabidopsis thaliana. We focused on the physicochemical properties of PCaP1 to understand its uniqueness in terms of structure and binding of metal ions. On fluorescence analysis, PCaP1 showed a signal of structural change in the presence of Cu2+. The near-UV CD spectra showed a marked change of PCaP1 in CuCl2 solution. The far-UV CD spectra showed the presence of -helices and the intrinsically unstructured region. However, addition of Cu2+ gave no change in the far-UV CD spectra. These results indicate that Cu2+ induced a change in the tertiary structure without changing the secondary structure. The protein was sensitive to proteinase in the presence of Cu2+, supporting that Cu2+ is involved in the structural change. The PCaP1 solution was titrated with CuCl2 and the change in the fluorescence spectrum was monitored to characterize Cu2+-binding properties. The obtained values of Kd for Cu2+ and the ligand-binding number were 10 µM and six ions per molecule, respectively. These findings indicate that PCaP1 has a high Cu2+-binding capacity with a relatively high affinity. PCaP1 lacks cysteine and histidine residues. A large number of glutamate residues may be involved in the Cu2+ binding.
The mitochondrial ATPase inhibitor, IF1, regulates the activity of F1Fo-ATPase. The inhibitory activity of IF1 is highly pH-dependent. The effective inhibition by IF1 requires a low pH. Under basic conditions, its activity markedly declines. The importance of His49 in the pH dependence of bovine IF1 is well-known. However, the residue is not conserved in yeast IF1. We previously showed that Glu21 is required for the pH dependence of yeast IF1, but the function of homologous Glu in mammalian IF1 is not clear. In this study, we examined the requirement for Glu26 of bovine IF1 (corresponding to Glu21 of yeast IF1) regarding its pH dependence by amino acid replacement. Three mutant proteins, E26A, H49K and the double mutant E26A/H49K, were overexpressed and purified. All mutants retained their inhibitory activity well at pH 8.2, although wild-type IF1 was ~10-fold less active at pH 8.2 than at 6.5. A covalent cross-linking study revealed that both wild-type IF1 and the E26A mutant formed a tetramer at pH 8.2, although H49K and E26A/H49K mutants did not. These results indicate that, in addition to His49, Glu26 participates in pH sensing in bovine IF1, and the mechanism of pH sensing mediated by Glu26 is different from the dimer–tetramer model proposed previously.
In this study, an RNA isolation method was developed using a biotinylated peptide nucleic acid (PNA) that is complementary to the target RNA. Using the biotinylated PNA method, we successfully isolated several RNAs from Escherichia coli and from human total RNA in pure form. Damage to the RNA appears to be negligible by this method because the method is rapid and does not require a high temperature treatment to facilitate RNA–PNA binding.
Quality control of proteins in the endoplasmic reticulum (ER) is achieved by two mechanisms, the productive folding mechanism, which is assisted by a number of ER-localized molecular chaperones and folding enzymes (collectively termed ER chaperones), and the ER-associated degradation (ERAD) mechanism, by which misfolded proteins are degraded by the ubiquitin-dependent proteasome system in the cytosol. Accumulation of unfolded proteins in the ER activates the unfolded protein response (UPR), resulting in transcriptional induction of ER chaperones and ERAD components. In mammals, three signalling pathways operate for the UPR, namely the IRE1-XBP1, PERK-ATF4 and ATF6 pathways. Analysis of mouse embryonic fibroblasts deficient in UPR signalling molecule indicates that transcriptional induction of ERAD components depends on the IRE1-XBP1 pathway. However, the molecular basis of this finding remains unclear. Here, we analysed the promoter of human HRD1, which encodes an E3 ubiquitin ligase, an important component of ERAD. We found that induction of HRD1 is mediated by two cis-acting elements, a canonical ER stress response element and a novel element we designate as UPR element II. The presence of UPR element II to which XBP1 but not ATF6 directly binds explains at least in part the dependency of HRD1 induction on the IRE1-XBP1 pathway.
Cathepsin K is known to play an important role in bone resorption, and it has the P2 specificity for proline. Rat cathepsin K has 88% identity with the human enzyme. However, it has been reported that its enzymatic activity for a Cbz-Leu-Arg-MCA substrate is lower than that of human cathepsin K, and that the rat enzyme is not well inhibited by human cathepsin K inhibitors. For this study, we prepared recombinant enzyme to investigate the substrate specificity of rat cathepsin K. Cleavage experiments using the fragment of type I collagen and peptidic libraries demonstrated that rat cathepsin K preferentially hydrolyses the substrates at the P2 Hyp position. Comparison of the S2 site between rat and human cathepsin K sequences indicated that two S2 residues at Ser134 and Val160 in rat are varied to Ala and Leu, respectively, in the human enzyme. Cleavage experiments using two single mutants, S134A and V160L, and one double mutant, S134A/V160L, of rat cathepsin K showed that all the rat mutants lost the P2 Hyp specificity. The information obtained from our comparative studies on rat and human cathepsin K should make a significant impact on developing specific inhibitors of human cathepsin K since rat is usually used as test species.
We previously demonstrated that the uptake of M180 amelogenin protein in dental epithelial cells (HAT-7) results in increased levels of amelogenin mRNA through enhanced mRNA stabilization. To determine the processes involved in the uptake of extracellular M180 amelogenin by cells and in amelogenin intracellular trafficking in the amelogenin protein-mediated amelogenin mRNA expression pathway, we investigated the effects of LAMP1 and LAMP3, which are candidate M180 amelogenin receptors, on M180 amelogenin uptake, localization and amelogenin mRNA induction by amelogenin protein, using anti-LAMP-1 and anti-LAMP-3 antibodies and siRNA analysis. The results indicate that LAMP3 blocking by anti-LAMP-3 decreases M180 amelogenin uptake, but does not affect amelogenin mRNA induction by amelogenin protein, suggesting that LAMP3 is related to amelogenin degradation. Down-regulation by siRNA of LAMP1, which is the receptor for small amelogenin protein (LRAP), does not affect M180 amelogenin uptake, localization or amelogenin mRNA induction by amelogenin protein. Thus, while LAMP1 is the specific receptor for LRAP, it is not a receptor for M180 amelogenin. These findings will aid further research into the understanding of M180 amelogenin function and expression.
Protein arginine methylation is one of the post-translational modifications which yield monomethyl and dimethyl (asymmetric or symmetric) arginines in proteins. In the present study, we investigated the status of protein arginine methylation during human diploid fibroblast senescence. When the expression of protein arginine methyltransferases (PRMTs), namely PRMT1, PRMT4, PRMT5 and PRMT6 was examined, a significant reduction was found in replicatively senescent cells as well as their catalytic activities against histone mixtures compared with the young cells. Furthermore, when the endogenous level of arginine-dimethylated proteins was determined, asymmetric modification (the product of type I PRMTs including PRMT1, PRMT4 and PRMT6) was markedly down-regulated. In contrast, both up- and down-regulations of symmetrically arginine-methylated proteins (the product of type II PRMTs including PRMT5) during replicative senescence were found. Furthermore, when young fibroblasts were induced to premature senescence by sub-cytotoxic H2O2 treatment, results similar to replicative senescence were obtained. Finally, we found that SV40-mediated immortalized WI-38 and HeLa cell lines maintained a higher level of asymmetrically modified proteins as well as type I PRMTs than young fibroblasts. These results suggest that the maintenance of asymmetric modification in the expressed target proteins of type I PRMTs might be critical for cellular proliferation.
Disaccharide-specific glycosidases (diglycosidases) are unique glycoside hydrolases, as their substrate specificities differ from those of monosaccharide-specific β-glycosidases (monoglycosidases), in spite of similarities in their sequences and reaction mechanisms. Diglycosidases selectively hydrolyse the β-glycosidic bond between glycone and aglycone of disaccharide glycosides, but do not cleave the bond between two saccharides, and barely hydrolyse monosaccharide glycosides. We analysed the substrate recognition mechanisms of diglycosidases by computational and experimental methods, using furcatin hydrolase (FH) (EC 3.2.1.161) derived from Viburnum furcatum. Amino acid sequence comparisons and model structure building revealed two residues, Ala419 and Ser504 of FH, as candidates determining the substrate specificity. These residues were specifically conserved in the diglycosidases. The model structure suggested that Ala419 is involved in the aglycone recognition, whereas Ser504 recognizes the external saccharide of the glycone. Mutations at these sites drastically decreased the diglycosidase activity. The mechanism by which the diglycosidases acquired their substrate specificity is discussed, based on these observations.
The unfolding of -momorcharin was systematically investigated using steady-state and time-resolved tryptophan fluorescence, circular dichroism and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. These spectroscopic studies demonstrated that -momorcharin unfolded through a compact folded intermediate state. The content of -helix was increased, Trp192 approached closer to the side of active site and its rotational motion was restricted by being equilibrated with 2–3 M of guanidine hydrochloride. Furthermore, the binding of ANS with -momorcharin was more suppressed to show that the hydrophobic parts would not be accessed to the protein surface but rather be sealed off in this specific conformation state. These results suggest that the structure of -momorcharin holds the more compact conformation as an incipient state for unfolding, which is the sharp contrast to β-momorcharin that gives the characteristics of the generally known molten globule state.
Transcription factor E1AF plays critical roles in neuronal development and tumour metastasis and is regulated by a number of signalling cascades, including the mitogen-activated protein kinase pathways. Accumulated evidence indicted that E1AF might contribute to cell survival in response to environment factors. Here, we provided evidence the cell cycle and apoptosis regulator E2F1 induces E1AF expression at the transcriptional level. DNA damage by etoposide causes E2F1-dependent induction of E1AF expression at transcriptional level. Furthermore, disruption of E1AF expression by E1AF RNAi decreased E2F1-induced apoptosis in response to etoposide. Thus, we conclude that activation of E1AF provides a means for E2F1 to induce cell apoptosis in response to DNA damage.
The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)–glutathione conjugate. On screening of protein transduction, optimized PEI–glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.
Calcitonin receptor-stimulating peptides (CRSPs) are new members of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family identified in pigs, dogs and other domestic animals, and CRSP-1 is an active ligand for the CT receptor (CT-R). We recently sequenced porcine CRSP genes (Crsps) and found similarity with the CT/CGRP gene (Ct/Cgrp) in sequence and genomic organization. In this study, we identified five Crsps, Crsp-1 to Crsp-5, in dogs. Crsp-1 has five exons with an exon–intron organization identical to that of porcine Crsp-1 or Crsp-2, while Crsp-2 and Crsp-3 have additional CT-2- and CT-3-coding exons like Ct/Cgrp. Crsp-2 was renamed as Ct-2/Crsp-2 because both CRSP-2 and CT-2 mRNAs were tissue-specifically expressed. Crsp-4 and Crsp-5 are presumably generated by retrotransposition. We postulate that Crsps were generated from the gene duplication of Ct/Cgrp, and gained their diversity during mammalian evolution. Among the canine CTs and CRSPs, CRSP-1, CT-1 and CT-2 are active ligands for the CT-R, but CRSP-2 and others are inactive. Canine CRSP-1 and CT-2 are expressed in the central and peripheral systems, while CT-1 is localized in the thyroid gland. These findings indicate that dogs can be used for an experimental model as analysing the physiological roles of the CT/CGRP/CRSP family.
The reactions of diol dehydratase with 3-unsaturated 1,2-diols and thioglycerol were investigated. Holodiol dehydratase underwent rapid and irreversible inactivation by either 3-butene-1,2-diol, 3-butyne-1,2-diol or thioglycerol without catalytic turnovers. In the inactivation, the Co–C bond of adenosylcobalamin underwent irreversible cleavage forming unidentified radicals and cob(II)alamin that resisted oxidation even in the presence of oxygen. Two moles of 5'-deoxyadenosine per mol of enzyme was formed as an inactivation product from the coenzyme adenosyl group. Inactivated holoenzymes underwent reactivation by diol dehydratase-reactivating factor in the presence of ATP, Mg2+ and adenosylcobalamin. It was thus concluded that these substrate analogues served as mechanism-based inactivators or pseudosubstrates, and that the coenzyme was damaged in the inactivation, whereas apoenzyme was not damaged. In the inactivation by 3-unsaturated 1,2-diols, product radicals stabilized by neighbouring unsaturated bonds might be unable to back-abstract the hydrogen atom from 5'-deoxyadenosine and then converted to unidentified products. In the inactivation by thioglycerol, a product radical may be lost by the elimination of sulphydryl group producing acrolein and unidentified sulphur compound(s). H2S or sulphide ion was not formed. The loss or stabilization of product radicals would result in the inactivation of holoenzyme, because the regeneration of the coenzyme becomes impossible.
We have been interested in partially folded proteins with marginal stability and activity, because they have a potential to be mature proteins by artificial evolution. A module is defined as a contiguous peptide chain forming a compact region in a globular protein. Modules may be used as building blocks to create partially folded proteins. Barnase, a ribonuclease consisting of 110 amino acids, has been divided into six modules (M1–M6), four peptide fragments, M12 (1–52), M123 (1–73), M1234 (1–88) and M12345 (1–98), have been constructed by progressive elongation of the modules from the N-terminus. Only M12345 (1–98) had a partially folded conformation, but it lacked detectable RNase activity. A mixture of M12345 (1–98) with M56 (89–110) showed weak but distinct RNase activity. Unfolded M12345 (1–96) was constructed by removal of two residues from the C-terminus of M12345 (1–98). The mixture of M12345 (1–96) with M56 (89–110) also showed RNase activity. Further, the interaction endowed M12345 (1–96) with conformational stability. We propose that N- and C-terminal fragments obtained by successive elongation of modules would interact to be a complex with marginal stability and activity, which would be used for creating a mature complex by artificial evolution.
The correction of an inactivated hygromycin resistance and enhanced green fluorescent protein (Hyg–EGFP) fusion gene by a several hundred-base single-stranded (ss) DNA fragment has been reported. In this study, the effectiveness of this type of gene correction was examined for various positions in the rpsL gene. Sense and anti-sense ssDNA fragments were prepared, and the gene correction efficiencies were determined by co-introduction of the target plasmid containing the gene with the ssDNA fragments. The gene correction efficiency varied (0.8–9.3%), depending on target positions and sense/anti-sense strands. Sense ssDNA fragments corrected the target gene with equal or higher efficiencies as compared to their anti-sense counterparts. The target positions corrected with high efficiency by the sense fragments also tended to be corrected efficiently by the anti-sense fragments. These results suggest that the sense ssDNA fragments are useful for the correction of mutated genes. The variation in the correction efficiency may depend on the sequence of the target position in double-stranded DNA.
The enzymes phosphoglucomutase (PGM) and phosphomannomutase (PMM) play an important role in the synthesis of extracellular polysaccharide. By colony hybridization of the fosmid library of Sphingomonas chungbukensis DJ77, an open reading frame (ORF-1) of 1,626 nucleotides, whose predicted product is highly homologous with other PGM proteins from several bacterial species, was identified. An additional open reading frame (ORF-2) of 1,437 nucleotides was identified, and its encoded protein shows a high level of similarity with the PGM/PMM protein family. The two genes were cloned into a bacterial expression vector pET-15b (+) and expressed in Escherichia coli as fusion proteins with (His)6-tag. Both recombinant proteins (designated as SP-1 and SP-2 for ORF-1 and ORF-2, respectively) exhibited PGM and PMM activities. The molecular masses of subunits of SP-1 and SP-2 were estimated to be around 58 and 51 kDa from SDS–PAGE, respectively. However, molecular masses of SP-1 and SP-2 in their native condition were determined to be approximately 59.5 and 105.4 kDa, according to non-denaturing PAGE, respectively. The SP-1 protein has a preference for glucose-1-phosphate rather than mannose-1-phosphate, while the preferred substrate of SP-2 is mannose-1-phosphate. Thus, the existence of two proteins with bifunctional PGM/PMM activities was first found S. chungbukensis DJ77.
by Jessica L. Dermody, Jonathan M. Dreyfuss, Judit Villén, Babatunde Ogundipe, Steven P. Gygi, Peter J. Park, Alfred S. Ponticelli, Claire L. Moore, Stephen Buratowski, Miriam E. Bucheli
The production of a functional mRNA is regulated at every step of transcription. An area not well-understood is the transition of RNA polymerase II from elongation to termination. The S. cerevisiae SR-like protein Npl3 functions to negatively regulate transcription termination by antagonizing the binding of polyA/termination proteins to the mRNA. In this study, Npl3 is shown to interact with the CTD and have a direct stimulatory effect on the elongation activity of the polymerase. The interaction is inhibited by phosphorylation of Npl3. In addition, Casein Kinase 2 was found to be required for the phosphorylation of Npl3 and affect its ability to compete against Rna15 (Cleavage Factor I) for binding to polyA signals. Our results suggest that phosphorylation of Npl3 promotes its dissociation from the mRNA/RNAP II, and contributes to the association of the polyA/termination factor Rna15. This work defines a novel role for Npl3 in elongation and its regulation by phosphorylation.
by Kellie J. Hall, Matthew T. Harper, Karen Gilio, Judith M. Cosemans, Johan W. M. Heemskerk, Alastair W. Poole
Background
PKCθ is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCθ−/− T cells exhibit reduced activation and PKCθ−/− mice are resistant to autoimmune disease, making PKCθ an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCθ positively regulates outside-in signalling through integrin αIIbβ3 in platelets, the role of PKCθ in GPVI-dependent signalling and functional activation of platelets has not been assessed.
Methodology/Principal Findings
In the present study we assessed static adhesion, cell spreading, granule secretion, integrin αIIbβ3 activation and platelet aggregation in washed mouse platelets lacking PKCθ. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCθ−/− platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCθ positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCθ−/− platelets also exhibited markedly enhanced GPVI-dependent α-granule secretion, although dense granule secretion was unaffected, suggesting that PKCθ differentially regulates these two granules. Inside-out regulation of αIIbβ3 activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s−1) was enhanced.
Conclusions/Significance
These data suggest that PKCθ is an important negative regulator of thrombus formation on collagen, potentially mediated by α-granule secretion and αIIbβ3 activation. PKCθ therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCθ inhibitors.
by Jeanne Farrell, Lavoisier Ramos, Martin Tresguerres, Margarita Kamenetsky, Lonny R. Levin, Jochen Buck
Background
Mammalian Soluble adenylyl cyclase (sAC, Adcy10, or Sacy) represents a source of the second messenger cAMP distinct from the widely studied, G protein-regulated transmembrane adenylyl cyclases. Genetic deletion of the second through fourth coding exons in Sacytm1Lex/Sacytm1Lex knockout mice results in a male sterile phenotype. The absence of any major somatic phenotype is inconsistent with the variety of somatic functions identified for sAC using pharmacological inhibitors and RNA interference.
Principal Findings
We now use immunological and molecular biological methods to demonstrate that somatic tissues express a previously unknown isoform of sAC, which utilizes a unique start site, and which ‘escapes’ the design of the Sacytm1Lex knockout allele.
Conclusions/Significance
These studies reveal increased complexity at the sAC locus, and they suggest that the known isoforms of sAC play a unique function in male germ cells.
Histone modifications in chromatin regulate gene expression. A transcriptional co-repressor complex containing LSD1–CoREST–HDAC1 (termed LCH hereafter for simplicity) represses transcription by coordinately removing histone modifications associated with transcriptional activation. RE1-silencing transcription factor (REST) recruits LCH to the promoters of neuron-specific genes, thereby silencing their transcription in non-neuronal tissues. ZNF198 is a member of a family of MYM-type zinc finger proteins that associate with LCH. Here, we show that ZNF198-like proteins are required for the repression of E-cadherin (a gene known to be repressed by LSD1), but not REST-responsive genes. ZNF198 binds preferentially to the intact LCH ternary complex, but not its individual subunits. ZNF198- and REST-binding to the LCH complex are mutually exclusive. ZNF198 associates with chromatin independently of LCH. Furthermore, modification of HDAC1 by small ubiquitin-like modifier (SUMO) in vitro weakens its interaction with CoREST whereas sumoylation of HDAC1 stimulates its binding to ZNF198. Finally, we mapped the LCH- and HDAC1–SUMO-binding domains of ZNF198 to tandem repeats of MYM-type zinc fingers. Therefore, our results suggest that ZNF198, through its multiple protein-protein interaction interfaces, helps to maintain the intact LCH complex on specific, non-REST-responsive promoters and may also prevent SUMO-dependent dissociation of HDAC1.
by Philippe A. Parone, Sandrine Da Cruz, Daniel Tondera, Yves Mattenberger, Dominic I. James, Pierre Maechler, François Barja, Jean-Claude Martinou
Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS). At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.
by Amelia Cimmino, Rosanna Capasso, Fabbri Muller, Irene Sambri, Lucia Masella, Marianna Raimo, Maria Luigia De Bonis, Stefania D'Angelo, Vincenzo Zappia, Patrizia Galletti, Diego Ingrosso
Background
Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free α-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal α-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s).
Methodology/Principal Findings
Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H2O2; 0.1–0.5 mM range). Results show that A) Cells overexpressing “wild type” human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H2O2 concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H2O2 treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase.
Conclusion/Significance
Apoptosis can be modulated by “on/off” switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein.
by Stephanie L. Timmerman, Jennifer S. Pfingsten, Jeffrey S. Kieft, Les A. Krushel
A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5′ leaders (1428 nt and 448 nt), both of which include the common 3′ exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5′ leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5′ leader are differentially regulated, in part by PTB1.
With increasing numbers of crystal structures of protein∶DNA and protein∶protein∶DNA complexes publically available, it is now possible to extract sufficient structural, physical-chemical and thermodynamic parameters to make general observations and predictions about their interactions. In particular, the properties of macromolecular assemblies of multiple proteins bound to DNA have not previously been investigated in detail.
Methodology/Principal Findings
We have performed computational structural analyses on macromolecular assemblies of multiple proteins bound to DNA using a variety of different computational tools: PISA; PROMOTIF; X3DNA; ReadOut; DDNA and DCOMPLEX. Additionally, we have developed and employed an algorithm for approximate collision detection and overlapping volume estimation of two macromolecules. An implementation of this algorithm is available at http://promoterplot.fmi.ch/Collision1/. The results obtained are compared with structural, physical-chemical and thermodynamic parameters from protein∶protein and single protein∶DNA complexes. Many of interface properties of multiple protein∶DNA complexes were found to be very similar to those observed in binary protein∶DNA and protein∶protein complexes. However, the conformational change of the DNA upon protein binding is significantly higher when multiple proteins bind to it than is observed when single proteins bind. The water mediated contacts are less important (found in less quantity) between the interfaces of components in ternary (protein∶protein∶DNA) complexes than in those of binary complexes (protein∶protein and protein∶DNA).The thermodynamic stability of ternary complexes is also higher than in the binary interactions. Greater specificity and affinity of multiple proteins binding to DNA in comparison with binary protein-DNA interactions were observed. However, protein-protein binding affinities are stronger in complexes without the presence of DNA.
Conclusions/Significance
Our results indicate that the interface properties: interface area; number of interface residues/atoms and hydrogen bonds; and the distribution of interface residues, hydrogen bonds, van der Walls contacts and secondary structure motifs are independent of whether or not a protein is in a binary or ternary complex with DNA. However, changes in the shape of the DNA reduce the off-rate of the proteins which greatly enhances the stability and specificity of ternary complexes compared to binary ones.
by Genc Basha, Gregory Lizée, Anna T. Reinicke, Robyn P. Seipp, Kyla D. Omilusik, Wilfred A. Jefferies
Background
Cross-presentation by dendritic cells (DCs) is a crucial prerequisite for effective priming of cytotoxic T-cell responses against bacterial, viral and tumor antigens; however, this antigen presentation pathway remains poorly defined.
Methodology/Principal Findings
In order to develop a comprehensive understanding of this process, we tested the hypothesis that the internalization of MHC class I molecules (MHC-I) from the cell surface is directly involved in cross-presentation pathway and the loading of antigenic peptides. Here we provide the first examination of the internalization of MHC-I in DCs and we demonstrate that the cytoplasmic domain of MHC-I appears to act as an addressin domain to route MHC-I to both endosomal and lysosomal compartments of DCs, where it is demonstrated that loading of peptides derived from exogenously-derived proteins occurs. Furthermore, by chasing MHC-I from the cell surface of normal and transgenic DCs expressing mutant forms of MHC-I, we observe that a tyrosine-based endocytic trafficking motif is required for the constitutive internalization of MHC-I molecules from the cell surface into early endosomes and subsequently deep into lysosomal peptide-loading compartments. Finally, our data support the concept that multiple pathways of peptide loading of cross-presented antigens may exist depending on the chemical nature and size of the antigen requiring processing.
Conclusions/Significance
We conclude that DCs have ‘hijacked’ and adapted a common vacuolar/endocytic intracellular trafficking pathway to facilitate MHC I access to the endosomal and lysosomal compartments where antigen processing and loading and antigen cross-presentation takes place.
by Lidia Ruiz, Magali Traskine, Irene Ferrer, Estrella Castro, Juan F. M. Leal, Marcelline Kaufman, Amancio Carnero
Background
P53 activation can trigger various outcomes, among them reversible growth arrest or cellular senescence. It is a live debate whether these outcomes are influenced by quantitative or qualitative mechanisms. Furthermore, the relative contribution of p53 to Ras-induced senescence is also matter of controversy.
Methodology/Principal Findings
This study compared situations in which different signals drove senescence with increasing levels of p53 activation. The study revealed that the levels of p53 activation do not determine the outcome of the response. This is further confirmed by the clustering of transcriptional patterns into two broad groups: p53-activated or p53-inactivated, i.e., growth and cellular arrest/senescence. Furthermore, while p53-dependent
