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Misfolding
Kinds of Misfolding Selected AbstractsProgress in the development of new treatments for combined Alzheimer's and Parkinson's diseasesDRUG DEVELOPMENT RESEARCH, Issue 3 2002Eliezer Masliah Abstract Misfolding of synaptic molecules such as amyloid , peptide and ,-synuclein has been proposed to play a key role in the mechanisms of neurodegeneration in Alzheimer's and Parkinson's disease, respectively. Notably, the majority of patients with Alzheimer's disease also have ,-synuclein-immunoreactive Lewy bodies, and a substantial proportion of them develop a form of parkinsonism also known as Lewy body disease, that defies conventional therapies. Thus, factors involved in the pathogenesis of Alzheimer's disease might promote the development of particularly recalcitrant forms of Lewy body disease. We have shown that the amyloid , peptide 1-42, of Alzheimer's disease, promotes the toxic conversion of ,-synuclein and accelerates ,-synuclein-dependent deficits in transgenic mice. Understanding the mechanisms promoting the toxic conversion of ,-synuclein is of critical importance for the design of rationale treatments for Lewy body disease and transgenic models hold the promise for the development of such novel therapies. In this context therapies aimed at: (1) reducing amyloid , peptide 1-42 production, (2) blocking toxic ,-synuclein oligomerization (e.g., ,-synuclein, antioxidants), (3) promoting ,-synuclein protofibril degradation, and (4) protecting neurons (e.g., anti-oxidants, neurotrophic agents) against toxic ,-synuclein aggregates might prove to be significantly useful in the treatment of Lewy body disease. We characterized ,-synuclein, the non-amyloidogenic homologue of ,-synuclein, as an inhibitor of aggregation of ,-synuclein. Our results raise the intriguing possibility that ,-synuclein might be a natural negative regulator of ,-synuclein aggregation, and that a similar class of endogenous factors might modulate the toxic conversion of other molecules involved in neurodegeneration. Such an anti-amyloidogenic property of ,-synuclein in combination with other treatments might also provide a novel strategy for the treatment of neurodegenerative disorders. Drug Dev. Res. 56:282,292, 2002. © 2002 Wiley-Liss, Inc. [source] Parallel protein folding with STAPLCONCURRENCY AND COMPUTATION: PRACTICE & EXPERIENCE, Issue 14 2005Shawna Thomas Abstract The protein-folding problem is a study of how a protein dynamically folds to its so-called native state,an energetically stable, three-dimensional conformation. Understanding this process is of great practical importance since some devastating diseases such as Alzheimer's and bovine spongiform encephalopathy (Mad Cow) are associated with the misfolding of proteins. We have developed a new computational technique for studying protein folding that is based on probabilistic roadmap methods for motion planning. Our technique yields an approximate map of a protein's potential energy landscape that contains thousands of feasible folding pathways. We have validated our method against known experimental results. Other simulation techniques, such as molecular dynamics or Monte Carlo methods, require many orders of magnitude more time to produce a single, partial trajectory. In this paper we report on our experiences parallelizing our method using STAPL (Standard Template Adaptive Parallel Library) that is being developed in the Parasol Lab at Texas A&M. An efficient parallel version will enable us to study larger proteins with increased accuracy. We demonstrate how STAPL enables portable efficiency across multiple platforms, ranging from small Linux clusters to massively parallel machines such as IBM's BlueGene/L, without user code modification. Copyright © 2005 John Wiley & Sons, Ltd. [source] Sulfonated molecules that bind a partially structured species of ,2 -microglobulin also influence refolding and fibrillogenesisELECTROPHORESIS, Issue 7 2008Chiara Carazzone Abstract Human ,2 -microglobulin (,2 -m) is a small amyloidogenic protein responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of ,2 -m through the binding to a small molecule, to possibly inhibit protein misfolding and amyloid fibril formation. The search of a strong ligand of this protein is extremely challenging: by using CE in affinity and refolding experiments we study the effect that previously selected sulfonated molecules have on the equilibrium between the native form and an ensemble of conformers populating the slow phase of ,2 -m folding. These data are correlated with the effect that the same molecules exert on in vitro fibrillogenesis experiments. [source] Dynamics, stability and iron-binding activity of frataxin clinical mutantsFEBS JOURNAL, Issue 14 2008Ana R. Correia Friedreich's ataxia results from a deficiency in the mitochondrial protein frataxin, which carries single point mutations in some patients. In the present study, we analysed the consequences of different disease-related mutations in vitro on the stability and dynamics of human frataxin. Two of the mutations, G130V and D122Y, were investigated for the first time. Analysis by CD spectroscopy demonstrated a substantial decrease in the thermodynamic stability of the variants during chemical and thermal unfolding (wild-type > W155R > I154F > D122Y > G130V), which was reversible in all cases. Protein dynamics was studied in detail and revealed that the mutants have distinct propensities towards aggregation. It was observed that the mutants have increased correlation times and different relative ratios between soluble and insoluble/aggregated protein. NMR showed that the clinical mutants retained a compact and relatively rigid globular core despite their decreased stabilities. Limited proteolysis assays coupled with LC-MS allowed the identification of particularly flexible regions in the mutants; interestingly, these regions included those involved in iron-binding. In agreement, the iron metallochaperone activity of the Friedreich's ataxia mutants was affected: some mutants precipitate upon iron binding (I154F and W155R) and others have a lower binding stoichiometry (G130V and D122Y). Our results suggest that, in heterozygous patients, the development of Friedreich's ataxia may result from a combination of reduced efficiency of protein folding and accelerated degradation in vivo, leading to lower than normal concentrations of frataxin. This hypothesis also suggests that, although quite different from other neurodegenerative diseases involving toxic aggregation, Friedreich's ataxia could also be linked to a process of protein misfolding due to specific destabilization of frataxin. [source] Full-length prion protein aggregates to amyloid fibrils and spherical particles by distinct pathwaysFEBS JOURNAL, Issue 9 2008Driss El Moustaine As limited structural information is available on prion protein (PrP) misfolding and aggregation, a causative link between the specific (supra)molecular structure of PrP and transmissible spongiform encephalopathies remains to be elucidated. In this study, high pressure was utilized, as an approach to perturb protein structure, to characterize different morphological and structural PrP aggregates. It was shown that full-length recombinant PrP undergoes ,-sheet aggregation on high-pressure-induced destabilization. By tuning the physicochemical conditions, the assembly process evolves through two distinct pathways leading to the irreversible formation of spherical particles or amyloid fibrils, respectively. When the PrP aggregation propensity is enhanced, high pressure induces the formation of a partially unfolded aggregated protein, AggHP, which relaxes at ambient pressure to form amorphous aggregates. The latter largely retain the native secondary structure. On prolonged incubation at high pressure, followed by depressurization, AggHP transforms to a monodisperse population of spherical particles of about 20 nm in diameter, characterized by an essentially ,-sheet secondary structure. When the PrP aggregation propensity is decreased, an oligomeric reaction intermediate, IHP, is formed under high pressure. After pressure release, IHP relaxes to the original native structure. However, on prolonged incubation at high pressure and subsequent depressurization, it transforms to amyloid fibrils. Structural evaluation, using optical spectroscopic methods, demonstrates that the conformation adopted by the subfibrillar oligomeric intermediate, IHP, constitutes a necessary prerequisite for the formation of amyloids. The use of high-pressure perturbation thus provides an insight into the molecular mechanism of the first stages of PrP misfolding into amyloids. [source] From HLA-B27 to spondyloarthritis: a journey through the ERIMMUNOLOGICAL REVIEWS, Issue 1 2010Robert A. Colbert Summary:, Almost four decades of research into the role of human leukocyte antigen-B27 (HLA-B27) in susceptibility to spondyloarthritis has yet to yield a convincing answer. New results from an HLA-B27 transgenic rat model now demonstrate quite convincingly that CD8+ T cells are not required for the inflammatory phenotype. Discoveries that the HLA-B27 heavy chain has a tendency to misfold during the assembly of class I complexes in the endoplasmic reticulum (ER) and to form aberrant disulfide-linked dimers after transport to the cell surface have forced the generation of new ideas about its role in disease pathogenesis. In transgenic rats, HLA-B27 misfolding generates ER stress and leads to activation of the unfolded protein response, which dramatically enhances the production of interleukin-23 (IL-23) in response to pattern recognition receptor agonists. These findings have led to the discovery of striking T-helper 17 cell activation and expansion in this animal model, consistent with results emerging from humans with spondyloarthritis and the discovery of IL23R as an additional susceptibility gene for ankylosing spondylitis. Together, these results suggest a novel link between HLA-B27 and the T-helper 17 axis through the consequences of protein misfolding and open new avenues of investigation as well as identifying new targets for therapeutic intervention in this group of diseases. [source] Etiopathogenic role of HLA-B27 alleles in ankylosing spondylitisINTERNATIONAL JOURNAL OF RHEUMATIC DISEASES, Issue 3 2005Nurullah AKKOC Abstract HLA-B27 is the major genetic susceptibility factor for ankylosing spondylitis (AS). However, its precise role in the pathogenesis of AS still remains unclear, even though its gene has been cloned and sequenced, and its crystallographic structure has been defined. Arthritogenic peptide and molecular mimicry hypotheses propose mechanisms related to an antigen-presenting function of HLA-B27 to be responsible for disease development. However, peculiar aspects of its immunobiology, such as its misfolding and heavy chain dimerization raise the possibility of involvement of pathogenic mechanisms unrelated to its physiological function. Moreover, HLA-B27 is not a single allele, but a family of 31 different alleles, named HLA-B*2701 to HLA-B*2727. Studies worldwide indicate that the relatively common alleles (subtypes) HLA-B*2705, B*2704, and B*2702 are strongly associated with AS, whereas HLA-B*2706 which is prevalent in South-east Asia and HLA-B*2709 which is prevalent on the Italian island of Sardinia, seem to lack such an association. The distinction between the disease-associated subtypes and those that are not associated, may provide clues to the actual role of HLA-B27 in disease pathogenesis. B*2706 differs from B*2704 by only two residues, and B*2709 differs from B*2705 by only one residue. Moreover, both B*2706 and B*2709 bind an endogenous peptide (derived from vasoactive intestinal peptide type 1 receptor) and also an exogenous peptide (latent membrane protein 2 of Epstein-Barr virus) but in two drastically diverse conformations. These recent X-ray diffraction studies of individual peptides in the context of different HLA-B27 alleles broaden our perception of the possible pathogenetic role of this molecule in the development of AS and related spondyloarthopathies. In summary, the pathogenetic role of HLA-B27 in AS seem to be quite heterogenous, and cannot be explained by a single mechanism, and new ideas have been raised based on the aberrant immunobiologic features of HLA-B27. [source] Serpin polymerization and its role in disease,The molecular basis of ,1 -antitrypsin deficiencyIUBMB LIFE, Issue 1 2009Anja S. Knaupp Abstract Protein aggregation is the cause of several human diseases. Understanding the molecular mechanisms involved in protein aggregation requires knowledge of the kinetics and structures populated during the reaction. Arguably, the best structurally characterized misfolding reaction is that of ,1 -antitrypsin. ,1 -Antitrypsin misfolding leads to both liver disease and emphysema and affect approximately 1 in 2000 of the population. This review will focus on the mechanism of ,1 -antitrypsin misfolding and the development of potential therapeutic strategies. © 2008 IUBMB IUBMB Life, 61(1): 1,5, 2009 [source] Unraveling the mysteries of protein folding and misfoldingIUBMB LIFE, Issue 12 2008Heath Ecroyd Abstract This mini-review focuses on the processes and consequences of protein folding and misfolding. The latter process often leads to protein aggregation and precipitation with the aggregates adopting either highly ordered (amyloid fibril) or disordered (amorphous) forms. In particular, the amyloid fibril is discussed because this form has gained considerable notoriety due to its close links to a variety of debilitating diseases including Alzheimer's, Parkinson's, Huntington's, and Creutzfeldt-Jakob diseases, and type-II diabetes. In each of these diseases a different protein forms fibrils, yet the fibrils formed have a very similar structure. The mechanism by which fibrils form, fibril structure, and the cytotoxicity associated with fibril formation are discussed. The generic nature of amyloid fibril structure suggests that a common target may be accessible to treat amyloid fibril-associated diseases. As such, the ability of some molecules, for example, the small heat-shock family of molecular chaperone proteins, to inhibit fibril formation is of interest due to their therapeutic potential. © 2008 IUBMB IUBMB Life, 60(12): 769,774, 2008 [source] Protein misfolding inside cells: The case of huntingtin and Huntington's diseaseIUBMB LIFE, Issue 11 2008Danny M. Hatters Abstract Huntington's disease is one of the several neurodegenerative diseases caused by dominant mutations that expand the number of glutamine codons within an existing poly-glutamine (polyQ) repeat sequence of a gene. An expanded polyQ sequence in the huntingtin gene is known to cause the huntingtin protein to aggregate and form intracellular inclusions as disease progresses. However, the role that polyQ-induced aggregation plays in disease is yet to be fully determined. This review focuses on key questions remaining for how the expanded polyQ sequences affect the aggregation properties of the huntingtin protein and the corresponding effects on cellular machinery. The scope includes the technical challenges that remain for rigorously assessing the effects of aggregation on the cellular machinery. © 2008 IUBMB IUBMB Life, 60(11): 724,728, 2008 [source] Protein folding in the post-genomic eraJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2002Jeannine M. Yon Abstract Protein folding is a topic of fundamental interest since it concerns the mechanisms by which the genetic message is translated into the three-dimensional and functional structure of proteins. In these post-genomic times, the knowledge of the fundamental principles are required in the exploitation of the information contained in the increasing number of sequenced genomes. Protein folding also has practical applications in the understanding of different pathologies and the development of novel therapeutics to prevent diseases associated with protein misfolding and aggregation. Significant advances have been made ranging from the Anfinsen postulate to the "new view" which describes the folding process in terms of an energy landscape. These new insights arise from both theoretical and experimental studies. The problem of folding in the cellular environment is briefly discussed. The modern view of misfolding and aggregation processes that are involved in several pathologies such as prion and Alzheimer diseases. Several approaches of structure prediction, which is a very active field of research, are described. [source] Contribution of glutamatergic signaling to nitrosative stress-induced protein misfolding in normal brain aging and neurodegenerative diseasesAGING CELL, Issue 3 2007Tomohiro Nakamura Summary Glutamatergic hyperactivity, associated with Ca2+ influx and consequent production of nitric oxide (NO), is potentially involved in both normal brain aging and age-related neurodegenerative disorders. Many neurodegenerative diseases are characterized by conformational changes in proteins that result in their misfolding and aggregation. Normal protein degradation by the ubiquitin-proteasome system can prevent accumulation of aberrantly folded proteins. Our recent studies have linked nitrosative stress to protein misfolding and neuronal cell death. In particular, molecular chaperones , such as protein disulfide isomerase, glucose regulated protein 78, and heat shock proteins , can provide neuroprotection from misfolded proteins by facilitating proper folding and thus preventing aggregation. Here, we present evidence for the hypothesis that NO contributes to normal brain aging and degenerative conditions by S-nitrosylating specific chaperones that would otherwise prevent accumulation of misfolded proteins. [source] Changes in endoplasmic reticulum stress proteins and aldolase A in cells exposed to dopamineJOURNAL OF NEUROCHEMISTRY, Issue 1 2008April A. Dukes Abstract In Parkinson's disease, oxidative stress is implicated in protein misfolding and aggregation, which may activate the unfolded protein response by the endoplasmic reticulum (ER). Dopamine (DA) can initiate oxidative stress via H2O2 formation by DA metabolism and by oxidation into DA quinone. We have previously shown that DA quinone induces oxidative protein modification, mitochondrial dysfunction in vitro, and dopaminergic cell toxicity in vivo and in vitro. In this study, we used cysteine- and lysine-reactive fluorescent dyes with 2D difference in-gel electrophoresis, mass spectrometry, and peptide mass fingerprint analysis to identify proteins in PC12 cell mitochondrial-enriched fractions that were altered in abundance following DA exposure (150 ,M, 16 h). Quantitative changes in proteins labeled with fluorescent dyes indicated increases in a subset of proteins after DA exposure: calreticulin, ERp29, ERp99, Grp58, Grp78, Grp94 and Orp150 (149,260%), and decreased levels of aldolase A (39,42%). Changes in levels of several proteins detected by 2D difference in-gel electrophoresis were confirmed by western blot. Using this unbiased proteomics approach, our findings demonstrated that in PC12 cells, DA exposure leads to a cellular response indicative of ER stress prior to the onset of cell death, providing a potential link between DA and the unfolded protein response in the pathogenesis of Parkinson's disease. [source] Neurotoxic species in prion disease: a role for PrP isoforms?JOURNAL OF NEUROCHEMISTRY, Issue 5 2007Christopher F. Harrison Abstract Prion diseases such as bovine spongiform encephalopathy in cattle and Creutzfeldt,Jakob disease in humans are associated with the misfolding and accumulation of an abnormal conformation of the host-encoded prion protein (PrP). Despite intensive research efforts conducted on PrP, the toxic agent involved in neurodegeneration is as yet unidentified. Several potential candidates have been proposed, each of which may be relevant to subsets of the broad array of prion diseases. In this study, we review current knowledge on neurotoxic PrP species, including the importance of a central hydrophobic domain for mediating neurotoxicty. [source] Potential implications of endogenous aldehydes in ,-amyloid misfolding, oligomerization and fibrillogenesisJOURNAL OF NEUROCHEMISTRY, Issue 5 2006Kun Chen Abstract Aldehydes are capable of inducing protein cross-linkage. An increase in aldehydes has been found in Alzheimer's disease. Formaldehyde and methylglyoxal are produced via deamination of, respectively, methylamine and aminoacetone catalyzed by semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6. The enzyme is located on the outer surface of the vasculature, where amyloidosis is often initiated. A high SSAO level has been identified as a risk factor for vascular disorders. Serum SSAO activity has been found to be increased in Alzheimer's patients. Malondialdehyde and 4-hydroxynonenal are derived from lipid peroxidation under oxidative stress, which is also associated with Alzheimer's disease. Aldehydes may potentially play roles in ,-amyloid aggregation related to the pathology of Alzheimer's disease. In the present study, thioflavin-T fluorometry, dynamic light scattering, circular dichroism spectroscopy and atomic force microscopy were employed to reveal the effect of endogenous aldehydes on ,-amyloid at different stages, i.e. ,-sheet formation, oligomerization and fibrillogenesis. Formaldehyde, methylglyoxal and malondialdehyde and, to a lesser extent, 4-hydroxynonenal are not only capable of enhancing the rate of formation of ,-amyloid ,-sheets, oligomers and protofibrils but also of increasing the size of the aggregates. The possible relevance to Alzheimer's disease of the effects of these aldehydes on ,-amyloid deposition is discussed. [source] Pathogenic mutations inactivate parkin by distinct mechanismsJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Iris H. Henn Abstract Loss of parkin function is the major cause of autosomal recessive Parkinson's disease (ARPD). A wide variety of parkin mutations have been identified in patients; however, the pathophysiological mechanisms leading to the inactivation of mutant parkin are poorly understood. In this study we characterized pathogenic C- and N-terminal parkin mutants and found distinct pathways of parkin inactivation. Deletion of the C terminus abrogated the association of parkin with cellular membranes and induced rapid misfolding and aggregation. Four N-terminal missense mutations, located within the ubiquitin-like domain (UBL), decrease the stability of parkin; as a consequence, these mutants are rapidly degraded by the proteasome. Furthermore, we present evidence that a smaller parkin species of 42 kDa, which is present in extracts prepared from human brain and cultured cells, originates from an internal start site and lacks the N-terminal UBL domain. [source] Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosisJOURNAL OF NEUROCHEMISTRY, Issue 5 2002Makoto Urushitani Abstract Accumulating evidence indicates that abnormal conformation of mutant superoxide dismutase 1 (SOD1) is an essential feature underlying the pathogenesis of mutant SOD1-linked familial amyotrophic lateral sclerosis (ALS). Here we investigated the role of ubiquitin-proteasome pathway in the mutant SOD1-related cell death and the effect of oxidative stress on the misfolding of mutant SOD1. Transient overexpression of ubiquitin with human SOD1 (wild-type, ala4val, gly85arg, gly93ala) in Neuro2A cells decreased the amount of mutant SOD1, but not of wild-type, while only mutants were co-immunoprecipitated with poly-ubiquitin. Proteasome inhibition by lactacystin augmented accumulation of mutant SOD1 in the non-ionic detergent-insoluble fraction. The spinal cord lysates from mutant SOD1 transgenic mice showed multiple carbonylated proteins, including mutant SOD1 with SDS-resistant dimer formation. Furthermore, the treatment of hSOD1-expressing cells with hydrogen peroxide promoted the oligomerization, and detergent-insolubility of mutant SOD1 alone, and the oxidized mutant SOD1 proteins were more heavily poly-ubiquitinated. In Neuro2A cells stably expressing human SOD1 protein, the proteasome function measured by chymotrypsin-like activity, was decreased over time without a quantitative alteration of the 20S proteasomal component. Finally, primary motor neurons from the mouse embryonic spinal cord were more vulnerable to lactacystin than non-motor neurons. These results indicate that the sustained expression of mutant SOD1 leads to proteasomal inhibition and motor neuronal death, which in part explains the pathogenesis of mutant SOD1-linked ALS. [source] Expression of mutant cartilage oligomeric matrix protein in human chondrocytes induces the pseudoachondroplasia phenotypeJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 4 2006Thomas M. Merritt Abstract Over 70 mutations in the cartilage oligomeric matrix protein (COMP), a large extracellular pentameric glycoprotein synthesized by chondrocytes, have been identified as causing two skeletal dysplasias: multiple epiphyseal dysplasia (MED/EDM1), and a dwarfing condition, pseudoachondroplasia (PSACH). These mutations induce misfolding of intracellular COMP, resulting in retention of the protein in the rough endoplasmic reticulum (rER) of chondrocytes. This accumulation of COMP in the rER creates the phenotypic enlarged rER cisternae in the cells, which is believed to compromise chondrocyte function and eventually cause cell death. To study the molecular mechanisms involved with the disease, we sought to develop an in vitro model that recapitulates the PSACH phenotype. Normal human chondrocytes were transfected with wildtype (wt-) COMP or with mutant COMP (D469del; mt-) recombinant adenoviruses and grown in a nonattachment redifferentiating culture system that provides an environment allowing formation of a differentiated chondrocyte nodule. Visualization of normal cells expressing COMP suggested the hallmarks of the PSACH phenotype. Mutant COMP expressed in normal cells was retained in enlarged rER cisternae, which also retained IX collagen (COL9) and matrilin-3 (MATN3). Although these proteins were secreted normally into the ECM of the wt-COMP nodules, reduced secretion of these proteins was observed in nodules composed of cells transfected with mt-COMP. The findings complement those found in chondrocytes from PSACH patient growth plates. This new model system allows for production of PSACH chondrocyte pathology in normal costochondral chondrocytes and can be used for future mechanistic and potential gene therapy studies. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [source] Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK-DnaJ-GrpE system in the management of protein misfolding under severe stress conditionsMOLECULAR MICROBIOLOGY, Issue 3 2004Mirna Mujacic Summary Escherichia coli Hsp31 is a homodimeric protein that exhibits chaperone activity in vitro and is a representative member of a recently recognized family of heat shock proteins (Hsps). To gain insights on Hsp31 cellular function, we deleted the hchA gene from the MC4100 chromosome and combined the resulting null allele with lesions in other cytoplasmic chaperones. Although the hchA mutant only exhibited growth defects when cultivated at 48°C, loss of Hsp31 had a strong deleterious effect on the ability of cells to survive and recover from transient exposure to 50°C, and led to the enhanced aggregation of a subset of host proteins at this temperature. The absence of Hsp31 did not significantly affect the ability of the ClpB-DnaK-DnaJ-GrpE system to clear thermally aggregated proteins at 30°C suggesting that Hsp31 does not possess disaggregase activity. Although it had no effect on the growth of groES30, ,clpB or ,ibpAB cells at high temperatures, the hchA deletion aggravated the temperature sensitive phenotype of dnaK756 and grpE280 mutants and led to increased aggregation in stressed dnaK756 cells. On the basis of biochemical, structural and genetic data, we propose that Hsp31 acts as a modified holding chaperone that captures early unfolding intermediates under prolonged conditions of severe stress and releases them when cells return to physiological conditions. This additional line of defence would complement the roles of DnaK-DnaJ-GrpE, ClpB and IbpB in the management of thermally induced cellular protein misfolding. [source] Protein misfolding in neurodegenerative diseasesNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 3 2004E. I. Agorogiannis A common pathogenic mechanism shared by diverse neurodegenerative disorders, like Alzheimer's disease, Parkinson's disease, Huntington's disease and transmissible spongiform encephalopathies, may be altered protein homeostasis leading to protein misfolding and aggregation of a wide variety of different proteins in the form of insoluble fibrils. Mutations in the genes encoding protein constituents of these aggregates have been linked to the corresponding diseases, thus a reasonable scenario of pathogenesis was based on misfolding of a neurone-specific protein that forms insoluble fibrils that subsequently kill neuronal cells. However, during the past 5 years accumulating evidence has revealed the neurotoxic role of prefibrillar intermediate forms (soluble oligomers and protofibrils) produced during fibril formation. Many think these may be the predominant neurotoxic species, whereas microscopically visible fibrillar aggregates may not be toxic. Large protein aggregates may rather be simply inactive, or even represent a protective state that sequesters and inactivates toxic oligomers and protofibrils. Further understanding of the biochemical mechanisms involved in protein misfolding and fibrillization may optimize the planning of common therapeutic approaches for neurodegenerative diseases, directed towards reversal of protein misfolding, blockade of protein oligomerization and interference with the action of toxic proteins. [source] One novel and one recurrent mutation in the PROS1 gene cause type I protein S deficiency in patients with pulmonary embolism associated with deep vein thrombosisAMERICAN JOURNAL OF HEMATOLOGY, Issue 10 2006Kazuhiro Mizukami Abstract We investigated the molecular basis of type I protein S (PS) deficiency in two unrelated Japanese families, in which both probands developed pulmonary embolism associated with deep vein thrombosis. Nucleotide sequencing of amplified DNA revealed distinct point mutations in the PROS1 gene of the probands, which were designated protein S Sapporo 1 and protein S Sapporo 2. Additional mutations in the PROS1 gene were excluded by DNA sequencing of all exons and intron/exon boundaries. In the 25-year-old Japanese male patient who carried protein S Sapporo 1, we identified a heterozygous A-to-T change in the invariant ag dinucleotide of the acceptor splice site of intron f of the PROS1 gene. This mutation is a novel splice site mutation that impairs normal mRNA splicing, leading to exon 7 skipping, which was confirmed by platelet mRNA analysis. Translation of this mutant transcript would result in a truncated protein that lacks the entire epidermal growth factor-like domain 3 of the PS molecule. In a 31-year-old Japanese male and his younger brother who each carried protein S Sapporo 2, we detected a previously described heterozygous T-to-C transition at nucleotide position 1147 in exon 10 of the PROS1 gene, which predicts an amino acid substitution of tryptophan by arginine at residue 342 in the laminin G1 domain of the PS molecule. Both mutations would cause misfolding of the PS protein, resulting in the impairment of secretion, which is consistent with the type I PS deficiency phenotype. Am. J. Hematol., 2006. © 2006 Wiley-Liss, Inc. [source] The dual role of a loop with low loop contact distance in folding and domain swappingPROTEIN SCIENCE, Issue 7 2002Apichart Linhananta Abstract , helices, , strands, and loops are the basic building blocks of protein structure. The folding kinetics of , helices and , strands have been investigated extensively. However, little is known about the formation of loops. Experimental studies show that for some proteins, the formation of a single loop is the rate-determining step for folding, whereas for others, a loop (or turn) can misfold to serve as the hinge loop region for domain-swapped species. Computer simulations of an all-atom model of fragment B of Staphylococcal protein A found that the formation of a single loop initiates the dominant folding pathway. On the other hand, the stability analysis of intermediates suggests that the same loop is a likely candidate to serve as a hinge loop for domain swapping. To interpret the simulation result, we developed a simple structural parameter: the loop contact distance (LCD), or the sequence distance of contacting residues between a loop and the rest of the protein. The parameter is applied to a number of other proteins, including SH3 domains and prion protein. The results suggest that a locally interacting loop (low LCD) can either promote folding or serve as the hinge region for domain swapping. Thus, there is an intimate connection between folding and domain swapping, a possible cause of misfolding and aggregation. [source] 26S proteasome regulatory particle mutants have increased oxidative stress toleranceTHE PLANT JOURNAL, Issue 1 2008Jasmina Kurepa Summary The 26S proteasome (26SP) is a multi-subunit, multi-catalytic protease that is responsible for most of the cytosolic and nuclear protein turnover. The 26SP is composed of two sub-particles, the 19S regulatory particle (RP) that binds and unfolds protein targets, and the 20S core particle (20SP) that degrades proteins into small peptides. Most 26SP targets are conjugated to a poly-ubiquitin (Ub) chain that serves as a degradation signal. However, some targets, such as oxidized proteins, do not require a poly-Ub tag for proteasomal degradation, and recent studies have shown that the main protease in this Ub-independent pathway is free 20SP. It is currently unknown how the ratio of 26SP- to 20SP-dependent proteolysis is controlled. Here we show that loss of function of the Arabidopsis RP subunits RPT2a, RPN10 and RPN12a leads to decreased 26SP accumulation, resulting in reduced rates of Ub-dependent proteolysis. In contrast, all three RP mutants have increased 20SP levels and thus enhanced Ub-independent protein degradation. As a consequence of this shift in proteolytic activity, mutant seedlings are hypersensitive to stresses that cause protein misfolding, and have increased tolerance to treatments that promote protein oxidation. Taken together, the data show that plant cells increase 20SP-dependent proteolysis when 26SP activity is impaired. [source] Oxidized/misfolded superoxide dismutase-1: the cause of all amyotrophic lateral sclerosis?ANNALS OF NEUROLOGY, Issue 6 2007Edor Kabashi PhD The identification in 1993 of superoxide dismutase-1 (SOD1) mutations as the cause of 10 to 20% of familial amyotrophic lateral sclerosis cases, which represents 1 to 2% of all amyotrophic lateral sclerosis (ALS) cases, prompted a substantial amount of research into the mechanisms of SOD1-mediated toxicity. Recent experiments have demonstrated that oxidation of wild-type SOD1 leads to its misfolding, causing it to gain many of the same toxic properties as mutant SOD1. In vitro studies of oxidized/misfolded SOD1 and in vivo studies of misfolded SOD1 have indicated that these protein species are selectively toxic to motor neurons, suggesting that oxidized/misfolded SOD1 could lead to ALS even in individuals who do not carry an SOD1 mutation. It has also been reported that glial cells secrete oxidized/misfolded mutant SOD1 to the extracellular environment, where it can trigger the selective death of motor neurons, offering a possible explanation for the noncell autonomous nature of mutant SOD1 toxicity and the rapid progression of disease once the first symptoms develop. Therefore, considering that sporadic (SALS) and familial ALS (FALS) cases are clinically indistinguishable, the toxic properties of mutated SOD1 are similar to that of oxidized/misfolded wild-type SOD1 (wtSOD1), and secreted/extracellular misfolded SOD1 is selectively toxic to motor neurons, we propose that oxidized/misfolded SOD1 is the cause of most forms of classic ALS and should be a prime target for the design of ALS treatments. Ann Neurol 2007 [source] HLA,B27 misfolding and the unfolded protein response augment interleukin-23 production and are associated with Th17 activation in transgenic ratsARTHRITIS & RHEUMATISM, Issue 9 2009Monica L. DeLay Objective To determine whether HLA,B27 misfolding and the unfolded protein response (UPR) result in cytokine dysregulation and whether this is associated with Th1 and/or Th17 activation in HLA,B27/human ,2 -microglobulin (Hu,2m),transgenic rats, an animal model of spondylarthritis. Methods Cytokine expression in lipopolysaccharide (LPS),stimulated macrophages was analyzed in the presence and absence of a UPR induced by chemical agents or by HLA,B27 up-regulation. Cytokine expression in colon tissue and in cells purified from the lamina propria was determined by real-time reverse transcription,polymerase chain reaction analysis, and differences in Th1 and Th17 CD4+ T cell populations were quantified after intracellular cytokine staining. Results Interleukin-23 (IL-23) was found to be synergistically up-regulated by LPS in macrophages undergoing a UPR induced by pharmacologic agents or by HLA,B27 misfolding. IL-23 was also increased in the colon tissue from B27/Hu,2m-transgenic rats concurrently with the development of intestinal inflammation, and IL-17, a downstream target of IL-23, exhibited robust up-regulation in a similar temporal pattern. IL-23 and IL-17 transcripts were localized to CD11+ antigen-presenting cells and CD4+ T cells, respectively, from the colonic lamina propria. Colitis was associated with a 6-fold expansion of CD4+ IL-17,expressing T cells. Conclusion The IL-23/IL-17 axis is strongly activated in the colon of B27/Hu,2m-transgenic rats with spondylarthritis-like disease. HLA,B27 misfolding and UPR activation in macrophages can result in enhanced induction of the pro-Th17 cytokine IL-23. These results suggest a possible link between HLA,B27 misfolding and immune dysregulation in this animal model, with implications for human disease. [source] HLA,B27 up-regulation causes accumulation of misfolded heavy chains and correlates with the magnitude of the unfolded protein response in transgenic rats: Implications for the pathogenesis of spondylarthritis-like diseaseARTHRITIS & RHEUMATISM, Issue 1 2007Matthew J. Turner Objective HLA,B27 is implicated in the pathogenesis of spondylarthritis (SpA), yet the molecular mechanisms are incompletely defined. HLA,B27 misfolding has been associated with endoplasmic reticulum stress and activation of the unfolded protein response (UPR) in macrophages from HLA,B27/human ,2 -microglobulin,transgenic (B27-transgenic) rats. This study was performed to assess the mechanisms that drive activation of the HLA,B27,induced UPR and to determine whether splenocytes respond in a similar manner. Methods Splenocytes were isolated and bone marrow macrophages were derived from B27-transgenic and wild-type rats. Cells were treated for up to 24 hours with cytokines that induce class I major histocompatibility complex expression. HLA,B27 expression and misfolding were assessed by real-time reverse transcription,polymerase chain reaction, flow cytometry, and immunoblotting. Activation of the UPR was measured by quantifying UPR target gene expression and X-box binding protein 1 messenger RNA (mRNA) splicing. Results HLA,B27 mRNA up-regulation was accompanied by a dramatic increase in the accumulation of misfolded heavy chains and preceded robust activation of the UPR in macrophages. When macrophages were treated with various cytokines, the magnitude of the UPR correlated strongly with the degree of HLA,B27 up-regulation. In contrast, B27-transgenic splenocytes exhibited only low-level differences in the expression of UPR target genes after exposure to interferon-, or concanavalin A, which resulted in minimal HLA,B27 up-regulation. Conclusion These results suggest that HLA,B27,associated activation of the UPR in macrophages is attributable to the accumulation of misfolded heavy chains, and that certain cell types may be more susceptible to the effects of HLA,B27 misfolding. Strategies that eliminate HLA,B27 up-regulation and/or the accumulation of misfolded heavy chains may be useful in evaluating the role of these events in the pathogenesis of SpA. [source] Aggregation characteristics of ovalbumin in ,-sheet conformation determined by spectroscopyBIOPOLYMERS, Issue 2 2002Raimon Sabaté Abstract Protein misfolding and aggregation are involved in a number of the so-called "conformational" diseases (e.g., transmissible spongiform encephalopathies and Alzheimer disease). The development of rational strategies to interfere with aggregation is a potential therapeutic approach that requires complete knowledge of the aggregation process. We studied the aggregation of ovalbumin in ,-sheet conformation using mainly the spectral changes in the spectra of two dyes (Congo Red and pinacyanol) caused by the aggregates. We assumed a linear model of polymerization that fit to the experimental data. The critical aggregation constant, concentration of half-aggregation, nucleation parameter, growth parameter, and number of aggregation and free energy changes (total and per residue) were determined as aggregation-related parameters. ,-Ovalbumin aggregates in a cooperative way. Moreover, the differences between such parameters obtained with Congo Red and pinacyanol suggest that each dye interacts with the protein in its own way. © 2002 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 67: 113,120, 2002 [source] Rational improvement of simvastatin synthase solubility in Escherichia coli leads to higher whole-cell biocatalytic activityBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Xinkai Xie Abstract Simvastatin is the active pharmaceutical ingredient of the blockbuster cholesterol lowering drug Zocor. We have previously developed an Escherichia coli based whole-cell biocatalytic platform towards the synthesis of simvastatin sodium salt (SS) starting from the precursor monacolin J sodium salt (MJSS). The centerpiece of the biocatalytic approach is the simvastatin synthase LovD, which is highly prone to misfolding and aggregation when overexpressed from E. coli. Increasing the solubility of LovD without decreasing its catalytic activity can therefore elevate the performance of the whole-cell biocatalyst. Using a combination of homology structural prediction and site-directed mutagenesis, we identified two cysteine residues in LovD that are responsible for nonspecific intermolecular crosslinking, which leads to oligomer formation and protein aggregation. Replacement of Cys40 and Cys60 with alanine residues resulted in marked gain in both protein solubility and whole-cell biocatalytic activities. Further mutagenesis experiments converting these two residues to small or polar natural amino acids showed that C40A and C60N are the most beneficial, affording 27% and 26% increase in whole cell activities, respectively. The double mutant C40A/C60N combines the individual improvements and displayed ,50% increase in protein solubility and whole-cell activity. Optimized fed-batch high-cell-density fermentation of the double mutant in an E. coli strain engineered for simvastatin production quantitatively (>99%) converted 45 mM MJSS to SS within 18 h, which represents a significant improvement over the performance of wild-type LovD under identical conditions. The high efficiency of the improved whole-cell platform renders the biocatalytic synthesis of SS an attractive substitute over the existing semisynthetic routes. Biotechnol. Bioeng. 2009;102: 20,28. © 2008 Wiley Periodicals, Inc. [source] Indispensable structure of solution additives to prevent inactivation of lysozyme for heating and refoldingBIOTECHNOLOGY PROGRESS, Issue 5 2009Tsuneyoshi Matsuoka Abstract This article investigates solution additives that prevent misfolding of lysozyme from heating treatment and during refolding processes. Comparison of heat treatment of native lysozyme and oxidative refolding from the reduced and denatured state of lysozyme in the presence of 44 different additives revealed an indispensable chemical structure for the additives to be effective against heat-induced misfolding and for refolding. The additives effective against heat treatment of native lysozyme possessed a main chain of the amino acid moiety. Amino acids that have esterificated and amidated carboxy groups prevented heat-induced misfoldings more effectively than amino acids themselves. On the other hand, the additives effective against oxidative refolding possessed a guanidium or ureido group. The former additives prevented hydrophobic interaction between the main chains of the unfolded polypeptide, while the latter additives increased the solubility of the aromatic and aliphatic side-chains. These data also support the fact that arginine (Arg) and Arg derivatives are versatile additives for both misfolding processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Cytoplasmic Overexpression, Folding, and Processing of Penicillin Acylase Precursor in EscherichiacoliBIOTECHNOLOGY PROGRESS, Issue 5 2005Yali Xu Penicillin acylase (PAC) precursor, proPAC, was overproduced in a soluble or insoluble form in the cytoplasm of Escherichia coli through the expression of the leader-less pac gene (ll-pac) devoid of the coding region for the signal peptide of PAC. Also, a portion of the overexpressed proPAC was further processed to form mature PAC, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E.coli. The cultivation performance for ll-pac expression was limited by several factors, including (1) misfolding of proPAC, resulting in the aggregation of insoluble proPAC as inclusion bodies, (2) intracellular proteolysis, leading to the degradation of the overexpressed gene products, and (3) inefficient PAC maturation, limiting the formation of active PAC. The effect of coexpression of various cytoplasmic chaperones, including trigger factor, GroEL/ES, DnaK/J-GrpE, and their combinations, on ll-pac expression was investigated. Intracellular proteolysis of the overexpressed gene products could be prevented by coexpression of GroEL/ES. On the other hand, coexpression of trigger factor appeared to be able to facilitate the folding of soluble proPAC and to improve PAC maturation. The roles of trigger factor and GroEL/ES could be coordinated to significantly improve ll-pac expression performance. DnaK/J-GrpE had an effect for solublization of proPAC and perhaps, similar to trigger factor, for improving PAC maturation. The ll-pac expression performance was also significantly improved through the simultaneous coexpression of DnaK/J-GrpE and GroEL/ES. The results of the study suggest that the folding and/or processing of proPAC could be a major issue limiting the overproduction of PAC in E. coli and the bottleneck could be eliminated through the coexpression of appropriate chaperone(s). [source] | ||||||||||||||||