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Proposed Role (proposed + role)
Selected AbstractsContractility of single human dermal myofibroblasts and fibroblastsCYTOSKELETON, Issue 2 2002Louise K. Wrobel Abstract Human dermal myofibroblasts, characterised by the expression of ,-smooth muscle actin, are part of the granulation tissue and implicated in the generation of contractile forces during normal wound healing and pathological contractures. We have compared the contractile properties of single human dermal fibroblasts and human dermal myofibroblasts by culturing them on flexible silicone elastomers. The flexibility of the silicone substratum permits the contractile forces exerted by the cells to be measured [Fray et al., 1998: Tissue Eng. 4:273,283], without changing their expression of ,-smooth muscle actin. The mean contractile force produced by myofibroblasts (2.2 ,N per cell) was not significantly different from that generated by fibroblasts (2.0 ,N per cell) when cultured on a substrata with a low elastomer stiffness. Forces produced by fibroblasts were unaffected by increases in elastomer stiffness, but forces measured for myofibroblasts increased to a mean value of 4.1 ,N/cell. This was associated with a higher proportion of myofibroblasts being able to produce wrinkles on elastomers of high stiffness compared to fibroblasts. We discuss the force measurements at the single cell level, for both fibroblast and myofibroblasts, in relation to the proposed role of myofibroblasts in wound healing and pathological contractures. Cell Motil. Cytoskeleton 52:82,90, 2002. © 2002 Wiley-Liss, Inc. [source] Serum angiotensin-converting enzyme and frequency of severe hypoglycaemia in Type 1 diabetes: does a relationship exist?DIABETIC MEDICINE, Issue 12 2007N. N. Zammitt Abstract Aims An association has been described between elevated serum angiotensin-converting enzyme (ACE) and an increased risk of severe hypoglycaemia (SH). To ascertain whether this reported association could be replicated in a different country, it was re-examined in 300 individuals with Type 1 diabetes. Methods People with Type 1 diabetes, none of whom was taking renin,angiotensin system blocking drugs, were recruited. Participants recorded the frequency with which they had experienced SH. Glycated haemoglobin (HbA1c) and serum ACE were measured. The difference in the incidence of SH between different quartiles of ACE activity and the relationship between serum ACE and SH were examined using non-parametric statistical tests and a negative binomial model. Results Data were obtained from 300 patients [158 male; HbA1c median (range) 8.2% (5.2,12.8%), median age 36 years (16,88); duration of diabetes 14.5 years (2,49)]. The incidence of SH was 0.93 episodes per patient year. The mean incidence of SH in the top and bottom quartiles of ACE activity was 0.5 and 1.7 episodes per patient year, respectively, but this difference was not statistically significant (P = 0.075). Spearman's test showed a very weak, although statistically significant, association between serum ACE level and SH incidence (r = 0.115, P = 0.047). The binomial model also showed a statistically significant (P = 0.002), but clinically weak, relationship between serum ACE and SH. Conclusions The present survey showed a weak relationship between serum ACE and the frequency of SH, the clinical relevance of which is unclear. This limits the proposed role for serum ACE as an index of risk for SH. [source] Localization of the mosaic transmembrane serine protease corin to heart myocytesFEBS JOURNAL, Issue 23 2000John D. Hooper Corin cDNA encodes an unusual mosaic type II transmembrane serine protease, which possesses, in addition to a trypsin-like serine protease domain, two frizzled domains, eight low-density lipoprotein (LDL) receptor domains, a scavenger receptor domain, as well as an intracellular cytoplasmic domain. In in vitro experiments, recombinant human corin has recently been shown to activate pro-atrial natriuretic peptide (ANP), a cardiac hormone essential for the regulation of blood pressure. Here we report the first characterization of corin protein expression in heart tissue. We generated antibodies to two different peptides derived from unique regions of the corin polypeptide, which detected immunoreactive corin protein of approximately 125,135 kDa in lysates from human heart tissues. Immunostaining of sections of human heart showed corin expression was specifically localized to the cross striations of cardiac myocytes, with a pattern of expression consistent with an integral membrane localization. Corin was not detected in sections of skeletal or smooth muscle. Corin has been suggested to be a candidate gene for the rare congenital heart disease, total anomalous pulmonary venous return (TAPVR) as the corin gene colocalizes to the TAPVR locus on human chromosome 4. However examination of corin protein expression in TAPVR heart tissue did not show evidence of abnormal corin expression. The demonstrated corin protein expression by heart myocytes supports its proposed role as the pro-ANP convertase, and thus a potentially critical mediator of major cardiovascular diseases including hypertension and congestive heart failure. [source] Estrogen and non-genomic upregulation of voltage-gated Na+ channel activity in MDA-MB-231 human breast cancer cells: Role in adhesion,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2010Scott P. Fraser External (but not internal) application of ,-estradiol (E2) increased the current amplitude of voltage-gated Na+ channels (VGSCs) in MDA-MB-231 human breast cancer (BCa) cells. The G-protein activator GTP-,-S, by itself, also increased the VGSC current whilst the G-protein inhibitor GDP-,-S decreased the effect of E2. Expression of GPR30 (a G-protein-coupled estrogen receptor) in MDA-MB-231 cells was confirmed by PCR, Western blot and immunocytochemistry. Importantly, G-1, a specific agonist for GPR30, also increased the VGSC current amplitude in a dose-dependent manner. Transfection and siRNA-silencing of GPR30 expression resulted in corresponding changes in GPR30 protein expression but only internally, and the response to E2 was not affected. The protein kinase A inhibitor, PKI, abolished the effect of E2, whilst forskolin, an adenylate cyclase activator, by itself, increased VGSC activity. On the other hand, pre-incubation of the MDA-MB-231 cells with brefeldin A (a trans -Golgi protein trafficking inhibitor) had no effect on the E2-induced increase in VGSC amplitude, indicating that such trafficking (,externalisation') of VGSC was not involved. Finally, acute application of E2 decreased cell adhesion whilst the specific VGSC blocker tetrodotoxin increased it. Co-application of E2 and tetrodotoxin inhibited the effect of E2 on cell adhesion, suggesting that the effect of E2 was mainly through VGSC activity. Pre-treatment of the cells with PKI abolished the effect of E2 on adhesion, consistent with the proposed role of PKA. Potential implications of the E2-induced non-genomic upregulation of VGSC activity for BCa progression are discussed. J. Cell. Physiol. 224: 527,539, 2010. © 2010 Wiley-Liss, Inc. [source] Cation/proton antiporter complements of bacteria: why so large and diverse?MOLECULAR MICROBIOLOGY, Issue 2 2009Terry A. Krulwich Summary Most bacterial genomes have five to nine distinct genes predicted to encode transporters that exchange cytoplasmic Na+ and/or K+ for H+ from outside the cell, i.e. monovalent cation/proton antiporters. By contrast, pathogens that live primarily inside host cells usually possess zero to one such antiporter while other stress-exposed bacteria exhibit even higher numbers. The monovalent cation/proton antiporters encoded by these diverse genes fall into at least eight different transporter protein families based on sequence similarity. They enable bacteria to meet challenges of high or fluctuating pH, salt, temperature or osmolarity, but we lack explanations for why so many antiporters are needed and for the value added by specific antiporter types in specific settings. In this issue of Molecular Microbiology, analyses of the pH dependence of cytoplasmic [Na+], [K+], pH and transmembrane electrical potential in the ,poly extremophile'Natranaerobius thermophilus are the context for assessment of the catalytic properties of 12 predicted monovalent cation/proton antiporters in the genome of this thermophilic haloalkaliphile. The results provide a profile of adaptations of the poly extremophilic anaerobe, including a proposed role of cytoplasmic buffering capacity. They also provide new perspectives on two large monovalent cation/proton antiporter families, the NhaC and the cation/proton antiporter-3 antiporter families. [source] An electrostatic network and long-range regulation of Src kinasesPROTEIN SCIENCE, Issue 11 2008Elif Ozkirimli Abstract The regulatory mechanism of Src tyrosine kinases includes conformational activation by a change in the catalytic domain tertiary structure and in domain,domain contacts between the catalytic domain and the SH2/SH3 regulatory domains. The kinase is activated when tyrosine phosphorylation occurs on the activation loop, but without phosphorylation of the C-terminal tail. Activation also occurs by allostery when contacts between the catalytic domain (CD) and the regulatory SH3 and SH2 domains are released as a result of exogenous protein binding. The aim of this work is to examine the proposed role of an electrostatic network in the conformational transition and to elucidate the molecular mechanism for long-range, allosteric conformational activation by using a combination of experimental enzyme kinetics and nonequilibrium molecular dynamics simulations. Salt dependence of the induction phase is observed in kinetic assays and supports the role of an electrostatic network in the transition. In addition, simulations provide evidence that allosteric activation involves a concerted motion coupling highly conserved residues, and spanning several nanometers from the catalytic site to the regulatory domain interface to communicate between the CD and the regulatory domains. [source] Voltage-activated proton currents in human lymphocytesTHE JOURNAL OF PHYSIOLOGY, Issue 1 2002Tom Schilling Voltage-activated proton currents are reported for the first time in human peripheral blood T and B lymphocytes and in the human leukaemic T cell line Jurkat E6-1. The properties of H+ currents studied using tight-seal voltage-clamp recording techniques were similar in all cells. Changing the pH gradient by one unit caused a 47 mV shift in the reversal potential, demonstrating high selectivity of the channels for protons. H+ current activation upon membrane depolarisation had a sigmoidal time course that could be fitted by a single exponential function after a brief delay. Increasing pHo shifted the activation threshold to more negative potentials, and increased both the H+ current amplitude and the rate of activation. In lymphocytes studied at pHi 6.0, the activation threshold was more negative and the H+ current density was three times larger than at pHi 7.0. Increasing the intracellular Ca2+ concentration to 1 ,m did not change H+ current amplitude or kinetics detectably. Extracellularly applied Zn2+ and Cd2+ inhibited proton currents, slowing activation and shifting the voltage-activation curve to more positive potentials. The H+ current amplitude was 100 times larger in CD19+ B lymphocytes and in Jurkat E6-1 cells than in CD3+ T lymphocytes. Following stimulation with the phorbol ester PMA, the H+ current density in peripheral blood T lymphocytes and Jurkat T cells increased. In contrast, the H+ current density of phorbol ester (PMA)-stimulated B lymphocytes was reduced and activation became slower. The pattern of expression of H+ channels in lymphocytes appears well suited to their proposed role of charge compensation during the respiratory burst. [source] PICKLE acts during germination to repress expression of embryonic traitsTHE PLANT JOURNAL, Issue 6 2005Hui-Chun Li Summary PICKLE (PKL) codes for a CHD3 chromatin remodeling factor that plays multiple roles in Arabidopsis growth and development. Previous analysis of the expression of genes that exhibit PKL -dependent regulation suggested that PKL acts during germination to repress expression of embryonic traits. In this study, we examined the expression of PKL protein to investigate when and where PKL acts to regulate development. A PKL:eGFP translational fusion is preferentially localized in the nucleus of cells, consistent with the proposed role for PKL as a chromatin remodeling factor. A steroid-inducible version of PKL [a fusion of PKL to the glucocorticoid receptor (PKL:GR)] was used to examine when PKL acts to repress expression of embryonic traits. We found that activation of PKL:GR during germination was sufficient to repress expression of embryonic traits in the primary roots of pkl seedlings, whereas activation of PKL:GR after germination had little effect. In contrast, we observed that PKL is required continuously after germination to repress expression of PHERES1, a type I MADS box gene that is normally expressed during early embryogenesis in wild-type plants. Thus, PKL acts at multiple points during development to regulate patterns of gene expression in Arabidopsis. [source] The VERNALIZATION INDEPENDENCE 4 gene encodes a novel regulator of FLOWERING LOCUS CTHE PLANT JOURNAL, Issue 5 2002Hua Zhang Summary The late-flowering, vernalization-responsive habit of many Arabidopsis ecotypes is mediated predominantly through repression of the floral programme by the FLOWERING LOCUS C (FLC) gene. To better understand this repressive mechanism, we have taken a genetic approach to identify novel genes that positively regulate FLC expression. We identified recessive mutations in a gene designated VERNALIZATION INDEPENDENCE 4 (VIP4), that confer early flowering and loss of FLC expression in the absence of cold. We cloned the VIP4 gene and found that it encodes a highly hydrophilic protein with similarity to proteins from yeasts, Drosophila, and Caenorhabditis elegans. Consistent with a proposed role as a direct activator of FLC, VIP4 is expressed throughout the plant in a pattern similar to that of FLC. However, unlike FLC, VIP4 RNA expression is not down-regulated in vernalized plants, suggesting that VIP4 is probably not sufficient to activate FLC, and that VIP4 is probably not directly involved in a vernalization mechanism. Epistasis analysis suggests that VIP4 could act in a separate pathway from previously identified FLC regulators, including FRIGIDA and the autonomous flowering promotion pathway gene LUMINIDEPENDENS. Mutants lacking detectable VIP4 expression flower earlier than FLC null mutants, suggesting that VIP4 regulates flowering-time genes in addition to FLC. Floral morphology is also disrupted in vip4 mutants; thus, VIP4 has multiple roles in development. [source] Deficiency of the type I interferon receptor protects mice from experimental lupus,ARTHRITIS & RHEUMATISM, Issue 11 2007Dina C. Nacionales Objective Systemic lupus erythematosus (SLE) is diagnosed according to a spectrum of clinical manifestations and autoantibodies associated with abnormal expression of type I interferon (IFN-I),stimulated genes (ISGs). The role of IFN-I in the pathogenesis of SLE remains uncertain, partly due to the lack of suitable animal models. The objective of this study was to examine the role of IFN-I signaling in the pathogenesis of murine lupus induced by 2,6,10,14-tetramethylpentadecane (TMPD). Methods IFN-I receptor,deficient (IFNAR,/,) 129Sv mice and wild-type (WT) 129Sv control mice were treated intraperitoneally with TMPD. The expression of ISGs was measured by real-time polymerase chain reaction. Autoantibody production was evaluated by immunofluorescence and enzyme-linked immunosorbent assay. Proteinuria and renal glomerular cellularity were measured and renal immune complexes were examined by immunofluorescence. Results Increased ISG expression was observed in the peripheral blood of TMPD-treated WT mice, but not in the peripheral blood of TMPD-treated IFNAR,/, mice. TMPD did not induce lupus-specific autoantibodies (anti-RNP, anti-Sm, anti,double-stranded DNA) in IFNAR,/, mice, whereas 129Sv controls developed these specificities. Although glomerular immune complexes were present in IFNAR,/, mice, proteinuria and glomerular hypercellularity did not develop, whereas these features of glomerulonephritis were found in the TMPD-treated WT controls. The clinical and serologic manifestations observed in TMPD-treated mice were strongly dependent on IFNAR signaling, which is consistent with the association of increased expression of ISGs with lupus-specific autoantibodies and nephritis in humans. Conclusion Similar to its proposed role in human SLE, signaling via the IFNAR is central to the pathogenesis of autoantibodies and glomerulonephritis in TMPD-induced lupus. This lupus model is the first animal model shown to recapitulate the "interferon signature" in peripheral blood. [source] Structure of aminopeptidase N from Escherichia coli complexed with the transition-state analogue aminophosphinic inhibitor PL250ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Marie-Claude Fournié-Zaluski Aminopeptidase N (APN; EC 3.4.11.2) purified from Escherichia coli has been crystallized with the optically pure aminophosphinic inhibitor PL250, H3N+ -CH(CH3)-P(O)(OH)-CH2 -CH(CH2Ph)-CONH-CH(CH2Ph)CO2,, which mimics the transition state of the hydrolysis reaction. PL250 inhibits APN with a Ki of 1.5,2.2,nM and its three-dimensional structure in complex with E. coli APN showed its interaction with the S1, S,1 and S,2 subsites of the catalytic site. In this structure, the Zn ion was shown to be pentacoordinated by His297, His301 and Glu320 of APN and the two O atoms of the phosphinic moiety of PL250. One of these O atoms is also involved in a hydrogen bond to Tyr381, supporting the proposed role of this amino acid in the stabilization of the transition state of the enzymatic process. The strength of the phosphinic zinc binding and the occupancy of the S,2 subsite account for the 100-fold increase in affinity of PL250 compared with the dipeptide-derived inhibitor bestatin (Ki = 4.1 × 10,6,M). Accordingly, the removal of the C-terminal phenylalanine of PL250 resulted in a large decrease in affinity (Ki = 2.17 × 10,7,M). Furthermore, it was observed that the C-terminal carboxyl group of the inhibitor makes no direct interactions with the amino acids of the APN active site. Interestingly, PL250 exhibits the same inhibitory potency for E. coli APN and for mammalian enzymes, suggesting that the structure of the complex could be used as a template for the rational design of various human APN inhibitors needed to study the role of this aminopeptidase in various pathologies. [source] Class III HD-Zip gene regulation, the golden fleece of ARGONAUTE activity?BIOESSAYS, Issue 9 2004John L. Bowman MicroRNAs, small noncoding RNAs, are implicated in gene regulation in both metazoans and plants. In plants, many of the targets of miRNA-mediated gene regulation encode transcription factors with functions in development, such as the Class III HD-Zip gene family whose members direct polarity establishment in leaves and vasculature. Three recent papers provide insight into how miRNAs, likely acting through a complex containing an Argonaute protein, regulate Class III HD-Zip gene expression in Arabidopsis and maize.1,3 While the precise biological activity of Argonaute proteins remains an enigma, ARGONAUTE1 in Arabidopsis is required for the proper regulation of miRNA165/166, which targets cleavage of Class III HD-Zip mRNAs. Consistent with their proposed role in negative regulation, expression of miRNA165/166 is complementary to that of Class III HD-Zip gene expression, but this is perturbed in agronaute1 mutants. Determining how these complementary patterns of expression are formed should lead us closer to an understanding of the molecular mechanisms by which asymmetry is established in developing leaves. BioEssays 26:938,942, 2004. © 2004 Wiley Periodicals, Inc. [source] RESEARCH ON PRISONERS , A COMPARISON BETWEEN THE IOM COMMITTEE RECOMMENDATIONS (2006) AND EUROPEAN REGULATIONSBIOETHICS, Issue 1 2010BERNICE S. ELGER ABSTRACT The Institute of Medicine (IOM) Committee on Ethical Considerations for Revisions to DHHS Regulations for Protection of Prisoners Involved in Research published its report in 2006. It was charged with developing an ethical framework for the conduct of research with prisoners and identifying the safeguards and conditions necessary to ensure that research with prisoners is conducted ethically. The recommendations contained in the IOM report differ from current European regulations in several ways, some being more restrictive and some less so. For example, the IOM report suggests limiting the percentage of prisoners that should be involved in a biomedical study to 50%, a limit that does not exist in Europe. However, the report does not specifically advise against research without a direct benefit to an individual prisoner: the European regulations are more restrictive than the IOM committee recommendations in this respect. The definition of minimal risk varies, as well as the proposed role of the minimal risk requirement and of the principle of subsidiarity (research that can only be done effectively in prisons). The IOM report proposes a number of thoughtful suggestions, which it would be beneficial to implement everywhere, such as registers of research on prisoners. The European regulations offer pragmatic solutions to several thorny issues. In summary, the IOM committee report represents an admirable effort to tackle the present inconsistencies and deficiencies of federal regulations in the US on research on prisoners (45 CFR 46 Subpart C). Nonetheless, before acting on the recommendations, US regulators might consider revisiting international guidelines such as those published by the Council for International Organizations of Medical Science (CIOMS) and the Declaration of Helsinki. [source] Identification by immunoblot of venom glycoproteins displaying immunoglobulin E-binding N -glycans as cross-reactive allergens in honeybee and yellow jacket venomCLINICAL & EXPERIMENTAL ALLERGY, Issue 3 2004W. Hemmer Summary Background IgE antibodies against carbohydrate epitopes have been identified recently as a major cause of in vitro double positivity to honeybee (HB) and vespid venom in patients with stinging-insect allergy. As these antibodies possibly have low clinical relevance they may be misleading in the diagnosis of venom allergy. Objective To confirm the role of carbohydrate epitopes in double positivity and to locate the responsible glycoallergens in HB and yellow jacket (YJ) venom by western blot. Methods Immunoblot inhibition using HB venom, YJ venom and two glycoprotein sources displaying 1-3-fucosylated N -glycans (i.e. oilseed rape (OSR) pollen, and the synthetic neo-glycoprotein fucosylated/xylosylated N -glycans from bromelain coupled to bovine serum albumin (MUXF-BSA)) as inhibitors were performed with sera from 15 double-positive patients with stinging-insect allergy. Additionally, reactivity with blotted hymenoptera venoms of a carbohydrate-specific rabbit antiserum against OSR pollen was investigated. Results Major venom glycoallergens binding with carbohydrate-specific human IgE and rabbit IgG were detected in HB venom at 42 (hyaluronidase (HYA)), 46, 65 and 95 kDa, and in YJ venom at 38 and 43 kDa (HYA). Antibody binding to these allergens was completely lost after periodate treatment. Glycans of HB phospholipase were bound by patients' IgE only after protein denaturation. In 10 of the 15 patients the reactivity was with the second venom because of carbohydrates alone. The high-molecular-weight glycoallergens identified in HB venom probably correspond to similar proteins described earlier, including allergens B and C. The 38-kDa YJ allergen might represent a homologue of V mac 3. Conclusions The data confirm the proposed role of carbohydrate-specific IgE in double positivity to HB and YJ venom and shed new light on some previously described minor hymenoptera allergens of uncertain clinical significance. The consideration of carbohydrate-specific IgE may allow to discriminate between patients with potentially relevant and patients with non-relevant double sensitization. [source] Characterization of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutaseFEBS JOURNAL, Issue 24 2000Elucidation of the roles of amino acids involved in substrate binding, catalysis The roles of a number of amino acids present at the active site of the monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe have been explored by site-directed mutagenesis. The amino acids examined could be divided broadly into those presumed from previous related structural studies to be important in the catalytic process (R14, S62 and E93) and those thought to be important in substrate binding (R94, R120 and R121). Most of these residues have not previously been studied by site-directed mutagenesis. All the mutants except R14 were expressed in an engineered null strain of Saccharomyces cerevisiae (S150-gpm::HIS) in good yield. The R14Q mutant was expressed in good yield in the transformed AH22 strain of S. cerevisiae. The S62A mutant was markedly unstable, preventing purification. The various mutants were purified to homogeneity and characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denaturation by guanidinium chloride, and stability of phosphorylated enzyme intermediate. In addition, the binding of substrate (3-phosphoglycerate) to wild-type, E93D and R120,121Q enzymes was measured by isothermal titration calorimetry. The results provide evidence for the proposed roles of each of these amino acids in the catalytic cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the enzyme using multidimensional NMR techniques. [source] Cell death and differentiation in the development of the endocardial cushion of the embryonic heartMICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2002Eltyeb Abdelwahid Abstract The transformation of the endocardial cushion into valves and septa is a critical step in cardiac morphogenesis as it initiates the development of the four-chambered heart. This transformation results from a region-specific balance between cellular proliferation, apoptosis, and differentiation. The development of the form and structure of the endocardial cushion is accompanied by precise patterns of abundant cell death having the morphological features of programmed cell death (apoptosis), which plays an important role in the elimination of redundant cells and in changes of phenotypic composition during histogenesis. Apoptosis is an essential process in morphogenesis as it balances mitosis in renewing tissues. It is controlled by one or more genetic programs that kill the targeted cell. However, the causes, role, and regulation of apoptosis in the developing endocardial cushion still remain to be determined. The clarification of the role of the apoptosis regulatory genes constitutes a major task in future studies of cell death in the developing heart. This new molecular histology of heart development awaits further experiments to clarify the interactive mechanisms that act to ensure the sculpting of the endocardial cushion into valves and septa by determining the size of the cushion cell populations. The relation between the expression of different factors and the modifications of the cushion region during cardiac development are reviewed. In addition, we review and summarize information on molecules identified in our experiments that imply the activity of a number of essential genes coinciding with the key steps in generating the overall architecture of the heart. We correlate their temporal and spatial expression with their proposed roles. Microsc. Res. Tech. 58:395,403, 2002. © 2002 Wiley-Liss, Inc. [source] Chaperonin-assisted folding of glutamine synthetase under nonpermissive conditions: Off-pathway aggregation propensity does not determine the co-chaperonin requirementPROTEIN SCIENCE, Issue 12 2000Paul A. Voziyan Abstract One of the proposed roles of the GroEL-GroES cavity is to provide an "infinite dilution" folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these "nonpermissive" folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these "low Mg2+" conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions. [source] | ||||||||||||||||