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Sample records for misfolded membrane proteins

  1. Misfolding of amyloidogenic proteins and their interactions with membranes.

    PubMed

    Relini, Annalisa; Marano, Nadia; Gliozzi, Alessandra

    2013-12-27

    In this paper, we discuss amyloidogenic proteins, their misfolding, resulting structures, and interactions with membranes, which lead to membrane damage and subsequent cell death. Many of these proteins are implicated in serious illnesses such as Alzheimer's disease and Parkinson's disease. Misfolding of amyloidogenic proteins leads to the formation of polymorphic oligomers and fibrils. Oligomeric aggregates are widely thought to be the toxic species, however, fibrils also play a role in membrane damage. We focus on the structure of these aggregates and their interactions with model membranes. Study of interactions of amlyoidogenic proteins with model and natural membranes has shown the importance of the lipid bilayer in protein misfolding and aggregation and has led to the development of several models for membrane permeabilization by the resulting amyloid aggregates. We discuss several of these models: formation of structured pores by misfolded amyloidogenic proteins, extraction of lipids, interactions with receptors in biological membranes, and membrane destabilization by amyloid aggregates perhaps analogous to that caused by antimicrobial peptides.

  2. Role of Lipids in Folding, Misfolding and Function of Integral Membrane Proteins.

    PubMed

    Hong, Heedeok

    2015-01-01

    The lipid bilayer that constitutes cell membranes imposes environmental constraints on the structure, folding and function of integral membrane proteins. The cell membrane is an enormously heterogeneous and dynamic system in its chemical composition and associated physical forces. The lipid compositions of cell membranes not only vary over the tree of life but also differ by subcellular compartments within the same organism. Even in the same subcellular compartment, the membrane composition shows strong temporal and spatial dependence on the environmental or biological cues. Hence, one may expect that the membrane protein conformations and their equilibria strongly depend on the physicochemical variables of the lipid bilayer. Contrary to this expectation, the structures of homologous membrane proteins belonging to the same family but from evolutionary distant organisms exhibit a striking similarity. Furthermore, the atomic structures of the same protein in different lipid environments are also very similar. This suggests that certain stable folds optimized for a specific function have been selected by evolution. On the other hand, there is growing evidence that, despite the overall stability of the protein folds, functions of certain membrane proteins require a particular lipid composition in the bulk bilayer or binding of specific lipid species. Here I discuss the specific and nonspecific modulation of folding, misfolding and function of membrane proteins by lipids and introduce several diseases that are caused by misfolding of membrane proteins.

  3. Simulations of membrane-bound diglycosylated human prion protein reveal potential protective mechanisms against misfolding.

    PubMed

    Cheng, Chin Jung; Koldsø, Heidi; Van der Kamp, Marc W; Schiøtt, Birgit; Daggett, Valerie

    2017-07-01

    Prion diseases are associated with the misfolding of the prion protein (PrP) from its normal cellular form (PrP(C) ) to its infectious scrapie form (PrP(S)(c) ). Post-translational modifications in PrP in vivo can play an important role in modulating the process of misfolding. To gain more insight into the effects of post-translational modifications in PrP structure and dynamics and to test the hypothesis that such modifications can interact with the protein, we have performed molecular dynamics simulations of diglycosylated human PrP(C) bound to a lipid bilayer via a glycophosphatidylinositol anchor. Multiple simulations were performed at three different pH ranges to explore pH effects on structure and dynamics. In contrast to simulations of protein-only PrP(C) , no large effects were observed upon lowering the pH of the system. The protein tilted toward the membrane surface in all of the simulations and the putative PrP(S)(c) oligomerization sites became inaccessible, thereby offering a possible protective mechanism against PrP(S)(c) -induced misfolding of PrP(C) . © 2017 International Society for Neurochemistry.

  4. The Safety Dance: Biophysics of Membrane Protein Folding and Misfolding in a Cellular Context

    PubMed Central

    Schlebach, Jonathan P.; Sanders, Charles R.

    2015-01-01

    Most biological processes require the production and degradation of proteins, a task that weighs heavily on the cell. Mutations that compromise the conformational stability of proteins place both specific and general burdens on cellular protein homeostasis (proteostasis) in ways that contribute to numerous diseases. Efforts to elucidate the chain of molecular events responsible for diseases of protein folding address one of the foremost challenges in biomedical science. However, relatively little is known about the processes by which mutations prompt the misfolding of α-helical membrane proteins, which rely on an intricate network of cellular machinery to acquire and maintain their functional structures within cellular membranes. In this review, we summarize the current understanding of the physical principles that guide membrane protein biogenesis and folding in the context of mammalian cells. Additionally, we explore how pathogenic mutations that influence biogenesis may differ from those that disrupt folding and assembly, as well as how this may relate to disease mechanisms and therapeutic intervention. These perspectives indicate an imperative for the use of information from structural, cellular, and biochemical studies of membrane proteins in the design of novel therapeutics and in personalized medicine. PMID:25420508

  5. Conformational Stability and Pathogenic Misfolding of the Integral Membrane Protein PMP22

    PubMed Central

    2016-01-01

    Despite broad biochemical relevance, our understanding of the physiochemical reactions that limit the assembly and cellular trafficking of integral membrane proteins remains superficial. In this work, we report the first experimental assessment of the relationship between the conformational stability of a eukaryotic membrane protein and the degree to which it is retained by cellular quality control in the secretory pathway. We quantitatively assessed both the conformational equilibrium and cellular trafficking of 12 variants of the α-helical membrane protein peripheral myelin protein 22 (PMP22), the intracellular misfolding of which is known to cause peripheral neuropathies associated with Charcot–Marie–Tooth disease (CMT). We show that the extent to which these mutations influence the energetics of Zn(II)-mediated PMP22 folding is proportional to the observed reduction in cellular trafficking efficiency. Strikingly, quantitative analyses also reveal that the reduction of motor nerve conduction velocities in affected patients is proportional to the extent of the mutagenic destabilization. This finding provides compelling evidence that the effects of these mutations on the energetics of PMP22 folding lie at the heart of the molecular basis of CMT. These findings highlight conformational stability as a key factor governing membrane protein biogenesis and suggest novel therapeutic strategies for CMT. PMID:26102530

  6. Protein folding and misfolding

    NASA Astrophysics Data System (ADS)

    Dobson, Christopher M.

    2003-12-01

    The manner in which a newly synthesized chain of amino acids transforms itself into a perfectly folded protein depends both on the intrinsic properties of the amino-acid sequence and on multiple contributing influences from the crowded cellular milieu. Folding and unfolding are crucial ways of regulating biological activity and targeting proteins to different cellular locations. Aggregation of misfolded proteins that escape the cellular quality-control mechanisms is a common feature of a wide range of highly debilitating and increasingly prevalent diseases.

  7. Misfolded Proteins and Retinal Dystrophies

    PubMed Central

    Lin, Jonathan H.; LaVail, Matthew M.

    2010-01-01

    Many mutations associated with retinal degeneration lead to the production of misfolded proteins by cells of the retina. Emerging evidence suggests that these abnormal proteins cause cell death by activating the Unfolded Protein Response, a set of conserved intracellular signaling pathways that detect protein misfolding within the endoplasmic reticulum and control protective and proapoptotic signal transduction pathways. Here, we review the misfolded proteins associated with select types of retinitis pigmentosa, Stargadt-like macular degeneration, and Doyne Honeycomb Retinal Dystrophy and discuss the role that endoplasmic reticulum stress and UPR signaling play in their pathogenesis. Last, we review new therapies for these diseases based on preventing protein misfolding in the retina. PMID:20238009

  8. Protein Misfolding Diseases.

    PubMed

    Hartl, F Ulrich

    2017-06-20

    The majority of protein molecules must fold into defined three-dimensional structures to acquire functional activity. However, protein chains can adopt a multitude of conformational states, and their biologically active conformation is often only marginally stable. Metastable proteins tend to populate misfolded species that are prone to forming toxic aggregates, including soluble oligomers and fibrillar amyloid deposits, which are linked with neurodegeneration in Alzheimer and Parkinson disease, and many other pathologies. To prevent or regulate protein aggregation, all cells contain an extensive protein homeostasis (or proteostasis) network comprising molecular chaperones and other factors. These defense systems tend to decline during aging, facilitating the manifestation of aggregate deposition diseases. This volume of the Annual Review of Biochemistry contains a set of three articles addressing our current understanding of the structures of pathological protein aggregates and their associated disease mechanisms. These articles also discuss recent insights into the strategies cells have evolved to neutralize toxic aggregates by sequestering them in specific cellular locations.

  9. Protein misfolding disorders and macroautophagy

    PubMed Central

    Menzies, Fiona M; Moreau, Kevin; Rubinsztein, David C

    2011-01-01

    A large group of diseases, termed protein misfolding disorders, share the common feature of the accumulation of misfolded proteins. The possibility of a common mechanism underlying either the pathogenesis or therapy for these diseases is appealing. Thus, there is great interest in the role of protein degradation via autophagy in such conditions where the protein is found in the cytoplasm. Here we review the growing evidence supporting a role for autophagic dysregulation as a contributing factor to protein accumulation and cellular toxicity in certain protein misfolding disorders and discuss the available evidence that upregulation of autophagy may be a valuable therapeutic strategy. PMID:21087849

  10. Binding affinity of amyloid oligomers to cellular membranes is a generic indicator of cellular dysfunction in protein misfolding diseases

    PubMed Central

    Evangelisti, Elisa; Cascella, Roberta; Becatti, Matteo; Marrazza, Giovanna; Dobson, Christopher M.; Chiti, Fabrizio; Stefani, Massimo; Cecchi, Cristina

    2016-01-01

    The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer’s disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca2+ across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane. PMID:27619987

  11. Binding affinity of amyloid oligomers to cellular membranes is a generic indicator of cellular dysfunction in protein misfolding diseases.

    PubMed

    Evangelisti, Elisa; Cascella, Roberta; Becatti, Matteo; Marrazza, Giovanna; Dobson, Christopher M; Chiti, Fabrizio; Stefani, Massimo; Cecchi, Cristina

    2016-09-13

    The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer's disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca(2+) across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane.

  12. Nanoimaging for protein misfolding diseases.

    PubMed

    Lyubchenko, Yuri L; Kim, Bo-Hyun; Krasnoslobodtsev, Alexey V; Yu, Junping

    2010-01-01

    Misfolding and aggregation of proteins are widespread phenomena leading to the development of numerous neurodegenerative disorders such as Parkinson's, Alzheimer's, and Huntington's diseases. Each of these diseases is linked to structural misfolding and aggregation of a particular protein. The aggregated forms of the protein induce the development of a particular disease at all levels, leading to neuronal dysfunction and loss. Because protein refolding is frequently accompanied by transient association of partially folded intermediates, the propensity to aggregate is considered a general characteristic of the majority of proteins. X-ray crystallography, nuclear magnetic resonance, electron microscopy, and atomic force microscopy have provided important information on the structure of aggregates. However, fundamental questions, such as why the misfolded conformation of the protein is formed, and why this state is important for self-assembly, remain unanswered. Although it is well known that the same protein under pathological conditions can lead to the formation of aggregates with diverse biological consequences, the conditions leading to misfolding and the formation of the disease prone complexes are unclear, complicating any development of efficient prevention of the diseases. Misfolded states exist transiently, so answering these questions requires the use of novel approaches and methods. Progress has been made during the past few years, when recently developed ensemble methods and single-molecule biophysics techniques were applied to the problem of the protein misfolding. In this review, the impacts of these studies on the understanding of the mechanisms of the protein self-assembly into aggregates and on the development of treatments of the diseases are discussed.

  13. Theory of protein misfolding and applications to misfolding diseases

    NASA Astrophysics Data System (ADS)

    Plotkin, Steven S.

    2009-03-01

    Physics-based algorithms can predict the misfolding mechanisms of proteins involved in aggregation-related diseases, including ALS and the Prion diseases. Predictions based on such an algorithm that we have developed, which employs both atomistic interactions and surface-area based coarse-graining, have been recently verified by immunological assays and point to diagnostic and therapeutic applications. I will describe the results of our misfolding theory, and discuss future directions towards drug research.

  14. Aggresomes: A Cellular Response to Misfolded Proteins

    PubMed Central

    Johnston, Jennifer A.; Ward, Cristina L.; Kopito, Ron R.

    1998-01-01

    Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins. PMID:9864362

  15. Transient misfolding dominates multidomain protein folding

    PubMed Central

    Borgia, Alessandro; Kemplen, Katherine R.; Borgia, Madeleine B.; Soranno, Andrea; Shammas, Sarah; Wunderlich, Bengt; Nettels, Daniel; Best, Robert B.; Clarke, Jane; Schuler, Benjamin

    2015-01-01

    Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result of evolutionary pressure to avoid misfolding and aggregation, particularly at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments and molecular simulations to investigate how misfolding between the immunoglobulin-like domains of titin is prevented. Surprisingly, we find that during refolding of tandem repeats, independent of sequence identity, more than half of all molecules transiently form a wide range of misfolded conformations. Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies. However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours. We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated. PMID:26572969

  16. Transient misfolding dominates multidomain protein folding

    NASA Astrophysics Data System (ADS)

    Borgia, Alessandro; Kemplen, Katherine R.; Borgia, Madeleine B.; Soranno, Andrea; Shammas, Sarah; Wunderlich, Bengt; Nettels, Daniel; Best, Robert B.; Clarke, Jane; Schuler, Benjamin

    2015-11-01

    Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result of evolutionary pressure to avoid misfolding and aggregation, particularly at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments and molecular simulations to investigate how misfolding between the immunoglobulin-like domains of titin is prevented. Surprisingly, we find that during refolding of tandem repeats, independent of sequence identity, more than half of all molecules transiently form a wide range of misfolded conformations. Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies. However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours. We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated.

  17. Protein Misfolding and Cardiac Disease

    PubMed Central

    Pattison, J. Scott; Robbins, Jeffrey

    2008-01-01

    Numerous neurodegenerative diseases are characterized by the accumulation of misfolded amyloidogenic proteins. Recent data indicate that a soluble pre-amyloid oligomer (PAO) may be the toxic entity in these diseases and the visible amyloid plaques, rather than causing the disease, may simply mark the terminal pathology. In prior studies, we observed PAO in the cardiomyocytes of many human heart failure samples. To test the hypothesis that cardiomyocyte-restricted expression of a known PAO is sufficient to cause heart failure, transgenic mice were created expressing polyglutamine repeats of 83 (PQ83) or 19 (PQ19).Long PQ repeats (>50) form PAOs and result in neurotoxicity in Huntington’s disease, whereas shorter PQ repeats are benign. PQ83 expression caused the intracellular accumulation of PAOs and aggregates leading to cardiomyocyte death and heart failure. Evidence of increased autophagy and necrosis accompanied the PQ83 cardiomyocyte pathology. The data confirm that protein misfolding resulting in intracellular PAO accumulation is sufficient to cause cardiomyocyte death and heart failure. PMID:18612262

  18. Cellular stress responses in protein misfolding diseases.

    PubMed

    Duennwald, Martin L

    2015-09-01

    Many human diseases, particularly neurodegenerative diseases, are associated with protein misfolding. Cellular protein quality control includes all processes that ensure proper protein folding and thus prevent the toxic consequences of protein misfolding. The heat shock response (HSR) and the unfolded protein response (UPR) are major stress response pathways within protein quality control that antagonize protein misfolding in the cytosol and the endoplasmic reticulum, respectively. Huntington's disease is an inherited neurodegenerative disease caused by the misfolding of an abnormally expanded polyglutamine (polyQ) region in the protein huntingtin (Htt), polyQHtt. Using Huntington's disease as a paradigm, I review here the central role of both the HSR and the UPR in defining the toxicity associated with polyQHtt in Huntington's disease. These findings may begin to unravel a previously unappreciated cooperation between different stress response pathways in cells expressing misfolded proteins and consequently in neurodegenerative diseases.

  19. Structural Determinants of Misfolding in Multidomain Proteins.

    PubMed

    Tian, Pengfei; Best, Robert B

    2016-05-01

    Recent single molecule experiments, using either atomic force microscopy (AFM) or Förster resonance energy transfer (FRET) have shown that multidomain proteins containing tandem repeats may form stable misfolded structures. Topology-based simulation models have been used successfully to generate models for these structures with domain-swapped features, fully consistent with the available data. However, it is also known that some multidomain protein folds exhibit no evidence for misfolding, even when adjacent domains have identical sequences. Here we pose the question: what factors influence the propensity of a given fold to undergo domain-swapped misfolding? Using a coarse-grained simulation model, we can reproduce the known propensities of multidomain proteins to form domain-swapped misfolds, where data is available. Contrary to what might be naively expected based on the previously described misfolding mechanism, we find that the extent of misfolding is not determined by the relative folding rates or barrier heights for forming the domains present in the initial intermediates leading to folded or misfolded structures. Instead, it appears that the propensity is more closely related to the relative stability of the domains present in folded and misfolded intermediates. We show that these findings can be rationalized if the folded and misfolded domains are part of the same folding funnel, with commitment to one structure or the other occurring only at a relatively late stage of folding. Nonetheless, the results are still fully consistent with the kinetic models previously proposed to explain misfolding, with a specific interpretation of the observed rate coefficients. Finally, we investigate the relation between interdomain linker length and misfolding, and propose a simple alchemical model to predict the propensity for domain-swapped misfolding of multidomain proteins.

  20. Structural Determinants of Misfolding in Multidomain Proteins

    PubMed Central

    Tian, Pengfei; Best, Robert B.

    2016-01-01

    Recent single molecule experiments, using either atomic force microscopy (AFM) or Förster resonance energy transfer (FRET) have shown that multidomain proteins containing tandem repeats may form stable misfolded structures. Topology-based simulation models have been used successfully to generate models for these structures with domain-swapped features, fully consistent with the available data. However, it is also known that some multidomain protein folds exhibit no evidence for misfolding, even when adjacent domains have identical sequences. Here we pose the question: what factors influence the propensity of a given fold to undergo domain-swapped misfolding? Using a coarse-grained simulation model, we can reproduce the known propensities of multidomain proteins to form domain-swapped misfolds, where data is available. Contrary to what might be naively expected based on the previously described misfolding mechanism, we find that the extent of misfolding is not determined by the relative folding rates or barrier heights for forming the domains present in the initial intermediates leading to folded or misfolded structures. Instead, it appears that the propensity is more closely related to the relative stability of the domains present in folded and misfolded intermediates. We show that these findings can be rationalized if the folded and misfolded domains are part of the same folding funnel, with commitment to one structure or the other occurring only at a relatively late stage of folding. Nonetheless, the results are still fully consistent with the kinetic models previously proposed to explain misfolding, with a specific interpretation of the observed rate coefficients. Finally, we investigate the relation between interdomain linker length and misfolding, and propose a simple alchemical model to predict the propensity for domain-swapped misfolding of multidomain proteins. PMID:27163669

  1. Nanoimaging for protein misfolding and related diseases

    PubMed Central

    Lyubchenko, Yuri L.; Sherman, Simon; Shlyakhtenko, Luda S.; Uversky, Vladimir N.

    2006-01-01

    Misfolding and aggregation of proteins is a common thread linking a number of important human health problems. The misfolded and aggregated proteins are inducers of cellular stress and activators of immunity in neurodegenerative diseases. They might posses clear cytotoxic properties, being responsible for the dysfunction and loss of cells in the affected organs. Despite the crucial importance of protein misfolding and abnormal interactions, very little is currently known about the molecular mechanism underlying these processes. Factors that lead to protein misfolding and aggregation in vitro are poorly understood, not to mention the complexities involved in the formation of protein nanoparticles with different morphologies (e.g. the nanopores) in vivo. A better understanding of the molecular mechanisms of misfolding and aggregation might facilitate development of the rational approaches to prevent pathologies mediated by protein misfolding. The conventional tools currently available to researchers can only provide an averaged picture of a living system, whereas much of the subtle or short-lived information is lost. We believe that the existing and emerging nanotools might help solving these problems by opening the entirely novel pathways for the development of early diagnostic and therapeutic approaches. This article summarizes recent advances of the nanoscience in detection and characterization of misfolded protein conformations. Based on these findings we outline our view on the nanoscience development towards identification intracellular nanomachines and/or multicomponent complexes critically involved in protein misfolding. PMID:16823798

  2. Enhanced Degradation of Misfolded Proteins Promotes Tumorigenesis.

    PubMed

    Chen, Liang; Brewer, Michael D; Guo, Lili; Wang, Ruoxing; Jiang, Peng; Yang, Xiaolu

    2017-03-28

    An adequate cellular capacity to degrade misfolded proteins is critical for cell survival and organismal health. A diminished capacity is associated with aging and neurodegenerative diseases; however, the consequences of an enhanced capacity remain undefined. Here, we report that the ability to clear misfolded proteins is increased during oncogenic transformation and is reduced upon tumor cell differentiation. The augmented capacity mitigates oxidative stress associated with oncogenic growth and is required for both the initiation and maintenance of malignant phenotypes. We show that tripartite motif-containing (TRIM) proteins select misfolded proteins for proteasomal degradation. The higher degradation power in tumor cells is attributed to the upregulation of the proteasome and especially TRIM proteins, both mediated by the antioxidant transcription factor Nrf2. These findings establish a critical role of TRIMs in protein quality control, connect the clearance of misfolded proteins to antioxidant defense, and suggest an intrinsic characteristic of tumor cells.

  3. Mechanisms of protein misfolding: Novel therapeutic approaches to protein-misfolding diseases

    NASA Astrophysics Data System (ADS)

    Salahuddin, Parveen; Siddiqi, Mohammad Khursheed; Khan, Sanaullah; Abdelhameed, Ali Saber; Khan, Rizwan Hasan

    2016-11-01

    In protein misfolding, protein molecule acquires wrong tertiary structure, thereby induces protein misfolding diseases. Protein misfolding can occur through various mechanisms. For instance, changes in environmental conditions, oxidative stress, dominant negative mutations, error in post-translational modifications, increase in degradation rate and trafficking error. All of these factors cause protein misfolding thereby leading to diseases conditions. Both in vitro and in vivo observations suggest that partially unfolded or misfolded intermediates are particularly prone to aggregation. These partially misfolded intermediates aggregate via the interaction with the complementary intermediates and consequently enhance oligomers formation that grows into fibrils and proto-fibrils. The amyloid fibrils for example, accumulate in the brain and central nervous system (CNS) as amyloid deposits in the Parkinson's disease (PD), Alzheimer's disease (AD), Prion disease and Amylo lateral Sclerosis (ALS). Furthermore, tau protein shows intrinsically disorder conformation; therefore its interaction with microtubule is impaired and this protein undergoes aggregation. This is also underlying cause of Alzheimers and other neurodegenerative diseases. Treatment of such misfolding maladies is considered as one of the most important challenges of the 21st century. Currently, several treatments strategies have been and are being discovered. These therapeutic interventions partly reversed or prevented the pathological state. More recently, a new approach was discovered, which employs nanobodies that targets multisteps in fibril formation pathway that may possibly completely cure these misfolding diseases. Keeping the above views in mind in the current review, we have comprehensively discussed the different mechanisms underlying protein misfolding thereby leading to diseases conditions and their therapeutic interventions.

  4. Molecular pathogenesis of protein misfolding diseases: pathological molecular environments versus quality control systems against misfolded proteins.

    PubMed

    Naiki, Hironobu; Nagai, Yoshitaka

    2009-12-01

    Diverse human diseases, including various neurodegenerative disorders and amyloidoses, are thought to result from the misfolding and aggregation of disease-causative proteins, and thus are collectively called protein misfolding diseases. Natively folded disease-causative proteins generally undergo a beta-sheet conformational transition through an energetically unfavourable process, and further polymerize into amyloid fibrils. In the case of beta(2)-microglobulin-related amyloidosis, an extracellular protein misfolding disease, many kinds of biological molecules including glycosaminoglycans, proteoglycans and lipids partially unfold beta(2)-microglobulin and catalyse its subsequent nucleus formation. After amyloid fibrils are formed, these biological molecules stabilize the beta(2)-microglobulin fibrils. In the polyglutamine neurodegenerative diseases, an intracellular protein misfolding disease, molecular chaperones as well as the ubiquitin-proteasome and autophagy-lysosome protein degradation systems, which are called the protein quality control systems, strictly regulate protein misfolding, aggregation and disease progression. A family of extracellular chaperones also binds to misfolded proteins and inhibit amyloid fibril formation in the extracellular space. Protein misfolding and aggregation may be an ideal therapeutic target for protein misfolding diseases in general.

  5. Protein misfolding and aggregation in Parkinson's disease.

    PubMed

    Tan, Jeanne M M; Wong, Esther S P; Lim, Kah-Leong

    2009-09-01

    Protein aggregation as a result of misfolding is a common theme underlying neurodegenerative diseases. In Parkinson's disease (PD), research on protein misfolding and aggregation has taken center stage following the association of alpha-synuclein gene mutations with familial forms of the disease, and importantly, the identification of the protein as a major component of Lewy bodies, a pathological hallmark of PD. Fueling this excitement is the subsequent identification of another PD-linked gene, parkin, as a ubiquitin ligase associated with the proteasome, a major intracellular protein degradation machinery that destroys unwanted, albeit mainly soluble, proteins. Notably, a role for parkin in the clearance of insoluble protein aggregates via macroautophagy has also been implicated by more recent studies. Paradoxically, like alpha-synuclein, parkin is also prone to misfolding, especially in the presence of age-related stress. Similarly, protein misfolding can also affect the function of other key PD-linked genes such as DJ-1, PINK1, and perhaps also LRRK2. Here, we discuss the role of protein misfolding and aggregation in PD, and how impairments of the various cellular protein quality systems could precipitate these events and lead to neuronal demise. Towards the end of our discussion, we also revisited the role of Lewy body formation in PD.

  6. Small molecules that target protein misfolding.

    PubMed

    Gavrin, Lori Krim; Denny, Rajiah Aldrin; Saiah, Eddine

    2012-12-27

    Protein misfolding is a process in which proteins are unable to attain or maintain their biologically active conformation. Factors contributing to protein misfolding include missense mutations and intracellular factors such as pH changes, oxidative stress, or metal ions. Protein misfolding is linked to a large number of diseases such as cystic fibrosis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and less familiar diseases such as Gaucher's disease, nephrogenic diabetes insipidus, and Creutzfeldt-Jakob disease. In this Perspective, we report on small molecules that bind to and stabilize the aberrant protein, thereby helping it to attain a native or near-native conformation and restoring its function. The following targets will be specifically discussed: transthyretin, p53, superoxide dismutase 1, lysozyme, serum amyloid A, prions, vasopressin receptor 2, and α-1-antitrypsin.

  7. Protein misfolding diseases: prospects of pharmacological treatment.

    PubMed

    Gámez, Alejandra; Yuste-Checa, Patricia; Brasil, Sandra; Briso-Montiano, Álvaro; Desviat, Lourdes R; Ugarte, Magdalena; Pérez-Cerdá, Celia; Pérez, Belén

    2017-07-03

    Protein misfolding has been linked to numerous inherited diseases. Loss- and gain-of-function mutations (common features of genetic diseases) may cause the destabilization of proteins, leading to alterations in their properties and/or cellular location, resulting in their incorrect functioning. Misfolded proteins can, however, be rescued via the use of proteostasis regulators and/or pharmacological chaperones, suggesting that treatments with small molecules might be developed for a range of genetic diseases. This work describes the potential of these small molecules in this respect, including for the treatment of PMM2-CDG. This article is protected by copyright. All rights reserved.

  8. Role of protein misfolding and proteostasis deficiency in protein misfolding diseases and aging.

    PubMed

    Cuanalo-Contreras, Karina; Mukherjee, Abhisek; Soto, Claudio

    2013-01-01

    The misfolding, aggregation, and tissue accumulation of proteins are common events in diverse chronic diseases, known as protein misfolding disorders. Many of these diseases are associated with aging, but the mechanism for this connection is unknown. Recent evidence has shown that the formation and accumulation of protein aggregates may be a process frequently occurring during normal aging, but it is unknown whether protein misfolding is a cause or a consequence of aging. To combat the formation of these misfolded aggregates cells have developed complex and complementary pathways aiming to maintain protein homeostasis. These protective pathways include the unfolded protein response, the ubiquitin proteasome system, autophagy, and the encapsulation of damaged proteins in aggresomes. In this paper we review the current knowledge on the role of protein misfolding in disease and aging as well as the implication of deficiencies in the proteostasis cellular pathways in these processes. It is likely that further understanding of the mechanisms involved in protein misfolding and the natural defense pathways may lead to novel strategies for treatment of age-dependent protein misfolding disorders and perhaps aging itself.

  9. Role of Protein Misfolding and Proteostasis Deficiency in Protein Misfolding Diseases and Aging

    PubMed Central

    Mukherjee, Abhisek; Soto, Claudio

    2013-01-01

    The misfolding, aggregation, and tissue accumulation of proteins are common events in diverse chronic diseases, known as protein misfolding disorders. Many of these diseases are associated with aging, but the mechanism for this connection is unknown. Recent evidence has shown that the formation and accumulation of protein aggregates may be a process frequently occurring during normal aging, but it is unknown whether protein misfolding is a cause or a consequence of aging. To combat the formation of these misfolded aggregates cells have developed complex and complementary pathways aiming to maintain protein homeostasis. These protective pathways include the unfolded protein response, the ubiquitin proteasome system, autophagy, and the encapsulation of damaged proteins in aggresomes. In this paper we review the current knowledge on the role of protein misfolding in disease and aging as well as the implication of deficiencies in the proteostasis cellular pathways in these processes. It is likely that further understanding of the mechanisms involved in protein misfolding and the natural defense pathways may lead to novel strategies for treatment of age-dependent protein misfolding disorders and perhaps aging itself. PMID:24348562

  10. Markov state models of protein misfolding

    NASA Astrophysics Data System (ADS)

    Sirur, Anshul; De Sancho, David; Best, Robert B.

    2016-02-01

    Markov state models (MSMs) are an extremely useful tool for understanding the conformational dynamics of macromolecules and for analyzing MD simulations in a quantitative fashion. They have been extensively used for peptide and protein folding, for small molecule binding, and for the study of native ensemble dynamics. Here, we adapt the MSM methodology to gain insight into the dynamics of misfolded states. To overcome possible flaws in root-mean-square deviation (RMSD)-based metrics, we introduce a novel discretization approach, based on coarse-grained contact maps. In addition, we extend the MSM methodology to include "sink" states in order to account for the irreversibility (on simulation time scales) of processes like protein misfolding. We apply this method to analyze the mechanism of misfolding of tandem repeats of titin domains, and how it is influenced by confinement in a chaperonin-like cavity.

  11. Protein Misfolding Cyclic Amplification of Infectious Prions.

    PubMed

    Moda, Fabio

    2017-01-01

    Transmissible spongiform encephalopathies, or prion diseases, are a group of incurable disorders caused by the accumulation of an abnormally folded prion protein (PrP(Sc)) in the brain. According to the "protein-only" hypothesis, PrP(Sc) is the infectious agent able to propagate the disease by acting as a template for the conversion of the correctly folded prion protein (PrP(C)) into the pathological isoform. Recently, the mechanism of PrP(C) conversion has been mimicked in vitro using an innovative technique named protein misfolding cyclic amplification (PMCA). This technology represents a great tool for studying diverse aspects of prion biology in the field of basic research and diagnosis. Moreover, PMCA can be expanded for the study of the misfolding process associated to other neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and frontotemporal lobar degeneration. © 2017 Elsevier Inc. All rights reserved.

  12. [Immunotherapy targeting misfolded proteins in neurodegenerative disease].

    PubMed

    Kanemaru, Kazutomi

    2013-04-01

    Aberrant protein aggregation is closely linked to the molecular pathogeneses of most neurodegenerative diseases. The major components of pathological aggregates have been characterized in various neurodegenerative diseases; for example, amyloid β-protein and phosphorylated tau in Alzheimer's disease, α-synuclein in Parkinson's disease, SOD1 or TDP-43 in amyotrophic lateral sclerosis, and huntingtin in Huntington's disease. These misfolded protein aggregates play a vital role in disease initiation and progression, and they have recently been shown to have prion-like spreading or seeded aggregation properties. Immunotherapy with specific monoclonal antibodies is a promising approach to clear misfolded protein aggregates and treat various neurodegenerative diseases; it is planned for use in clinical trials in the near future.

  13. Protein Misfolding and Retinal Degeneration

    PubMed Central

    Tzekov, Radouil; Stein, Linda; Kaushal, Shalesh

    2011-01-01

    The retina is a highly complex and specialized organ that performs preliminary analysis of visual information. Composed of highly metabolically active tissue, the retina requires a precise and well-balanced means of maintaining its functional activity during extended periods of time. Maintenance and regulation of a vast array of different structural and functional proteins is required for normal function of the retina. This process is referred to as protein homeostasis and involves a variety of activities, including protein synthesis, folding, transport, degradation, elimination, and recycling. Deregulation of any of these activities can lead to malfunctioning of the retina, from subtle subclinical signs to severe retinal degenerative diseases leading to blindness. Examples of retinal degenerative diseases caused by disruption of protein homeostasis include retinitis pigmentosa and Stargardt’s disease. A detailed discussion of the role of disruption in protein homeostasis in these and other retinal diseases is presented, followed by examples of some existing and potential treatments. PMID:21825021

  14. Unraveling protein misfolding diseases using model systems

    PubMed Central

    Peffer, Sara; Cope, Kimberly; Morano, Kevin A

    2015-01-01

    Experimental model systems have long been used to probe the causes, consequences and mechanisms of pathology leading to human disease. Ideally, such information can be exploited to inform the development of therapeutic strategies or treatments to combat disease progression. In the case of protein misfolding diseases, a wide range of model systems have been developed to investigate different aspects of disorders including Huntington's disease, Parkinson's disease, Alzheimer's disease as well as amyotrophic lateral sclerosis. Utility of these systems broadly correlates with evolutionary complexity: small animal models such as rodents and the fruit fly are appropriate for pharmacological modeling and cognitive/behavioral assessment, the roundworm Caenorhabditis elegans allows analysis of tissue-specific disease features, and unicellular organisms such as the yeast Saccharomyces cerevisiae and the bacterium Escherichia coli are ideal for molecular studies. In this chapter, we highlight key advances in our understanding of protein misfolding/unfolding disease provided by model systems. PMID:28031870

  15. Unraveling protein misfolding diseases using model systems.

    PubMed

    Peffer, Sara; Cope, Kimberly; Morano, Kevin A

    2015-09-01

    Experimental model systems have long been used to probe the causes, consequences and mechanisms of pathology leading to human disease. Ideally, such information can be exploited to inform the development of therapeutic strategies or treatments to combat disease progression. In the case of protein misfolding diseases, a wide range of model systems have been developed to investigate different aspects of disorders including Huntington's disease, Parkinson's disease, Alzheimer's disease as well as amyotrophic lateral sclerosis. Utility of these systems broadly correlates with evolutionary complexity: small animal models such as rodents and the fruit fly are appropriate for pharmacological modeling and cognitive/behavioral assessment, the roundworm Caenorhabditis elegans allows analysis of tissue-specific disease features, and unicellular organisms such as the yeast Saccharomyces cerevisiae and the bacterium Escherichia coli are ideal for molecular studies. In this chapter, we highlight key advances in our understanding of protein misfolding/unfolding disease provided by model systems.

  16. Chaperones and cardiac misfolding protein diseases.

    PubMed

    Christians, Elisabeth S; Mustafi, Soumyajit B; Benjamin, Ivor J

    2014-05-01

    Cardiomyocytes are best known for their spontaneous beating activity, large cell size, and low regenerative capacity during adulthood. The mechanical activity of cardiomyocytes depends on a sophisticated contractile apparatus comprised of sarcomeres whose rhythmic contraction relies on Ca(2+) transients with a high level of energy consumption. Hence the proper folding and assembly of the sarcomeric and other accessory proteins involved in those diverse functions (i.e., structural, mechanical, energy exchange and production) is critical for muscle mechanics. Chaperone proteins assist other polypeptides to reach their proper conformation, activity and/or location. Consequently, chaperone-like functions are important for the healthy heart but assume greater relevance during cardiac diseases when such chaperone proteins are recruited: 1) to protect cardiac cells against adverse effects during the pathological transition, and 2) to mitigate certain pathogenic mechanisms per se. Protein misfolding is observed as a consequence of inappropriate intracellular environment with acquired conditions (e.g., ischemia/reperfusion and redox imbalance) or because of mutations, which can modify primary to quaternary protein structures. In this review, we discuss the importance of cardiac chaperones while emphasizing the genetic origin (modification of gene/protein sequence) of cardiac protein misfolding and their consequences on the cardiomyocytes leading to organ dysfunction and failure.

  17. Protein folding and misfolding: mechanism and principles.

    PubMed

    Englander, S Walter; Mayne, Leland; Krishna, Mallela M G

    2007-11-01

    Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors

  18. Protein aggregation and misfolding: good or evil?

    NASA Astrophysics Data System (ADS)

    Pastore, Annalisa; Temussi, Pierandrea

    2012-06-01

    Protein aggregation and misfolding have important implications in an increasing number of fields ranging from medicine to biology to nanotechnology and material science. The interest in understanding this field has accordingly increased steadily over the last two decades. During this time the number of publications that have been dedicated to protein aggregation has increased exponentially, tackling the problem from several different and sometime contradictory perspectives. This review is meant to summarize some of the highlights that come from these studies and introduce this topical issue on the subject. The factors that make a protein aggregate and the cellular strategies that defend from aggregation are discussed together with the perspectives that the accumulated knowledge may open.

  19. Small heat shock proteins and protein-misfolding diseases.

    PubMed

    Laskowska, Ewa; Matuszewska, Ewelina; Kuczyńska-Wiśnik, Dorota

    2010-02-01

    Small heat shock proteins (sHsps) are molecular chaperones ubiquitously distributed in numerous species, from bacteria to humans. A conserved C-terminal "alpha-crystallin" domain organized in a beta-sheet sandwich and oligomeric structure are common features of sHsps. sHsps protect cells against many kinds of stresses including heat shock, oxidative and osmotic stress. sHsps recognize unfolded proteins, prevent their irreversible aggregation and facilitate refolding of bound substrates in cooperation with ATP-dependent molecular chaperones (Hsp70/Hsp40). Mammalian sHsps (HSPBs) are multifunctional proteins involved in many cellular processes including those which are not directly related to protein folding and aggregation. HSPBs participate in cell development and cancerogenesis, regulate apoptosis and control cytoskeletal architecture. Recent data revealed that HSPBs also play an important role in membrane stabilization. Mutation in HSPB genes have been identified, which are responsible for the development of cataract, desmin related myopathy and neuropathies. HSPBs are often found as components of protein aggregates associated with protein-misfolding disorders, such as Parkinson's, Alzheimer's, Alexander's and prion diseases. It is supposed that the presence of HSPBs in intra- or extracellular protein deposits is a consequence of the chaperone activity of HSPBs, however more studies are needed to reveal the exact function of HSPBs during the formation (or removal) of disease-related aggregates.

  20. Protein degradation and protection against misfolded or damaged proteins

    NASA Astrophysics Data System (ADS)

    Goldberg, Alfred L.

    2003-12-01

    The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.

  1. Molecular and cellular aspects of protein misfolding and disease.

    PubMed

    Herczenik, Eszter; Gebbink, Martijn F B G

    2008-07-01

    Proteins are essential elements for life. They are building blocks of all organisms and the operators of cellular functions. Humans produce a repertoire of at least 30,000 different proteins, each with a different role. Each protein has its own unique sequence and shape (native conformation) to fulfill its specific function. The appearance of incorrectly shaped (misfolded) proteins occurs on exposure to environmental changes. Protein misfolding and the subsequent aggregation is associated with various, often highly debilitating, diseases for which no sufficient cure is available yet. In the first part of this review we summarize the structural composition of proteins and the current knowledge of underlying forces that lead proteins to lose their native structure. In the second and third parts we describe the molecular and cellular mechanisms that are associated with protein misfolding in disease. Finally, in the last part we portray recent efforts to develop treatments for protein misfolding diseases.

  2. Protein misfolding cyclic amplification of infectious prions

    PubMed Central

    Morales, Rodrigo; Duran-Aniotz, Claudia; Diaz-Espinoza, Rodrigo; Camacho, Manuel V; Soto, Claudio

    2014-01-01

    Prions are proteinaceous infectious agents responsible for the transmission of prion diseases. The lack of a procedure for cultivating prions in the laboratory has been a major limitation to the study of the unorthodox nature of this infectious agent and the molecular mechanism by which the normal prion protein (PrPC) is converted into the abnormal isoform (PrPSc). Protein misfolding cyclic amplification (PMCA ), described in detail in this protocol, is a simple, fast and efficient methodology to mimic prion replication in the test tube. PMCA involves incubating materials containing minute amounts of infectious prions with an excess of PrPC and boosting the conversion by cycles of sonication to fragment the converting units, thereby leading to accelerated prion replication. PMCA is able to detect the equivalent of a single molecule of infectious PrPSc and propagate prions that maintain high infectivity, strain properties and species specificity. A single PMCA assay takes little more than 3 d to replicate a large amount of prions, which could take years in an in vivo situation. Since its invention 10 years ago, PMCA has helped to answer fundamental questions about this intriguing infectious agent and has been broadly applied in research areas that include the food industry, blood bank safety and human and veterinary disease diagnosis. PMID:22743831

  3. Prions and the potential transmissibility of protein misfolding diseases.

    PubMed

    Kraus, Allison; Groveman, Bradley R; Caughey, Byron

    2013-01-01

    Prions, or infectious proteins, represent a major frontier in the study of infectious agents. The prions responsible for mammalian transmissible spongiform encephalopathies (TSEs) are due primarily to infectious self-propagation of misfolded prion proteins. TSE prion structures remain ill-defined, other than being highly structured, self-propagating, and often fibrillar protein multimers with the capacity to seed, or template, the conversion of their normal monomeric precursors into a pathogenic form. Purified TSE prions usually take the form of amyloid fibrils, which are self-seeding ultrastructures common to many serious protein misfolding diseases such as Alzheimer's, Parkinson's, Huntington's and Lou Gehrig's (amytrophic lateral sclerosis). Indeed, recent reports have now provided evidence of prion-like propagation of several misfolded proteins from cell to cell, if not from tissue to tissue or individual to individual. These findings raise concerns that various protein misfolding diseases might have spreading, prion-like etiologies that contribute to pathogenesis or prevalence.

  4. NALP3 inflammasome activation in protein misfolding diseases.

    PubMed

    Shi, Fushan; Kouadir, Mohammed; Yang, Yang

    2015-08-15

    Protein-misfolding diseases, such as Alzheimer's disease, type 2 diabetes, Prion diseases, and Parkinson's disease, are characterized by inflammatory reactions. In all these diseases, IL-1β (Interlukine-1β) has been shown to be an important regulator, and the misfolded proteins are proved to be triggers of the release of IL-1β. Recently, several reports demonstrated that the inflammasome activation is involved in the progress of the misfolded protein diseases, and that the inflammasome can recognize pathogenic proteins leading to the release of IL-1β. In this review, we discuss the role of inflammasome in the pathogenesis of misfolded protein diseases and the potential of inflammasome-targeting therapeutic interventions in the management of these diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. A universal method for detection of amyloidogenic misfolded proteins.

    PubMed

    Yam, Alice Y; Wang, Xuemei; Gao, Carol Man; Connolly, Michael D; Zuckermann, Ronald N; Bleu, Thieu; Hall, John; Fedynyshyn, Joseph P; Allauzen, Sophie; Peretz, David; Salisbury, Cleo M

    2011-05-24

    Diseases associated with the misfolding of endogenous proteins, such as Alzheimer's disease and type II diabetes, are becoming increasingly prevalent. The pathophysiology of these diseases is not totally understood, but mounting evidence suggests that the misfolded protein aggregates themselves may be toxic to cells and serve as key mediators of cell death. As such, an assay that can detect aggregates in a sensitive and selective fashion could provide the basis for early detection of disease, before cellular damage occurs. Here we report the evolution of a reagent that can selectively capture diverse misfolded proteins by interacting with a common supramolecular feature of protein aggregates. By coupling this enrichment tool with protein specific immunoassays, diverse misfolded proteins and sub-femtomole amounts of oligomeric aggregates can be detected in complex biological matrices. We anticipate that this near-universal approach for quantitative misfolded protein detection will become a useful research tool for better understanding amyloidogenic protein pathology as well as serve as the basis for early detection of misfolded protein diseases.

  6. Protein folding and misfolding in the neurodegenerative disorders: a review.

    PubMed

    Bolshette, N B; Thakur, K K; Bidkar, A P; Trandafir, C; Kumar, P; Gogoi, R

    2014-03-01

    Protein misfolding is an intrinsic aspect of normal folding within the complex cellular environment. Its effects are minimized in living system by the action of a range of protective mechanisms including molecular chaperones and quality control systems. According to the current growing research, protein misfolding is a recognized key feature of most neurodegenerative diseases. Extensive biochemical, neuropathological, and genetic evidence suggest that the cerebral accumulation of amyloid fibrils is the central event in the pathogenesis of neurodegenerative disorders. In the first part of this review we have discussed the general course of action of folding and misfolding of the proteins. Later part of this review gives an outline regarding the role of protein misfolding in the molecular and cellular mechanisms in the pathogenesis of Alzheimer's and Parkinson along with their treatment possibilities. Finally, we have mentioned about the recent findings in neurodegenerative diseases. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  7. Protein-misfolding diseases and chaperone-based therapeutic approaches.

    PubMed

    Chaudhuri, Tapan K; Paul, Subhankar

    2006-04-01

    A large number of neurodegenerative diseases in humans result from protein misfolding and aggregation. Protein misfolding is believed to be the primary cause of Alzheimer's disease, Parkinson's disease, Huntington's disease, Creutzfeldt-Jakob disease, cystic fibrosis, Gaucher's disease and many other degenerative and neurodegenerative disorders. Cellular molecular chaperones, which are ubiquitous, stress-induced proteins, and newly found chemical and pharmacological chaperones have been found to be effective in preventing misfolding of different disease-causing proteins, essentially reducing the severity of several neurodegenerative disorders and many other protein-misfolding diseases. In this review, we discuss the probable mechanisms of several protein-misfolding diseases in humans, as well as therapeutic approaches for countering them. The role of molecular, chemical and pharmacological chaperones in suppressing the effect of protein misfolding-induced consequences in humans is explained in detail. Functional aspects of the different types of chaperones suggest their uses as potential therapeutic agents against different types of degenerative diseases, including neurodegenerative disorders.

  8. Highly Efficient Protein Misfolding Cyclic Amplification

    PubMed Central

    Ostapchenko, Valeriy G.; Savtchenk, Regina; Alexeeva, Irina; Rohwer, Robert G.; Baskakov, Ilia V.

    2011-01-01

    Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrPC into PrPSc in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrPC may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrPC into PrPSc from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrPSc by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrPC susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrPSc in vitro. PMID:21347353

  9. Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins

    PubMed Central

    Hoffstrom, Benjamin G.; Kaplan, Anna; Letso, Reka; Schmid, Ralf; Turmel, Gregory J.; Lo, Donald C.; Stockwell, Brent R.

    2010-01-01

    A hallmark of many neurodegenerative diseases is accumulation of misfolded proteins within neurons, leading to cellular dysfunction and cell death. Although several mechanisms have been proposed to link protein misfolding to cellular toxicity, the connection remains enigmatic. Here, we report a cell death pathway involving protein disulfide isomerase (PDI), a protein chaperone that catalyzes isomerization, reduction, and oxidation of disulfides. Through a small-molecule-screening approach, we discovered five structurally distinct compounds that prevent apoptosis induced by mutant huntingtin protein. Using modified Huisgen cycloaddition chemistry, we then identified PDI as the molecular target of these small molecules. Expression of polyglutamine-expanded huntingtin exon 1 in PC12 cells caused PDI to accumulate at mitochondrial-associated-ER-membranes and trigger apoptotic cell death, via mitochondrial outer membrane permeabilization. Inhibiting PDI in rat brain cells suppressed the toxicity of mutant huntingtin exon1 and Aβ peptides processed from the amyloid precursor protein. This pro-apoptotic function of PDI provides a new mechanism linking protein misfolding and apoptotic cell death. PMID:21079601

  10. Cellular proteostasis: degradation of misfolded proteins by lysosomes

    PubMed Central

    Jackson, Matthew P.

    2016-01-01

    Proteostasis refers to the regulation of the cellular concentration, folding, interactions and localization of each of the proteins that comprise the proteome. One essential element of proteostasis is the disposal of misfolded proteins by the cellular pathways of protein degradation. Lysosomes are an important site for the degradation of misfolded proteins, which are trafficked to this organelle by the pathways of macroautophagy, chaperone-mediated autophagy and endocytosis. Conversely, amyloid diseases represent a failure in proteostasis, in which proteins misfold, forming amyloid deposits that are not degraded effectively by cells. Amyloid may then exacerbate this failure by disrupting autophagy and lysosomal proteolysis. However, targeting the pathways that regulate autophagy and the biogenesis of lysosomes may present approaches that can rescue cells from the deleterious effects of amyloidogenic proteins. PMID:27744333

  11. Protein aggregation, misfolding and consequential human neurodegenerative diseases.

    PubMed

    Sami, Neha; Rahman, Safikur; Kumar, Vijay; Zaidi, Sobia; Islam, Asimul; Ali, Sher; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2017-11-01

    Proteins are major components of the biological functions in a cell. Biology demands that a protein must fold into its stable three-dimensional structure to become functional. In an unfavorable cellular environment, protein may get misfolded resulting in its aggregation. These conformational disorders are directly related to the tissue damage resulting in cellular dysfunction giving rise to different diseases. This way, several neurodegenerative diseases such as Alzheimer, Parkinson Huntington diseases and amyotrophic lateral sclerosis are caused. Misfolding of the protein is prevented by innate molecular chaperones of different classes. It is envisaged that work on this line is likely to translate the knowledge into the development of possible strategies for early diagnosis and efficient management of such related human diseases. The present review deals with the human neurodegenerative diseases caused due to the protein misfolding highlighting pathomechanisms and therapeutic intervention.

  12. Type 2 diabetes as a protein misfolding disease.

    PubMed

    Mukherjee, Abhisek; Morales-Scheihing, Diego; Butler, Peter C; Soto, Claudio

    2015-07-01

    Type 2 diabetes (T2D) is a highly prevalent and chronic metabolic disorder. Recent evidence suggests that formation of toxic aggregates of the islet amyloid polypeptide (IAPP) might contribute to β-cell dysfunction and disease. However, the mechanism of protein aggregation and associated toxicity remains unclear. Misfolding, aggregation, and accumulation of diverse proteins in various organs is the hallmark of the group of protein misfolding disorders (PMDs), including highly prevalent illnesses affecting the central nervous system (CNS) such as Alzheimer's disease (AD) and Parkinson's disease (PD). In this review we discuss the current understanding of the mechanisms implicated in the formation of protein aggregates in the endocrine pancreas and associated toxicity in the light of the long-standing knowledge from neurodegenerative disorders associated with protein misfolding. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Type 2 Diabetes as a Protein Misfolding Disease

    PubMed Central

    Mukherjee, Abhisek; Morales-Scheihing, Diego; Butler, Peter C.; Soto, Claudio

    2015-01-01

    Type 2 diabetes is a highly prevalent and chronic metabolic disorder. Recent evidence suggests that formation of toxic aggregates of the islet amyloid polypeptide (IAPP) might contribute to β-cell dysfunction and disease. However, the mechanism of protein aggregation and associated toxicity is still unclear. Misfolding, aggregation and accumulation of diverse proteins in different organs is the hallmark in the group of protein misfolding disorders (PMDs), including highly prevalent illnesses affecting the central nervous system such as Alzheimer’s and Parkinson’s diseases. In this review we will discuss the current understanding of the mechanisms implicated in the formation of protein aggregates in pancreas and associated toxicity in the light of the longstanding knowledge from neurodegenerative disorders associated with protein misfolding. PMID:25998900

  14. Protein misfolding, aggregation, and autophagy after brain ischemia.

    PubMed

    Luo, Tianfei; Park, Yujung; Sun, Xin; Liu, Chunli; Hu, Bingren

    2013-12-01

    Ischemic brain injury is a common disorder linked to a variety of diseases. Significant progress has been made in our understanding of the underlying mechanisms. Previous studies show that protein misfolding, aggregation, and multiple organelle damage are major pathological events in postischemic neurons. The autophagy pathway is the chief route for bulk degradation of protein aggregates and damaged organelles. The latest studies suggest that impairment of autophagy contributes to abnormal protein aggregation and organelle damages after brain ischemia. This article reviews recent studies of protein misfolding, aggregation, and impairment of autophagy after brain ischemia.

  15. Single-molecule approaches to prion protein misfolding.

    PubMed

    Yu, Hao; Dee, Derek R; Woodside, Michael T

    2013-01-01

    The structural conversion of the prion protein PrP into a transmissible, misfolded form is the central element of prion disease, yet there is little consensus as to how it occurs. Key aspects of conversion into the diseased state remain unsettled, from details about the earliest stages of misfolding such as the involvement of partially- or fully-unfolded intermediates to the structure of the infectious state. Part of the difficulty in understanding the structural conversion arises from the complexity of the underlying energy landscapes. Single molecule methods provide a powerful tool for probing complex folding pathways as in prion misfolding, because they allow rare and transient events to be observed directly. We discuss recent work applying single-molecule probes to study misfolding in prion proteins, and what it has revealed about the folding dynamics of PrP that may underlie its unique behavior. We also discuss single-molecule studies probing the interactions that stabilize non-native structures within aggregates, pointing the way to future work that may help identify the microscopic events triggering pathogenic conversion. Although single-molecule approaches to misfolding are relatively young, they have a promising future in prion science.

  16. Rare ER protein misfolding-mistrafficking disorders: Therapeutic developments.

    PubMed

    Hegde, Ramanath Narayana; Subramanian, Advait; Pothukuchi, Prathyush; Parashuraman, Seetharaman; Luini, Alberto

    2017-04-01

    The presence of a functional protein at the appropriate location in the cell is the result of the processes of transcription, translation, folding and trafficking to the correct destination. There are numerous diseases that are caused by protein misfolding, mainly due to mutations in the respective gene. The consequences of this misfolding may be that proteins effectively lose their function, either by being removed by the cellular quality control machinery or by accumulating at the incorrect intracellular or extracellular location. A number of mutations that lead to protein misfolding and affect trafficking to the final destination, e.g. Cystic fibrosis, Wilson's disease, and Progressive Familial Intrahepatic 1 cholestasis, result in proteins that retain partial function if their folding and trafficking is restored either by molecular or pharmacological means. In this review, we discuss several mutant proteins within this class of misfolding diseases and provide an update on the status of molecular and therapeutic developments and potential therapeutic strategies being developed to counter these diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Prions and the Potential Transmissibility of Protein Misfolding Diseases*

    PubMed Central

    Kraus, Allison; Groveman, Bradley R.; Caughey, Byron

    2016-01-01

    Prions, or infectious proteins, represent a major frontier in the study of infectious agents. The prions responsible for mammalian transmissible spongiform encephalopathies (TSEs) are due primarily to infectious self-propagation of misfolded prion proteins. TSE prion structures remain ill-defined, other than being highly structured, self-propagating, and often fibrillar protein multimers with the capacity to seed, or template, the conversion of their normal monomeric precursors into a pathogenic form. Purified TSE prions usually take the form of amyloid fibrils, which are self-seeding ultrastructures common to many serious protein misfolding diseases such as Alzheimer’s, Parkinson’s, Huntington’s and Lou Gehrig’s (amytrophic lateral sclerosis). Indeed, recent reports have now provided evidence of prion-like propagation of several misfolded proteins from cell to cell, if not from tissue to tissue or individual to individual. These findings raise concerns that various protein misfolding diseases might have spreading, prion-like etiologies that contribute to pathogenesis or prevalence. PMID:23808331

  18. [Spreading of protein misfolding: A new paradigm in neurology].

    PubMed

    Hauw, J-J; Haïk, S; Duyckaerts, C

    2015-12-01

    Protein misfolding and spreading ("transconformation") are being better understood. Described in Prions diseases, this new paradigm in the field of neurodegenerative disorders and brain aging also implies sporadic inclusion myositis, type 2 diabetes, some cancers, sickle cell disease... Misfolding is transmitted from a protein or peptide to a normally folded one. Often associated with a stress of the endoplasmic reticulum, it may spread along the neurites, following anterograde or retrograde axonal transport. In the central nervous system, it occurs in a few cells and there is invasion of adjacent cells by cell-to-cell spread. Three varieties of protein misfolding occur along neuroanatomical pathways. It can be a 'centripetal' process. The synucleinopathy of Parkinson disease has been carefully studied: the changes first occur in cardiac or enteric plexuses... and reach later on the mesencephalon and neocortex. Thus, skin biopsy might prove a diagnostic tool. Protein misfolding may also occur along 'centrifugal' pathways, from motor cortex to peripheral motor neurons. Examples are provided by SOD and pTDP-43 in Amyotrophic Lateral Sclerosis. Amyloid β peptide in cerebral aging and Alzheimer's disease also spread from occipital cortex to the brainstem. Lastly, the propagation may remain 'central' for TDP-43 in behavioral variant frontotemporal dementia, following only pathways of the encephalic neural network. This has to be confirmed, however, since the spreading of some proteins (such as tau or Aβ peptides) has been considered central for a long time and has proved today to involve extracerebral tissues. The complex mechanisms of protein misfolding, still in analysis, include the involvement of chaperone proteins, the formation of very toxic labile proteins molecules (oligomers?), and provide a number of new therapeutic perspectives. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  19. A central role for calcineurin in protein misfolding neurodegenerative diseases.

    PubMed

    Shah, Syed Zahid Ali; Hussain, Tariq; Zhao, Deming; Yang, Lifeng

    2017-03-01

    Accumulation of misfolded/unfolded aggregated proteins in the brain is a hallmark of many neurodegenerative diseases affecting humans and animals. Dysregulation of calcium (Ca(2+)) and disruption of fast axonal transport (FAT) are early pathological events that lead to loss of synaptic integrity and axonal degeneration in early stages of neurodegenerative diseases. Dysregulated Ca(2+) in the brain is triggered by accumulation of misfolded/unfolded aggregated proteins in the endoplasmic reticulum (ER), a major Ca(2+) storing organelle, ultimately leading to neuronal dysfunction and apoptosis. Calcineurin (CaN), a Ca(2+)/calmodulin-dependent serine/threonine phosphatase, has been implicated in T cells activation through the induction of nuclear factor of activated T cells (NFAT). In addition to the involvement of several other signaling cascades, CaN has been shown to play a role in early synaptic dysfunction and neuronal death. Therefore, inhibiting hyperactivated CaN in early stages of disease might be a promising therapeutic strategy for treating patients with protein misfolding diseases. In this review, we briefly summarize the structure of CaN, inhibition mechanisms by which immunosuppressants inhibit CaN, role of CaN in maintaining neuronal and synaptic integrity and homeostasis and the role played by CaN in protein unfolding/misfolding neurodegenerative diseases.

  20. Emerging novel concept of chaperone therapies for protein misfolding diseases

    PubMed Central

    SUZUKI, Yoshiyuki

    2014-01-01

    Chaperone therapy is a newly developed molecular therapeutic approach to protein misfolding diseases. Among them we found unstable mutant enzyme proteins in a few lysosomal diseases, resulting in rapid intracellular degradation and loss of function. Active-site binding low molecular competitive inhibitors (chemical chaperones) paradoxically stabilized and enhanced the enzyme activity in somatic cells by correction of the misfolding of enzyme protein. They reached the brain through the blood-brain barrier after oral administration, and corrected pathophysiology of the disease. In addition to these inhibitory chaperones, non-competitive chaperones without inhibitory bioactivity are being developed. Furthermore molecular chaperone therapy utilizing the heat shock protein and other chaperone proteins induced by small molecules has been experimentally tried to handle abnormally accumulated proteins as a new approach particularly to neurodegenerative diseases. These three types of chaperones are promising candidates for various types of diseases, genetic or non-genetic, and neurological or non-neurological, in addition to lysosomal diseases. PMID:24814990

  1. Emerging novel concept of chaperone therapies for protein misfolding diseases.

    PubMed

    Suzuki, Yoshiyuki

    2014-01-01

    Chaperone therapy is a newly developed molecular therapeutic approach to protein misfolding diseases. Among them we found unstable mutant enzyme proteins in a few lysosomal diseases, resulting in rapid intracellular degradation and loss of function. Active-site binding low molecular competitive inhibitors (chemical chaperones) paradoxically stabilized and enhanced the enzyme activity in somatic cells by correction of the misfolding of enzyme protein. They reached the brain through the blood-brain barrier after oral administration, and corrected pathophysiology of the disease. In addition to these inhibitory chaperones, non-competitive chaperones without inhibitory bioactivity are being developed. Furthermore molecular chaperone therapy utilizing the heat shock protein and other chaperone proteins induced by small molecules has been experimentally tried to handle abnormally accumulated proteins as a new approach particularly to neurodegenerative diseases. These three types of chaperones are promising candidates for various types of diseases, genetic or non-genetic, and neurological or non-neurological, in addition to lysosomal diseases.

  2. Protein misfolding in disease and small molecule therapies.

    PubMed

    Gomes, Cláudio M

    2012-01-01

    A large number of human disorders are caused by defects in protein folding resulting from genetic mutations or adverse physiological conditions, and these are collectively referred to protein misfolding diseases. Such disorders imply dysfunction of a cellular process either as a result of a toxic gain of function due to protein aggregation, or loss of function due to protein instability, inefficient folding or defective trafficking. For a number of cases, drugs acting directly on the affected protein have been found to prevent misfolding and rescue function. This brief review will illustrate molecular mechanisms through which small molecules acting as folding correctors can prevent excessive protein buildup or recover faulty protein conformers, thus acting as effective therapeutic pharmacological chaperones. As background, the principles underlying the thermodynamics and kinetics of the protein folding reaction will be overviewed, as well as pathways leading to the formation of misfolding. The mechanism of action of small molecule correctors will then be discussed in light of these basic principles using illustrative examples referring to drugs that are effective over proteins involved in trafficking and folding diseases, amyloid aggregation disorders and metabolic deficiencies. An outlook on synergistic effects between different folding correctors and their combination with proteostasis regulators will also be addressed, as a relevant strategy towards the design of more effective therapies against protein folding diseases.

  3. Inhibiting toxic aggregation of amyloidogenic proteins: a therapeutic strategy for protein misfolding diseases.

    PubMed

    Cheng, Biao; Gong, Hao; Xiao, Hongwen; Petersen, Robert B; Zheng, Ling; Huang, Kun

    2013-10-01

    The deposition of self-assembled amyloidogenic proteins is associated with multiple diseases, including Alzheimer's disease, Parkinson's disease and type 2 diabetes mellitus. The toxic misfolding and self-assembling of amyloidogenic proteins are believed to underlie protein misfolding diseases. Novel drug candidates targeting self-assembled amyloidogenic proteins represent a potential therapeutic approach for protein misfolding diseases. In this perspective review, we provide an overview of the recent progress in identifying inhibitors that block the aggregation of amyloidogenic proteins and the clinical applications thereof. Compounds such as polyphenols, certain short peptides, and monomer- or oligomer-specific antibodies, can interfere with the self-assembly of amyloidogenic proteins, prevent the formation of oligomers, amyloid fibrils and the consequent cytotoxicity. Some inhibitors have been tested in clinical trials for treating protein misfolding diseases. Inhibitors that target the aggregation of amyloidogenic proteins bring new hope to therapy for protein misfolding diseases. © 2013.

  4. Deubiquitinase activity is required for the proteasomal degradation of misfolded cytosolic proteins upon heat-stress

    PubMed Central

    Fang, Nancy N.; Zhu, Mang; Rose, Amalia; Wu, Kuen-Phon; Mayor, Thibault

    2016-01-01

    Elimination of misfolded proteins is crucial for proteostasis and to prevent proteinopathies. Nedd4/Rsp5 emerged as a major E3-ligase involved in multiple quality control pathways that target misfolded plasma membrane proteins, aggregated polypeptides and cytosolic heat-induced misfolded proteins for degradation. It remained unclear how in one case cytosolic heat-induced Rsp5 substrates are destined for proteasomal degradation, whereas other Rsp5 quality control substrates are otherwise directed to lysosomal degradation. Here we find that Ubp2 and Ubp3 deubiquitinases are required for the proteasomal degradation of cytosolic misfolded proteins targeted by Rsp5 after heat-shock (HS). The two deubiquitinases associate more with Rsp5 upon heat-stress to prevent the assembly of K63-linked ubiquitin on Rsp5 heat-induced substrates. This activity was required to promote the K48-mediated proteasomal degradation of Rsp5 HS-induced substrates. Our results indicate that ubiquitin chain editing is key to the cytosolic protein quality control under stress conditions. PMID:27698423

  5. Identifying Unstable Regions of Proteins Involved in Misfolding Diseases

    NASA Astrophysics Data System (ADS)

    Guest, Will; Cashman, Neil; Plotkin, Steven

    2009-05-01

    Protein misfolding is a necessary step in the pathogenesis of many diseases, including Creutzfeldt-Jakob disease (CJD) and familial amyotrophic lateral sclerosis (fALS). Identifying unstable structural elements in their causative proteins elucidates the early events of misfolding and presents targets for inhibition of the disease process. An algorithm was developed to calculate the Gibbs free energy of unfolding for all sequence-contiguous regions of a protein using three methods to parameterize energy changes: a modified G=o model, changes in solvent-accessible surface area, and all-atoms molecular dynamics. The entropic effects of disulfide bonds and post-translational modifications are treated analytically. It incorporates a novel method for finding local dielectric constants inside a protein to accurately handle charge effects. We have predicted the unstable parts of prion protein and superoxide dismutase 1, the proteins involved in CJD and fALS respectively, and have used these regions as epitopes to prepare antibodies that are specific to the misfolded conformation and show promise as therapeutic agents.

  6. Hemin as a generic and potent protein misfolding inhibitor

    SciTech Connect

    Liu, Yanqin; Carver, John A.; Ho, Lam H.; Elias, Abigail K.; Musgrave, Ian F.; Pukala, Tara L.

    2014-11-14

    Highlights: • Hemin prevents Aβ42, α-synuclein and RCM-κ-casein forming amyloid fibrils. • Hemin inhibits the β-sheet structure formation of Aβ42. • Hemin reduces the cell toxicity caused by fibrillar Aβ42. • Hemin dissociates partially formed Aβ42 fibrils. • Hemin prevents amorphous aggregation by ADH, catalase and γs-crystallin. - Abstract: Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer’s disease, Parkinson’s disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.

  7. Hemin as a generic and potent protein misfolding inhibitor.

    PubMed

    Liu, Yanqin; Carver, John A; Ho, Lam H; Elias, Abigail K; Musgrave, Ian F; Pukala, Tara L

    2014-11-14

    Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer's disease, Parkinson's disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Modeling protein misfolding in charcot-marie-tooth disease.

    PubMed

    Theocharopoulou, Georgia; Vlamos, Panayiotis

    2015-01-01

    Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder. Recent advancements in molecular biology have elucidated the molecular bases of this genetically heterogeneous neuropathy. Still, the major challenge lies in determining the individual contributions by malfunctions of proteins to the disease's pathology. This paper reviews the identified molecular mechanisms underlying major forms of CMT disease. A growing body of evidence has highlighted the role of protein misfolding in demyelinating peripheral neuropathies and neurodegenerative diseases. Several hypotheses have been proposed to explain how misfolded aggregates induce neuronal damage. Current research focuses on developing novel therapeutic targets which aim to prevent, or even reverse the formation of protein aggregation. Interestingly, the role of the cellular defence mechanisms against accumulation of misfolded proteins may play a key role leading to novel strategies for treatment accelerating the clearance of their toxic early aggregates. Based on these findings we propose a model for describing in terms of a formal computer language, the biomolecular processes involving proteins associated with CMT disease.

  9. Frustration in the energy landscapes of multidomain protein misfolding

    PubMed Central

    Zheng, Weihua; Schafer, Nicholas P.; Wolynes, Peter G.

    2013-01-01

    Frustration from strong interdomain interactions can make misfolding a more severe problem in multidomain proteins than in single-domain proteins. On the basis of bioinformatic surveys, it has been suggested that lowering the sequence identity between neighboring domains is one of nature’s solutions to the multidomain misfolding problem. We investigate folding of multidomain proteins using the associative-memory, water-mediated, structure and energy model (AWSEM), a predictive coarse-grained protein force field. We find that reducing sequence identity not only decreases the formation of domain-swapped contacts but also decreases the formation of strong self-recognition contacts between β-strands with high hydrophobic content. The ensembles of misfolded structures that result from forming these amyloid-like interactions are energetically disfavored compared with the native state, but entropically favored. Therefore, these ensembles are more stable than the native ensemble under denaturing conditions, such as high temperature. Domain-swapped contacts compete with self-recognition contacts in forming various trapped states, and point mutations can shift the balance between the two types of interaction. We predict that multidomain proteins that lack these specific strong interdomain interactions should fold reliably. PMID:23319605

  10. Frustration in the energy landscapes of multidomain protein misfolding.

    PubMed

    Zheng, Weihua; Schafer, Nicholas P; Wolynes, Peter G

    2013-01-29

    Frustration from strong interdomain interactions can make misfolding a more severe problem in multidomain proteins than in single-domain proteins. On the basis of bioinformatic surveys, it has been suggested that lowering the sequence identity between neighboring domains is one of nature's solutions to the multidomain misfolding problem. We investigate folding of multidomain proteins using the associative-memory, water-mediated, structure and energy model (AWSEM), a predictive coarse-grained protein force field. We find that reducing sequence identity not only decreases the formation of domain-swapped contacts but also decreases the formation of strong self-recognition contacts between β-strands with high hydrophobic content. The ensembles of misfolded structures that result from forming these amyloid-like interactions are energetically disfavored compared with the native state, but entropically favored. Therefore, these ensembles are more stable than the native ensemble under denaturing conditions, such as high temperature. Domain-swapped contacts compete with self-recognition contacts in forming various trapped states, and point mutations can shift the balance between the two types of interaction. We predict that multidomain proteins that lack these specific strong interdomain interactions should fold reliably.

  11. Protein misfolding and endoplasmic reticulum stress in chronic lung disease.

    PubMed

    Wei, James; Rahman, Sadaf; Ayaub, Ehab A; Dickhout, Jeffrey G; Ask, Kjetil

    2013-04-01

    The pathogenesis of chronic lung disorders is poorly understood but is often thought to arise because of repeated injuries derived from exposure to exogenous or endogenous stress factors. Protein-misfolding events have been observed in a variety of genetic and nongenetic chronic lung disorders and may contribute to both the initiation and the progression of lung disease through endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Evidence indicates that exposure to common lung irritants such as cigarette smoke, environmental pollutants, and infectious viral or bacterial agents can induce ER stress and protein misfolding. Although the UPR is thought to be a molecular mechanism involved in the repair and restoration of protein homeostasis or "proteostasis," prolonged activation of the UPR may lead to compromised cellular functions, cellular transformation, or cell death. Here, we review literature that associates protein-misfolding events with ER stress and UPR activation and discuss how this basic molecular repair mechanism may contribute to the initiation and progression of various genetic and nongenetic chronic lung diseases.

  12. Cytosolic proteostasis through importing of misfolded proteins into mitochondria.

    PubMed

    Ruan, Linhao; Zhou, Chuankai; Jin, Erli; Kucharavy, Andrei; Zhang, Ying; Wen, Zhihui; Florens, Laurence; Li, Rong

    2017-03-16

    Loss of proteostasis underlies ageing and neurodegeneration characterized by the accumulation of protein aggregates and mitochondrial dysfunction. Although many neurodegenerative-disease-associated proteins can be found in mitochondria, it remains unclear how mitochondrial dysfunction and protein aggregation could be related. In dividing yeast cells, protein aggregates that form under stress or during ageing are preferentially retained by the mother cell, in part through tethering to mitochondria, while the disaggregase Hsp104 helps to dissociate aggregates and thereby enables refolding or degradation of misfolded proteins. Here we show that, in yeast, cytosolic proteins prone to aggregation are imported into mitochondria for degradation. Protein aggregates that form under heat shock contain both cytosolic and mitochondrial proteins and interact with the mitochondrial import complex. Many aggregation-prone proteins enter the mitochondrial intermembrane space and matrix after heat shock, and some do so even without stress. Timely dissolution of cytosolic aggregates requires the mitochondrial import machinery and proteases. Blocking mitochondrial import but not proteasome activity causes a marked delay in the degradation of aggregated proteins. Defects in cytosolic Hsp70s leads to enhanced entry of misfolded proteins into mitochondria and elevated mitochondrial stress. We term this mitochondria-mediated proteostasis mechanism MAGIC (mitochondria as guardian in cytosol) and provide evidence that it may exist in human cells.

  13. Nanoprobing of misfolding and interactions of amyloid β 42 protein.

    PubMed

    Kim, Bo-Hyun; Lyubchenko, Yuri L

    2014-05-01

    The assembly of amyloid β (Aβ) proteins into nanostructures is currently considered a major pathway of Alzheimer's disease development, but the molecular mechanisms of this self-assembly process remains unclear. Recently, we showed that single-molecule AFM force spectroscopy (SMFS) is capable of probing the dynamics and interaction between Aβ40 peptides, and these studies allowed us to shed new light on transiently existing Aβ40 misfolding states. In this study, we applied the same SMFS approach to characterize the misfolding of Aβ42 peptide, the most toxic Aβ alloform. The quantitative analysis of SMFS data demonstrated that Aβ interaction leads to the formation of dimers with a lifetime in the range of a second. Interaction via C-terminal segments prevailed at pH 7, but interaction within the peptide center prevailed at acidic pH levels. The difference in the misfolding properties for Aβ40 and Aβ42 peptides and the mechanisms of amyloid nanoassembly are discussed. Despite decades of intense research, Alzheimer's disease still remains incurable. This novel study focuses on the assembly of amyloid β proteins into nanostructures, which is a key mechanism in Alzheimer's disease development. Single molecule atomic force spectroscopy is utilized to shed light on the molecular mechanisms of this self-assembly process. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Cross Currents in Protein Misfolding Disorders: Interactions and Therapy

    PubMed Central

    Morales, Rodrigo; Green, Kristi M.; Soto, Claudio

    2009-01-01

    Protein Misfolding Disorders (PMDs) are a group of diseases characterized by the accumulation of abnormally folded proteins. Despite the wide range of proteins and tissues involved, PMDs share similar molecular and pathogenic mechanisms. Several epidemiological, clinical and experimental reports have described the co-existence of PMDs, suggesting a possible cross-talk between them. A better knowledge of the molecular basis of PMDs could have important implications for understanding the mechanism by which the diseases appear and progress and ultimately to develop novel strategies for treatment. Due to their similar molecular mechanisms, common therapeutic strategies could be applied for the diseases in this group. PMID:19702573

  15. Cadmium Causes Misfolding and Aggregation of Cytosolic Proteins in Yeast.

    PubMed

    Jacobson, Therese; Priya, Smriti; Sharma, Sandeep K; Andersson, Stefanie; Jakobsson, Sofia; Tanghe, Robbe; Ashouri, Arghavan; Rauch, Sebastien; Goloubinoff, Pierre; Christen, Philipp; Tamás, Markus J

    2017-09-01

    Cadmium is a highly poisonous metal and is classified as a human carcinogen. While its toxicity is undisputed, the underlying in vivo molecular mechanisms are not fully understood. Here, we demonstrate that cadmium induces aggregation of cytosolic proteins in living Saccharomyces cerevisiae cells. Cadmium primarily targets proteins in the process of synthesis or folding, probably by interacting with exposed thiol groups in not-yet-folded proteins. On the basis of in vitro and in vivo data, we show that cadmium-aggregated proteins form seeds that increase the misfolding of other proteins. Cells that cannot efficiently protect the proteome from cadmium-induced aggregation or clear the cytosol of protein aggregates are sensitized to cadmium. Thus, protein aggregation may contribute to cadmium toxicity. This is the first report on how cadmium causes misfolding and aggregation of cytosolic proteins in vivo The proposed mechanism might explain not only the molecular basis of the toxic effects of cadmium but also the suggested role of this poisonous metal in the pathogenesis of certain protein-folding disorders. Copyright © 2017 American Society for Microbiology.

  16. Cyclic Amplification of Prion Protein Misfolding

    PubMed Central

    Barria, Marcelo A; Gonzalez-Romero, Dennisse; Soto, Claudio

    2014-01-01

    Protein Misfolfing Cyclic amplification (PMCA) is a technique that take advantage of the nucleation-dependent prion replication process to accelerate the conversion of PrPC into PrPSc in the test tube. PMCA uses ultrasound waves to fragment the PrPSc polymers, increasing the amount of seeds present in the infected sample without affecting their ability to act as conversion nucleus. Over the past 5 years PMCA has became an invaluable technique to study diverse aspects of prions. The PMCA technology has been used by several groups to understand the molecular mechanism of prion replication, the cellular factors involved in prion propagation, the intriguing phenomena of prion strains and species barriers, to detect PrPSc in tissues and biological fluids and to screen for inhibitors against prion replication. In this article we describe a detailed protocol of the PMCA technique, highlighting some of the important technical aspects to obtain a successful and reproducible application of the technology. PMID:22528092

  17. Distinct role of hydration water in protein misfolding and aggregation revealed by fluctuating thermodynamics analysis.

    PubMed

    Chong, Song-Ho; Ham, Sihyun

    2015-04-21

    Protein aggregation in aqueous cellular environments is linked to diverse human diseases. Protein aggregation proceeds through a multistep process initiated by conformational transitions, called protein misfolding, of monomer species toward aggregation-prone structures. Various forms of aggregate species are generated through the association of misfolded monomers including soluble oligomers and amyloid fibrils. Elucidating the molecular mechanisms and driving forces involved in the misfolding and subsequent association has been a central issue for understanding and preventing protein aggregation diseases such as Alzheimer's, Parkinson's, and type II diabetes. In this Account, we provide a thermodynamic perspective of the misfolding and aggregation of the amyloid-beta (Aβ) protein implicated in Alzheimer's disease through the application of fluctuating thermodynamics. This approach "dissects" the conventional thermodynamic characterization of the end states into the one of the fluctuating processes connecting them, and enables one to analyze variations in the thermodynamic functions that occur during the course of protein conformational changes. The central quantity in this approach is the solvent-averaged effective energy, f = Eu + Gsolv, comprising the protein potential energy (Eu) and the solvation free energy (Gsolv), whose time variation reflects the protein dynamics on the free energy landscape. Protein configurational entropy is quantified by the magnitude of fluctuations in f. We find that misfolding of the Aβ monomer when released from a membrane environment to an aqueous phase is driven by favorable changes in protein potential energy and configurational entropy, but it is also accompanied by an unfavorable increase in solvation free energy. The subsequent dimerization of the misfolded Aβ monomers occurs in two steps. The first step, where two widely separated monomers come into contact distance, is driven by water-mediated attraction, that is, by a

  18. Ubx4 modulates cdc48 activity and influences degradation of misfolded proteins of the endoplasmic reticulum.

    PubMed

    Alberts, Sven M; Sonntag, Caroline; Schäfer, Antje; Wolf, Dieter H

    2009-06-12

    Misfolded proteins of the secretory pathway are recognized in the endoplasmic reticulum (ER), retrotranslocated into the cytoplasm, and degraded by the ubiquitin-proteasome system. Right after retrotranslocation and polyubiquitination, they are extracted from the cytosolic side of the ER membrane through a complex consisting of the AAA ATPase Cdc48 (p97 in mammals), Ufd1, and Npl4. This complex delivers misfolded proteins to the proteasome for final degradation. Extraction, delivery, and processing of ERAD (ER-associated degradation) substrates to the proteasome requires additional cofactors of Cdc48. Here we characterize the UBX domain containing protein Ubx4 (Cui1) as a crucial factor for the degradation of polyubiquitinated proteins via ERAD. Ubx4 modulates the Cdc48-Ufd1-Npl4 complex to guarantee its correct function. Mutant variants of Ubx4 lead to defective degradation of misfolded proteins and accumulation of polyubiquitinated proteins bound to Cdc48. We show the requirement of the UBX domain of Ubx4 for its function in ERAD. The observation that Ubx2 and Ubx4 are not found together in one complex with Cdc48 suggests several distinct steps in modulating the activity and localization of Cdc48 in ERAD.

  19. Protein misfolding cyclic amplification (PMCA): Current status and future directions.

    PubMed

    Saá, Paula; Cervenakova, Larisa

    2015-09-02

    Transmissible spongiform encephalopathies (TSEs) most commonly known as prion diseases are invariably fatal neurological disorders that affect humans and animals. These disorders differ from other neurodegenerative conformational diseases caused by the accumulation in the brain of misfolded proteins, sometimes with amyloid properties, in their ability to infect susceptible species by various routes. While the infectious properties of amyloidogenic proteins, other than misfolded prion protein (PrP(TSE)), are currently under scrutiny, their potential to transmit from cell to cell, one of the intrinsic properties of the prion, has been recently shown in vitro and in vivo. Over the decades, various cell culture and laboratory animal models have been developed to study TSEs. These assays have been widely used in a variety of applications but showed to be time consuming and entailed elevated costs. Novel economic and fast alternatives became available with the development of in vitro assays that are based on the property of conformationally abnormal PrP(TSE) to recruit normal cellular PrP(C) to misfold. These include the cell-free conversion assay, protein misfolding cyclic amplification (PMCA) and quaking induced conversion assay (QuIC), of which the PMCA has been the only technology shown to generate infectious prions. Moreover, it allows indefinite amplification of PrP(TSE) with strain-specific biochemical and biological properties of the original molecules and under certain conditions may give rise to new spontaneously generated prions. The method also allows addressing the species barrier phenomena and assessing possible risks of animal-to-animal and animal-to-human transmission. Additionally, its unprecedented sensitivity has made possible the detection of as little as one infectious dose of PrP(TSE) and the biochemical identification of this protein in different tissues and biological fluids, including blood, cerebral spinal fluid (CSF), semen, milk, urine and

  20. Chemical and/or Biological Therapeutic Strategies to Ameliorate Protein Misfolding Diseases

    PubMed Central

    Ong, Derrick Sek Tong; Kelly, Jeffery W.

    2010-01-01

    Inheriting a mutant misfolding-prone protein that cannot be efficiently folded in a given cell type(s) results in a spectrum of human loss-of-function misfolding diseases. The inability of the biological protein maturation pathways to adapt to a specific misfolding-prone protein also contributes to pathology. Chemical and biological therapeutic strategies are presented that restore protein homeostasis, or proteostasis, either by enhancing the biological capacity of the proteostasis network or through small molecule stabilization of a specific misfolding-prone protein. Herein, we review the recent literature on therapeutic strategies to ameliorate protein misfolding diseases that function through either of these mechanisms, or a combination thereof, and provide our perspective on the promise of alleviating protein misfolding diseases by taking advantage of proteostasis adaptation. PMID:21146391

  1. Chemical and/or biological therapeutic strategies to ameliorate protein misfolding diseases.

    PubMed

    Ong, Derrick Sek Tong; Kelly, Jeffery W

    2011-04-01

    Inheriting a mutant misfolding-prone protein that cannot be efficiently folded in a given cell type(s) results in a spectrum of human loss-of-function misfolding diseases. The inability of the biological protein maturation pathways to adapt to a specific misfolding-prone protein also contributes to pathology. Chemical and biological therapeutic strategies are presented that restore protein homeostasis, or proteostasis, either by enhancing the biological capacity of the proteostasis network or through small molecule stabilization of a specific misfolding-prone protein. Herein, we review the recent literature on therapeutic strategies to ameliorate protein misfolding diseases that function through either of these mechanisms, or a combination thereof, and provide our perspective on the promise of alleviating protein misfolding diseases by taking advantage of proteostasis adaptation. Copyright © 2010 Elsevier Ltd. All rights reserved.

  2. Misfolded proteins impose a dosage-dependent fitness cost and trigger a cytosolic unfolded protein response in yeast

    PubMed Central

    Dion, Michael F.; Budnik, Bogdan A.; Wang, Stephanie M.; Hartl, Daniel L.; Drummond, D. Allan

    2011-01-01

    Evolving lineages face a constant intracellular threat: most new coding sequence mutations destabilize the folding of the encoded protein. Misfolded proteins form insoluble aggregates and are hypothesized to be intrinsically cytotoxic. Here, we experimentally isolate a fitness cost caused by toxicity of misfolded proteins. We exclude other costs of protein misfolding, such as loss of functional protein or attenuation of growth-limiting protein synthesis resources, by comparing growth rates of budding yeast expressing folded or misfolded variants of a gratuitous protein, YFP, at equal levels. We quantify a fitness cost that increases with misfolded protein abundance, up to as much as a 3.2% growth rate reduction when misfolded YFP represents less than 0.1% of total cellular protein. Comparable experiments on variants of the yeast gene orotidine-5′-phosphate decarboxylase (URA3) produce similar results. Quantitative proteomic measurements reveal that, within the cell, misfolded YFP induces coordinated synthesis of interacting cytosolic chaperone proteins in the absence of a wider stress response, providing evidence for an evolved modular response to misfolded proteins in the cytosol. These results underscore the distinct and evolutionarily relevant molecular threat of protein misfolding, independent of protein function. Assuming that most misfolded proteins impose similar costs, yeast cells express almost all proteins at steady-state levels sufficient to expose their encoding genes to selection against misfolding, lending credibility to the recent suggestion that such selection imposes a global constraint on molecular evolution. PMID:21187411

  3. Chaperonins fight aminoglycoside-induced protein misfolding and promote short-term tolerance in Escherichia coli.

    PubMed

    Goltermann, Lise; Good, Liam; Bentin, Thomas

    2013-04-12

    For almost half of a century, we have known that aminoglycoside antibiotics corrupt ribosomes, causing translational misreading, yet it remains unclear whether or not misreading triggers protein misfolding, and possible effects of chaperone action on drug susceptibilities are poorly understood. Here, we show that aminoglycosides cause cytosolic protein misfolding and that chaperonin GroEL/GroES overexpression counters this defect. During aminoglycoside exposure to exponential cultures, chaperonin overexpression protected the bacterial membrane potential, rescued cell growth, and facilitated survival, whereas inhibition of chaperonin expression sensitized bacteria. Overexpression of the DnaK/DnaJ/GrpE chaperone system similarly facilitated survival but did not promote growth of aminoglycoside-treated bacteria. Inhibition of chaperonin expression sensitized bacteria to aminoglycosides as measured by reduced minimum inhibitory concentrations, whereas GroEL/GroES overexpression did not increase minimum inhibitory concentrations. Our observations establish misfolding of cytosolic proteins as an effect of aminoglycoside action and reveal that chaperones, chaperonins in particular, help bacteria cope during early exposure to these drugs.

  4. Direct observation of multiple misfolding pathways in a single prion protein molecule.

    PubMed

    Yu, Hao; Liu, Xia; Neupane, Krishna; Gupta, Amar Nath; Brigley, Angela M; Solanki, Allison; Sosova, Iveta; Woodside, Michael T

    2012-04-03

    Protein misfolding is a ubiquitous phenomenon associated with a wide range of diseases. Single-molecule approaches offer a powerful tool for deciphering the mechanisms of misfolding by measuring the conformational fluctuations of a protein with high sensitivity. We applied single-molecule force spectroscopy to observe directly the misfolding of the prion protein PrP, a protein notable for having an infectious misfolded state that is able to propagate by recruiting natively folded PrP. By measuring folding trajectories of single PrP molecules held under tension in a high-resolution optical trap, we found that the native folding pathway involves only two states, without evidence for partially folded intermediates that have been proposed to mediate misfolding. Instead, frequent but fleeting transitions were observed into off-pathway intermediates. Three different misfolding pathways were detected, all starting from the unfolded state. Remarkably, the misfolding rate was even higher than the rate for native folding. A mutant PrP with higher aggregation propensity showed increased occupancy of some of the misfolded states, suggesting these states may act as intermediates during aggregation. These measurements of individual misfolding trajectories demonstrate the power of single-molecule approaches for characterizing misfolding directly by mapping out nonnative folding pathways.

  5. Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia.

    PubMed

    Buhimschi, Irina A; Nayeri, Unzila A; Zhao, Guomao; Shook, Lydia L; Pensalfini, Anna; Funai, Edmund F; Bernstein, Ira M; Glabe, Charles G; Buhimschi, Catalin S

    2014-07-16

    Preeclampsia is a pregnancy-specific disorder of unknown etiology and a leading contributor to maternal and perinatal morbidity and mortality worldwide. Because there is no cure other than delivery, preeclampsia is the leading cause of iatrogenic preterm birth. We show that preeclampsia shares pathophysiologic features with recognized protein misfolding disorders. These features include urine congophilia (affinity for the amyloidophilic dye Congo red), affinity for conformational state-dependent antibodies, and dysregulation of prototype proteolytic enzymes involved in amyloid precursor protein (APP) processing. Assessment of global protein misfolding load in pregnancy based on urine congophilia (Congo red dot test) carries diagnostic and prognostic potential for preeclampsia. We used conformational state-dependent antibodies to demonstrate the presence of generic supramolecular assemblies (prefibrillar oligomers and annular protofibrils), which vary in quantitative and qualitative representation with preeclampsia severity. In the first attempt to characterize the preeclampsia misfoldome, we report that the urine congophilic material includes proteoforms of ceruloplasmin, immunoglobulin free light chains, SERPINA1, albumin, interferon-inducible protein 6-16, and Alzheimer's β-amyloid. The human placenta abundantly expresses APP along with prototype APP-processing enzymes, of which the α-secretase ADAM10, the β-secretases BACE1 and BACE2, and the γ-secretase presenilin-1 were all up-regulated in preeclampsia. The presence of β-amyloid aggregates in placentas of women with preeclampsia and fetal growth restriction further supports the notion that this condition should join the growing list of protein conformational disorders. If these aggregates play a pathophysiologic role, our findings may lead to treatment for preeclampsia.

  6. Solubility and supersaturation-dependent protein misfolding revealed by ultrasonication.

    PubMed

    Lin, Yuxi; Lee, Young-Ho; Yoshimura, Yuichi; Yagi, Hisashi; Goto, Yuji

    2014-02-25

    Although alcohols are useful cosolvents for producing amyloid fibrils, the underlying mechanism of alcohol-dependent fibrillation is unclear. We studied the alcohol-induced fibrillation of hen egg-white lysozyme at various concentrations of ethanol, 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Under the conditions where the alcohol-denatured lysozyme retained metastability, ultrasonication effectively triggered fibrillation. The optimal alcohol concentration depended on the alcohol species. HFIP showed a sharp maximum at 12-16%. For TFE, a broad maximum at 40-80% was observed. Ethanol exhibited only an increase in fibrillation above 60%. These profiles were opposite to the equilibrium solubility of lysozyme in water/alcohol mixtures. The results indicate that although fibrillation is determined by solubility, supersaturation prevents conformational transitions and ultrasonication is highly effective in minimizing an effect of supersaturation. We propose an alcohol-dependent protein misfolding funnel useful for examining amyloidogenicity. This misfolding funnel will apply to fibrillation under physiological conditions where biological environments play important roles in decreasing the solubility.

  7. Protein misfolding in neurodegenerative diseases: implications and strategies.

    PubMed

    Sweeney, Patrick; Park, Hyunsun; Baumann, Marc; Dunlop, John; Frydman, Judith; Kopito, Ron; McCampbell, Alexander; Leblanc, Gabrielle; Venkateswaran, Anjli; Nurmi, Antti; Hodgson, Robert

    2017-01-01

    A hallmark of neurodegenerative proteinopathies is the formation of misfolded protein aggregates that cause cellular toxicity and contribute to cellular proteostatic collapse. Therapeutic options are currently being explored that target different steps in the production and processing of proteins implicated in neurodegenerative disease, including synthesis, chaperone-assisted folding and trafficking, and degradation via the proteasome and autophagy pathways. Other therapies, like mTOR inhibitors and activators of the heat shock response, can rebalance the entire proteostatic network. However, there are major challenges that impact the development of novel therapies, including incomplete knowledge of druggable disease targets and their mechanism of action as well as a lack of biomarkers to monitor disease progression and therapeutic response. A notable development is the creation of collaborative ecosystems that include patients, clinicians, basic and translational researchers, foundations and regulatory agencies to promote scientific rigor and clinical data to accelerate the development of therapies that prevent, reverse or delay the progression of neurodegenerative proteinopathies.

  8. Integrated organotypic slice cultures and RT-QuIC (OSCAR) assay: implications for translational discovery in protein misfolding diseases

    USDA-ARS?s Scientific Manuscript database

    Protein misfolding is a key pathological event in neurodegenerative diseases like prion diseases, synucleinopathies, and tauopathies that are collectively termed protein misfolding disorders (PMD). Prions are a prototypic model to study protein aggregation biology and therapeutic development. Attemp...

  9. Distribution of Misfolded Prion Protein Seeding Activity Alone Does Not Predict Regions of Neurodegeneration

    PubMed Central

    Alibhai, James; Blanco, Richard A.; Barria, Marcelo A.; Piccardo, Pedro; Caughey, Byron; Perry, V. Hugh; Freeman, Tom C.; Manson, Jean C.

    2016-01-01

    Protein misfolding is common across many neurodegenerative diseases, with misfolded proteins acting as seeds for "prion-like" conversion of normally folded protein to abnormal conformations. A central hypothesis is that misfolded protein accumulation, spread, and distribution are restricted to specific neuronal populations of the central nervous system and thus predict regions of neurodegeneration. We examined this hypothesis using a highly sensitive assay system for detection of misfolded protein seeds in a murine model of prion disease. Misfolded prion protein (PrP) seeds were observed widespread throughout the brain, accumulating in all brain regions examined irrespective of neurodegeneration. Importantly, neither time of exposure nor amount of misfolded protein seeds present determined regions of neurodegeneration. We further demonstrate two distinct microglia responses in prion-infected brains: a novel homeostatic response in all regions and an innate immune response restricted to sites of neurodegeneration. Therefore, accumulation of misfolded prion protein alone does not define targeting of neurodegeneration, which instead results only when misfolded prion protein accompanies a specific innate immune response. PMID:27880767

  10. Targeting unfolded protein response signaling pathways to ameliorate protein misfolding diseases.

    PubMed

    Ryno, Lisa M; Wiseman, R Luke; Kelly, Jeffery W

    2013-06-01

    Protein homeostasis (or proteostasis) within the endoplasmic reticulum (ER) is regulated by the unfolded protein response (UPR). The UPR consists of three integrated signaling pathways activated by the accumulation of misfolded proteins within the ER lumen. Activation of the UPR alters ER proteostasis through translational attenuation of new protein synthesis and transcriptional remodeling of ER proteostasis pathways, providing a mechanism to adapt ER proteostasis in response to cellular stress. The capacity of the UPR to alter ER proteostasis suggests that exogenous manipulation of UPR signaling pathways offers therapeutic promise to alter the fate of pathologic proteins associated with human protein misfolding diseases. Here, we discuss the therapeutic potential of exogenous UPR activation to treat human disease and highlight specific small molecule approaches for regulating UPR signaling that could be beneficial to treat protein misfolding diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Protein misfolding in the endoplasmic reticulum as a conduit to human disease.

    PubMed

    Wang, Miao; Kaufman, Randal J

    2016-01-21

    In eukaryotic cells, the endoplasmic reticulum is essential for the folding and trafficking of proteins that enter the secretory pathway. Environmental insults or increased protein synthesis often lead to protein misfolding in the organelle, the accumulation of misfolded or unfolded proteins - known as endoplasmic reticulum stress - and the activation of the adaptive unfolded protein response to restore homeostasis. If protein misfolding is not resolved, cells die. Endoplasmic reticulum stress and activation of the unfolded protein response help to determine cell fate and function. Furthermore, endoplasmic reticulum stress contributes to the aetiology of many human diseases.

  12. Fast Protein Translation Can Promote Co- and Posttranslational Folding of Misfolding-Prone Proteins.

    PubMed

    Trovato, Fabio; O'Brien, Edward P

    2017-05-09

    Chemical kinetic modeling has previously been used to predict that fast-translating codons can enhance cotranslational protein folding by helping to avoid misfolded intermediates. Consistent with this prediction, protein aggregation in yeast and worms was observed to increase when translation was globally slowed down, possibly due to increased cotranslational misfolding. Observation of similar behavior in molecular simulations would confirm predictions from the simpler chemical kinetic model and provide a molecular perspective on cotranslational folding, misfolding, and the impact of translation speed on these processes. All-atom simulations cannot reach the timescales relevant to protein synthesis, and most conventional structure-based coarse-grained models do not allow for nonnative structure formation. Here, we introduce a protocol to incorporate misfolding using the functional forms of publicly available force fields. With this model we create two artificial proteins that are capable of undergoing structural transitions between a native and a misfolded conformation and simulate their synthesis by the ribosome. Consistent with the chemical kinetic predictions, we find that rapid synthesis of misfolding-prone nascent-chain segments increases the fraction of folded proteins by kinetically partitioning more molecules through on-pathway intermediates, decreasing the likelihood of sampling misfolded conformations. Novel to this study, to our knowledge, we observe that differences in protein dynamics, arising from different translation-elongation schedules, can persist long after the nascent protein has been released from the ribosome, and that a sufficient level of energetic frustration is needed for fast-translating codons to be beneficial for folding. These results provide further evidence that fast-translating codons can be as biologically important as pause sites in coordinating cotranslational folding. Copyright © 2017 Biophysical Society. Published by Elsevier

  13. Cystic fibrosis – a multiorgan protein misfolding disease

    PubMed Central

    Fraser-Pitt, Douglas; O’Neil, Deborah

    2015-01-01

    Cystic fibrosis (CF) is a heterogeneous multiorgan disease caused by mutations in the CFTR gene leading to misfolding (and other defects) and consequent dysfunction of CFTR protein. The majority of mutations cause a severe CF phenotype, and people with this condition will require a wide variety of medical interventions and therapies throughout their lives to address the symptoms of their condition. CF affects many different organ systems, but the most serious consequence of the disease is degeneration of lung function due to chronic respiratory infection and colonization of the airways with opportunistic microbial pathogens. Improvements in therapeutics, particularly the effective use of antibiotics, have led to significant gradual increases in life expectancy. There remains, however, a continuing need for newer, safer and more effective antimicrobials and mucolytic agents to maintain and improve our ability to combat CF lung infections before other curative approaches which target the root cause of the disease become available. PMID:28031875

  14. Protein misfolding specifies recruitment to cytoplasmic inclusion bodies.

    PubMed

    Bersuker, Kirill; Brandeis, Michael; Kopito, Ron R

    2016-04-25

    Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington's disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs.

  15. Molecular chaperones in targeting misfolded proteins for ubiquitin-dependent degradation.

    PubMed

    Kriegenburg, Franziska; Ellgaard, Lars; Hartmann-Petersen, Rasmus

    2012-02-01

    The accumulation of misfolded proteins presents a considerable threat to the health of individual cells and has been linked to severe diseases, including neurodegenerative disorders. Considering that, in nature, cells often are exposed to stress conditions that may lead to aberrant protein conformational changes, it becomes clear that they must have an efficient quality control apparatus to refold or destroy misfolded proteins. In general, cells rely on molecular chaperones to seize and refold misfolded proteins. If the native state is unattainable, misfolded proteins are targeted for degradation via the ubiquitin-proteasome system. The specificity of this proteolysis is generally provided by E3 ubiquitin-protein ligases, hundreds of which are encoded in the human genome. However, rather than binding the misfolded proteins directly, most E3s depend on molecular chaperones to recognize the misfolded protein substrate. Thus, by delegating substrate recognition to chaperones, E3s deftly utilize a pre-existing cellular system for selectively targeting misfolded proteins. Here, we review recent advances in understanding the interplay between molecular chaperones and the ubiquitin-proteasome system in the cytosol, nucleus, endoplasmic reticulum and mitochondria. © 2011 The Authors Journal compilation © 2011 FEBS.

  16. Protein Folding and Misfolding, Endoplasmic Reticulum Stress in Neurodegenerative Diseases: in Trace of Novel Drug Targets.

    PubMed

    Penke, Botond; Bogár, Ferenc; Fülöp, Lívia

    2016-01-01

    Alzheimer's disease (AD) is characterized by severe cognitive impairment and memory loss. AD is classified both into the "protein conformational" and the "endoplasmic reticulum-mitochondria stress" disorders. AD is a very complex, multifactorial disease of heterogeneous genetic and environmental background. The amyloid hypothesis of AD cannot fully explain the various clinical forms of the disease. Protein folding and misfolding in the endoplasmic reticulum (ER), and accumulation of several misfolded proteins (β-amyloid, Tau, alpha-synuclein, etc.) in ER and mitochondria (MT) may play a key role in the development of AD. Functional degradation of the synapse and the synapse holding neurites represents the first step in the pathogenesis of neurodegeneration. MT and ER are tightly coupled both physically and functionally with a special lipid raft called mitochondria-associated ER-membrane (MAM). MAM is crucial for Ca(2+) signalling and metabolic regulation of the cell. In turn, the impairment of ER-MT interplay is a common mechanism of different neurodegenerative diseases. In this review, we discuss recent findings focusing on the protein conformational and metabolic dysfunction, and the role of MAM and ER-MT crosstalk in neurodegeneration.

  17. Proteomics: a strategy to understand the novel targets in protein misfolding and cancer therapy

    PubMed Central

    Nagaraj, Nagathihalli S; Singh, Om V; Merchant, Nipun B

    2014-01-01

    Proteins carry out important functions as they fold themselves. Protein misfolding occurs during different biochemical processes and may lead to the development of diseases such as cancer, which is characterized by genetic instability. The cancer microenvironment exposes malignant cells to a variety of stressful conditions that may further promote protein misfolding. Tumor development and progression often arises from mutations that interfere with the appropriate function of tumor-suppressor proteins and oncogenes. These may be due to alteration of catalytic activity of the protein, loss of binding sites for effector proteins or alterations of the native folded protein conformation. Src family kinases, p53, mTOR and C-terminus of HSC70 interacting protein (CHIPs) are some examples associated with protein misfolding and tumorigenesis. Molecular chaperones, such as heat-shock protein (HSP)70 and HSP90, assist protein folding and recognize target misfolded proteins for degradation. It is likely that this misfolding in cancer is linked by common principles, and may, therefore, present an exciting possibility to identify common targets for therapeutic intervention. Here we aim to review a number of examples that show how alterations in the folding of tumor-suppressor proteins or oncogenes lead to tumorigenesis. The possibility of targeting the targets to repair or degrade protein misfolding in cancer therapy is discussed. PMID:20653514

  18. Proteomics: a strategy to understand the novel targets in protein misfolding and cancer therapy.

    PubMed

    Nagaraj, Nagathihalli S; Singh, Om V; Merchant, Nipun B

    2010-08-01

    Proteins carry out important functions as they fold themselves. Protein misfolding occurs during different biochemical processes and may lead to the development of diseases such as cancer, which is characterized by genetic instability. The cancer microenvironment exposes malignant cells to a variety of stressful conditions that may further promote protein misfolding. Tumor development and progression often arises from mutations that interfere with the appropriate function of tumor-suppressor proteins and oncogenes. These may be due to alteration of catalytic activity of the protein, loss of binding sites for effector proteins or alterations of the native folded protein conformation. Src family kinases, p53, mTOR and C-terminus of HSC70 interacting protein (CHIPs) are some examples associated with protein misfolding and tumorigenesis. Molecular chaperones, such as heat-shock protein (HSP)70 and HSP90, assist protein folding and recognize target misfolded proteins for degradation. It is likely that this misfolding in cancer is linked by common principles, and may, therefore, present an exciting possibility to identify common targets for therapeutic intervention. Here we aim to review a number of examples that show how alterations in the folding of tumor-suppressor proteins or oncogenes lead to tumorigenesis. The possibility of targeting the targets to repair or degrade protein misfolding in cancer therapy is discussed.

  19. Nanotools for Megaproblems: Probing Protein Misfolding Diseases Using Nanomedicine Modus Operandi

    PubMed Central

    Uversky, Vladimir N.; Kabanov, Alexander V.; Lyubchenko, Yuri L.

    2007-01-01

    Misfolding and self-assembly of proteins in nanoaggregates of different sizes and morphologies (nanoensembles, primary nanofilaments, nanorings, filaments, protofibrils, fibrils, etc.) is a common theme unifying a number of human pathologies termed protein misfolding diseases. Recent studies highlight increasing recognition of the public health importance of protein misfolding diseases, including various neurodegenerative disorders and amyloidoses. It is understood now that the first essential elements in the vast majority of neurodegenerative processes are misfolded and aggregated proteins. Altogether, the accumulation of abnormal protein nanoensembles exerts toxicity by disrupting intracellular transport, overwhelming protein degradation pathways, and/or disturbing vital cell functions. In addition, the formation of inclusion bodies is known to represent a major problem in the production of recombinant therapeutic proteins. Formulation of these therapeutic proteins into delivery systems and their in vivo delivery are often complicated by protein association. Thus, protein folding abnormalities and subsequent events underlie a multitude of human pathologies and difficulties with protein therapeutic applications. The field of medicine therefore can be greatly advanced by establishing a fundamental understanding of key factors leading to misfolding and self-assembly responsible for various protein folding pathologies. This article overviews protein misfolding diseases and outlines some novel and advanced nanotechnologies, including nanoimaging techniques, nanotoolboxes and nanocontainers, complemented by appropriate ensemble techniques, all focused on the ultimate goal to establish etiology and to diagnose, prevent, and cure these devastating disorders. PMID:17022621

  20. Strain-dependent profile of misfolded prion protein aggregates

    PubMed Central

    Morales, Rodrigo; Hu, Ping Ping; Duran-Aniotz, Claudia; Moda, Fabio; Diaz-Espinoza, Rodrigo; Chen, Baian; Bravo-Alegria, Javiera; Makarava, Natallia; Baskakov, Ilia V.; Soto, Claudio

    2016-01-01

    Prions are composed of the misfolded prion protein (PrPSc) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrPSc aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrPSc aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrPSc aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrPSc aggregates and the incubation periods for the strains studied. The relative presence of PrPSc in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrPSc aggregates in prion-induced neurodegeneration. PMID:26877167

  1. Strain-dependent profile of misfolded prion protein aggregates.

    PubMed

    Morales, Rodrigo; Hu, Ping Ping; Duran-Aniotz, Claudia; Moda, Fabio; Diaz-Espinoza, Rodrigo; Chen, Baian; Bravo-Alegria, Javiera; Makarava, Natallia; Baskakov, Ilia V; Soto, Claudio

    2016-02-15

    Prions are composed of the misfolded prion protein (PrP(Sc)) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrP(Sc) aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrP(Sc) aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrP(Sc) aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrP(Sc) aggregates and the incubation periods for the strains studied. The relative presence of PrP(Sc) in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrP(Sc) aggregates in prion-induced neurodegeneration.

  2. Single-molecule fluorescence reveals sequence-specific misfolding in multidomain proteins

    PubMed Central

    Borgia, Madeleine B.; Borgia, Alessandro; Best, Robert B.; Steward, Annette; Nettels, Daniel; Wunderlich, Bengt; Schuler, Benjamin; Clarke, Jane

    2011-01-01

    A large range of debilitating medical conditions1 are linked to protein misfolding, which may compete with productive folding particularly in proteins containing multiple domains2. With 75% of the eukaryotic proteome consisting of multidomain proteins, how is inter-domain misfolding avoided? It has been proposed that maintaining low sequence identity between covalently linked domains is a mechanism to avoid misfolding3. Here we use single-molecule Förster Resonance Energy Transfer (FRET) experiments4,5 to detect and quantify rare misfolding events in tandem Ig domains from the I-band of titin under native conditions. About 5.5% of molecules with identical domains misfold during refolding in vitro and form a surprisingly stable state with an unfolding half time of several days. Tandem arrays of immunoglobulin-like (Ig-like) domains in humans exhibit significantly lower sequence identity between neighbouring domains than between non-adjacent domains3. In particular, the sequence identity of neighbouring domains has been found to be preferentially below 40%3. Interestingly we observe no misfolding for a tandem of naturally neighbouring domains with low sequence identity (24%), whereas misfolding occurs between domains which are 42% identical. Coarse-grained molecular simulations predict the formation of domain-swapped structures, which are in excellent agreement with the observed transfer efficiency of the misfolded species. We infer that the interactions underlying misfolding are very specific and result in a sequence-specific domain swapping mechanism. Diversifying the sequence between neighbouring domains appears to be a successful evolutionary strategy to avoid misfolding in multidomain proteins. PMID:21623368

  3. Single-molecule fluorescence reveals sequence-specific misfolding in multidomain proteins.

    PubMed

    Borgia, Madeleine B; Borgia, Alessandro; Best, Robert B; Steward, Annette; Nettels, Daniel; Wunderlich, Bengt; Schuler, Benjamin; Clarke, Jane

    2011-05-29

    A large range of debilitating medical conditions is linked to protein misfolding, which may compete with productive folding particularly in proteins containing multiple domains. Seventy-five per cent of the eukaryotic proteome consists of multidomain proteins, yet it is not understood how interdomain misfolding is avoided. It has been proposed that maintaining low sequence identity between covalently linked domains is a mechanism to avoid misfolding. Here we use single-molecule Förster resonance energy transfer to detect and quantify rare misfolding events in tandem immunoglobulin domains from the I band of titin under native conditions. About 5.5 per cent of molecules with identical domains misfold during refolding in vitro and form an unexpectedly stable state with an unfolding half-time of several days. Tandem arrays of immunoglobulin-like domains in humans show significantly lower sequence identity between neighbouring domains than between non-adjacent domains. In particular, the sequence identity of neighbouring domains has been found to be preferentially below 40 per cent. We observe no misfolding for a tandem of naturally neighbouring domains with low sequence identity (24 per cent), whereas misfolding occurs between domains that are 42 per cent identical. Coarse-grained molecular simulations predict the formation of domain-swapped structures that are in excellent agreement with the observed transfer efficiency of the misfolded species. We infer that the interactions underlying misfolding are very specific and result in a sequence-specific domain-swapping mechanism. Diversifying the sequence between neighbouring domains seems to be a successful evolutionary strategy to avoid misfolding in multidomain proteins.

  4. Misfolded Proteins in Alzheimer’s Disease and Type II Diabetes

    PubMed Central

    DeToma, Alaina S.; Salamekh, Samer; Ramamoorthy, Ayyalusamy; Lim, Mi Hee

    2011-01-01

    This review presents descriptions of two amyloidogenic proteins, amyloid-β (Aβ) peptides and islet amyloid polypeptide (IAPP), whose misfolding propensities are implicated in Alzheimer’s disease (AD) and type II diabetes, respectively. Protein misfolding diseases share similarities, as well as some unique protein-specific traits, that could contribute to the initiation and/or development of their associated conditions. Aβ and IAPP are representative amyloidoses and used to highlight some of the primary considerations for studying misfolded proteins associated with human diseases. Among these factors, their physiological formation, aggregation, interactions with metal ions and other protein partners, and toxicity are presented. Small molecules that target and modulate the metal-Aβ interaction and neurotoxicity are included to illustrate one of the current approaches for studying the complex nature of misfolded proteins at the molecular level. PMID:21818468

  5. Protein misfolding occurs by slow diffusion across multiple barriers in a rough energy landscape

    PubMed Central

    Yu, Hao; Dee, Derek R.; Liu, Xia; Brigley, Angela M.; Sosova, Iveta; Woodside, Michael T.

    2015-01-01

    The timescale for the microscopic dynamics of proteins during conformational transitions is set by the intrachain diffusion coefficient, D. Despite the central role of protein misfolding and aggregation in many diseases, it has proven challenging to measure D for these processes because of their heterogeneity. We used single-molecule force spectroscopy to overcome these challenges and determine D for misfolding of the prion protein PrP. Observing directly the misfolding of individual dimers into minimal aggregates, we reconstructed the energy landscape governing nonnative structure formation. Remarkably, rather than displaying multiple pathways, as typically expected for aggregation, PrP dimers were funneled into a thermodynamically stable misfolded state along a single pathway containing several intermediates, one of which blocked native folding. Using Kramers’ rate theory, D was found to be 1,000-fold slower for misfolding than for native folding, reflecting local roughening of the misfolding landscape, likely due to increased internal friction. The slow diffusion also led to much longer transit times for barrier crossing, allowing transition paths to be observed directly for the first time to our knowledge. These results open a new window onto the microscopic mechanisms governing protein misfolding. PMID:26109573

  6. Protein misfolding occurs by slow diffusion across multiple barriers in a rough energy landscape.

    PubMed

    Yu, Hao; Dee, Derek R; Liu, Xia; Brigley, Angela M; Sosova, Iveta; Woodside, Michael T

    2015-07-07

    The timescale for the microscopic dynamics of proteins during conformational transitions is set by the intrachain diffusion coefficient, D. Despite the central role of protein misfolding and aggregation in many diseases, it has proven challenging to measure D for these processes because of their heterogeneity. We used single-molecule force spectroscopy to overcome these challenges and determine D for misfolding of the prion protein PrP. Observing directly the misfolding of individual dimers into minimal aggregates, we reconstructed the energy landscape governing nonnative structure formation. Remarkably, rather than displaying multiple pathways, as typically expected for aggregation, PrP dimers were funneled into a thermodynamically stable misfolded state along a single pathway containing several intermediates, one of which blocked native folding. Using Kramers' rate theory, D was found to be 1,000-fold slower for misfolding than for native folding, reflecting local roughening of the misfolding landscape, likely due to increased internal friction. The slow diffusion also led to much longer transit times for barrier crossing, allowing transition paths to be observed directly for the first time to our knowledge. These results open a new window onto the microscopic mechanisms governing protein misfolding.

  7. A revisited folding reporter for quantitative assay of protein misfolding and aggregation in mammalian cells.

    PubMed

    Gregoire, Simpson; Kwon, Inchan

    2012-10-01

    Protein misfolding and aggregation play important roles in many physiological processes. These include pathological protein aggregation in neurodegenerative diseases and biopharmaceutical protein aggregation during production in mammalian cells. To develop a simple non-invasive assay for protein misfolding and aggregation in mammalian cells, the folding reporter green fluorescent protein (GFP) system, originally developed for bacterial cells, was evaluated. As a folding reporter, GFP was fused to the C-terminus of a panel of human copper/zinc superoxide dismutase (SOD1) mutants with varying misfolding/aggregation propensities. Flow cytometric analysis of transfected HEK293T and NSC-34 cells revealed that the mean fluorescence intensities of the cells expressing GFP fusion of SOD1 variants exhibited an inverse correlation with the misfolding/aggregation propensities of the four SOD1 variants. Our results support the hypothesis that the extent of misfolding/aggregation of a target protein in mammalian cells can be quantitatively estimated by measuring the mean fluorescence intensity of the cells expressing GFP fusion. The assay method developed herein will facilitate the understanding of aggregation process of SOD1 variants and the identification of aggregation inhibitors. The method also has great promise for misfolding/aggregation studies of other proteins in mammalian cells. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Diabetic Risk Factors Promote Islet Amyloid Polypeptide Misfolding by a Common, Membrane-mediated Mechanism

    PubMed Central

    Okada, Alan K.; Teranishi, Kazuki; Isas, J. Mario; Bedrood, Sahar; Chow, Robert H.; Langen, Ralf

    2016-01-01

    The current diabetes epidemic is associated with a diverse set of risk factors including obesity and exposure to plastics. Notably, significant elevations of negatively charged amphiphilic molecules are observed in obesity (e.g. free fatty acids and phosphatidic acid) and plastics exposure (monophthalate esters). It remains unclear whether these factors share pathogenic mechanisms and whether links exist with islet amyloid polypeptide (IAPP) misfolding, a process central to β-cell dysfunction and death. Using a combination of fluorescence, circular dichroism and electron microscopy, we show that phosphatidic acid, oleic acid, and the phthalate metabolite MBzP partition into neutral membranes and enhance IAPP misfolding. The elevation of negative charge density caused by the presence of the risk factor molecules stabilizes a common membrane-bound α-helical intermediate that, in turn, facilitates IAPP misfolding. This shared mechanism points to a critical role for the membrane-bound intermediate in disease pathogenesis, making it a potential target for therapeutic intervention. PMID:27531121

  9. Detecting selection for negative design in proteins through an improved model of the misfolded state.

    PubMed

    Minning, Jonas; Porto, Markus; Bastolla, Ugo

    2013-07-01

    Proteins that need to be structured in their native state must be stable both against the unfolded ensemble and against incorrectly folded (misfolded) conformations with low free energy. Positive design targets the first type of stability by strengthening native interactions. The second type of stability is achieved by destabilizing interactions that occur frequently in the misfolded ensemble, a strategy called negative design. Here, we investigate negative design adopting a statistical mechanical model of the misfolded ensemble, which improves the usual Gaussian approximation by taking into account the third moment of the energy distribution and contact correlations. Applying this model, we detect and quantify selection for negative design in most natural proteins, and we analytically design protein sequences that are stable both against unfolding and against misfolding. Copyright © 2013 Wiley Periodicals, Inc.

  10. Impact of translational error-induced and error-free misfolding on the rate of protein evolution

    PubMed Central

    Yang, Jian-Rong; Zhuang, Shi-Mei; Zhang, Jianzhi

    2010-01-01

    What determines the rate of protein evolution is a fundamental question in biology. Recent genomic studies revealed a surprisingly strong anticorrelation between the expression level of a protein and its rate of sequence evolution. This observation is currently explained by the translational robustness hypothesis in which the toxicity of translational error-induced protein misfolding selects for higher translational robustness of more abundant proteins, which constrains sequence evolution. However, the impact of error-free protein misfolding has not been evaluated. We estimate that a non-negligible fraction of misfolded proteins are error free and demonstrate by a molecular-level evolutionary simulation that selection against protein misfolding results in a greater reduction of error-free misfolding than error-induced misfolding. Thus, an overarching protein-misfolding-avoidance hypothesis that includes both sources of misfolding is superior to the translational robustness hypothesis. We show that misfolding-minimizing amino acids are preferentially used in highly abundant yeast proteins and that these residues are evolutionarily more conserved than other residues of the same proteins. These findings provide unambiguous support to the role of protein-misfolding-avoidance in determining the rate of protein sequence evolution. PMID:20959819

  11. Impact of translational error-induced and error-free misfolding on the rate of protein evolution.

    PubMed

    Yang, Jian-Rong; Zhuang, Shi-Mei; Zhang, Jianzhi

    2010-10-19

    What determines the rate of protein evolution is a fundamental question in biology. Recent genomic studies revealed a surprisingly strong anticorrelation between the expression level of a protein and its rate of sequence evolution. This observation is currently explained by the translational robustness hypothesis in which the toxicity of translational error-induced protein misfolding selects for higher translational robustness of more abundant proteins, which constrains sequence evolution. However, the impact of error-free protein misfolding has not been evaluated. We estimate that a non-negligible fraction of misfolded proteins are error free and demonstrate by a molecular-level evolutionary simulation that selection against protein misfolding results in a greater reduction of error-free misfolding than error-induced misfolding. Thus, an overarching protein-misfolding-avoidance hypothesis that includes both sources of misfolding is superior to the translational robustness hypothesis. We show that misfolding-minimizing amino acids are preferentially used in highly abundant yeast proteins and that these residues are evolutionarily more conserved than other residues of the same proteins. These findings provide unambiguous support to the role of protein-misfolding-avoidance in determining the rate of protein sequence evolution.

  12. Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies.

    PubMed

    Ciechanover, Aaron; Kwon, Yong Tae

    2015-03-13

    Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.

  13. Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies

    PubMed Central

    Ciechanover, Aaron; Kwon, Yong Tae

    2015-01-01

    Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons. PMID:25766616

  14. Redox reactions induced by nitrosative stress mediate protein misfolding and mitochondrial dysfunction in neurodegenerative diseases.

    PubMed

    Gu, Zezong; Nakamura, Tomohiro; Lipton, Stuart A

    2010-06-01

    Overstimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors accounts, at least in part, for excitotoxic neuronal damage, potentially contributing to a wide range of acute and chronic neurologic diseases. Neurodegenerative disorders including Alzheimer's disease (AD) and Parkinson's disease (PD), manifest deposits of misfolded or aggregated proteins, and result from synaptic injury and neuronal death. Recent studies have suggested that nitrosative stress due to generation of excessive nitric oxide (NO) can mediate excitotoxicity in part by triggering protein misfolding and aggregation, and mitochondrial fragmentation in the absence of genetic predisposition. S-Nitrosylation, or covalent reaction of NO with specific protein thiol groups, represents a convergent signal pathway contributing to NO-induced protein misfolding and aggregation, compromised dynamics of mitochondrial fission-fusion process, thus leading to neurotoxicity. Here, we review the effect of S-nitrosylation on protein function under excitotoxic conditions, and present evidence suggesting that NO contributes to protein misfolding and aggregation via S-nitrosylating protein-disulfide isomerase or the E3 ubiquitin ligase parkin, and mitochondrial fragmentation through beta-amyloid-related S-nitrosylation of dynamin-related protein-1. Moreover, we also discuss that inhibition of excessive NMDA receptor activity by memantine, an uncompetitive/fast off-rate (UFO) drug can ameliorate excessive production of NO, protein misfolding and aggregation, mitochondrial fragmentation, and neurodegeneration.

  15. Rheumatoid Rescue of Misfolded Cellular Proteins by MHC Class II Molecules: A New Hypothesis for Autoimmune Diseases.

    PubMed

    Arase, Hisashi

    2016-01-01

    Misfolded proteins localized in the endoplasmic reticulum are degraded promptly and thus are not transported outside cells. However, misfolded proteins in the endoplasmic reticulum are rescued from protein degradation upon association with major histocompatibility complex (MHC) class II molecules and are transported to the cell surface by MHC class II molecules without being processed to peptides. Studies on the misfolded proteins rescued by MHC class II molecules have revealed that misfolded proteins associated with MHC class II molecules are specific targets for autoantibodies produced in autoimmune diseases. Furthermore, a strong correlation has been observed between autoantibody binding to misfolded proteins associated with MHC class II molecules and the autoimmune disease susceptibility conferred by each MHC class II allele. These new insights into MHC class II molecules suggest that misfolded proteins rescued from protein degradation by MHC class II molecules are recognized as "neo-self" antigens by immune system and are involved in autoimmune diseases as autoantibody targets.

  16. Unraveling the Molecular Mechanism of pH-Induced Misfolding and Oligomerization of the Prion Protein.

    PubMed

    Singh, Jogender; Udgaonkar, Jayant B

    2016-03-27

    The misfolding of the prion protein (PrP) to aggregated forms is linked to several neurodegenerative diseases. Misfolded oligomeric forms of PrP are associated with neurotoxicity and/or infectivity, but the molecular mechanism by which they form is still poorly understood. A reduction in pH is known to be a key factor that triggers misfolded oligomer formation by PrP, but the residues whose protonation is linked with misfolding remain unidentified. The structural consequences of the protonation of these residues also remain to be determined. In the current study, amino acid residues whose protonation is critical for PrP misfolding and oligomerization have been identified using site-directed mutagenesis and misfolding/oligomerization assays. It is shown that the protonation of either H186 or D201, which mimics the effects of pathogenic mutations (H186R and D201N) at both residue sites, is critically linked to the stability, misfolding and oligomerization of PrP. Hydrogen-deuterium exchange studies coupled with mass spectrometry show that the protonation of either H186 or D201 leads to the same common structural change: increased structural dynamics in helix 1 and that in the loop between helix 1 and β-strand 2. It is shown that the protonation of either of these residues is sufficient for accelerating misfolded oligomer formation, most likely because the protonation of either residue causes the same structural perturbation. Hence, the increased structural dynamics in helix 1 and that in the loop between helix 1 and β-strand 2 appear to play an early critical role in acid-induced misfolding of PrP. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Protein folding, misfolding, and aggregation. Formation of inclusion bodies and aggresomes.

    PubMed

    Markossian, K A; Kurganov, B I

    2004-09-01

    In this review the mechanisms of protein folding, misfolding, and aggregation as well as the mechanisms of cell defense against toxic protein aggregates are considered. Misfolded and aggregated proteins in cells are exposed to chaperone-mediated refolding and are degraded by proteasomes if refolding is impossible. Proteolysis-stable protein aggregates accumulate, forming inclusion bodies. In eucaryotic cells, protein aggregates form structures in the pericentrosomal area that have been termed "aggresomes". Formation of aggresomes in cells is a general cellular response to the presence of misfolded proteins when the degrading capacity of the cells is exceeded. The role of aggresomes in disturbance of the proteasomal system operation and in cellular death, particularly in the so-called "protein conformational diseases", is discussed.

  18. Structure-Based Prediction of Unstable Regions in Proteins: Applications to Protein Misfolding Diseases

    NASA Astrophysics Data System (ADS)

    Guest, Will; Cashman, Neil; Plotkin, Steven

    2009-03-01

    Protein misfolding is a necessary step in the pathogenesis of many diseases, including Creutzfeldt-Jakob disease (CJD) and familial amyotrophic lateral sclerosis (fALS). Identifying unstable structural elements in their causative proteins elucidates the early events of misfolding and presents targets for inhibition of the disease process. An algorithm was developed to calculate the Gibbs free energy of unfolding for all sequence-contiguous regions of a protein using three methods to parameterize energy changes: a modified G=o model, changes in solvent-accessible surface area, and solution of the Poisson-Boltzmann equation. The entropic effects of disulfide bonds and post-translational modifications are treated analytically. It incorporates a novel method for finding local dielectric constants inside a protein to accurately handle charge effects. We have predicted the unstable parts of prion protein and superoxide dismutase 1, the proteins involved in CJD and fALS respectively, and have used these regions as epitopes to prepare antibodies that are specific to the misfolded conformation and show promise as therapeutic agents.

  19. Molecular interaction between type 2 diabetes and Alzheimer's disease through cross-seeding of protein misfolding.

    PubMed

    Moreno-Gonzalez, I; Edwards Iii, G; Salvadores, N; Shahnawaz, M; Diaz-Espinoza, R; Soto, C

    2017-09-01

    Numerous epidemiological studies have shown a significantly higher risk for development of Alzheimer's disease (AD) in patients affected by type 2 diabetes (T2D), but the molecular mechanism responsible for this association is presently unknown. Both diseases are considered protein misfolding disorders associated with the accumulation of protein aggregates; amyloid-beta (Aβ) and tau in the brain during AD, and islet amyloid polypeptide (IAPP) in pancreatic islets in T2D. Formation and accumulation of these proteins follows a seeding-nucleation model, where a misfolded aggregate or 'seed' promotes the rapid misfolding and aggregation of the native protein. Our underlying hypothesis is that misfolded IAPP produced in T2D potentiates AD pathology by cross-seeding Aβ, providing a molecular explanation for the link between these diseases. Here, we examined how misfolded IAPP affects Aβ aggregation and AD pathology in vitro and in vivo. We observed that addition of IAPP seeds accelerates Aβ aggregation in vitro in a seeding-like manner and the resulting fibrils are composed of both peptides. Transgenic animals expressing both human proteins exhibited exacerbated AD-like pathology compared with AD transgenic mice or AD transgenic animals with type 1 diabetes (T1D). Remarkably, IAPP colocalized with amyloid plaques in brain parenchymal deposits, suggesting that these peptides may directly interact and aggravate the disease. Furthermore, inoculation of pancreatic IAPP aggregates into the brains of AD transgenic mice resulted in more severe AD pathology and significantly greater memory impairments than untreated animals. These data provide a proof-of-concept for a new disease mechanism involving the interaction of misfolded proteins through cross-seeding events which may contribute to accelerate or exacerbate disease pathogenesis. Our findings could shed light on understanding the linkage between T2D and AD, two of the most prevalent protein misfolding disorders.

  20. Protein misfolding and aggregation research: some thoughts on improving quality and utility.

    PubMed

    Murphy, Regina M; Roberts, Christopher J

    2013-01-01

    Once misfolded and aggregated proteins were as interesting as yesterday's trash, just a bothersome byproduct of productive activities. Today, they attract sustained interest from both basic researchers and practicing engineers. In the burgeoning biopharmaceutical industry, protein misfolding and aggregation pose significant challenges to the economic manufacture of safe and effective protein products. In the clinic, protein aggregates are believed to be pathological agents in a number of serious neurodegenerative disorders, such as Alzheimer's and Parkinson's. Over the past few years, the quantity of research into biotechnological aspects of protein misfolding and aggregation has skyrocketed. However, the quality of the published work is quite variable. In this brief opinion piece, we describe what we believe are some key features of high-quality publications in protein aggregation. We focus on experimental studies that may also have a kinetic modeling component. © 2013 American Institute of Chemical Engineers.

  1. Molecular Mechanism of the Misfolding and Oligomerization of the Prion Protein: Current Understanding and Its Implications.

    PubMed

    Singh, Jogender; Udgaonkar, Jayant B

    2015-07-28

    Prion diseases, also known as transmissible spongiform encephalopathies, make up a group of fatal neurodegenerative disorders linked with the misfolding and aggregation of the prion protein (PrP). Although it is not yet understood how the misfolding of PrP induces neurodegeneration, it is widely accepted that the formation of misfolded prion protein (termed PrP(Sc)) is both the triggering event in the disease and the main component of the infectious agent responsible for disease transmission. Despite the clear involvement of PrP(Sc) in prion diseases, the exact composition of PrP(Sc) is not yet well-known. Recent studies show that misfolded oligomers of PrP could, however, be responsible for neurotoxicity and/or infectivity in the prion diseases. Hence, understanding the molecular mechanism of formation of the misfolded oligomers of PrP is critical for developing an understanding about the prion diseases and for developing anti-prion therapeutics. This review discusses recent advances in understanding the molecular mechanism of misfolded oligomer formation by PrP and its implications for the development of anti-prion therapeutics.

  2. Glucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins.

    PubMed

    Das, Indrajit; Png, Chin Wen; Oancea, Iulia; Hasnain, Sumaira Z; Lourie, Rohan; Proctor, Martina; Eri, Rajaraman D; Sheng, Yong; Crane, Denis I; Florin, Timothy H; McGuckin, Michael A

    2013-06-03

    Endoplasmic reticulum (ER) stress in intestinal secretory cells has been linked with colitis in mice and inflammatory bowel disease (IBD). Endogenous intestinal glucocorticoids are important for homeostasis and glucocorticoid drugs are efficacious in IBD. In Winnie mice with intestinal ER stress caused by misfolding of the Muc2 mucin, the glucocorticoid dexamethasone (DEX) suppressed ER stress and activation of the unfolded protein response (UPR), substantially restoring goblet cell Muc2 production. In mice lacking inflammation, a glucocorticoid receptor antagonist increased ER stress, and DEX suppressed ER stress induced by the N-glycosylation inhibitor, tunicamycin (Tm). In cultured human intestinal secretory cells, in a glucocorticoid receptor-dependent manner, DEX suppressed ER stress and UPR activation induced by blocking N-glycosylation, reducing ER Ca(2+) or depleting glucose. DEX up-regulated genes encoding chaperones and elements of ER-associated degradation (ERAD), including EDEM1. Silencing EDEM1 partially inhibited DEX's suppression of misfolding-induced ER stress, showing that DEX enhances ERAD. DEX inhibited Tm-induced MUC2 precursor accumulation, promoted production of mature mucin, and restored ER exit and secretion of Winnie mutant recombinant Muc2 domains, consistent with enhanced protein folding. In IBD, glucocorticoids are likely to ameliorate ER stress by promoting correct folding of secreted proteins and enhancing removal of misfolded proteins from the ER.

  3. Spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness to stress

    PubMed Central

    Escusa-Toret, Stéphanie; Vonk, Willianne I. M.; Frydman, Judith

    2014-01-01

    The extensive links between proteotoxic stress, protein aggregation and pathologies ranging from aging to neurodegeneration underscore the importance of understanding how cells manage protein misfolding. Using live-cell imaging, we here determine the fate of stress-induced misfolded proteins from their initial appearance until their elimination. Upon denaturation, misfolded proteins are sequestered from the bulk cytoplasm into dynamic ER-associated puncta that move and coalesce into larger structures in an energy-dependent but cytoskeleton-independent manner. These puncta, which we name Q-bodies, concentrate different misfolded and stress-denatured proteins en-route to degradation, but do not contain amyloid aggregates, which localize instead to the IPOD. Q-body formation and clearance depends on an intact cortical ER and a complex chaperone network that is affected by rapamycin and impaired during chronological aging. Importantly, Q-body formation enhances cellular fitness during stress. We conclude that spatial sequestration of misfolded proteins in Q-bodies is an early quality control strategy occurring synchronously with degradation to clear the cytoplasm from potentially toxic species. PMID:24036477

  4. Protein quality control in the ER: the recognition of misfolded proteins.

    PubMed

    Määttänen, Pekka; Gehring, Kalle; Bergeron, John J M; Thomas, David Y

    2010-07-01

    The mechanism, in molecular terms of protein quality control, specifically of how the cell recognizes and discriminates misfolded proteins, remains a challenge. In the secretory pathway the folding status of glycoproteins passing through the endoplasmic reticulum is marked by the composition of the N-glycan. The different glycoforms are recognized by specialized lectins. The folding sensor UGGT acts as an unusual molecular chaperone and covalently modifies the Man9 N-glycan of a misfolded protein by adding a glucose moiety and converts it to Glc1Man9 that rebinds the lectin calnexin. However, further links between the folding status of a glycoprotein and the composition of the N-glycan are unclear. There is little unequivocal evidence for other proteins in the ER recognizing the N-glycan and also acting as molecular chaperones. Nevertheless, based upon a few examples, we suggest that this function is carried out by individual proteins in several different complexes. Thus, calnexin binds the protein disulfide isomerase ERp57, that acts upon Glc1Man9 glycoproteins. In another example the protein disulfide isomerase ERdj5 binds specifically to EDEM (which is probably a mannosidase) and a lectin OS9, and reduces the disulfide bonds of bound glycoproteins destined for ERAD. Thus the glycan recognition is performed by a lectin and the chaperone function performed by a specific partner protein that can recognize misfolded proteins. We predict that this will be a common arrangement of proteins in the ER and that members of protein foldase families such as PDI and PPI will bind specifically to lectins in the ER. Molecular chaperones BiP and GRp94 will assist in the folding of proteins bound in these complexes as well as in the folding of non-glycoproteins.

  5. Y145Stop is sufficient to induce de novo generation prions using protein misfolding cyclic amplification.

    PubMed

    Abdallah, Ahmed; Wang, Ping; Richt, Juergen A; Sreevatsan, Srinand

    2012-01-01

    A point mutation in Prnp that converts tyrosine (Y) at position 145 into a stop codon leading to a truncated prion molecule as found in an inherited transmissible spongiform encephalopathy (TSE), Gertsmann-Sträussler-Scheincker syndrome, suggests that the N-terminus of the molecule (spanning amino acids 23-144) likely plays a critical role in prion misfolding as well as in protein-protein interactions. We hypothesized that Y145Stop molecule represents an unstable part of the prion protein that is prone to spontaneous misfolding. Utilizing protein misfolding cyclic amplification (PMCA) we show that the recombinant polypeptide corresponding to the Y145Stop of sheep and deer PRNP can be in vitro converted to PK-resistant PrP (Sc) in presence or absence of preexisting prions. In contrast, recombinant protein full-length PrP (C) did not show a propensity for spontaneous conformational conversion to protease resistant isoforms. Further, we show that seeded or spontaneously misfolded Y145Stop molecules can efficiently convert purified mammalian PrP (C) into protease resistant isoforms. These results establish that the N-terminus of PrP (C) molecule corresponding to residues 23-144 plays a role in seeding and misfolding of mammalian prions.

  6. Spatially organized aggregation of misfolded proteins as cellular stress defense strategy.

    PubMed

    Miller, Stephanie B M; Mogk, Axel; Bukau, Bernd

    2015-04-10

    An evolutionary conserved response of cells to proteotoxic stress is the organized sequestration of misfolded proteins into subcellular deposition sites. In Saccharomyces cerevisiae, three major sequestration sites for misfolded proteins exist, IPOD (insoluble protein deposit), INQ (intranuclear quality control compartment) [former JUNQ (juxtanuclear quality control compartment)] and CytoQ. IPOD is perivacuolar and predominantly sequesters amyloidogenic proteins. INQ and CytoQs are stress-induced deposits for misfolded proteins residing in the nucleus and the cytosol, respectively, and requiring cell-compartment-specific aggregases, nuclear Btn2 and cytosolic Hsp42 for formation. The organized aggregation of misfolded proteins is proposed to serve several purposes collectively increasing cellular fitness and survival under proteotoxic stress. These include (i) shielding of cellular processes from interference by toxic protein conformers, (ii) reducing the substrate burden for protein quality control systems upon immediate stress, (iii) orchestrating chaperone and protease functions for efficient repair or degradation of damaged proteins [this involves initial extraction of aggregated molecules via the Hsp70/Hsp104 bi-chaperone system followed by either refolding or proteasomal degradation or removal of entire aggregates by selective autophagy (aggrephagy) involving the adaptor protein Cue5] and (iv) enabling asymmetric retention of protein aggregates during cell division, thereby allowing for damage clearance in daughter cells. Regulated protein aggregation thus serves cytoprotective functions vital for the maintenance of cell integrity and survival even under adverse stress conditions and during aging.

  7. Requirements for Mutant and Wild-Type Prion Protein Misfolding In Vitro

    PubMed Central

    Noble, Geoffrey P.; Walsh, Daniel J.; Miller, Michael B.; Jackson, Walker S.; Supattapone, Surachai

    2015-01-01

    Misfolding of the prion protein (PrP) plays a central role in the pathogenesis of infectious, sporadic, and inherited prion diseases. Here we use a chemically defined prion propagation system to study misfolding of the pathogenic PrP mutant D177N in vitro. This mutation causes PrP to misfold spontaneously in the absence of cofactor molecules in a process dependent on time, temperature, pH, and intermittent sonication. Spontaneously misfolded mutant PrP is able to template its unique conformation onto wild-type PrP substrate in a process that requires a phospholipid activity distinct from that required for the propagation of infectious prions. Similar results were obtained with a second pathogenic PrP mutant, E199K, but not with the polymorphic substitution M128V. Moreover, wild-type PrP inhibits mutant PrP misfolding in a dose-dependent manner, and cofactor molecules can antagonize this effect. These studies suggest that interactions between mutant PrP, wild-type PrP, and other cellular factors may control the rate of PrP misfolding in inherited prion diseases. PMID:25584902

  8. Ubiquitin ligase ITCH recruitment suppresses the aggregation and cellular toxicity of cytoplasmic misfolded proteins

    PubMed Central

    Chhangani, Deepak; Upadhyay, Arun; Amanullah, Ayeman; Joshi, Vibhuti; Mishra, Amit

    2014-01-01

    The protein quality control (QC) system protects cells against cellular toxicity induced by misfolded proteins and maintains overall cellular fitness. Inefficient clearance of or failure to degrade damaged proteins causes several diseases, especially age-linked neurodegenerative disorders. Attenuation of misfolded protein degradation under severe stress conditions leads to the rapid over-accumulation of toxic proteinaceous aggregates in the cytoplasmic compartment. However, the precise cytoplasmic quality control degradation mechanism is unknown. In the present study, we demonstrate that the Nedd4-like E3 ubiquitin ligase ITCH specifically interacts with mutant bona fide misfolded proteins and colocalizes with their perinuclear aggregates. In a cell culture model, we demonstrate ITCH recruitment by cytoplasmic inclusions containing polyglutamine-expanded huntingtin or ataxin-3 proteins. Transient overexpression of ITCH dramatically induced the degradation of thermally denatured misfolded luciferase protein. Partial depletion of ITCH increased the rate of aggregate formation and cell death generated by expanded polyglutamine proteins. Finally, we demonstrate that overexpression of ITCH alleviates the cytotoxic potential of expanded polyglutamine proteins and reduces aggregation. These observations indicate that ITCH is involved in the cytosolic quality control pathway and may help to explain how abnormal proteins are targeted by QC ubiquitin-protein ligases. PMID:24865853

  9. A molecular imaging biosensor detects in vivo protein folding and misfolding.

    PubMed

    Sheahan, Anjali V; Sekar, Thillai V; Chen, Kai; Paulmurugan, Ramasamy; Massoud, Tarik F

    2016-07-01

    Aberrant protein folding represents the molecular basis of many important human diseases. Although the discovery of new anti-misfolding drugs is a major priority in molecular therapeutics, there is currently no generalizable protein folding assay for use in cell-based high throughput screening (HTS) of chemical libraries, or for in vivo imaging. We molecularly engineered a bioluminescence-based biosensor composed of rationally split Firefly luciferase reporter fragments flanking a test protein, and used this in a protein-fragment complementation assay to quantitatively measure folding of the test protein. We comprehensively validated this biosensor in vitro, in cells, and by optically imaging protein folding and misfolding in living mice using several test proteins including enhanced green fluorescent protein, Renilla luciferase, Gaussia luciferase, and SIRT1. Applications of this novel biosensor are potentially far-reaching in both cell-based HTS approaches to discover new anti-misfolding drugs, and when using the same biosensor in validation studies of drug candidates in small animal models. Novel anti-misfolding drugs are needed as molecular therapeutics for many diseases. We developed first in vivo imaging protein folding biosensor to aid drug discovery. Biosensor created by flanking a test protein with rationally split Firefly luciferase. Biosensor validated by detecting folding of test proteins EGFP, Rluc, Gluc, and SIRT1. Generalizable molecular biosensor for translational applications in drug screening.

  10. Underlying mechanisms and chemical/biochemical therapeutic approaches to ameliorate protein misfolding neurodegenerative diseases.

    PubMed

    Hekmatimoghaddam, Seyedhossein; Zare-Khormizi, Mohamad Reza; Pourrajab, Fatemeh

    2016-02-22

    Protein misfolding and inclusion body formations are common events in neurodegenerative diseases characterized by deposition of misfolded proteins inside or outside of neurons, and are commonly referred to as "protein misfolding neurodegenerative diseases" (PMNDs). These phenotypically diverse but biochemically similar aggregates suggest a highly conserved molecular mechanism of pathogenesis. These challenges are magnified by presence of mutations that render individual proteins subject to misfolding and/or aggregation. Cell proteostasis network and molecular chaperoning are maintaining cell proteome to preserve the protein folding, refolding, oligomerization, or disaggregation, and play formidable tasks to maintain the health of organism in the face of developmental changes, environmental insults, and rigors of aging. Maintenance of cell proteome requires the orchestration of major pathways of the cellular proteostasis network (heat shock response (HSR) in the cytosol and the unfolded protein response (UPR) in the endoplasmic reticulum). Proteostasis responses culminate in transcriptional and post-transcriptional programs that up-regulate the homeostatic mechanisms. Proteostasis is strongly influenced by the general properties of individual proteins for folding, misfolding, and aggregation. We examine a growing body of evidence establishing that when cellular proteostasis goes awry, it can be reestablished by deliberate chemical and biological interventions. We first try to introduce some new chemical approaches to prevent the misfolding or aggregation of specific proteins via direct binding interactions. We then start with approaches that employ chemicals or biological agents to enhance the general capacity of the proteostasis network. We finish with evidence that synergy is achieved with the combination of mechanistically distinct approaches to reestablish organ proteostasis. © 2016 BioFactors, 2016. © 2016 International Union of Biochemistry and Molecular

  11. Probing Protein Fluctuations, Folding and Misfolding at Single-molecule Resolution

    NASA Astrophysics Data System (ADS)

    Deniz, Ashok

    2010-03-01

    The conformational fluctuations and folding of proteins are key for their function in cells and organisms. Conversely, misfolding and aggregation can cause disease, although amyloids with functional significance are also being identified. To better understand these aspects of protein biophysics, we utilize single-molecule fluorescence and complementary methods to directly study complex protein dynamics, structural distributions, and conformational transitions. In one example, we used these methods to investigate disorder and disorder-to-order transitions in intrinsically disordered proteins (IDPs). IDPs are an interesting class of proteins which are relatively unstructured in isolation, but can often fold by interacting with binding partners. These complex systems are increasingly found to play major roles in biology and disease. In one case, we used a combination of single-molecule FRET (smFRET), coincidence and correlation analyses to probe the native structural features of a yeast protein Sup35, whose amyloid state is believed to be used in a beneficial context in yeast. We find that the monomeric protein populates a compact and rapidly fluctuating ensemble of conformations. In another case, we studied the binding-coupled folding of the IDP alpha-synuclein, whose misfolding and aggregation have been linked to Parkinson's disease. Single-molecule measurements directly revealed a complex multi-state folding landscape for this protein. Observations of a transient folding intermediate using microfluidic mixing, and links to misfolding and aggregation will also be discussed. Our results highlight single-molecule methodology that is broadly applicable to map protein folding and misfolding landscapes.

  12. gp78 functions downstream of Hrd1 to promote degradation of misfolded proteins of the endoplasmic reticulum

    PubMed Central

    Zhang, Ting; Xu, Yue; Liu, Yanfen; Ye, Yihong

    2015-01-01

    Eukaryotic cells eliminate misfolded proteins from the endoplasmic reticulum (ER) via a conserved process termed ER-associated degradation (ERAD). Central regulators of the ERAD system are membrane-bound ubiquitin ligases, which are thought to channel misfolded proteins through the ER membrane during retrotranslocation. Hrd1 and gp78 are mammalian ubiquitin ligases homologous to Hrd1p, an ubiquitin ligase essential for ERAD in Saccharomyces cerevisiae. However, the functional relevance of these proteins to Hrd1p is unclear. In this paper, we characterize the gp78-containing ubiquitin ligase complex and define its functional interplay with Hrd1 using biochemical and recently developed CRISPR-based genetic tools. Our data show that transient inactivation of the gp78 complex by short hairpin RNA–mediated gene silencing causes significant stabilization of both luminal and membrane ERAD substrates, but unlike Hrd1, which plays an essential role in retrotranslocation and ubiquitination of these ERAD substrates, knockdown of gp78 does not affect either of these processes. Instead, gp78 appears to act downstream of Hrd1 to promote ERAD via cooperation with the BAG6 chaperone complex. We conclude that the Hrd1 complex forms an essential retrotranslocation module that is evolutionarily conserved, but the mammalian ERAD system uses additional ubiquitin ligases to assist Hrd1 during retrotranslocation. PMID:26424800

  13. Protein Post-Translational Modifications and Misfolding: New Concepts in Heart Failure

    PubMed Central

    del Monte, Federica; Agnetti, Giulio

    2015-01-01

    A new concept in the field of heart failure research points to a role of misfolded proteins, forming pre-amyloid oligomers (PAOs), in cardiac toxicity. This is largely based on few studies reporting the presence of PAOs, similar to those observed in neurodegenerative disease, in experimental models and human heart failure. As the majority of proteinopathies are sporadic in nature, protein post-translational modifications (PTMs) likely play a major role in this growing class of diseases. In fact, PTMs are known regulators of protein folding and of the formation of amyloid species in well-established proteinopathies. Proteomics has been instrumental in identifying both chemical and enzymatic PTMs, with a potential impact on protein mis-/folding. Here we provide the basics on how proteins fold along with a few examples of PTMs known to modulate protein misfolding and aggregation, with particular focus on the heart. Due to its innovative content and the growing awareness of the toxicity of misfolded proteins an “Alzheimer’s theory of heart failure” is timely. Moreover, the continuous innovations in proteomic technologies will help pinpoint PTMs that could contribute to the process. This nuptial between biology and technology could greatly assist in identifying biomarkers with increased specificity as well as more effective therapies. PMID:24946239

  14. Inhibition of protein misfolding/aggregation using polyglutamine binding peptide QBP1 as a therapy for the polyglutamine diseases.

    PubMed

    Popiel, H Akiko; Takeuchi, Toshihide; Burke, James R; Strittmatter, Warren J; Toda, Tatsushi; Wada, Keiji; Nagai, Yoshitaka

    2013-07-01

    Protein misfolding and aggregation in the brain have been recognized to be crucial in the pathogenesis of various neurodegenerative diseases, including Alzheimer's, Parkinson's, and the polyglutamine (polyQ) diseases, which are collectively called the "protein misfolding diseases". In the polyQ diseases, an abnormally expanded polyQ stretch in the responsible proteins causes the proteins to misfold and aggregate, eventually resulting in neurodegeneration. Hypothesizing that polyQ protein misfolding and aggregation could be inhibited by molecules specifically binding to the expanded polyQ stretch, we identified polyQ binding peptide 1 (QBP1). We show that QBP1 does, indeed, inhibit misfolding and aggregation of the expanded polyQ protein in vitro. Furthermore overexpression of QBP1 by the crossing of transgenic animals inhibits neurodegeneration in Drosophila models of the polyQ diseases. We also introduce our attempts to deliver QBP1 into the brain by administration using viral vectors and protein transduction domains. Interestingly, recent data suggest that QBP1 can also inhibit the misfolding/aggregation of proteins responsible for other protein misfolding diseases, highlighting the potential of QBP1 as a general therapeutic molecule for a wide range of neurodegenerative diseases. We hope that in the near future, aggregation inhibitor-based drugs will be developed and bring relief to patients suffering from these currently intractable protein misfolding diseases.

  15. A protein family under 'stress' - serpin stability, folding and misfolding.

    PubMed

    Devlin, Glyn L; Bottomley, Stephen P

    2005-01-01

    The native fold of inhibitory serpins (serpin proteinase inhibitors) is metastable and therefore does not represent the most stable conformation that the primary sequence encodes for. The most stable form is adopted when the reactive centre loop (RCL) inserts, as the fourth strand, into the A b -sheet. Currently a serpin can adopt at least four more stable conformations, termed the cleaved, delta, latent and polymeric states. The accessibility of these alternative low energy folds renders the serpin molecule susceptible to mutations that can result in dysfunction and pathology. Here, we discuss the means by which the serpin can attain and preserve this metastable conformation. We also consider the triggers for misfolding to these more stable states and the mechanisms by which it occurs.

  16. [Molecular therapy targeting protein misfolding and aggregation for the polyglutamine diseases].

    PubMed

    Nagai, Yoshitaka

    2009-11-01

    Abnormal aggregation and deposition of misfolded proteins have been recognized as a common molecular pathogenesis of various neurodegenerative diseases including Alzheimer's, Parkinson's, and the polyglutamine (polyQ) diseases. The polyQ diseases, including Huntington's disease and various spinocerebellar ataxias, are caused by abnormal expansions of the polyQ stretch (> 35-40) within disease-causative proteins, which are thought to trigger their misfolding and aggregation, leading to their deposition as inclusion bodies, and eventually resulting in neurodegeneration. We found that the expanded polyQ protein undergoes a conformational transition to a beta-sheet dominant structure in the monomeric state, triggering cytotoxicity, and subsequently resulting in formation of insoluble amyloid-like fibrillar aggregates. Targeting misfolding and aggregation of the expanded polyQ protein, we demonstrated that QBP1 (PolyQ-Binding Peptide 1: SNWKWWPGIFD) prevents the toxic beta-sheet transition and aggregation of the expanded polyQ protein in vitro and suppresses polyQ-induced neurodegeneration in Drosophila. From high-throughput screening of a chemical compound library (46,000), we have identified approximately 100 polyQ aggregate inhibitors as therapeutic candidates so far. We also found that 17-AAG, an HSF1-activating compound, suppresses polyQ-induced neurodegeneration in Drosophila through induction of endogenous molecular chaperones. We propose that our therapeutic strategy targeting protein misfolding and aggregation can also be applied to other neurodegenerative diseases.

  17. Transport of misfolded endoplasmic reticulum proteins to the cell surface by MHC class II molecules

    PubMed Central

    Jiang, Yan; Arase, Noriko

    2013-01-01

    Nascent MHC class II molecules are associated with the invariant chain and are transported to the endolysosomal pathway, where MHC class II molecules acquire peptide antigens. On the other hand, misfolded endoplasmic reticulum (ER) proteins are generally degraded in the cells and are neither expressed on the cell surface nor secreted. Here, we found that MHC class II molecules associate with some misfolded ER proteins via the peptide-binding groove in competition with invariant chain. The misfolded proteins associated with MHC class II molecules are transported intact to the cell surface without processing to peptides. Furthermore, these complexes efficiently stimulate antigen-specific B cells. These findings reveal that MHC class II molecules function as a chaperone for the cell surface expression of misfolded ER proteins. In addition, we suggest that MHC class II molecules present not only peptides but also intact host-cell-derived proteins on the cell surface. These findings provide new insights into the function of MHC class II molecules. PMID:23334921

  18. Role of local and nonlocal interactions in folding and misfolding of globular proteins

    NASA Astrophysics Data System (ADS)

    Kumar, Adesh; Baruah, Anupaul; Biswas, Parbati

    2017-02-01

    A Monte Carlo simulation based sequence design method is proposed to study the role of the local and the nonlocal interactions with varying secondary structure content in protein folding, misfolding and unfolding. A statistical potential is developed from the compilation of a data set of proteins, which accounts for the respective contribution of local and the nonlocal interactions. Sequences are designed through a combination of positive and negative design by a Monte Carlo simulation in the sequence space. The weights of the local and the nonlocal interactions are tuned appropriately to study the role of the local and the nonlocal interactions in the folding, unfolding and misfolding of the designed sequences. Results suggest that the nonlocal interactions are the primary determinant of protein folding while the local interactions may be required but not always necessary. The nonlocal interactions mainly guide the polypeptide chain to form compact structures but do not differentiate between the native-like conformations, while the local interactions stabilize the target conformation against the native-like competing conformations. The study concludes that the local interactions govern the fold-misfold transition, while the nonlocal interactions regulate the fold-unfold transition of proteins. However, for proteins with predominantly β-sheet content, the nonlocal interactions control both fold-misfold and fold-unfold transitions.

  19. Dealing with misfolded proteins: examining the neuroprotective role of molecular chaperones in neurodegeneration.

    PubMed

    Ali, Yousuf O; Kitay, Brandon M; Zhai, R Grace

    2010-10-08

    Human neurodegenerative diseases arise from a wide array of genetic and environmental factors. Despite the diversity in etiology, many of these diseases are considered "conformational" in nature, characterized by the accumulation of pathological, misfolded proteins. These misfolded proteins can induce cellular stress by overloading the proteolytic machinery, ultimately resulting in the accumulation and deposition of aggregated protein species that are cytotoxic. Misfolded proteins may also form aberrant, non-physiological protein-protein interactions leading to the sequestration of other normal proteins essential for cellular functions. The progression of such disease may therefore be viewed as a failure of normal protein homeostasis, a process that involves a network of molecules regulating the synthesis, folding, translocation and clearance of proteins. Molecular chaperones are highly conserved proteins involved in the folding of nascent proteins, and the repair of proteins that have lost their typical conformations. These functions have therefore made molecular chaperones an active area of investigation within the field of conformational diseases. This review will discuss the role of molecular chaperones in neurodegenerative diseases, highlighting their functional classification, regulation, and therapeutic potential for such diseases.

  20. Dealing with Misfolded Proteins: Examining the Neuroprotective Role of Molecular Chaperones in Neurodegeneration

    PubMed Central

    Ali, Yousuf O.; Kitay, Brandon M.; Zhai, R. Grace

    2011-01-01

    Human neurodegenerative diseases arise from a wide array of genetic and environmental factors. Despite the diversity in etiology, many of these diseases are considered "conformational" in nature, characterized by the accumulation of pathological, misfolded proteins. These misfolded proteins can induce cellular stress by overloading the proteolytic machinery, ultimately resulting in the accumulation and deposition of aggregated protein species that are cytotoxic. Misfolded proteins may also form aberrant, non-physiological protein-protein interactions leading to the sequestration of other normal proteins essential for cellular functions. The progression of such disease may therefore be viewed as a failure of normal protein homeostasis, a process that involves a network of molecules regulating the synthesis, folding, translocation and clearance of proteins. Molecular chaperones are highly conserved proteins involved in the folding of nascent proteins, and the repair of proteins that have lost their typical conformations. These functions have therefore made molecular chaperones an active area of investigation within the field of conformational diseases. This review will discuss the role of molecular chaperones in neurodegenerative diseases, highlighting their functional classification, regulation, and therapeutic potential for such diseases. PMID:20938400

  1. Understanding the frustration arising from the competition between function, misfolding, and aggregation in a globular protein.

    PubMed

    Gianni, Stefano; Camilloni, Carlo; Giri, Rajanish; Toto, Angelo; Bonetti, Daniela; Morrone, Angela; Sormanni, Pietro; Brunori, Maurizio; Vendruscolo, Michele

    2014-09-30

    Folding and function may impose different requirements on the amino acid sequences of proteins, thus potentially giving rise to conflict. Such a conflict, or frustration, can result in the formation of partially misfolded intermediates that can compromise folding and promote aggregation. We investigate this phenomenon by studying frataxin, a protein whose normal function is to facilitate the formation of iron-sulfur clusters but whose mutations are associated with Friedreich's ataxia. To characterize the folding pathway of this protein we carry out a Φ-value analysis and use the resulting structural information to determine the structure of the folding transition state, which we then validate by a second round of rationally designed mutagenesis. The analysis of the transition-state structure reveals that the regions involved in the folding process are highly aggregation-prone. By contrast, the regions that are functionally important are partially misfolded in the transition state but highly resistant to aggregation. Taken together, these results indicate that in frataxin the competition between folding and function creates the possibility of misfolding, and that to prevent aggregation the amino acid sequence of this protein is optimized to be highly resistant to aggregation in the regions involved in misfolding.

  2. Protein misfolding detected early in pathogenesis of transgenic mouse model of Huntington disease using amyloid seeding assay.

    PubMed

    Gupta, Sharad; Jie, Shy'Ann; Colby, David W

    2012-03-23

    Huntington disease (HD) is one of several fatal neurodegenerative disorders associated with misfolded proteins. Here, we report a novel method for the sensitive detection of misfolded huntingtin (HTT) isolated from the brains of transgenic (Tg) mouse models of HD and humans with HD using an amyloid seeding assay (ASA), which is based on the propensity of misfolded proteins to act as a seed and shorten the nucleation-associated lag phase in the kinetics of amyloid formation in vitro. Using synthetic polyglutamine peptides as the substrate for amyloid formation, we found that partially purified misfolded HTT obtained from end-stage brain tissue of two Tg HD mouse models and brain tissue of post-mortem human HD patients was capable of specifically accelerating polyglutamine amyloid formation compared with unseeded reactions and controls. Alzheimer and prion disease brain tissues did not do so, demonstrating the specificity of the ASA. It is unclear whether early intermediates or later conformational species in the protein misfolding process act as seeds in the ASA for HD. However, we were able to detect misfolded protein in the brains of YAC128 mice early in disease pathogenesis (11 weeks of age), whereas large inclusion bodies have not been observed in the brains of these mice by histology until 78 weeks of age, much later in the pathogenic process. The sensitive detection of misfolded HTT protein early in the disease pathogenesis in the YAC128 Tg mouse model strengthens the argument for a causative role of protein misfolding in HD.

  3. Histochemical approaches to assess cell-to-cell transmission of misfolded proteins in neurodegenerative diseases

    PubMed Central

    Natale, G.; Pompili, E.; Biagioni, F.; Paparelli, S.; Lenzi, P.; Fornai, F.

    2013-01-01

    Formation, aggregation and transmission of abnormal proteins are common features in neurodegenerative disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease. The mechanisms underlying protein alterations in neurodegenerative diseases remain controversial. Novel findings highlighted altered protein clearing systems as common biochemical pathways which generate protein misfolding, which in turn causes protein aggregation and protein spreading. In fact, proteinaceous aggregates are prone to cell-tocell propagation. This is reminiscent of what happens in prion disorders, where the prion protein misfolds thus forming aggregates which spread to neighbouring cells. For this reason, the term prionoids is currently used to emphasize how several misfolded proteins are transmitted in neurodegenerative diseases following this prion-like pattern. Histochemical techniques including the use of specific antibodies covering both light and electron microscopy offer a powerful tool to describe these phenomena and investigate specific molecular steps. These include: prion like protein alterations; glycation of prion-like altered proteins to form advanced glycation end-products (AGEs); mechanisms of extracellular secretion; interaction of AGEs with specific receptors placed on neighbouring cells (RAGEs). The present manuscript comments on these phenomena aimed to provide a consistent scenario of the available histochemical approaches to dissect each specific step. PMID:23549464

  4. Identification of Human Proteins That Modify Misfolding and Proteotoxicity of Pathogenic Ataxin-1

    PubMed Central

    Riechers, Sean-Patrick; Muehlenberg, Katja; Möller, Angeli; Reinhardt, Anita; Vinayagam, Arunachalam; Schaefer, Martin H.; Boutros, Michael; Tricoire, Hervé; Andrade-Navarro, Miguel A.; Wanker, Erich E.

    2012-01-01

    Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1–interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo. PMID:22916034

  5. Methionine Oxidation Perturbs the Structural Core of the Prion Protein and Suggests a Generic Misfolding Pathway*

    PubMed Central

    Younan, Nadine D.; Nadal, Rebecca C.; Davies, Paul; Brown, David R.; Viles, John H.

    2012-01-01

    Oxidative stress and misfolding of the prion protein (PrPC) are fundamental to prion diseases. We have therefore probed the effect of oxidation on the structure and stability of PrPC. Urea unfolding studies indicate that H2O2 oxidation reduces the thermodynamic stability of PrPC by as much as 9 kJ/mol. 1H-15N NMR studies indicate methionine oxidation perturbs key hydrophobic residues on one face of helix-C as follows: Met-205, Val-209, and Met-212 together with residues Val-160 and Tyr-156. These hydrophobic residues pack together and form the structured core of the protein, stabilizing its ternary structure. Copper-catalyzed oxidation of PrPC causes a more significant alteration of the structure, generating a monomeric molten globule species that retains its native helical content. Further copper-catalyzed oxidation promotes extended β-strand structures that lack a cooperative fold. This transition from the helical molten globule to β-conformation has striking similarities to a misfolding intermediate generated at low pH. PrP may therefore share a generic misfolding pathway to amyloid fibers, irrespective of the conditions promoting misfolding. Our observations support the hypothesis that oxidation of PrP destabilizes the native fold of PrPC, facilitating the transition to PrPSc. This study gives a structural and thermodynamic explanation for the high levels of oxidized methionine in scrapie isolates. PMID:22654104

  6. Ligand binding and hydration in protein misfolding: insights from studies of prion and p53 tumor suppressor proteins.

    PubMed

    Silva, Jerson L; Vieira, Tuane C R G; Gomes, Mariana P B; Bom, Ana Paula Ano; Lima, Luis Mauricio T R; Freitas, Monica S; Ishimaru, Daniella; Cordeiro, Yraima; Foguel, Debora

    2010-02-16

    Protein misfolding has been implicated in a large number of diseases termed protein- folding disorders (PFDs), which include Alzheimer's disease, Parkinson's disease, transmissible spongiform encephalopathies, familial amyloid polyneuropathy, Huntington's disease, and type II diabetes. In these diseases, large quantities of incorrectly folded proteins undergo aggregation, destroying brain cells and other tissues. The interplay between ligand binding and hydration is an important component of the formation of misfolded protein species. Hydration drives various biological processes, including protein folding, ligand binding, macromolecular assembly, enzyme kinetics, and signal transduction. The changes in hydration and packing, both when proteins fold correctly or when folding goes wrong, leading to PFDs, are examined through several biochemical, biophysical, and structural approaches. Although in many cases the binding of a ligand such as a nucleic acid helps to prevent misfolding and aggregation, there are several examples in which ligands induce misfolding and assembly into amyloids. This occurs simply because the formation of structured aggregates (such as protofibrillar and fibrillar amyloids) involves decreases in hydration, formation of a hydrogen-bond network in the secondary structure, and burying of nonpolar amino acid residues, processes that also occur in the normal folding landscape. In this Account, we describe the present knowledge of the folding and misfolding of different proteins, with a detailed emphasis on mammalian prion protein (PrP) and tumoral suppressor protein p53; we also explore how ligand binding and hydration together influence the fate of the proteins. Anfinsen's paradigm that the structure of a protein is determined by its amino acid sequence is to some extent contradicted by the observation that there are two isoforms of the prion protein with the same sequence: the cellular and the misfolded isoform. The cellular isoform of PrP has a

  7. Degradation of misfolded proteins by autophagy: is it a strategy for Huntington's disease treatment?

    PubMed

    Lin, Fang; Qin, Zheng-Hong

    2013-01-01

    Autophagy is a degradation pathway for long-lived cytoplasmic proteins, protein complexes, or damaged organelles. The accumulation and aggregation of misfolded proteins are hallmarks of several neurodegenerative diseases. Many researchers have reported that autophagy degrades disease-causing misfolded and aggregated proteins, including mutant huntingtin (Htt) in Huntington's disease, mutant synuclein in familial Parkingson's disease, mutant Cu, Zn-Superoxide dismutase (SOD1) in familial amyotrophic lateral sclerosis. In this review, we will bring up new evidence to elucidate the involvement of autophagy in degradation of mutant Htt, discuss the mechanisms regulating the degradation of mutant Htt by autophagy and the therapeutic effects of drugs that enhance autophagy to improve clearance of mutant Htt. We propose that enhancement of autophagy by drugs may be a strategy to treat or retard progression of Huntington's disease.

  8. Effect of interactions with the chaperonin cavity on protein folding and misfolding.

    PubMed

    Sirur, Anshul; Knott, Michael; Best, Robert B

    2014-04-14

    Recent experimental and computational results have suggested that attractive interactions between a chaperonin and an enclosed substrate can have an important effect on the protein folding rate: it appears that folding may even be slower inside the cavity than under unconfined conditions, in contrast to what we would expect from excluded volume effects on the unfolded state. Here we examine systematically the dependence of the protein stability and folding rate on the strength of such attractive interactions between the chaperonin and substrate, by using molecular simulations of model protein systems in an idealised attractive cavity. Interestingly, we find a maximum in stability, and a rate which indeed slows down at high attraction strengths. We have developed a simple phenomenological model which can explain the variations in folding rate and stability due to differing effects on the free energies of the unfolded state, folded state, and transition state; changes in the diffusion coefficient along the folding coordinate are relatively small, at least for our simplified model. In order to investigate a possible role for these attractive interactions in folding, we have studied a recently developed model for misfolding in multidomain proteins. We find that, while encapsulation in repulsive cavities greatly increases the fraction of misfolded protein, sufficiently strong attractive protein-cavity interactions can strongly reduce the fraction of proteins reaching misfolded traps.

  9. A review on protein misfolding, aggregation and strategies to prevent related ailments.

    PubMed

    Shamsi, Tooba Naz; Athar, Teeba; Parveen, Romana; Fatima, Sadaf

    2017-07-23

    This review aims to highlight the fundamental mechanism of protein misfolding leading to protein aggregation and associated diseases. It also aims to anticipate novel therapeutic strategies with which to prevent or treat these highly debilitating conditions linked to these pathologies. The failure of a protein to correctly fold de novo or to remain correctly folded can have profound consequences on a living system especially when the cellular quality control processes fail to eliminate the rogue proteins. The core cause of over 20 different human diseases which have now been designated as 'conformational diseases' including neurodegenerative diseases such as Alzheimer's disease (AD), Huntington's disease (HD) and Parkinson's disease (PD) etc. A comprehensive study on protein misfolding, aggregation, and the outcomes of the effects of cytotoxic aggregates will lead to understand the aggregation-mediated cell toxicity and serves as a foundation for future research in development of promising therapies and drugs. This review has also shed light on the mechanism of protein misfolding which leads to its aggregation and hence the neurodegeneration. From these considerations, one could also envisage the possibility that protein aggregation may be exploited by nature to perform specific physiological functions in differing biological contexts. Copyright © 2017. Published by Elsevier B.V.

  10. Molecular Cross-talk between Misfolded Proteins in Animal Models of Alzheimer’s and Prion Diseases

    PubMed Central

    Morales, Rodrigo; Estrada, Lisbell D.; Diaz-Espinoza, Rodrigo; Morales-Scheihing, Diego; Jara, Maria C.; Castilla, Joaquin; Soto, Claudio

    2010-01-01

    The central event in Protein Misfolding Disorders (PMDs) is the accumulation of a misfolded form of a naturally expressed protein. Despite the diversity of clinical symptoms associated to different PMDs, many similarities in their mechanism suggest that distinct pathologies may cross-talk at the molecular level. The main goal of this study was to analyze the interaction of the protein misfolding processes implicated in Alzheimer’s and prion diseases. For this purpose we inoculated prions in an Alzheimer’s transgenic mouse model that develop typical amyloid plaques and followed the progression of pathological changes over time. Our findings show a dramatic acceleration and exacerbation of both pathologies. The onset of prion disease symptoms in transgenic mice appeared significantly faster with a concomitant increase on the level of misfolded prion protein in the brain. A striking increase in amyloid plaque deposition was observed in prion infected mice compared with their non-inoculated counterparts. Histological and biochemical studies showed the association of the two misfolded proteins in the brain and in vitro experiments showed that protein misfolding can be enhanced by a cross-seeding mechanism. These results suggest a profound interaction between Alzheimer’s and prion pathologies, indicating that one protein misfolding process may be an important risk factor for the development of a second one. Our findings may have important implications to understand the origin and progression of PMDs. PMID:20357103

  11. Mechanistic basis for the recognition of a misfolded protein by the molecular chaperone Hsp90.

    PubMed

    Oroz, Javier; Kim, Jin Hae; Chang, Bliss J; Zweckstetter, Markus

    2017-04-01

    The critical toxic species in over 40 human diseases are misfolded proteins. Their interaction with molecular chaperones such as Hsp90, which preferentially interacts with metastable proteins, is essential for the blocking of disease progression. Here we used nuclear magnetic resonance (NMR) spectroscopy to determine the three-dimensional structure of the misfolded cytotoxic monomer of the amyloidogenic human protein transthyretin, which is characterized by the release of the C-terminal β-strand and perturbations of the A-B loop. The misfolded transthyretin monomer, but not the wild-type protein, binds to human Hsp90. In the bound state, the Hsp90 dimer predominantly populates an open conformation, and transthyretin retains its globular structure. The interaction surface for the transthyretin monomer comprises the N-terminal and middle domains of Hsp90 and overlaps with that of the Alzheimer's-disease-related protein tau. Taken together, the data suggest that Hsp90 uses a mechanism for the recognition of aggregation-prone proteins that is largely distinct from those of other Hsp90 clients.

  12. Adenosine triphosphate (ATP) reduces amyloid-β protein misfolding in vitro.

    PubMed

    Coskuner, Orkid; Murray, Ian V J

    2014-01-01

    Alzheimer's disease (AD) is a devastating disease of aging that initiates decades prior to clinical manifestation and represents an impending epidemic. Two early features of AD are metabolic dysfunction and changes in amyloid-β protein (Aβ) levels. Since levels of ATP decrease over the course of the disease and Aβ is an early biomarker of AD, we sought to uncover novel linkages between the two. First and remarkably, a GxxxG motif is common between both Aβ (oligomerization motif) and nucleotide binding proteins (Rossmann fold). Second, ATP was demonstrated to protect against Aβ mediated cytotoxicity. Last, there is structural similarity between ATP and amyloid binding/inhibitory compounds such as ThioT, melatonin, and indoles. Thus, we investigated whether ATP alters misfolding of the pathologically relevant Aβ42. To test this hypothesis, we performed computational and biochemical studies. Our computational studies demonstrate that ATP interacts strongly with Tyr10 and Ser26 of Aβ fibrils in solution. Experimentally, both ATP and ADP reduced Aβ misfolding at physiological intracellular concentrations, with thresholds at ~500 μM and 1 mM respectively. This inhibition of Aβ misfolding is specific; requiring Tyr10 of Aβ and is enhanced by magnesium. Last, cerebrospinal fluid ATP levels are in the nanomolar range and decreased with AD pathology. This initial and novel finding regarding the ATP interaction with Aβ and reduction of Aβ misfolding has potential significance to the AD field. It provides an underlying mechanism for published links between metabolic dysfunction and AD. It also suggests a potential role of ATP in AD pathology, as the occurrence of misfolded extracellular Aβ mirrors lowered extracellular ATP levels. Last, the findings suggest that Aβ conformation change may be a sensor of metabolic dysfunction.

  13. Yeast cells reveal the misfolding and the cellular mislocalization of the human BRCA1 protein.

    PubMed

    Thouvenot, Pierre; Fourrière, Lou; Dardillac, Elodie; Ben Yamin, Barbara; Lescure, Aurianne; Lejour, Vincent; Heiligenstein, Xavier; Boulé, Jean-Baptiste; Romao, Maryse; Raposo-Benedetti, Graça; Lopez, Bernard S; Nicolas, Alain; Millot, Gaël A

    2016-12-01

    Understanding the effect of an ever-growing number of human variants detected by genome sequencing is a medical challenge. The yeast Saccharomyces cerevisiae model has held attention for its capacity to monitor the functional impact of missense mutations found in human genes, including the BRCA1 breast and ovarian cancer susceptibility gene. When expressed in yeast, the wild-type full-length BRCA1 protein forms a single nuclear aggregate and induces a growth inhibition. Both events are modified by pathogenic mutations of BRCA1. However, the biological processes behind these events in yeast remain to be determined. Here, we show that the BRCA1 nuclear aggregation and the growth inhibition are sensitive to misfolding effects induced by missense mutations. Moreover, misfolding mutations impair the nuclear targeting of BRCA1 in yeast cells and in a human cell line. In conclusion, we establish a connection between misfolding and nuclear transport impairment, and we illustrate that yeast is a suitable model to decipher the effect of misfolding mutations. © 2016. Published by The Company of Biologists Ltd.

  14. Dual Functional Small Molecule Probes as Fluorophore and Ligand for Misfolding Proteins

    PubMed Central

    Zhang, Xueli; Ran, Chongzhao

    2013-01-01

    Misfolding of a protein is a destructive process for variety of diseases that include neurodegenerative diseases such as Alzheimer’s disease, Parkinson disease, Huntington disease, mad cow disease, amyotrophic lateral sclerosis (ALS), and frontal temporal dementia (FTD), and other non-CNS diseases such as diabetes, cystic fibrosis, and lysosomal storage diseases. Formation of various misfunctional large assembles of the misfolded protein is the primary consequence. To detect the formation of the aggregated species is very important for not only basic mechanism research but also very crucial for diagnosis of the diseases. In this review, we updated references related to the new development of the dual functional fluorescent small molecule probes for detecting the aggregated proteins in vitro and in vivo. PMID:24363605

  15. Partially Unfolded Forms of the Prion Protein Populated under Misfolding-promoting Conditions

    PubMed Central

    Moulick, Roumita; Das, Ranabir; Udgaonkar, Jayant B.

    2015-01-01

    The susceptibility of the cellular prion protein (PrPC) to convert to an alternative misfolded conformation (PrPSc), which is the key event in the pathogenesis of prion diseases, is indicative of a conformationally flexible native (N) state. In the present study, hydrogen-deuterium exchange (HDX) in conjunction with mass spectrometry and nuclear magnetic resonance spectroscopy were used for the structural and energetic characterization of the N state of the full-length mouse prion protein, moPrP(23–231), under conditions that favor misfolding. The kinetics of HDX of 34 backbone amide hydrogens in the N state were determined at pH 4. In contrast to the results of previous HDX studies on the human and Syrian hamster prion proteins at a higher pH, various segments of moPrP were found to undergo different extents of subglobal unfolding events at pH 4, a pH at which the protein is known to be primed to misfold to a β-rich conformation. No residual structure around the disulfide bond was observed for the unfolded state at pH 4. The N state of the prion protein was observed to be at equilibrium with at least two partially unfolded forms (PUFs). These PUFs, which are accessed by stochastic fluctuations of the N state, have altered surface area exposure relative to the N state. One of these PUFs resembles a conformation previously implicated to be an initial intermediate in the conversion of monomeric protein into misfolded oligomer at pH 4. PMID:26306043

  16. The two faces of protein misfolding: gain- and loss-of-function in neurodegenerative diseases.

    PubMed

    Winklhofer, Konstanze F; Tatzelt, Jörg; Haass, Christian

    2008-01-23

    The etiologies of neurodegenerative diseases may be diverse; however, a common pathological denominator is the formation of aberrant protein conformers and the occurrence of pathognomonic proteinaceous deposits. Different approaches coming from neuropathology, genetics, animal modeling and biophysics have established a crucial role of protein misfolding in the pathogenic process. However, there is an ongoing debate about the nature of the harmful proteinaceous species and how toxic conformers selectively damage neuronal populations. Increasing evidence indicates that soluble oligomers are associated with early pathological alterations, and strikingly, oligomeric assemblies of different disease-associated proteins may share common structural features. A major step towards the understanding of mechanisms implicated in neuronal degeneration is the identification of genes, which are responsible for familial variants of neurodegenerative diseases. Studies based on these disease-associated genes illuminated the two faces of protein misfolding in neurodegeneration: a gain of toxic function and a loss of physiological function, which can even occur in combination. Here, we summarize how these two faces of protein misfolding contribute to the pathomechanisms of Alzheimer's disease, frontotemporal lobar degeneration, Parkinson's disease and prion diseases.

  17. The two faces of protein misfolding: gain- and loss-of-function in neurodegenerative diseases

    PubMed Central

    Winklhofer, Konstanze F; Tatzelt, Jörg; Haass, Christian

    2008-01-01

    The etiologies of neurodegenerative diseases may be diverse; however, a common pathological denominator is the formation of aberrant protein conformers and the occurrence of pathognomonic proteinaceous deposits. Different approaches coming from neuropathology, genetics, animal modeling and biophysics have established a crucial role of protein misfolding in the pathogenic process. However, there is an ongoing debate about the nature of the harmful proteinaceous species and how toxic conformers selectively damage neuronal populations. Increasing evidence indicates that soluble oligomers are associated with early pathological alterations, and strikingly, oligomeric assemblies of different disease-associated proteins may share common structural features. A major step towards the understanding of mechanisms implicated in neuronal degeneration is the identification of genes, which are responsible for familial variants of neurodegenerative diseases. Studies based on these disease-associated genes illuminated the two faces of protein misfolding in neurodegeneration: a gain of toxic function and a loss of physiological function, which can even occur in combination. Here, we summarize how these two faces of protein misfolding contribute to the pathomechanisms of Alzheimer's disease, frontotemporal lobar degeneration, Parkinson's disease and prion diseases. PMID:18216876

  18. Gene therapy for misfolding protein diseases of the central nervous system.

    PubMed

    San Sebastian, Waldy; Samaranch, Lluis; Kells, Adrian P; Forsayeth, John; Bankiewicz, Krystof S

    2013-07-01

    Protein aggregation as a result of misfolding is a common theme underlying neurodegenerative diseases. Accordingly, most recent studies aim to prevent protein misfolding and/or aggregation as a strategy to treat these pathologies. For instance, state-of-the-art approaches, such as silencing protein overexpression by means of RNA interference, are being tested with positive outcomes in preclinical models of animals overexpressing the corresponding protein. Therapies designed to treat central nervous system diseases should provide accurate delivery of the therapeutic agent and long-term or chronic expression by means of a nontoxic delivery vehicle. After several years of technical advances and optimization, gene therapy emerges as a promising approach able to fulfill those requirements. In this review we will summarize the latest improvements achieved in gene therapy for central nervous system diseases associated with protein misfolding (e.g., amyotrophic lateral sclerosis, Alzheimer's, Parkinson's, Huntington's, and prion diseases), as well as the most recent approaches in this field to treat these pathologies.

  19. Transcriptional induction of the heat shock protein B8 mediates the clearance of misfolded proteins responsible for motor neuron diseases.

    PubMed

    Crippa, Valeria; D'Agostino, Vito G; Cristofani, Riccardo; Rusmini, Paola; Cicardi, Maria E; Messi, Elio; Loffredo, Rosa; Pancher, Michael; Piccolella, Margherita; Galbiati, Mariarita; Meroni, Marco; Cereda, Cristina; Carra, Serena; Provenzani, Alessandro; Poletti, Angelo

    2016-03-10

    Neurodegenerative diseases (NDs) are often associated with the presence of misfolded protein inclusions. The chaperone HSPB8 is upregulated in mice, the human brain and muscle structures affected during NDs progression. HSPB8 exerts a potent pro-degradative activity on several misfolded proteins responsible for familial NDs forms. Here, we demonstrated that HSPB8 also counteracts accumulation of aberrantly localized misfolded forms of TDP-43 and its 25 KDa fragment involved in most sporadic cases of Amyotrophic Lateral Sclerosis (sALS) and of Fronto Lateral Temporal Dementia (FLTD). HSPB8 acts with BAG3 and the HSP70/HSC70-CHIP complex enhancing the autophagic removal of misfolded proteins. We performed a high-through put screening (HTS) to find small molecules capable of inducing HSPB8 in neurons for therapeutic purposes. We identified two compounds, colchicine and doxorubicin, that robustly up-regulated HSPB8 expression. Both colchicine and doxorubicin increased the expression of the master regulator of autophagy TFEB, the autophagy linker p62/SQSTM1 and the autophagosome component LC3. In line, both drugs counteracted the accumulation of TDP-43 and TDP-25 misfolded species responsible for motoneuronal death in sALS. Thus, analogs of colchicine and doxorubicin able to induce HSPB8 and with better safety and tolerability may result beneficial in NDs models.

  20. Transcriptional induction of the heat shock protein B8 mediates the clearance of misfolded proteins responsible for motor neuron diseases

    PubMed Central

    Crippa, Valeria; D’Agostino, Vito G.; Cristofani, Riccardo; Rusmini, Paola; Cicardi, Maria E.; Messi, Elio; Loffredo, Rosa; Pancher, Michael; Piccolella, Margherita; Galbiati, Mariarita; Meroni, Marco; Cereda, Cristina; Carra, Serena; Provenzani, Alessandro; Poletti, Angelo

    2016-01-01

    Neurodegenerative diseases (NDs) are often associated with the presence of misfolded protein inclusions. The chaperone HSPB8 is upregulated in mice, the human brain and muscle structures affected during NDs progression. HSPB8 exerts a potent pro-degradative activity on several misfolded proteins responsible for familial NDs forms. Here, we demonstrated that HSPB8 also counteracts accumulation of aberrantly localized misfolded forms of TDP-43 and its 25 KDa fragment involved in most sporadic cases of Amyotrophic Lateral Sclerosis (sALS) and of Fronto Lateral Temporal Dementia (FLTD). HSPB8 acts with BAG3 and the HSP70/HSC70-CHIP complex enhancing the autophagic removal of misfolded proteins. We performed a high-through put screening (HTS) to find small molecules capable of inducing HSPB8 in neurons for therapeutic purposes. We identified two compounds, colchicine and doxorubicin, that robustly up-regulated HSPB8 expression. Both colchicine and doxorubicin increased the expression of the master regulator of autophagy TFEB, the autophagy linker p62/SQSTM1 and the autophagosome component LC3. In line, both drugs counteracted the accumulation of TDP-43 and TDP-25 misfolded species responsible for motoneuronal death in sALS. Thus, analogs of colchicine and doxorubicin able to induce HSPB8 and with better safety and tolerability may result beneficial in NDs models. PMID:26961006

  1. Proteostasis impairment in protein-misfolding and -aggregation diseases.

    PubMed

    Hipp, Mark S; Park, Sae-Hun; Hartl, F Ulrich

    2014-09-01

    Cells possess an extensive network of components to safeguard proteome integrity and maintain protein homeostasis (proteostasis). When this proteostasis network (PN) declines in performance, as may be the case during aging, newly synthesized proteins are no longer able to fold efficiently and metastable proteins lose their functionally active conformations, particularly under conditions of cell stress. Apart from loss-of-function effects, a critical consequence of PN deficiency is the accumulation of cytotoxic protein aggregates, which are also associated with many age-dependent neurodegenerative diseases and other medical disorders. Here we discuss recent evidence that the chronic production of aberrantly folded and aggregated proteins in these diseases is harmful by overtaxing PN capacity, setting in motion a vicious cycle of increasing proteome imbalance that eventually leads to PN collapse and cell death. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Misfolded proteins activate Factor XII in humans, leading to kallikrein formation without initiating coagulation

    PubMed Central

    Maas, Coen; Govers-Riemslag, José W.P.; Bouma, Barend; Schiks, Bettina; Hazenberg, Bouke P.C.; Lokhorst, Henk M.; Hammarström, Per; ten Cate, Hugo; de Groot, Philip G.; Bouma, Bonno N.; Gebbink, Martijn F.B.G.

    2008-01-01

    When blood is exposed to negatively charged surface materials such as glass, an enzymatic cascade known as the contact system becomes activated. This cascade is initiated by autoactivation of Factor XII and leads to both coagulation (via Factor XI) and an inflammatory response (via the kallikrein-kinin system). However, while Factor XII is important for coagulation in vitro, it is not important for physiological hemostasis, so the physiological role of the contact system remains elusive. Using patient blood samples and isolated proteins, we identified a novel class of Factor XII activators. Factor XII was activated by misfolded protein aggregates that formed by denaturation or by surface adsorption, which specifically led to the activation of the kallikrein-kinin system without inducing coagulation. Consistent with this, we found that Factor XII, but not Factor XI, was activated and kallikrein was formed in blood from patients with systemic amyloidosis, a disease marked by the accumulation and deposition of misfolded plasma proteins. These results show that the kallikrein-kinin system can be activated by Factor XII, in a process separate from the coagulation cascade, and point to a protective role for Factor XII following activation by misfolded protein aggregates. PMID:18725990

  3. Can misfolded proteins be beneficial? The HAMLET case.

    PubMed

    Pettersson-Kastberg, Jenny; Aits, Sonja; Gustafsson, Lotta; Mossberg, Anki; Storm, Petter; Trulsson, Maria; Persson, Filip; Mok, K Hun; Svanborg, Catharina

    2009-01-01

    By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain. Here we present HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) as an example where partial unfolding and the incorporation of cofactor create a complex with new, beneficial properties. Native alpha-lactalbumin functions as a substrate specifier in lactose synthesis, but when partially unfolded the protein binds oleic acid and forms the tumoricidal HAMLET complex. When the properties of HAMLET were first described they were surprising, as protein folding intermediates and especially amyloid-forming protein intermediates had been regarded as toxic conformations, but since then structural studies have supported functional diversity arising from a change in fold. The properties of HAMLET suggest a mechanism of structure-function variation, which might help the limited number of human protein genes to generate sufficient structural diversity to meet the diverse functional demands of complex organisms.

  4. Mistranslation-induced protein misfolding as a dominant constraint on coding-sequence evolution

    PubMed Central

    Drummond, D. Allan; Wilke, Claus O.

    2009-01-01

    Summary The biological causes of selective pressures on coding-sequence evolution remain controversial, despite the surprising consistency of covariation between common measures of evolutionary change (substitution rates) and gene expression (mRNA levels, codon usage) across taxa. We carry out a unified analysis which reveals these conserved patterns in E. coli, yeast, worm, fly, mouse, and human, and suggests that all trends stem largely from a unified underlying selective pressure. In metazoans, these trends are strongest in tissues composed of neurons, whose structure and lifetime confer extreme sensitivity to protein misfolding. We propose, and demonstrate using a molecular-level evolutionary simulation, that selection against toxicity of misfolded proteins generated by ribosome errors suffices to create all the observed covariation. The mechanistic model of molecular evolution which emerges yields testable biochemical predictions, calls into question use of nonsynonymous-to-synonymous substitution ratios (Ka/Ks) to detect functional selection, and suggests how mistranslation may contribute to neurodegenerative disease. PMID:18662548

  5. De Novo Generation of a Unique Cervid Prion Strain Using Protein Misfolding Cyclic Amplification

    PubMed Central

    Meyerett-Reid, Crystal; Wyckoff, A. Christy; Spraker, Terry; Pulford, Bruce; Bender, Heather

    2017-01-01

    ABSTRACT Substantial evidence supports the hypothesis that prions are misfolded, infectious, insoluble, and protease-resistant proteins (PrPRES) devoid of instructional nucleic acid that cause transmissible spongiform encephalopathies (TSEs). Protein misfolding cyclic amplification (PMCA) has provided additional evidence that PrPRes acts as a template that can convert the normal cellular prion protein (PrPC) present in uninfected normal brain homogenate (NBH) into the infectious misfolded PrPRES isoform. Human PrPC has been shown to spontaneously convert to a misfolded pathological state causing sporadic Creutzfeldt-Jakob disease (sCJD). Several investigators have reported spontaneous generation of prions by in vitro assays, including PMCA. Here we tested the rate of de novo generation of cervid prions in our laboratory using our standard PMCA protocol and NBH from transgenic mice expressing cervid PrPC (TgCerPrP mice). We generated de novo prions in rounds 4, 5, and 7 at low cumulative rates of 1.6, 5.0, and 6.7%, respectively. The prions caused infectious chronic wasting disease (CWD) upon inoculation into normal uninfected TgCerPrP mice and displayed unique biochemical characteristics compared to other cervid prion strains. We conclude that PMCA of cervid PrPC from normal brain homogenate spontaneously generated a new cervid prion strain. These data support the potential for cervids to develop sporadic CWD. IMPORTANCE CWD is the only known TSE that affects free-ranging wildlife, specifically cervids such as elk, deer, moose, caribou, and reindeer. CWD has become endemic in both free-ranging and captive herds in North America, South Korea, and, most recently, northern Europe. The prion research community continues to debate the origins of CWD. Original foci of CWD emergence in Colorado and Wyoming coincident with the sheep TSE scrapie suggest that scrapie prions may have adapted to cervids to cause CWD. However, emerging evidence supports the idea that cervid Pr

  6. De Novo Generation of a Unique Cervid Prion Strain Using Protein Misfolding Cyclic Amplification.

    PubMed

    Meyerett-Reid, Crystal; Wyckoff, A Christy; Spraker, Terry; Pulford, Bruce; Bender, Heather; Zabel, Mark D

    2017-01-01

    Substantial evidence supports the hypothesis that prions are misfolded, infectious, insoluble, and protease-resistant proteins (PrP(RES)) devoid of instructional nucleic acid that cause transmissible spongiform encephalopathies (TSEs). Protein misfolding cyclic amplification (PMCA) has provided additional evidence that PrPRes acts as a template that can convert the normal cellular prion protein (PrP(C)) present in uninfected normal brain homogenate (NBH) into the infectious misfolded PrP(RES) isoform. Human PrP(C) has been shown to spontaneously convert to a misfolded pathological state causing sporadic Creutzfeldt-Jakob disease (sCJD). Several investigators have reported spontaneous generation of prions by in vitro assays, including PMCA. Here we tested the rate of de novo generation of cervid prions in our laboratory using our standard PMCA protocol and NBH from transgenic mice expressing cervid PrP(C) (TgCerPrP mice). We generated de novo prions in rounds 4, 5, and 7 at low cumulative rates of 1.6, 5.0, and 6.7%, respectively. The prions caused infectious chronic wasting disease (CWD) upon inoculation into normal uninfected TgCerPrP mice and displayed unique biochemical characteristics compared to other cervid prion strains. We conclude that PMCA of cervid PrP(C) from normal brain homogenate spontaneously generated a new cervid prion strain. These data support the potential for cervids to develop sporadic CWD. IMPORTANCE CWD is the only known TSE that affects free-ranging wildlife, specifically cervids such as elk, deer, moose, caribou, and reindeer. CWD has become endemic in both free-ranging and captive herds in North America, South Korea, and, most recently, northern Europe. The prion research community continues to debate the origins of CWD. Original foci of CWD emergence in Colorado and Wyoming coincident with the sheep TSE scrapie suggest that scrapie prions may have adapted to cervids to cause CWD. However, emerging evidence supports the idea that cervid

  7. ALS-related misfolded protein management in motor neurons and muscle cells.

    PubMed

    Galbiati, Mariarita; Crippa, Valeria; Rusmini, Paola; Cristofani, Riccardo; Cicardi, Maria Elena; Giorgetti, Elisa; Onesto, Elisa; Messi, Elio; Poletti, Angelo

    2014-12-01

    Amyotrophic Lateral Sclerosis (ALS) is the most common form of adult-onset motor neuron disease. It is now considered a multi-factorial and multi-systemic disorder in which alterations of the crosstalk between neuronal and non-neuronal cell types might influence the course of the disease. In this review, we will provide evidence that dysfunctions of affected muscle cells are not only a marginal consequence of denervation associated to motor neurons loss, but a direct consequence of cell muscle toxicity of mutant SOD1. In muscle, the misfolded state of mutant SOD1 protein, unlike in motor neurons, does not appear to have direct effects on protein aggregation and mitochondrial functionality. Muscle cells are, in fact, more capable than motor neurons to handle misfolded proteins, suggesting that mutant SOD1 toxicity in muscle is not mediated by classical mechanisms of intracellular misfolded proteins accumulation. Several recent works indicate that a higher activation of molecular chaperones and degradative systems is present in muscle cells, which for this reason are possibly able to better manage misfolded mutant SOD1. However, several alterations in gene expression and regenerative potential of skeletal muscles have also been reported as a consequence of the expression of mutant SOD1 in muscle. Whether these changes in muscle cells are causative of ALS or a consequence of motor neuron alterations is not yet clear, but their elucidation is very important, since the understanding of the mechanisms involved in mutant SOD1 toxicity in muscle may facilitate the design of treatments directed toward this specific tissue to treat ALS or at least to delay disease progression.

  8. A computational combinatorial approach identifies a protein inhibitor of superoxide dismutase 1 misfolding, aggregation, and cytotoxicity.

    PubMed

    Banerjee, Victor; Oren, Ofek; Ben-Zeev, Efrat; Taube, Ran; Engel, Stanislav; Papo, Niv

    2017-09-22

    Molecular agents that specifically bind and neutralize misfolded and toxic superoxide dismutase 1 (SOD1) mutant proteins may find application in attenuating the disease progression of familial amyotrophic lateral sclerosis. However, high structural similarities between the wild-type and mutant SOD1 proteins limit the utility of this approach. Here we addressed this challenge by converting a promiscuous natural human IgG-binding domain, the hyperthermophilic variant of protein G (HTB1), into a highly specific aggregation inhibitor (designated HTB1M) of two familial amyotrophic lateral sclerosis-linked SOD1 mutants, SOD1(G93A) and SOD1(G85R) We utilized a computational algorithm for mapping protein surfaces predisposed to HTB1 intermolecular interactions to construct a focused HTB1 library, complemented with an experimental platform based on yeast surface display for affinity and specificity screening. HTB1M displayed high binding specificity toward SOD1 mutants, inhibited their amyloid aggregation in vitro, prevented the accumulation of misfolded proteins in living cells, and reduced the cytotoxicity of SOD1(G93A) expressed in motor neuron-like cells. Competition assays and molecular docking simulations suggested that HTB1M binds to SOD1 via both its α-helical and β-sheet domains at the native dimer interface that becomes exposed upon mutated SOD1 misfolding and monomerization. Our results demonstrate the utility of computational mapping of the protein-protein interaction potential for designing focused protein libraries to be used in directed evolution. They also provide new insight into the mechanism of conversion of broad-spectrum immunoglobulin-binding proteins, such as HTB1, into target-specific proteins, thereby paving the way for the development of new selective drugs targeting the amyloidogenic proteins implicated in a variety of human diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Misfolded Proteins: From Little Villains to Little Helpers in the Fight Against Cancer

    PubMed Central

    Brüning, Ansgar; Jückstock, Julia

    2015-01-01

    The application of cytostatic drugs targeting the high proliferation rates of cancer cells is currently the most commonly used treatment option in cancer chemotherapy. However, severe side effects and resistance mechanisms may occur as a result of such treatment, possibly limiting the therapeutic efficacy of these agents. In recent years, several therapeutic strategies have been developed that aim at targeting not the genomic integrity and replication machinery of cancer cells but instead their protein homeostasis. During malignant transformation, the cancer cell proteome develops vast aberrations in the expression of mutated proteins, oncoproteins, drug- and apoptosis-resistance proteins, etc. A complex network of protein quality-control mechanisms, including chaperoning by heat shock proteins (HSPs), not only is essential for maintaining the extravagant proteomic lifestyle of cancer cells but also represents an ideal cancer-specific target to be tackled. Furthermore, the high rate of protein synthesis and turnover in certain types of cancer cells can be specifically directed by interfering with the proteasomal and autophagosomal protein recycling and degradation machinery, as evidenced by the clinical application of proteasome inhibitors. Since proteins with loss of their native conformation are prone to unspecific aggregations and have proved to be detrimental to normal cellular function, specific induction of misfolded proteins by HSP inhibitors, proteasome inhibitors, hyperthermia, or inducers of endoplasmic reticulum stress represents a new method of cancer cell killing exploitable for therapeutic purposes. This review describes drugs – approved, repurposed, or under investigation – that can be used to accumulate misfolded proteins in cancer cells, and particularly focuses on the molecular aspects that lead to the cytotoxicity of misfolded proteins in cancer cells. PMID:25759792

  10. In vitro amplification of H-type atypical bovine spongiform encephalopathy by protein misfolding cyclic amplification

    PubMed Central

    O‘Connor, Matthew J.; Bishop, Keith; Workman, Robert G.; Maddison, Ben C.

    2017-01-01

    ABSTRACT The in vitro amplification of prions by serial protein misfolding cyclic amplification has been shown to detect PrPSc to levels at least as sensitive as rodent bioassay but in a fraction of the time. Bovine spongiform encephalopathy is a zoonotic prion disease in cattle and has been shown to occur in 3 distinct forms, classical BSE (C-BSE) and 2 atypical BSE forms (L-BSE and H-BSE). Atypical forms are usually detected in asymptomatic, older cattle and are suggested to be spontaneous forms of the disease. Here, we show the development of a serial protein misfolding cyclic amplification method for the detection of H-BSE. The assay could detect PrPSc from 3 distinct experimental isolates of H-BSE, could detect PrPSc in as little as 1×10−12 g of brain material and was highly specific. Additionally, the product of serial protein misfolding cyclic amplification at all dilutions of seed analyzed could be readily distinguished from L-BSE, which did not amplify, and C-BSE, which had PrPSc with distinct protease K-resistance and protease K-resistant PrPSc molecular weights. PMID:28281929

  11. Antibodies and protein misfolding: From structural research tools to therapeutic strategies.

    PubMed

    De Genst, Erwin; Messer, Anne; Dobson, Christopher M

    2014-11-01

    Protein misfolding disorders, including the neurodegenerative conditions Alzheimer's disease (AD) and Parkinson's disease (PD) represent one of the major medical challenges or our time. The underlying molecular mechanisms that govern protein misfolding and its links with disease are very complex processes, involving the formation of transiently populated but highly toxic molecular species within the crowded environment of the cell and tissue. Nevertheless, much progress has been made in understanding these events in recent years through innovative experiments and therapeutic strategies, and in this review we present an overview of the key roles of antibodies and antibody fragments in these endeavors. We discuss in particular how these species are being used in combination with a variety of powerful biochemical and biophysical methodologies, including a range of spectroscopic and microscopic techniques applied not just in vitro but also in situ and in vivo, both to gain a better understanding of the mechanistic nature of protein misfolding and aggregation and also to design novel therapeutic strategies to combat the family of diseases with which they are associated. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. Copyright © 2014. Published by Elsevier B.V.

  12. Cellular misfolded proteins rescued from degradation by MHC class II molecules are possible targets for autoimmune diseases.

    PubMed

    Arase, Noriko; Arase, Hisashi

    2015-11-01

    The major function of major histocompatibility complex (MHC) class II molecules is the presentation of peptide antigens to helper T cells. However, when misfolded proteins are associated with MHC class II molecules in the endoplasmic reticulum, they are transported to the cell surface by MHC class II molecules without processing to peptides. Of note, misfolded proteins complexed with MHC class II molecules are specifically recognized by autoantibodies produced in patients with autoimmune diseases such as rheumatoid arthritis and antiphospholipid syndrome. Furthermore, autoantibody binding to misfolded proteins complexed with MHC class II molecules is associated with the susceptibility to autoimmune diseases conferred by each MHC class II allele. Therefore, misfolded proteins rescued from degradation by MHC class II molecules may be recognized as 'neo-self' antigens by the immune system and be involved in the pathogenicity of autoimmune diseases.

  13. A Bystander Mechanism Explains the Specific Phenotype of a Broadly Expressed Misfolded Protein

    PubMed Central

    Klabonski, Lauren; Senthilkumar, Lakshana; Gidalevitz, Tali

    2016-01-01

    Misfolded proteins in transgenic models of conformational diseases interfere with proteostasis machinery and compromise the function of many structurally and functionally unrelated metastable proteins. This collateral damage to cellular proteins has been termed 'bystander' mechanism. How a single misfolded protein overwhelms the proteostasis, and how broadly-expressed mutant proteins cause cell type-selective phenotypes in disease are open questions. We tested the gain-of-function mechanism of a R37C folding mutation in an endogenous IGF-like C.elegans protein DAF-28. DAF-28(R37C) is broadly expressed, but only causes dysfunction in one specific neuron, ASI, leading to a distinct developmental phenotype. We find that this phenotype is caused by selective disruption of normal biogenesis of an unrelated endogenous protein, DAF-7/TGF-β. The combined deficiency of DAF-28 and DAF-7 biogenesis, but not of DAF-28 alone, explains the gain-of-function phenotype—deficient pro-growth signaling by the ASI neuron. Using functional, fluorescently-tagged protein, we find that, in animals with mutant DAF-28/IGF, the wild-type DAF-7/TGF-β is mislocalized to and accumulates in the proximal axon of the ASI neuron. Activation of two different branches of the unfolded protein response can modulate both the developmental phenotype and DAF-7 mislocalization in DAF-28(R37C) animals, but appear to act through divergent mechanisms. Our finding that bystander targeting of TGF-β explains the phenotype caused by a folding mutation in an IGF-like protein suggests that, in conformational diseases, bystander misfolding may specify the distinct phenotypes caused by different folding mutations. PMID:27926939

  14. Monitoring protein misfolding by site-specific labeling of proteins in vivo.

    PubMed

    Hsieh, Tzung-yang; Nillegoda, Nadinath B; Tyedmers, Jens; Bukau, Bernd; Mogk, Axel; Kramer, Günter

    2014-01-01

    Incorporating fluorescent amino acids by suppression of the TAG amber codon is a useful tool for site-specific labeling of proteins and visualizing their localization in living cells. Here we use a plasmid encoded orthogonal tRNA/aminoacyl-tRNA synthetase pair to site-specifically label firefly luciferase with the environmentally sensitive fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2- aminopropanoic acid (ANAP) and explore the detectability of conformational changes in labeled luciferase in the yeast cytoplasm. We find that ANAP labeling efficiency is greatly increased in [PSI+] cells and show that analysis of the ANAP fluorescence emission by confocal imaging allows for tracking the thermal unfolding and aggregation of luciferase in vivo. Furthermore we demonstrate that flow cytometry can be used to study conformational changes in luciferase and chaperone-mediated refolding in quantitative terms and at the level of single cells. This experimental setup for the first time allows for the direct analysis of the folding state of a protein in living cells and may serve as valuable new tool for examining mechanisms of protein folding, misfolding and aggregation.

  15. Monitoring Protein Misfolding by Site-Specific Labeling of Proteins In Vivo

    PubMed Central

    Hsieh, Tzung-yang; Nillegoda, Nadinath B.; Tyedmers, Jens; Bukau, Bernd; Mogk, Axel; Kramer, Günter

    2014-01-01

    Incorporating fluorescent amino acids by suppression of the TAG amber codon is a useful tool for site-specific labeling of proteins and visualizing their localization in living cells. Here we use a plasmid encoded orthogonal tRNA/aminoacyl-tRNA synthetase pair to site-specifically label firefly luciferase with the environmentally sensitive fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2- aminopropanoic acid (ANAP) and explore the detectability of conformational changes in labeled luciferase in the yeast cytoplasm. We find that ANAP labeling efficiency is greatly increased in [PSI+] cells and show that analysis of the ANAP fluorescence emission by confocal imaging allows for tracking the thermal unfolding and aggregation of luciferase in vivo. Furthermore we demonstrate that flow cytometry can be used to study conformational changes in luciferase and chaperone-mediated refolding in quantitative terms and at the level of single cells. This experimental setup for the first time allows for the direct analysis of the folding state of a protein in living cells and may serve as valuable new tool for examining mechanisms of protein folding, misfolding and aggregation. PMID:24915041

  16. Novel insights into protein misfolding diseases revealed by ion mobility-mass spectrometry.

    PubMed

    Williams, Danielle M; Pukala, Tara L

    2013-01-01

    Amyloid disorders incorporate a wide range of human diseases arising from the failure of a specific peptide or protein to adopt, or remain in, its native functional conformational state. These pathological conditions, such as Parkinson's disease, Alzheimer's disease and Huntington's disease are highly debilitating, exact enormous costs on both individuals and society, and are predicted to increase in prevalence. Consequently, they form the focus of a topical and rich area of current scientific research. A major goal in attempts to understand and treat protein misfolding diseases is to define the structures and interactions of protein species intermediate between fully folded and aggregated, and extract a description of the aggregation process. This has proven a difficult task due to the inability of traditional structural biology approaches to analyze structurally heterogeneous systems. Continued developments in instrumentation and analytical approaches have seen ion mobility-mass spectrometry (IM-MS) emerge as a complementary approach for protein structure determination, and in some cases, a structural biology tool in its own right. IM-MS is well suited to the study of protein misfolding, and has already yielded significant structural information for selected amyloidogenic systems during the aggregation process. This review describes IM-MS for protein structure investigation, and provides a summary of current research highlighting how this methodology has unequivocally and unprecedentedly provided structural and mechanistic detail pertaining to the oligomerization of a variety of disease related proteins. Copyright © 2013 Wiley Periodicals, Inc.

  17. The push-and-pull hypothesis in protein unfolding, misfolding and aggregation.

    PubMed

    de Oliveira, Guilherme A P; Silva, Jerson L

    2017-03-29

    The combination of biophysical and structural techniques has allowed the visualization of species classified as dry molten-globule states. Further destabilization causes these structures to follow through a wet-globule stage to reach an unfolded chain. We have recently combined small angle X-ray scattering and nuclear magnetic resonance to observe these species, and we introduce a push-and-pull hypothesis to explain the dissimilar actions of urea and high pressure on proteins. The implications of these molten-globule states are further discussed in light of their potential physiological and pathological roles, especially in protein misfolding diseases.

  18. Neurodegenerative diseases linked to misfolded proteins and their therapeutic approaches: A review.

    PubMed

    Khanam, Hena; Ali, Abad; Asif, Mohd; Shamsuzzaman

    2016-11-29

    Neurodegenerative diseases, such as Alzheimer's, Parkinson's, Creutzfeldt-Jacob, Huntington's diseases and amyotrophic lateral sclerosis, are mainly characterized by the massive deposition of misfolded protein aggregates consequent to aberrant production or overexpression of specific proteins. The development of new therapeutics for the treatment of neurodegenerative pathophysiologies currently stands at a crossroads. This presents an opportunity to transition future drug discovery efforts to target disease modification, an area in which much still remains unknown. In this review we examine recent progress in the area of neurodegenerative drug discovery, focusing on some of the most common targets. Copyright © 2016. Published by Elsevier Masson SAS.

  19. A network of ubiquitin ligases is important for the dynamics of misfolded protein aggregates in yeast.

    PubMed

    Theodoraki, Maria A; Nillegoda, Nadinath B; Saini, Jagdeep; Caplan, Avrom J

    2012-07-06

    Quality control ubiquitin ligases promote degradation of misfolded proteins by the proteasome. If the capacity of the ubiquitin/proteasome system is exceeded, then misfolded proteins accumulate in aggregates that are cleared by the autophagic system. To identify components of the ubiquitin/proteasome system that protect against aggregation, we analyzed a GFP-tagged protein kinase, Ste11ΔN(K444R)-GFP, in yeast strains deleted for 14 different ubiquitin ligases. We show that deletion of almost all of these ligases affected the proteostatic balance in untreated cells such that Ste11ΔN(K444R)-GFP aggregation was changed significantly compared with the levels found in wild type cells. By contrast, aggregation was increased significantly in only six E3 deletion strains when Ste11ΔN(K444R)-GFP folding was impaired due to inhibition of the molecular chaperone Hsp90 with geldanamycin. The increase in aggregation of Ste11ΔN(K444R)-GFP due to deletion of UBR1 and UFD4 was partially suppressed by deletion of UBR2 due to up-regulation of Rpn4, which controls proteasome activity. Deletion of UBR1 in combination with LTN1, UFD4, or DOA10 led to a marked hypersensitivity to azetidine 2-carboxylic acid, suggesting some redundancy in the networks of quality control ubiquitin ligases. Finally, we show that Ubr1 promotes clearance of protein aggregates when the autophagic system is inactivated. These results provide insight into the mechanics by which ubiquitin ligases cooperate and provide feedback regulation in the clearance of misfolded proteins.

  20. Proteins with RNA Chaperone Activity: A World of Diverse Proteins with a Common Task—Impediment of RNA Misfolding

    PubMed Central

    Semrad, Katharina

    2011-01-01

    Proteins with RNA chaperone activity are ubiquitous proteins that play important roles in cellular mechanisms. They prevent RNA from misfolding by loosening misfolded structures without ATP consumption. RNA chaperone activity is studied in vitro and in vivo using oligonucleotide- or ribozyme-based assays. Due to their functional as well as structural diversity, a common chaperoning mechanism or universal motif has not yet been identified. A growing database of proteins with RNA chaperone activity has been established based on evaluation of chaperone activity via the described assays. Although the exact mechanism is not yet understood, it is more and more believed that disordered regions within proteins play an important role. This possible mechanism and which proteins were found to possess RNA chaperone activity are discussed here. PMID:21234377

  1. Shape matters: the complex relationship between aggregation and toxicity in protein-misfolding diseases.

    PubMed

    Ries, Heidrun Maja; Nussbaum-Krammer, Carmen

    2016-10-15

    A particular subgroup of protein-misfolding diseases, comprising Alzheimer's and Parkinson's disease, involves amyloidogenic proteins that can form alternative pathogenic conformations with a high tendency to self-assemble into oligomeric and fibrillar species. Although misfolded proteins have been clearly linked to disease, the exact nature of the toxic species remains highly controversial. Increasing evidence suggests that there is little correlation between the occurrence of macroscopic protein deposits and toxic phenotypes in affected cells and tissues. In this article, we recap amyloid aggregation pathways, describe prion-like propagation, elaborate on detrimental interactions of protein aggregates with the cellular protein quality control system and discuss why some aggregates are toxic, whereas others seem to be beneficial. On the basis of recent studies on prion strains, we reason that the specific aggregate conformation and the resulting individual interaction with the cellular environment might be the major determinant of toxicity. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  2. The ER stress sensor PERK luminal domain functions as a molecular chaperone to interact with misfolded proteins

    SciTech Connect

    Wang, Peng; Li, Jingzhi; Sha, Bingdong

    2016-11-29

    PERK is one of the major sensor proteins which can detect the protein-folding imbalance generated by endoplasmic reticulum (ER) stress. It remains unclear how the sensor protein PERK is activated by ER stress. It has been demonstrated that the PERK luminal domain can recognize and selectively interact with misfolded proteins but not native proteins. Moreover, the PERK luminal domain may function as a molecular chaperone to directly bind to and suppress the aggregation of a number of misfolded model proteins. The data strongly support the hypothesis that the PERK luminal domain can interact directly with misfolded proteins to induce ER stress signaling. To illustrate the mechanism by which the PERK luminal domain interacts with misfolded proteins, the crystal structure of the human PERK luminal domain was determined to 3.2 Å resolution. Two dimers of the PERK luminal domain constitute a tetramer in the asymmetric unit. Superimposition of the PERK luminal domain molecules indicated that the β-sandwich domain could adopt multiple conformations. It is hypothesized that the PERK luminal domain may utilize its flexible β-sandwich domain to recognize and interact with a broad range of misfolded proteins.

  3. The ER stress sensor PERK luminal domain functions as a molecular chaperone to interact with misfolded proteins.

    PubMed

    Wang, Peng; Li, Jingzhi; Sha, Bingdong

    2016-12-01

    PERK is one of the major sensor proteins which can detect the protein-folding imbalance generated by endoplasmic reticulum (ER) stress. It remains unclear how the sensor protein PERK is activated by ER stress. It has been demonstrated that the PERK luminal domain can recognize and selectively interact with misfolded proteins but not native proteins. Moreover, the PERK luminal domain may function as a molecular chaperone to directly bind to and suppress the aggregation of a number of misfolded model proteins. The data strongly support the hypothesis that the PERK luminal domain can interact directly with misfolded proteins to induce ER stress signaling. To illustrate the mechanism by which the PERK luminal domain interacts with misfolded proteins, the crystal structure of the human PERK luminal domain was determined to 3.2 Å resolution. Two dimers of the PERK luminal domain constitute a tetramer in the asymmetric unit. Superimposition of the PERK luminal domain molecules indicated that the β-sandwich domain could adopt multiple conformations. It is hypothesized that the PERK luminal domain may utilize its flexible β-sandwich domain to recognize and interact with a broad range of misfolded proteins.

  4. A mechanistic insight into protein-ligand interaction, folding, misfolding, aggregation and inhibition of protein aggregates: An overview.

    PubMed

    Chandel, Tajalli Ilm; Zaman, Masihuz; Khan, Mohsin Vahid; Ali, Maroof; Rabbani, Gulam; Ishtikhar, Mohd; Khan, Rizwan Hasan

    2017-09-08

    This review article summarises the possible mechanisms of the protein-ligand interaction, folding, misfolding, aggregation and inhibition of protein aggregates. Under certain stressed condition the folding process deviates from its path and results into misfolding and aggregation of proteins. So aggregates have to be inhibited in order to cure the diseases. In some cases of protein-ligand interaction studies we have seen that the interaction of a protein with more than one ligand may show both type of quenching mechanisms i.e. dynamic as well as static quenching rather than single type of quenching mechanism, that result can be entirely different by the result of binding study utilising single ligand. So, likewise it is hypothesized that if the aggregates are inhibited by using more than one inhibitor may give more fruitful results rather than application of single inhibitor in inhibition and disaggregation of the preformed aggregates. Therefore, we have hypothesized mechanisms for the inhibition of protein aggregates that may assist in curing the neurodegenerative diseases. Thus, besides the mechanism of protein-ligand interaction, folding, misfolding and aggregation; the hypothesized mechanisms for the inhibition of protein aggregates may show new route to researchers either directly or indirectly in treating the diseases. Copyright © 2017. Published by Elsevier B.V.

  5. Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases

    PubMed Central

    Park, Hyun-Joo; Tailor, Chetankumar; Che, Clare; Kamani, Mustafa; Spitalny, George; Binnington, Beth

    2016-01-01

    Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon), to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin) containing genetically inactivated (± an N-terminal polyleucine tail) A subunit can, within 2–4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF) mutant (5-10x), F508delCFTR Golgi maturation (<10x), cell surface expression (20x) and chloride transport (2x) in F508del CFTR transfected cells and patient-derived F508delCFTR bronchiolar epithelia, without apparent cytopathology. These toxoids also increase glucocerobrosidase (GCC) in N370SGCC Gaucher Disease fibroblasts (3x), another ERAD–exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases. PMID:27935997

  6. Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases.

    PubMed

    Adnan, Humaira; Zhang, Zhenbo; Park, Hyun-Joo; Tailor, Chetankumar; Che, Clare; Kamani, Mustafa; Spitalny, George; Binnington, Beth; Lingwood, Clifford

    2016-01-01

    Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon), to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin) containing genetically inactivated (± an N-terminal polyleucine tail) A subunit can, within 2-4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF) mutant (5-10x), F508delCFTR Golgi maturation (<10x), cell surface expression (20x) and chloride transport (2x) in F508del CFTR transfected cells and patient-derived F508delCFTR bronchiolar epithelia, without apparent cytopathology. These toxoids also increase glucocerobrosidase (GCC) in N370SGCC Gaucher Disease fibroblasts (3x), another ERAD-exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases.

  7. Region-specific protein misfolding cyclic amplification reproduces brain tropism of prion strains.

    PubMed

    Privat, Nicolas; Levavasseur, Etienne; Yildirim, Serfildan; Hannaoui, Samia; Brandel, Jean-Philippe; Laplanche, Jean-Louis; Béringue, Vincent; Seilhean, Danielle; Haïk, Stéphane

    2017-10-06

    Human prion diseases such as Creutzfeldt-Jakob disease are transmissible brain proteinopathies, characterized by the accumulation of a misfolded isoform of the host cellular prion protein (PrP) in the brain. According to the prion model, prions are defined as proteinaceous infectious particles composed solely of this abnormal isoform of PrP (PrP(Sc)). Even in the absence of genetic material, various prion strains can be propagated in experimental models. They can be distinguished by the pattern of disease they produce and especially by the localization of PrP(Sc) deposits within the brain and the spongiform lesions they induce. The mechanisms involved in this strain-specific targeting of distinct brain regions still are a fundamental, unresolved question in prion research. To address this question, we exploited a prion conversion in vitro assay, protein misfolding cyclic amplification (PMCA), by using experimental scrapie and human prion strains as seeds and specific brain regions from mice and humans as substrates. We show here that region-specific PMCA in part reproduces the specific brain targeting observed in experimental, acquired, and sporadic Creutzfeldt-Jakob diseases. Furthermore, we provide evidence that, in addition to cellular prion protein, other region- and species-specific molecular factors influence the strain-dependent prion conversion process. This important step toward understanding prion strain propagation in the human brain may impact research on the molecular factors involved in protein misfolding and the development of ultrasensitive methods for diagnosing prion disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Protein misfolding and the pathogenesis of ABCA4-associated retinal degenerations

    PubMed Central

    Zhang, Ning; Tsybovsky, Yaroslav; Kolesnikov, Alexander V.; Rozanowska, Malgorzata; Swider, Malgorzata; Schwartz, Sharon B.; Stone, Edwin M.; Palczewska, Grazyna; Maeda, Akiko; Kefalov, Vladimir J.; Jacobson, Samuel G.; Cideciyan, Artur V.; Palczewski, Krzysztof

    2015-01-01

    Mutations in the ABCA4 gene are a common cause of autosomal recessive retinal degeneration. All mouse models to date are based on knockouts of Abca4, even though the disease is often caused by missense mutations such as the complex allele L541P;A1038V (PV). We now show that the PV mutation causes severe human disease whereas the V mutation alone causes mild disease. Mutant ABCA4 proteins expressed heterologously in mammalian cells retained normal cellular localization. However, basal and all-trans-retinal-stimulated ATPase activities were reduced substantially for P and PV but only mildly for V. Electron microscopy revealed marked structural changes and misfolding for the P and PV mutants but few changes for the V mutant, consistent with the disease severity difference in patients. We generated Abca4PV/PV knock-in mice homozygous for the complex PV allele to investigate the effects of this misfolding mutation in vivo. Mutant ABCA4 RNA levels approximated WT ABCA4 RNA levels but, surprisingly, only trace amounts of mutant ABCA4 protein were noted in the retina. RNA sequencing of WT, Abca4−/− and Abca4PV/PV mice revealed mild gene expression alterations in the retina and RPE. Similar to Abca4−/− mice, Abca4PV/PV mice showed substantial A2E and lipofuscin accumulation in their RPE cells but no retinal degeneration up to 12 months of age. Thus, rapid degradation of this large misfolded mutant protein in mouse retina caused little detectable photoreceptor degeneration. These findings suggest likely differences in the unfolded protein response between murine and human photoreceptors and support development of therapies directed at increasing this capability in patients. PMID:25712131

  9. Molecular chaperones are nanomachines that catalytically unfold misfolded and alternatively folded proteins.

    PubMed

    Mattoo, Rayees U H; Goloubinoff, Pierre

    2014-09-01

    By virtue of their general ability to bind (hold) translocating or unfolding polypeptides otherwise doomed to aggregate, molecular chaperones are commonly dubbed "holdases". Yet, chaperones also carry physiological functions that do not necessitate prevention of aggregation, such as altering the native states of proteins, as in the disassembly of SNARE complexes and clathrin coats. To carry such physiological functions, major members of the Hsp70, Hsp110, Hsp100, and Hsp60/CCT chaperone families act as catalytic unfolding enzymes or unfoldases that drive iterative cycles of protein binding, unfolding/pulling, and release. One unfoldase chaperone may thus successively convert many misfolded or alternatively folded polypeptide substrates into transiently unfolded intermediates, which, once released, can spontaneously refold into low-affinity native products. Whereas during stress, a large excess of non-catalytic chaperones in holding mode may optimally prevent protein aggregation, after the stress, catalytic disaggregases and unfoldases may act as nanomachines that use the energy of ATP hydrolysis to repair proteins with compromised conformations. Thus, holding and catalytic unfolding chaperones can act as primary cellular defenses against the formation of early misfolded and aggregated proteotoxic conformers in order to avert or retard the onset of degenerative protein conformational diseases.

  10. Applying chaperones to protein-misfolding disorders: molecular chaperones against α-synuclein in Parkinson's disease.

    PubMed

    Chaari, Ali; Hoarau-Véchot, Jessica; Ladjimi, Moncef

    2013-09-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of a protein called α-synuclein (α-syn) into inclusions known as lewy bodies (LB) within neurons. This accumulation is also due to insufficient formation and activity of dopamine produced in certain neurons within the substantia nigra. Lewy bodies are the pathological hallmark of the idiopathic disorder and the cascade that allows α-synuclein to misfold, aggregate and form these inclusions has been the subject of intensive research. Targeting these early steps of oligomerization is one of the main therapeutic approaches in order to develop neurodegenerative-modifying agents. Because the folding and refolding of alpha synuclein is the key point of this cascade, we are interested in this review to summarize the role of some molecular chaperones proteins such as Hsp70, Hsp90 and small heat shock proteins (sHsp) and Hsp 104. Hsp70 and its co-chaperone, Hsp70 and small heat shock proteins can prevent neurodegeneration by preventing α-syn misfolding, oligomerization and aggregation in vitro and in Parkinson disease animal models. Hsp104 is able to resolve disordered protein aggregates and cross beta amyloid conformers. Together, these chaperones have a complementary effect and can be a target for therapeutic intervention in PD. Published by Elsevier B.V.

  11. Prion versus doppel protein misfolding: new insights from replica-exchange molecular dynamics simulations.

    PubMed

    Baillod, Pascal; Garrec, Julian; Tavernelli, Ivano; Rothlisberger, Ursula

    2013-11-26

    The doppel (Dpl) and prion (PrP) proteins share a very similar fold (three helices and two short β-strands), while they differ significantly in sequence (only 25% homologous) and in disease-related β-rich conformations that occur for PrP only. In a previous study [Baillod, P., et al. (2012) Biochemistry 51, 9891-9899], we investigated the misfolding and rare, β-rich folds of monomeric PrP with replica-exchange molecular dynamics (REMD) simulations. In the work presented here, we perform analogous simulations for Dpl with the aim of comparing the two systems and characterizing possible specificities of PrP for misfolding and amyloidogenesis. Our extensive simulations, which allow us to overcome high energy barriers via the REMD approach, sample several β-rich folds, some of which are stable at room temperature, for both proteins. Per residue secondary structure propensities reveal that novel β-sheets of Dpl and PrP are formed by amino acids belonging to the helices that are the least stable in the respective native structure, H1 for Dpl and H2 and H3 for PrP, in agreement with experimental data. Using a specific clustering method that allows discrimination against different β-strand arrangements, seven β-rich folds could be characterized for PrP and five for Dpl, which are clearly distinct and share only one single similar fold. A major difference between the two proteins is found in the free energy barriers leading to misfolded structures: they are approximately 3 times higher for Dpl than for PrP. This suggests that the difference in amyloidogenic behavior between PrP and Dpl might be due to kinetic reasons.

  12. Luminescent conjugated oligothiophenes: optical dyes for revealing pathological hallmarks of protein misfolding diseases

    NASA Astrophysics Data System (ADS)

    Hammarström, Per; Lindgren, Mikael; Nilsson, K. Peter R.

    2010-08-01

    Luminescent conjugated polymers (LCPs) have been frequently utilized for optical biosensors. The detection schemes of these sensors are employing the light harvesting properties or the conformation sensitive optical properties of the conjugated polymers. LCPs have been utilized as colorimetric and fluorescent sensing elements for the recording of biological processes. However, LCPs have several limitations for being used as real time in vivo imaging agents. In this regard, novel thiophene based molecular scaffolds, denoted luminescent conjugated oligothiophenes (LCOs) have been developed. These LCOs are chemically defined molecules having distinct side chain functionalizations and a precise number of thiophene units. Herein the utilization of LCOs as specific ligands for the pathological hallmarks underlying protein misfolding diseases, such as Alzheimer's disease, is described. The use of the conformation sensitive optical properties of the LCOs for spectral separation of these pathological entities in a diversity of in vitro, ex vivo or in vivo systems is demonstrated. The protein aggregates are easily identified due to the conformation-dependent emission profile from the LCOs and spectral assignment of protein aggregates can be obtained. Overall, these probes will offer practical research tools for studying protein misfolding diseases and facilitate the study of the molecular mechanism underlying these disorders.

  13. Epidemic spreading model to characterize misfolded proteins propagation in aging and associated neurodegenerative disorders.

    PubMed

    Iturria-Medina, Yasser; Sotero, Roberto C; Toussaint, Paule J; Evans, Alan C

    2014-11-01

    Misfolded proteins (MP) are a key component in aging and associated neurodegenerative disorders. For example, misfolded Amyloid-ß (Aß) and tau proteins are two neuropathogenic hallmarks of Alzheimer's disease. Mechanisms underlying intra-brain MP propagation/deposition remain essentially uncharacterized. Here, is introduced an epidemic spreading model (ESM) for MP dynamics that considers propagation-like interactions between MP agents and the brain's clearance response across the structural connectome. The ESM reproduces advanced Aß deposition patterns in the human brain (explaining 46∼56% of the variance in regional Aß loads, in 733 subjects from the ADNI database). Furthermore, this model strongly supports a) the leading role of Aß clearance deficiency and early Aß onset age during Alzheimer's disease progression, b) that effective anatomical distance from Aß outbreak region explains regional Aß arrival time and Aß deposition likelihood, c) the multi-factorial impact of APOE e4 genotype, gender and educational level on lifetime intra-brain Aß propagation, and d) the modulatory impact of Aß propagation history on tau proteins concentrations, supporting the hypothesis of an interrelated pathway between Aß pathophysiology and tauopathy. To our knowledge, the ESM is the first computational model highlighting the direct link between structural brain networks, production/clearance of pathogenic proteins and associated intercellular transfer mechanisms, individual genetic/demographic properties and clinical states in health and disease. In sum, the proposed ESM constitutes a promising framework to clarify intra-brain region to region transference mechanisms associated with aging and neurodegenerative disorders.

  14. Implications of 3D domain swapping for protein folding, misfolding and function.

    PubMed

    Rousseau, Frederic; Schymkowitz, Joost; Itzhaki, Laura S

    2012-01-01

    Three-dimensional domain swapping is the process by which two identical protein chains exchange a part of their structure to form an intertwined dimer or higher-order oligomer. The phenomenon has been observed in the crystal structures of a range of different proteins. In this chapter we review the experiments that have been performed in order to understand the sequence and structural determinants of domain-swapping and these show how the general principles obtained can be used to engineer proteins to domain swap. We discuss the role of domain swapping in regulating protein function and as one possible mechanism of protein misfolding that can lead to aggregation and disease. We also review a number of interesting pathways of macromolecular assembly involving β-strand insertion or complementation that are related to the domain-swapping phenomenon.

  15. Kinetic modelling indicates that fast-translating codons can coordinate cotranslational protein folding by avoiding misfolded intermediates

    NASA Astrophysics Data System (ADS)

    O'Brien, Edward P.; Vendruscolo, Michele; Dobson, Christopher M.

    2014-01-01

    It has been observed for several proteins that slowing down the rate at which individual codons are translated can increase their probability of cotranslational protein folding, while speeding up codon translation can decrease it. Here we investigate whether or not this inverse relationship between translation speed and the cotranslational folding probability is a general phenomenon or if other scenarios are possible. We first derive chemical kinetic equations that relate individual codon translation rates to the probability that a domain will fold, populate an intermediate or misfold, and examine the cotranslational folding scenarios that are possible within these models. We find that speeding up codon translation through misfolding-prone segments can, in some cases, increase the folding probability of a domain immediately before the nascent protein is released from the ribosome and decrease its chances of misfolding. Thus, for some proteins fast-translating codons could be as important as slow-translating codons in coordinating cotranslational protein folding.

  16. Model systems of protein-misfolding diseases reveal chaperone modifiers of proteotoxicity

    PubMed Central

    2016-01-01

    ABSTRACT Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases. PMID:27491084

  17. Model systems of protein-misfolding diseases reveal chaperone modifiers of proteotoxicity.

    PubMed

    Brehme, Marc; Voisine, Cindy

    2016-08-01

    Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases. © 2016. Published by The Company of Biologists Ltd.

  18. Protein misfolding and aggregation in Alzheimer's disease and type 2 diabetes mellitus.

    PubMed

    Ashraf, Ghulam M; Greig, Nigel H; Khan, Taqi A; Hassan, Iftekhar; Tabrez, Shams; Shakil, Shazi; Sheikh, Ishfaq A; Zaidi, Syed K; Akram, Mohammad; Jabir, Nasimudeen R; Firoz, Chelaprom K; Naeem, Aabgeena; Alhazza, Ibrahim M; Damanhouri, Ghazi A; Kamal, Mohammad A

    2014-01-01

    In general, proteins can only execute their various biological functions when they are appropriately folded. Their amino acid sequence encodes the relevant information required for correct three-dimensional folding, with or without the assistance of chaperones. The challenge associated with understanding protein folding is currently one of the most important aspects of the biological sciences. Misfolded protein intermediates form large polymers of unwanted aggregates and are involved in the pathogenesis of many human diseases, including Alzheimer's disease (AD) and Type 2 diabetes mellitus (T2DM). AD is one of the most prevalent neurological disorders and has worldwide impact; whereas T2DM is considered a metabolic disease that detrementally influences numerous organs, afflicts some 8% of the adult population, and shares many risk factors with AD. Research data indicates that there is a widespread conformational change in the proteins involved in AD and T2DM that form β-sheet like motifs. Although conformation of these β-sheets is common to many functional proteins, the transition from α-helix to β-sheet is a typical characteristic of amyloid deposits. Any abnormality in this transition results in protein aggregation and generation of insoluble fibrils. The abnormal and toxic proteins can interact with other native proteins and consequently catalyze their transition into the toxic state. Both AD and T2DM are prevalent in the aged population. AD is characterized by the accumulation of amyloid-β (Aβ) in brain, while T2DM is characterized by the deposition of islet amyloid polypeptide (IAPP, also known as amylin) within beta-cells of the pancreas. T2DM increases pathological angiogenesis and immature vascularisation. This also leads to chronic cerebral hypoperfusion, which results in dysfunction and degeneration of neuroglial cells. With an abundance of common mechanisms underpinning both disorders, a significant question that can be posed is whether T2DM leads

  19. Protein misfolding and aggregation in Alzheimer’s disease and Type 2 Diabetes Mellitus

    PubMed Central

    Ashraf, Ghulam Md; Greig, Nigel H.; Khan, Taqi Ahmad; Hassan, Iftekhar; Tabrez, Shams; Shakil, Shazi; Sheikh, Ishfaq Ahmed; Zaidi, Syed Kashif; Wali, Mohammad Akram; Jabir, Nasimudeen R.; Firoz, C.K.; Naeem, Aabgeena; Alhazza, Ibrahim M.; Damanhouri, Ghazi A.; Kamal, Mohammad Amjad

    2016-01-01

    In general, proteins can only execute their various biological functions when they are appropriately folded. Their amino acid sequence encodes the relevant information required for correct three-dimensional folding, with or without the assistance of chaperones. The challenge associated with understanding protein folding is currently one of the most important aspects of the biological sciences. Misfolded protein intermediates form large polymers of unwanted aggregates and are involved in the pathogenesis of many human diseases, including Alzheimer’s disease (AD) and Type 2 diabetes mellitus (T2DM). AD is one of the most prevalent neurological disorders and has worldwide impact; whereas T2DM is considered a metabolic disease that detrementally influences numerous organs, afflicts some 8% of the adult population, and shares many risk factors with AD. Research data indicates that there is a widespread conformational change in the proteins involved in AD and T2DM that form β-sheets like motifs. Although conformation of these β-sheets is common to many functional proteins, the transition from α-helix to β-sheet is a typical characteristic of amyloid deposits. Any abnormality in this transition results in protein aggregation and generation of insoluble fibrils. The abnormal and toxic proteins can interact with other native proteins and consequently catalyze their transition into the toxic state. Both AD and T2DM are prevalent in the aged population. AD is characterized by the accumulation of amyloid-β (Aβ) in brain, while T2DM is characterized by the deposition of islet amyloid polypeptide (IAPP, also known as amylin) within beta-cells of the pancreas. T2DM increases pathological angiogenesis and immature vascularisation. This also leads to chronic cerebral hypoperfusion, which results in dysfunction and degeneration of neuroglial cells. With an abundance of common mechanisms underpinning both disorders, a significant question that can be posed is whether T2DM

  20. Single-particle electron microscopy structure of UDP-glucose:glycoprotein glucosyltransferase suggests a selectivity mechanism for misfolded proteins.

    PubMed

    Calles-Garcia, Daniel; Yang, Meng; Soya, Naoto; Melero, Roberto; Ménade, Marie; Ito, Yukishige; Vargas, Javier; Lukacs, Gergely L; Kollman, Justin M; Kozlov, Guennadi; Gehring, Kalle

    2017-07-07

    The enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) mediates quality control of glycoproteins in the endoplasmic reticulum by attaching glucose to N-linked glycan of misfolded proteins. As a sensor, UGGT ensures that misfolded proteins are recognized by the lectin chaperones and do not leave the secretory pathway. The structure of UGGT and the mechanism of its selectivity for misfolded proteins have been unknown for 25 years. Here, we used negative-stain electron microscopy and small-angle X-ray scattering to determine the structure of UGGT from Drosophila melanogaster at 18-Å resolution. Three-dimensional reconstructions revealed a cage-like structure with a large central cavity. Particle classification revealed flexibility that precluded determination of a high-resolution structure. Introduction of biotinylation sites into a fungal UGGT expressed in Escherichia coli allowed identification of the catalytic and first thioredoxin-like domains. We also used hydrogen-deuterium exchange mass spectrometry to map the binding site of an accessory protein, Sep15, to the first thioredoxin-like domain. The UGGT structural features identified suggest that the central cavity contains the catalytic site and is lined with hydrophobic surfaces. This enhances the binding of misfolded substrates with exposed hydrophobic residues and excludes folded proteins with hydrophilic surfaces. In conclusion, we have determined the UGGT structure, which enabled us to develop a plausible functional model of the mechanism for UGGT's selectivity for misfolded glycoproteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Effect of fullerenol surface chemistry on nanoparticle binding-induced protein misfolding

    NASA Astrophysics Data System (ADS)

    Radic, Slaven; Nedumpully-Govindan, Praveen; Chen, Ran; Salonen, Emppu; Brown, Jared M.; Ke, Pu Chun; Ding, Feng

    2014-06-01

    Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation -- a common strategy for solubilizing and functionalizing fullerene -- on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and dynamics of ubiquitin. We found that all derivatives bound to the model protein. Specifically, the more hydrophilic nanoparticles with a higher number of hydroxyl groups bound to the surface of the protein via hydrogen bonds, which stabilized the protein without inducing large conformational changes in the protein structure. In contrast, fullerene derivatives with a smaller number of hydroxyl groups buried their hydrophobic surface inside the protein, thereby causing protein denaturation. Overall, our results revealed a distinct role of surface chemistry on nanoparticle-protein binding and binding-induced protein misfolding.Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation -- a common strategy for solubilizing and functionalizing fullerene -- on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and

  2. GCK-MODY diabetes as a protein misfolding disease: the mutation R275C promotes protein misfolding, self-association and cellular degradation.

    PubMed

    Negahdar, Maria; Aukrust, Ingvild; Molnes, Janne; Solheim, Marie H; Johansson, Bente B; Sagen, Jørn V; Dahl-Jørgensen, Knut; Kulkarni, Rohit N; Søvik, Oddmund; Flatmark, Torgeir; Njølstad, Pål R; Bjørkhaug, Lise

    2014-01-25

    GCK-MODY, dominantly inherited mild hyperglycemia, is associated with more than 600 mutations in the glucokinase gene. Different molecular mechanisms have been shown to explain GCK-MODY. Here, we report a Pakistani family harboring the glucokinase mutation c.823C>T (p.R275C). The recombinant and in cellulo expressed mutant pancreatic enzyme revealed slightly increased enzyme activity (kcat) and normal affinity for α-D-glucose, and resistance to limited proteolysis by trypsin comparable with wild-type. When stably expressed in HEK293 cells and MIN6 β-cells (at different levels), the mutant protein appeared misfolded and unstable with a propensity to form dimers and aggregates. Its degradation rate was increased, involving the lysosomal and proteasomal quality control systems. On mutation, a hydrogen bond between the R275 side-chain and the carbonyl oxygen of D267 is broken, destabilizing the F260-L271 loop structure and the protein. This promotes the formation of dimers/aggregates and suggests that an increased cellular degradation is the molecular mechanism by which R275C causes GCK-MODY. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  3. Identification and Structural Characterization of the Precursor Conformation of the Prion Protein which Directly Initiates Misfolding and Oligomerization.

    PubMed

    Moulick, Roumita; Udgaonkar, Jayant B

    2017-03-24

    To identify and structurally characterize the precursor conformation of the prion protein (PrP), from which misfolding and aggregation directly commence, has been a long-standing goal. Misfolding converts the α-helical, non-pathogenic functional form of PrP to pathogenic, β-structured oligomeric and amyloidogenic forms, which are the cause of prion diseases. Susceptibility to sporadic prion disease correlates well with the propensity of PrP to misfold to cytotoxic, proteinase K resistant oligomeric conformations at low pH. In this study, mutagenesis at the hydrophobic core of the mouse PrP has been shown to stabilize a monomeric unfolding intermediate (I), which is populated significantly at equilibrium at low pH. Importantly, the rate of formation of β-structured oligomers at low pH is found to correlate well with the extent to which this intermediate is populated. The misfolding process is limited by the dimerization of I, indicating that I is the monomeric precursor conformation that directly initiates misfolding. Structural and thermodynamic characterization by native-state hydrogen-deuterium exchange mass spectrometry studies indicate that the precursor conformation is a partially unfolded form of PrP that forms under misfolding-prone solvent conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Protein Misfolding in Prion and Prion-Like Diseases: Reconsidering a Required Role for Protein Loss-of-Function.

    PubMed

    Leighton, Patricia L A; Allison, W Ted

    2016-07-06

    Prion disease research has contributed much toward understanding other neurodegenerative diseases, including recent demonstrations that Alzheimer's disease (AD) and other neurodegenerative diseases are prion-like. Prion-like diseases involve the spread of degeneration between individuals and/or among cells or tissues via template directed misfolding, wherein misfolded protein conformers propagate disease by causing normal proteins to misfold. Here we use the premise that AD, amyotrophic lateral sclerosis, Huntington's disease, and other similar diseases are prion-like and ask: Can we apply knowledge gained from studies of these prion-like diseases to resolve debates about classical prion diseases? We focus on controversies about what role(s) protein loss-of-function might have in prion diseases because this has therapeutic implications, including for AD. We examine which loss-of-function events are recognizable in prion-like diseases by considering the normal functions of the proteins before their misfolding and aggregation. We then delineate scenarios wherein gain-of-function and/or loss-of-function would be necessary or sufficient for neurodegeneration. We consider roles of PrPC loss-of-function in prion diseases and in AD, and conclude that the conventional wisdom that prion diseases are 'toxic gain-of-function diseases' has limitations. While prion diseases certainly have required gain-of-function components, we propose that disease phenotypes are predominantly caused by deficits in the normal physiology of PrPC and its interaction partners as PrPC converts to PrPSc. In this model, gain-of-function serves mainly to spread disease, and loss-of-function directly mediates neuron dysfunction. We propose experiments and predictions to assess our conclusion. Further study on the normal physiological roles of these key proteins is warranted.

  5. Glycoform-independent prion conversion by highly efficient, cell-based, protein misfolding cyclic amplification

    PubMed Central

    Moudjou, Mohammed; Chapuis, Jérôme; Mekrouti, Mériem; Reine, Fabienne; Herzog, Laetitia; Sibille, Pierre; Laude, Hubert; Vilette, Didier; Andréoletti, Olivier; Rezaei, Human; Dron, Michel; Béringue, Vincent

    2016-01-01

    Prions are formed of misfolded assemblies (PrPSc) of the variably N-glycosylated cellular prion protein (PrPC). In infected species, prions replicate by seeding the conversion and polymerization of host PrPC. Distinct prion strains can be recognized, exhibiting defined PrPSc biochemical properties such as the glycotype and specific biological traits. While strain information is encoded within the conformation of PrPSc assemblies, the storage of the structural information and the molecular requirements for self-perpetuation remain uncertain. Here, we investigated the specific role of PrPC glycosylation status. First, we developed an efficient protein misfolding cyclic amplification method using cells expressing the PrPC species of interest as substrate. Applying the technique to PrPC glycosylation mutants expressing cells revealed that neither PrPC nor PrPSc glycoform stoichiometry was instrumental to PrPSc formation and strainness perpetuation. Our study supports the view that strain properties, including PrPSc glycotype are enciphered within PrPSc structural backbone, not in the attached glycans. PMID:27384922

  6. Glycoform-independent prion conversion by highly efficient, cell-based, protein misfolding cyclic amplification.

    PubMed

    Moudjou, Mohammed; Chapuis, Jérôme; Mekrouti, Mériem; Reine, Fabienne; Herzog, Laetitia; Sibille, Pierre; Laude, Hubert; Vilette, Didier; Andréoletti, Olivier; Rezaei, Human; Dron, Michel; Béringue, Vincent

    2016-07-07

    Prions are formed of misfolded assemblies (PrP(Sc)) of the variably N-glycosylated cellular prion protein (PrP(C)). In infected species, prions replicate by seeding the conversion and polymerization of host PrP(C). Distinct prion strains can be recognized, exhibiting defined PrP(Sc) biochemical properties such as the glycotype and specific biological traits. While strain information is encoded within the conformation of PrP(Sc) assemblies, the storage of the structural information and the molecular requirements for self-perpetuation remain uncertain. Here, we investigated the specific role of PrP(C) glycosylation status. First, we developed an efficient protein misfolding cyclic amplification method using cells expressing the PrP(C) species of interest as substrate. Applying the technique to PrP(C) glycosylation mutants expressing cells revealed that neither PrP(C) nor PrP(Sc) glycoform stoichiometry was instrumental to PrP(Sc) formation and strainness perpetuation. Our study supports the view that strain properties, including PrP(Sc) glycotype are enciphered within PrP(Sc) structural backbone, not in the attached glycans.

  7. Pathogenic mechanisms of prion protein, amyloid-β and α-synuclein misfolding: the prion concept and neurotoxicity of protein oligomers.

    PubMed

    Ugalde, Cathryn L; Finkelstein, David I; Lawson, Victoria A; Hill, Andrew F

    2016-10-01

    Proteinopathies represent a group of diseases characterized by the unregulated misfolding and aggregation of proteins. Accumulation of misfolded protein in the central nervous system (CNS) is associated with neurodegenerative diseases, such as the transmissible spongiform encephalopathies (or prion diseases), Alzheimer's disease, and the synucleinopathies (the most common of which is Parkinson's disease). Of these, the pathogenic mechanisms of prion diseases are particularly striking where the transmissible, causative agent of disease is the prion, or proteinaceous infectious particle. Prions are composed almost exclusively of PrP(Sc) ; a misfolded isoform of the normal cellular protein, PrP(C) , which is found accumulated in the CNS in disease. Today, mounting evidence suggests other aggregating proteins, such as amyloid-β (Aβ) and α-synuclein (α-syn), proteins associated with Alzheimer's disease and synucleinopathies, respectively, share similar biophysical and biochemical properties with PrP(Sc) that influences how they misfold, aggregate, and propagate in disease. In this regard, the definition of a 'prion' may ultimately expand to include other pathogenic proteins. Unifying knowledge of folded proteins may also reveal common mechanisms associated with other features of disease that are less understood, such as neurotoxicity. This review discusses the common features Aβ and α-syn share with PrP and neurotoxic mechanisms associated with these misfolded proteins. Several proteins are known to misfold and accumulate in the central nervous system causing a range of neurodegenerative diseases, such as Alzheimer's, Parkinson's, and the prion diseases. Prions are transmissible misfolded conformers of the prion protein, PrP, which seed further generation of infectious proteins. Similar effects have recently been observed in proteins associated with Alzheimer's disease and the synucleinopathies, leading to the proposition that the definition of a 'prion' may

  8. Distinguishing between sequential and nonsequentially folded proteins: implications for folding and misfolding.

    PubMed Central

    Tsai, C. J.; Maizel, J. V.; Nussinov, R.

    1999-01-01

    We describe here an algorithm for distinguishing sequential from nonsequentially folding proteins. Several experiments have recently suggested that most of the proteins that are synthesized in the eukaryotic cell may fold sequentially. This proposed folding mechanism in vivo is particularly advantageous to the organism. In the absence of chaperones, the probability that a sequentially folding protein will misfold is reduced significantly. The problem we address here is devising a procedure that would differentiate between the two types of folding patterns. Footprints of sequential folding may be found in structures where consecutive fragments of the chain interact with each other. In such cases, the folding complexity may be viewed as being lower. On the other hand, higher folding complexity suggests that at least a portion of the polypeptide backbone folds back upon itself to form three-dimensional (3D) interactions with noncontiguous portion(s) of the chain. Hence, we look at the mechanism of folding of the molecule via analysis of its complexity, that is, through the 3D interactions formed by contiguous segments on the polypeptide chain. To computationally splice the structure into consecutively interacting fragments, we either cut it into compact hydrophobic folding units or into a set of hypothetical, transient, highly populated, contiguous fragments ("building blocks" of the structure). In sequential folding, successive building blocks interact with each other from the amino to the carboxy terminus of the polypeptide chain. Consequently, the results of the parsing differentiate between sequentially vs. nonsequentially folded chains. The automated assessment of the folding complexity provides insight into both the likelihood of misfolding and the kinetic folding rate of the given protein. In terms of the funnel free energy landscape theory, a protein that truly follows the mechanism of sequential folding, in principle, encounters smoother free energy barriers

  9. Arabidopsis AtPARK13, Which Confers Thermotolerance, Targets Misfolded Proteins*

    PubMed Central

    Basak, Indranil; Pal, Ramavati; Patil, Ketan S.; Dunne, Aisling; Ho, Hsin-Pin; Lee, Sungsu; Peiris, Diluka; Maple-Grødem, Jodi; Odell, Mark; Chang, Emmanuel J.; Larsen, Jan Petter; Møller, Simon G.

    2014-01-01

    Mutations in HTRA2/Omi/PARK13 have been implicated in Parkinson disease (PD). PARK13 is a neuroprotective serine protease; however, little is known about how PARK13 confers stress protection and which protein targets are directly affected by PARK13. We have reported that Arabidopsis thaliana represents a complementary PD model, and here we demonstrate that AtPARK13, similar to human PARK13 (hPARK13), is a mitochondrial protease. We show that the expression/accumulation of AtPARK13 transcripts are induced by heat stress but not by other stress conditions, including oxidative stress and metals. Our data show that elevated levels of AtPARK13 confer thermotolerance in A. thaliana. Increased temperatures accelerate protein unfolding, and we demonstrate that although AtPARK13 can act on native protein substrates, unfolded proteins represent better AtPARK13 substrates. The results further show that AtPARK13 and hPARK13 can degrade the PD proteins α-synuclein (SNCA) and DJ-1/PARK7 directly, without autophagy involvement, and that misfolded SNCA and DJ-1 represent better substrates than their native counterparts. Comparative proteomic profiling revealed AtPARK13-mediated proteome changes, and we identified four proteins that show altered abundance in response to AtPARK13 overexpression and elevated temperatures. Our study not only suggests that AtPARK13 confers thermotolerance by degrading misfolded protein targets, but it also provides new insight into possible roles of this protease in neurodegeneration. PMID:24719325

  10. Heavy Metals and Metalloids As a Cause for Protein Misfolding and Aggregation

    PubMed Central

    Tamás, Markus J.; Sharma, Sandeep K.; Ibstedt, Sebastian; Jacobson, Therese; Christen, Philipp

    2014-01-01

    While the toxicity of metals and metalloids, like arsenic, cadmium, mercury, lead and chromium, is undisputed, the underlying molecular mechanisms are not entirely clear. General consensus holds that proteins are the prime targets; heavy metals interfere with the physiological activity of specific, particularly susceptible proteins, either by forming a complex with functional side chain groups or by displacing essential metal ions in metalloproteins. Recent studies have revealed an additional mode of metal action targeted at proteins in a non-native state; certain heavy metals and metalloids have been found to inhibit the in vitro refolding of chemically denatured proteins, to interfere with protein folding in vivo and to cause aggregation of nascent proteins in living cells. Apparently, unfolded proteins with motile backbone and side chains are considerably more prone to engage in stable, pluridentate metal complexes than native proteins with their well-defined 3D structure. By interfering with the folding process, heavy metal ions and metalloids profoundly affect protein homeostasis and cell viability. This review describes how heavy metals impede protein folding and promote protein aggregation, how cells regulate quality control systems to protect themselves from metal toxicity and how metals might contribute to protein misfolding disorders. PMID:24970215

  11. Heavy metals and metalloids as a cause for protein misfolding and aggregation.

    PubMed

    Tamás, Markus J; Sharma, Sandeep K; Ibstedt, Sebastian; Jacobson, Therese; Christen, Philipp

    2014-02-25

    While the toxicity of metals and metalloids, like arsenic, cadmium, mercury, lead and chromium, is undisputed, the underlying molecular mechanisms are not entirely clear. General consensus holds that proteins are the prime targets; heavy metals interfere with the physiological activity of specific, particularly susceptible proteins, either by forming a complex with functional side chain groups or by displacing essential metal ions in metalloproteins. Recent studies have revealed an additional mode of metal action targeted at proteins in a non-native state; certain heavy metals and metalloids have been found to inhibit the in vitro refolding of chemically denatured proteins, to interfere with protein folding in vivo and to cause aggregation of nascent proteins in living cells. Apparently, unfolded proteins with motile backbone and side chains are considerably more prone to engage in stable, pluridentate metal complexes than native proteins with their well-defined 3D structure. By interfering with the folding process, heavy metal ions and metalloids profoundly affect protein homeostasis and cell viability. This review describes how heavy metals impede protein folding and promote protein aggregation, how cells regulate quality control systems to protect themselves from metal toxicity and how metals might contribute to protein misfolding disorders.

  12. Techniques for Monitoring Protein Misfolding and Aggregation in Vitro and in Living Cells

    PubMed Central

    Gregoire, Simpson; Irwin, Jacob; Kwon, Inchan

    2012-01-01

    Protein misfolding and aggregation have been considered important in understanding many neurodegenerative diseases and recombinant biopharmaceutical production. Therefore, various traditional and modern techniques have been utilized to monitor protein aggregation in vitro and in living cells. Fibril formation, morphology and secondary structure content of amyloidogenic proteins in vitro have been monitored by molecular probes, TEM/AFM, and CD/FTIR analyses, respectively. Protein aggregation in living cells has been qualitatively or quantitatively monitored by numerous molecular folding reporters based on either fluorescent protein or enzyme. Aggregation of a target protein is directly correlated to the changes in fluorescence or enzyme activity of the folding reporter fused to the target protein, which allows non-invasive monitoring aggregation of the target protein in living cells. Advances in the techniques used to monitor protein aggregation in vitro and in living cells have greatly facilitated the understanding of the molecular mechanism of amyloidogenic protein aggregation associated with neurodegenerative diseases, optimizing culture conditions to reduce aggregation of biopharmaceuticals expressed in living cells, and screening of small molecule libraries in the search for protein aggregation inhibitors. PMID:23565019

  13. Mechanism of copper(II)-induced misfolding of Parkinson's disease protein.

    PubMed

    Rose, Frisco; Hodak, Miroslav; Bernholc, Jerzy

    2011-01-01

    α-synuclein (aS) is a natively unfolded pre-synaptic protein found in all Parkinson's disease patients as the major component of fibrillar plaques. Metal ions, and especially Cu(II), have been demonstrated to accelerate aggregation of aS into fibrillar plaques, the precursors to Lewy bodies. In this work, copper binding to aS is investigated by a combination of quantum and molecular mechanics simulations. Starting from the experimentally observed attachment site, several optimized structures of Cu-binding geometries are examined. The most energetically favorable attachment results in significant allosteric changes, making aS more susceptible to misfolding. Indeed, an inverse kinematics investigation of the configuration space uncovers a dynamically stable β-sheet conformation of Cu-aS that serves as a nucleation point for a second β-strand. Based on these findings, we propose an atomistic mechanism of copper-induced misfolding of aS as an initial event in the formation of Lewy bodies and thus in PD pathogenesis.

  14. Introducing a rigid loop structure from deer into mouse prion protein increases its propensity for misfolding in vitro.

    PubMed

    Kyle, Leah M; John, Theodore R; Schätzl, Hermann M; Lewis, Randolph V

    2013-01-01

    Prion diseases are fatal neurodegenerative disorders characterized by misfolding of the cellular prion protein (PrP(c)) into the disease-associated isoform (PrP(Sc)) that has increased β-sheet content and partial resistance to proteolytic digestion. Prion diseases from different mammalian species have varying propensities for transmission upon exposure of an uninfected host to the infectious agent. Chronic Wasting Disease (CWD) is a highly transmissible prion disease that affects free ranging and farmed populations of cervids including deer, elk and moose, as well as other mammals in experimental settings. The molecular mechanisms allowing CWD to maintain comparatively high transmission rates have not been determined. Previous work has identified a unique structural feature in cervid PrP, a rigid loop between β-sheet 2 and α-helix 2 on the surface of the protein. This study was designed to test the hypothesis that the rigid loop has a direct influence on the misfolding process. The rigid loop was introduced into murine PrP as the result of two amino acid substitutions: S170N and N174T. Wild-type and rigid loop murine PrP were expressed in E. coli and purified. Misfolding propensity was compared for the two proteins using biochemical techniques and cell free misfolding and conversion systems. Murine PrP with a rigid loop misfolded in cell free systems with greater propensity than wild type murine PrP. In a lipid-based conversion assay, rigid loop PrP converted to a PK resistant, aggregated isoform at lower concentrations than wild-type PrP. Using both proteins as substrates in real time quaking-induced conversion, rigid loop PrP adopted a misfolded isoform more readily than wild type PrP. Taken together, these findings may help explain the high transmission rates observed for CWD within cervids.

  15. Chemical and Biological Approaches for Adapting Proteostasis to Ameliorate Protein Misfolding and Aggregation Diseases–Progress and Prognosis

    PubMed Central

    Lindquist, Susan L.; Kelly, Jeffery W.

    2011-01-01

    Maintaining the proteome to preserve the health of an organism in the face of developmental changes, environmental insults, infectious diseases, and rigors of aging is a formidable task. The challenge is magnified by the inheritance of mutations that render individual proteins subject to misfolding and/or aggregation. Maintenance of the proteome requires the orchestration of protein synthesis, folding, degradation, and trafficking by highly conserved/deeply integrated cellular networks. In humans, no less than 2000 genes are involved. Stress sensors detect the misfolding and aggregation of proteins in specific organelles and respond by activating stress-responsive signaling pathways. These culminate in transcriptional and posttranscriptional programs that up-regulate the homeostatic mechanisms unique to that organelle. Proteostasis is also strongly influenced by the general properties of protein folding that are intrinsic to every proteome. These include the kinetics and thermodynamics of the folding, misfolding, and aggregation of individual proteins. We examine a growing body of evidence establishing that when cellular proteostasis goes awry, it can be reestablished by deliberate chemical and biological interventions. We start with approaches that employ chemicals or biological agents to enhance the general capacity of the proteostasis network. We then introduce chemical approaches to prevent the misfolding or aggregation of specific proteins through direct binding interactions. We finish with evidence that synergy is achieved with the combination of mechanistically distinct approaches to reestablish organismal proteostasis. PMID:21900404

  16. Chemical and biological approaches for adapting proteostasis to ameliorate protein misfolding and aggregation diseases: progress and prognosis.

    PubMed

    Lindquist, Susan L; Kelly, Jeffery W

    2011-12-01

    Maintaining the proteome to preserve the health of an organism in the face of developmental changes, environmental insults, infectious diseases, and rigors of aging is a formidable task. The challenge is magnified by the inheritance of mutations that render individual proteins subject to misfolding and/or aggregation. Maintenance of the proteome requires the orchestration of protein synthesis, folding, degradation, and trafficking by highly conserved/deeply integrated cellular networks. In humans, no less than 2000 genes are involved. Stress sensors detect the misfolding and aggregation of proteins in specific organelles and respond by activating stress-responsive signaling pathways. These culminate in transcriptional and posttranscriptional programs that up-regulate the homeostatic mechanisms unique to that organelle. Proteostasis is also strongly influenced by the general properties of protein folding that are intrinsic to every proteome. These include the kinetics and thermodynamics of the folding, misfolding, and aggregation of individual proteins. We examine a growing body of evidence establishing that when cellular proteostasis goes awry, it can be reestablished by deliberate chemical and biological interventions. We start with approaches that employ chemicals or biological agents to enhance the general capacity of the proteostasis network. We then introduce chemical approaches to prevent the misfolding or aggregation of specific proteins through direct binding interactions. We finish with evidence that synergy is achieved with the combination of mechanistically distinct approaches to reestablish organismal proteostasis.

  17. Protein misfolding in Dictyostelium: Using a freak of nature to gain insight into a universal problem.

    PubMed

    Malinovska, Liliana; Alberti, Simon

    2015-01-01

    Prion-like proteins can undergo conformational rearrangements from an intrinsically disordered to a highly ordered amyloid state. This ability to change conformation is encoded in distinctive domains, termed prion domains (PrDs). Previous work suggests that PrDs change conformation to affect protein function and create phenotypic diversity. More recent work shows that PrDs can also undergo many weak interactions when disordered, allowing them to organize the intracellular space into dynamic compartments. However, mutations within PrDs and altered aggregation properties have also been linked to age-related diseases in humans. Thus, the physiological role of prion-like proteins, the mechanisms regulating their conformational promiscuity and the links to disease are still unclear. Here, we summarize recent work with prion-like proteins in Dictyostelium discoideum. This work was motivated by the finding that D. discoideum has the highest content of prion-like proteins of all organisms investigated to date. Surprisingly, we find that endogenous and exogenous prion-like proteins remain soluble in D. discoideum and do not misfold and aggregate. We provide evidence that this is due to specific adaptations in the protein quality control machinery, which may allow D. discoideum to tolerate its highly aggregation-prone proteome. We predict that D. discoideum will be an important model to study the function of prion-like proteins and their mechanistic links to disease.

  18. Characterization of protein quality control components via dual reporter-containing misfolded cytosolic model substrates.

    PubMed

    Amm, Ingo; Kawan, Mona; Wolf, Dieter H

    2016-12-15

    Protein misfolding and protein aggregation are causes of severe diseases as neurodegenerative disorders, diabetes and cancer. Therefore, the cell has to constantly monitor the folding status of its proteome. Chaperones and components of the ubiquitin-proteasome system are key players in the cellular protein quality control process. In order to characterize components of the protein quality control system in a well-established model eukaryote - the yeast Saccharomyces cerevisiae - we established new cytosolic model substrates based on firefly luciferase and β-isopropylmalate dehydrogenase (Leu2). The use of these two different enzymes arranged in tandem as reporters enabled us to analyse the folding status and the degradation propensity of these new model substrates in yeast cells mutated in components of the cellular protein quality control system. The Hsp70 chaperone system known to be essential in the cellular protein quality control was chosen as a model for showing the high value of the luciferase-based model substrates in the characterization of components of the cytosolic protein quality control system in yeast. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Different misfolding mechanisms converge on common conformational changes: human prion protein pathogenic mutants Y218N and E196K.

    PubMed

    Cheng, Chin Jung; Daggett, Valerie

    2014-01-01

    Prion diseases are caused by misfolding and aggregation of the prion protein (PrP). Pathogenic mutations such as Y218N and E196K are known to cause Gerstmann-Sträussler-Scheinker syndrome and Creutzfeldt-Jakob disease, respectively. Here we describe molecular dynamics simulations of these mutant proteins to better characterize the detailed conformational effects of these sequence substitutions. Our results indicate that the mutations disrupt the wild-type native PrP(C) structure and cause misfolding. Y218N reduced hydrophobic packing around the X-loop (residues 165-171), and E196K abolished an important wild-type salt bridge. While differences in the mutation site led PrP mutants to misfold along different pathways, we observed multiple traits of misfolding that were common to both mutants. Common traits of misfolding included: 1) detachment of the short helix (HA) from the PrP core; 2) exposure of side chain F198; and 3) formation of a nonnative strand at the N-terminus. The effect of the E196K mutation directly abolished the wild-type salt bridge E196-R156, which further destabilized the F198 hydrophobic pocket and HA. The Y218N mutation propagated its effect by increasing the HB-HC interhelical angle, which in turn disrupted the packing around F198. Furthermore, a nonnative contact formed between E221 and S132 on the S1-HA loop, which offered a direct mechanism for disrupting the hydrophobic packing between the S1-HA loop and HC. While there were common misfolding features shared between Y218N and E196K, the differences in the orientation of HB and HC and the X-loop conformation might provide a structural basis for identifying different prion strains.

  20. Structural effects of multiple pathogenic mutations suggest a model for the initiation of misfolding of the prion protein.

    PubMed

    Singh, Jogender; Udgaonkar, Jayant B

    2015-06-22

    A molecular understanding of the prion diseases requires delineation of the origin of misfolding of the prion protein (PrP). An understanding of how different disease-linked mutations affect the structure and dynamics of native monomeric PrP can provide a clue about how misfolding commences. In this study, hydrogen-deuterium exchange mass spectrometry was used to show that several disease-linked mutant variants, which are thermodynamically destabilized, share a common structural perturbation in their native states: helix 1 is destabilized to an extent that correlates well with the destabilization of the native protein. The mutant variants misfold and form oligomers faster than does the wild-type protein, at rates that increase exponentially with the extent to which helix 1 is destabilized in the native protein. It appears, therefore, that the loss of helix 1 structure marks the beginning of PrP misfolding and oligomerization. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Protein misfolding cyclic amplification induces the conversion of recombinant prion protein to PrP oligomers causing neuronal apoptosis.

    PubMed

    Yuan, Zhen; Yang, Lifeng; Chen, Baian; Zhu, Ting; Hassan, Mohammad Farooque; Yin, Xiaomin; Zhou, Xiangmei; Zhao, Deming

    2015-06-01

    The formation of neurotoxic prion protein (PrP) oligomers is thought to be a key step in the development of prion diseases. Recently, it was determined that the sonication and shaking of recombinant PrP can convert PrP monomers into β-state oligomers. Herein, we demonstrate that β-state oligomeric PrP can be generated through protein misfolding cyclic amplification from recombinant full-length hamster, human, rabbit, and mutated rabbit PrP, and that these oligomers can be used for subsequent research into the mechanisms of PrP-induced neurotoxicity. We have characterized protein misfolding cyclic amplification-induced monomer-to-oligomer conversion of PrP from three species using western blotting, circular dichroism, size-exclusion chromatography, and resistance to proteinase K (PK) digestion. We have further shown that all of the resulting β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrP(C) in the neurons, whereas the corresponding monomeric PrP were not toxic. In addition, we found that this toxicity is the result of oligomer-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3 in both wild-type and PrP(-/-) cortical neurons. It is our hope that these results may contribute to our understanding of prion transformation within the brain. We found that β-state oligomeric PrPs can be generated through protein misfolding cyclic amplification (PMCA) from recombinant full-length hamster, human, rabbit, and mutated rabbit PrP. β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrP(C) in the neurons, while the corresponding monomeric PrPs were not toxic. This toxicity is the result of oligomers-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3. These results may contribute to our understanding of prion transformation within the brain. © 2015 International Society for Neurochemistry.

  2. Highly efficient amplification of chronic wasting disease agent by protein misfolding cyclical amplification with beads (PMCAb)

    USGS Publications Warehouse

    Johnson, Chad J.; Aiken, Judd M.; McKenzie, Debbie; Samuel, Michael D.; Pedersen, Joel A.

    2012-01-01

    Protein misfolding cyclic amplification (PMCA) has emerged as an important technique for detecting low levels of pathogenic prion protein in biological samples. The method exploits the ability of the pathogenic prion protein to convert the normal prion protein to a proteinase K-resistant conformation. Inclusion of Teflon® beads in the PMCA reaction (PMCAb) has been previously shown to increase the sensitivity and robustness of detection for the 263 K and SSLOW strains of hamster-adapted prions. Here, we demonstrate that PMCAb with saponin dramatically increases the sensitivity of detection for chronic wasting disease (CWD) agent without compromising the specificity of the assay (i.e., no false positive results). Addition of Teflon® beads increased the robustness of the PMCA reaction, resulting in a decrease in the variability of PMCA results. Three rounds of serial PMCAb allowed detection of CWD agent from a 6.7×10−13 dilution of 10% brain homogenate (1.3 fg of source brain). Titration of the same brain homogenate in transgenic mice expressing cervid prion protein (Tg(CerPrP)1536+/−mice) allowed detection of CWD agent from the 10−6 dilution of 10% brain homogenate. PMCAb is, thus, more sensitive than bioassay in transgenic mice by a factor exceeding 105. Additionally, we are able to amplify CWD agent from brain tissue and lymph nodes of CWD-positive white-tailed deer having Prnp alleles associated with reduced disease susceptibility.

  3. Highly Efficient Amplification of Chronic Wasting Disease Agent by Protein Misfolding Cyclic Amplification with Beads (PMCAb)

    PubMed Central

    Johnson, Chad J.; Aiken, Judd M.; McKenzie, Debbie; Samuel, Michael D.; Pedersen, Joel A.

    2012-01-01

    Protein misfolding cyclic amplification (PMCA) has emerged as an important technique for detecting low levels of pathogenic prion protein in biological samples. The method exploits the ability of the pathogenic prion protein to convert the normal prion protein to a proteinase K-resistant conformation. Inclusion of Teflon® beads in the PMCA reaction (PMCAb) has been previously shown to increase the sensitivity and robustness of detection for the 263 K and SSLOW strains of hamster-adapted prions. Here, we demonstrate that PMCAb with saponin dramatically increases the sensitivity of detection for chronic wasting disease (CWD) agent without compromising the specificity of the assay (i.e., no false positive results). Addition of Teflon® beads increased the robustness of the PMCA reaction, resulting in a decrease in the variability of PMCA results. Three rounds of serial PMCAb allowed detection of CWD agent from a 6.7×10−13 dilution of 10% brain homogenate (1.3 fg of source brain). Titration of the same brain homogenate in transgenic mice expressing cervid prion protein (Tg(CerPrP)1536+/− mice) allowed detection of CWD agent from the 10−6 dilution of 10% brain homogenate. PMCAb is, thus, more sensitive than bioassay in transgenic mice by a factor exceeding 105. Additionally, we are able to amplify CWD agent from brain tissue and lymph nodes of CWD-positive white-tailed deer having Prnp alleles associated with reduced disease susceptibility. PMID:22514738

  4. Conformational analysis of misfolded protein aggregation by FRET and live-cell imaging techniques.

    PubMed

    Kitamura, Akira; Nagata, Kazuhiro; Kinjo, Masataka

    2015-03-16

    Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster/fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and bimolecular fluorescence complementation (BiFC) have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes), and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis.

  5. IRE1 directs proteasomal and lysosomal degradation of misfolded rhodopsin

    PubMed Central

    Chiang, Wei-Chieh; Messah, Carissa; Lin, Jonathan H.

    2012-01-01

    Endoplasmic reticulum (ER) is responsible for folding of secreted and membrane proteins in eukaryotic cells. Disruption of ER protein folding leads to ER stress. Chronic ER stress can cause cell death and is proposed to underlie the pathogenesis of many human diseases. Inositol-requiring enzyme 1 (IRE1) directs a key unfolded protein response signaling pathway that controls the fidelity of ER protein folding. IRE1 signaling may be particularly helpful in preventing chronic ER stress and cell injury by alleviating protein misfolding in the ER. To examine this, we used a chemical-genetic approach to selectively activate IRE1 in mammalian cells and tested how artificial IRE1 signaling affected the fate of misfolded P23H rhodopsin linked to photoreceptor cell death. We found that IRE1 signaling robustly promoted the degradation of misfolded P23H rhodopsin without affecting its wild-type counterpart. We also found that IRE1 used both proteasomal and lysosomal degradation pathways to remove P23H rhodopsin. Surprisingly, when one degradation pathway was compromised, IRE1 signaling could still promote misfolded rhodopsin degradation using the remaining pathway. Last, we showed that IRE1 signaling also reduced levels of several other misfolded rhodopsins with lesser effects on misfolded cystic fibrosis transmembrane conductance regulator. Our findings reveal the diversity of proteolytic mechanisms used by IRE1 to eliminate misfolded rhodopsin. PMID:22219383

  6. Inactivation of template-directed misfolding of infectious prion protein by ozone.

    PubMed

    Ding, Ning; Neumann, Norman F; Price, Luke M; Braithwaite, Shannon L; Balachandran, Aru; Belosevic, Miodrag; El-Din, Mohamed Gamal

    2012-02-01

    Misfolded prions (PrP(Sc)) are well known for their resistance to conventional decontamination processes. The potential risk of contamination of the water environment, as a result of disposal of specified risk materials (SRM), has raised public concerns. Ozone is commonly utilized in the water industry for inactivation of microbial contaminants and was tested in this study for its ability to inactivate prions (263K hamster scrapie = PrP(Sc)). Treatment variables included initial ozone dose (7.6 to 25.7 mg/liter), contact time (5 s and 5 min), temperature (4°C and 20°C), and pH (pH 4.4, 6.0, and 8.0). Exposure of dilute suspensions of the infected 263K hamster brain homogenates (IBH) (0.01%) to ozone resulted in the in vitro destruction of the templating properties of PrP(Sc), as measured by the protein misfolding cyclic amplification (PMCA) assay. The highest levels of prion inactivation (≥4 log(10)) were observed with ozone doses of 13.0 mg/liter, at pH 4.4 and 20°C, resulting in a CT (the product of residual ozone concentration and contact time) value as low as 0.59 mg · liter(-1) min. A comparison of ozone CT requirements among various pathogens suggests that prions are more susceptible to ozone degradation than some model bacteria and protozoa and that ozone treatment may be an effective solution for inactivating prions in water and wastewater.

  7. Inactivation of Template-Directed Misfolding of Infectious Prion Protein by Ozone

    PubMed Central

    Ding, Ning; Price, Luke M.; Braithwaite, Shannon L.; Balachandran, Aru; Belosevic, Miodrag

    2012-01-01

    Misfolded prions (PrPSc) are well known for their resistance to conventional decontamination processes. The potential risk of contamination of the water environment, as a result of disposal of specified risk materials (SRM), has raised public concerns. Ozone is commonly utilized in the water industry for inactivation of microbial contaminants and was tested in this study for its ability to inactivate prions (263K hamster scrapie = PrPSc). Treatment variables included initial ozone dose (7.6 to 25.7 mg/liter), contact time (5 s and 5 min), temperature (4°C and 20°C), and pH (pH 4.4, 6.0, and 8.0). Exposure of dilute suspensions of the infected 263K hamster brain homogenates (IBH) (0.01%) to ozone resulted in the in vitro destruction of the templating properties of PrPSc, as measured by the protein misfolding cyclic amplification (PMCA) assay. The highest levels of prion inactivation (≥4 log10) were observed with ozone doses of 13.0 mg/liter, at pH 4.4 and 20°C, resulting in a CT (the product of residual ozone concentration and contact time) value as low as 0.59 mg · liter−1 min. A comparison of ozone CT requirements among various pathogens suggests that prions are more susceptible to ozone degradation than some model bacteria and protozoa and that ozone treatment may be an effective solution for inactivating prions in water and wastewater. PMID:22138993

  8. S-Nitrosylation and uncompetitive/fast off-rate (UFO) drug therapy in neurodegenerative disorders of protein misfolding.

    PubMed

    Nakamura, T; Lipton, S A

    2007-07-01

    Although activation of glutamate receptors is essential for normal brain function, excessive activity leads to a form of neurotoxicity known as excitotoxicity. Key mediators of excitotoxic damage include overactivation of N-methyl-D-aspartate (NMDA) receptors, resulting in excessive Ca(2+) influx with production of free radicals and other injurious pathways. Overproduction of free radical nitric oxide (NO) contributes to acute and chronic neurodegenerative disorders. NO can react with cysteine thiol groups to form S-nitrosothiols and thus change protein function. S-nitrosylation can result in neuroprotective or neurodestructive consequences depending on the protein involved. Many neurodegenerative diseases manifest conformational changes in proteins that result in misfolding and aggregation. Our recent studies have linked nitrosative stress to protein misfolding and neuronal cell death. Molecular chaperones - such as protein-disulfide isomerase, glucose-regulated protein 78, and heat-shock proteins - can provide neuroprotection by facilitating proper protein folding. Here, we review the effect of S-nitrosylation on protein function under excitotoxic conditions, and present evidence that NO contributes to degenerative conditions by S-nitrosylating-specific chaperones that would otherwise prevent accumulation of misfolded proteins and neuronal cell death. In contrast, we also review therapeutics that can abrogate excitotoxic damage by preventing excessive NMDA receptor activity, in part via S-nitrosylation of this receptor to curtail excessive activity.

  9. Toward therapeutic targets for SCA3: Insight into the role of Machado-Joseph disease protein ataxin-3 in misfolded proteins clearance.

    PubMed

    Li, Xiaoling; Liu, Hongmei; Fischhaber, Paula L; Tang, Tie-Shan

    2015-09-01

    Machado-Joseph disease (MJD, also known as spinocerebellar ataxia type 3, SCA3), an autosomal dominant neurological disorder, is caused by an abnormal expanded polyglutamine (polyQ) repeat in the ataxin-3 protein. The length of the expanded polyQ stretch correlates positively with the severity of the disease and inversely with the age at onset. To date, we cannot fully explain the mechanism underlying neurobiological abnormalities of this disease. Yet, accumulating reports have demonstrated the functions of ataxin-3 protein in the chaperone system, ubiquitin-proteasome system, and aggregation-autophagy, all of which suggest a role of ataxin-3 in the clearance of misfolded proteins. Notably, the SCA3 pathogenic form of ataxin-3 (ataxin-3(exp)) impairs the misfolded protein clearance via mechanisms that are either dependent or independent of its deubiquitinase (DUB) activity, resulting in the accumulation of misfolded proteins and the progressive loss of neurons in SCA3. Some drugs, which have been used as activators/inducers in the chaperone system, ubiquitin-proteasome system, and aggregation-autophagy, have been demonstrated to be efficacious in the relief of neurodegeneration diseases like Huntington's disease (HD), Parkinson's (PD), Alzheimer's (AD) as well as SCA3 in animal models and clinical trials, putting misfolded protein clearance on the list of potential therapeutic targets. Here, we undertake a comprehensive review of the progress in understanding the physiological functions of ataxin-3 in misfolded protein clearance and how the polyQ expansion impairs misfolded protein clearance. We then detail the preclinical studies targeting the elimination of misfolded proteins for SCA3 treatment. We close with future considerations for translating these pre-clinical results into therapies for SCA3 patients. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Small heat shock proteins sequester misfolding proteins in near-native conformation for cellular protection and efficient refolding

    PubMed Central

    Ungelenk, Sophia; Moayed, Fatemeh; Ho, Chi-Ting; Grousl, Tomas; Scharf, Annette; Mashaghi, Alireza; Tans, Sander; Mayer, Matthias P.; Mogk, Axel; Bukau, Bernd

    2016-01-01

    Small heat shock proteins (sHsp) constitute an evolutionary conserved yet diverse family of chaperones acting as first line of defence against proteotoxic stress. sHsps coaggregate with misfolded proteins but the molecular basis and functional implications of these interactions, as well as potential sHsp specific differences, are poorly explored. In a comparative analysis of the two yeast sHsps, Hsp26 and Hsp42, we show in vitro that model substrates retain near-native state and are kept physically separated when complexed with either sHsp, while being completely unfolded when aggregated without sHsps. Hsp42 acts as aggregase to promote protein aggregation and specifically ensures cellular fitness during heat stress. Hsp26 in contrast lacks aggregase function but is superior in facilitating Hsp70/Hsp100-dependent post-stress refolding. Our findings indicate the sHsps of a cell functionally diversify in stress defence, but share the working principle to promote sequestration of misfolding proteins for storage in native-like conformation. PMID:27901028

  11. Amyloidogenicity of p53: a hidden link between protein misfolding and cancer.

    PubMed

    Gong, Hao; Yang, Xin; Zhao, Yudan; Petersen, Robert B; Liu, Xinran; Liu, Yang; Huang, Kun

    2015-01-01

    Pathogenic aggregation is closely associated with various protein misfolding diseases such as type 2 diabetes mellitus and Alzheimer's disease. Amyloidogenic proteins that have a propensity to assemble into amyloid oligomers and fibrils form the aggregates. The tumor suppressor p53, a transcription factor that regulates the cell cycle and apoptosis, is also amyloidogenic. In tumor models, both wild type and mutant p53 proteins show aggregation kinetics and morphology similar to those of classical amyloidogenic proteins, such as β-amyloid peptide and α- synuclein. Wild type p53 loses its anticancer activity when it aggregates, while p53 mutants with enhanced amyloidogenicity show accelerated aggregation. So far, amyloidogenic p53 mutations have been implicated in more than ten different types of cancer, suggesting a connection between p53 aggregation and cancer. Therefore, inhibition of both inherent and mutation induced p53 aggregation may stabilize p53 in a functional conformation and provide a novel approach to cancer prevention and treatment. Here, we summarize recent findings on carcinogenic aggregation of wild type p53 and its clinical mutants, structure-dependent amyloidogenesis of p53, and several promising strategies based on inhibition of p53 aggregation are also discussed.

  12. The role of HSP70 and its co-chaperones in protein misfolding, aggregation and disease.

    PubMed

    Duncan, Emma J; Cheetham, Michael E; Chapple, J Paul; van der Spuy, Jacqueline

    2015-01-01

    Molecular chaperones and their associated co-chaperones are essential in health and disease as they are key facilitators of protein folding, quality control and function. In particular, the HSP70 molecular chaperone networks have been associated with neurodegenerative diseases caused by aberrant protein folding. The pathogenesis of these disorders usually includes the formation of deposits of misfolded, aggregated protein. HSP70 and its co-chaperones have been recognised as potent modulators of inclusion formation and cell survival in cellular and animal models of neurodegenerative disease. In has become evident that the HSP70 chaperone machine functions not only in folding, but also in proteasome mediated degradation of neurodegenerative disease proteins. Thus, there has been a great deal of interest in the potential manipulation of molecular chaperones as a therapeutic approach for many neurodegenerations. Furthermore, mutations in several HSP70 co-chaperones and putative co-chaperones have been identified as causing inherited neurodegenerative and cardiac disorders, directly linking the HSP70 chaperone system to human disease.

  13. Garbage on, garbage off: new insights into plasma membrane protein quality control.

    PubMed

    MacGurn, Jason A

    2014-08-01

    Maintenance of cellular protein quality - by restoring misfolded proteins to their native state and by targeting terminally misfolded or damaged proteins for degradation - is a critical function of all cells. To ensure protein quality, cells have evolved various organelle-specific quality control mechanisms responsible for recognizing and responding to misfolded proteins at different subcellular locations of the cell. Recently, several publications have begun to elucidate mechanisms of quality control that operate at the plasma membrane (PM), recognizing misfolded PM proteins and targeting their endocytic trafficking and lysosomal degradation. Here, I discuss these recent developments in our understanding of PM quality control mechanisms and how they relate to global protein quality control strategies in the cell.

  14. The Volumetric Diversity of Misfolded Prion Protein Oligomers Revealed by Pressure Dissociation*

    PubMed Central

    Torrent, Joan; Lange, Reinhard; Rezaei, Human

    2015-01-01

    Protein oligomerization has been associated with a wide range of diseases. High pressure approaches offer a powerful tool for deciphering the underlying molecular mechanisms by revealing volume changes associated with the misfolding and assembly reactions. We applied high pressure to induce conformational changes in three distinct β-sheet-rich oligomers of the prion protein PrP, a protein characterized by a variety of infectious quaternary structures that can propagate stably and faithfully and cause diseases with specific phenotypic traits. We show that pressure induces dissociation of the oligomers and leads to a lower volume monomeric PrP state that refolds into the native conformation after pressure release. By measuring the different pressure and temperature sensitivity of the tested PrP oligomers, we demonstrate significantly different void volumes in their quaternary structure. In addition, by focusing on the kinetic and energetic behavior of the pressure-induced dissociation of one specific PrP oligomer, we reveal a large negative activation volume and an increase in both apparent activation enthalpy and entropy. This suggests a transition state ensemble that is less structured and significantly more hydrated than the oligomeric state. Finally, we found that site-specific fluorescent labeling allows monitoring of the transient population of a kinetic intermediate in the dissociation reaction. Our results indicate that defects in atomic packing may deserve consideration as a new factor that influences differences between PrP assemblies and that could be relevant also for explaining the origin of prion strains. PMID:26126829

  15. Novel insight into streptozotocin-induced diabetic rats from the protein misfolding perspective.

    PubMed

    Leyva-García, Edgar; Lara-Martínez, Reyna; Morán-Zanabria, Liborio; Revilla-Monsalve, Cristina; Jiménez-García, Luis Felipe; Oviedo, Norma; Murata, Chiharu; Garrido-Magaña, Eulalia; Altamirano-Bustamante, Nelly F; Altamirano-Bustamante, Myriam M

    2017-09-14

    Protein folding is a process of self-assembly defined by the sequence of the amino acids of the protein involved. Additionally, proteins tend to unfold, misfold and aggregate due to both intrinsic and extrinsic causes. Human islet amyloid polypeptide (hIAPP) aggregation is an early step in diabetes mellitus. However, the aggregation of rat IAPP (rIAPP) remains an open question. Adult female Sprague-Dawley rats weighing 150-250 g were divided into two groups. The experimental group (streptozotocin [STZ]) (n = 21) received an intraperitoneal injection of a single dose of 40 mg/kg STZ. We used the mouse anti-IAPP antibody and the anti-amyloid oligomer antibody to study the temporal course of rIAPP oligomerization during STZ-induced diabetes using a wide array of methods, strategies and ideas derived from biochemistry, cell biology, and proteomic medicine. Here, we demonstrated the tendency of rIAPP to aggregate and trigger cooperative processes of self-association or hetero-assembly that lead to the formation of amyloid oligomers (trimers and hexamers). Our results are the first to demonstrate the role of rIAPP amyloid oligomers in the development of STZ-induced diabetes in rats. The IAPP amyloid oligomers are biomarkers of the onset and progression of diabetes and could play a role as therapeutic targets.

  16. Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process*

    PubMed Central

    Tsai, Chai-jui; Aslam, Kiran; Drendel, Holli M.; Asiago, Josephat M.; Goode, Kourtney M.; Paul, Lake N.; Rochet, Jean-Christophe; Hazbun, Tony R.

    2015-01-01

    The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxic shift reprogramming and has glyoxalase activity. We show that substoichiometric concentrations of Hsp31 can abrogate aggregation of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of α-synuclein (αSyn), a target of the chaperone activity of human DJ-1, an Hsp31 homolog. We demonstrate that Hsp31 is able to suppress the in vitro fibrillization or aggregation of αSyn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of αSyn cytoplasmic foci, and yeast cells were rescued from αSyn-generated proteotoxicity upon Hsp31 overexpression. Moreover, we showed that Hsp31 protein levels are increased by H2O2, in the diauxic phase of normal growth conditions, and in cells under αSyn-mediated proteotoxic stress. We show that Hsp31 chaperone activity and not the methylglyoxalase activity or the autophagy pathway drives the protective effects. We also demonstrate reduced aggregation of the Sup35 prion domain, PrD-Sup35, as visualized by fluorescent protein fusions. In addition, Hsp31 acts on its substrates prior to the formation of large aggregates because Hsp31 does not mutually localize with prion aggregates, and it prevents the formation of detectable in vitro αSyn fibrils. These studies establish that the protective role of Hsp31 against cellular stress is achieved by chaperone activity that intervenes early in the protein misfolding process and is effective on a wide spectrum of substrate proteins, including αSyn and prion proteins. PMID:26306045

  17. Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process.

    PubMed

    Tsai, Chai-Jui; Aslam, Kiran; Drendel, Holli M; Asiago, Josephat M; Goode, Kourtney M; Paul, Lake N; Rochet, Jean-Christophe; Hazbun, Tony R

    2015-10-09

    The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxic shift reprogramming and has glyoxalase activity. We show that substoichiometric concentrations of Hsp31 can abrogate aggregation of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of α-synuclein (αSyn), a target of the chaperone activity of human DJ-1, an Hsp31 homolog. We demonstrate that Hsp31 is able to suppress the in vitro fibrillization or aggregation of αSyn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of αSyn cytoplasmic foci, and yeast cells were rescued from αSyn-generated proteotoxicity upon Hsp31 overexpression. Moreover, we showed that Hsp31 protein levels are increased by H2O2, in the diauxic phase of normal growth conditions, and in cells under αSyn-mediated proteotoxic stress. We show that Hsp31 chaperone activity and not the methylglyoxalase activity or the autophagy pathway drives the protective effects. We also demonstrate reduced aggregation of the Sup35 prion domain, PrD-Sup35, as visualized by fluorescent protein fusions. In addition, Hsp31 acts on its substrates prior to the formation of large aggregates because Hsp31 does not mutually localize with prion aggregates, and it prevents the formation of detectable in vitro αSyn fibrils. These studies establish that the protective role of Hsp31 against cellular stress is achieved by chaperone activity that intervenes early in the protein misfolding process and is effective on a wide spectrum of substrate proteins, including αSyn and prion proteins. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Functional characterization of NAT/NCS2 proteins of Aspergillus brasiliensis reveals a genuine xanthine-uric acid transporter and an intrinsically misfolded polypeptide.

    PubMed

    Krypotou, Emilia; Scazzocchio, Claudio; Diallinas, George

    2015-02-01

    The Nucleobase-Ascorbate Transporter (NAT) family includes members in nearly all domains of life. Functionally characterized NAT transporters from bacteria, fungi, plants and mammals are ion-coupled symporters specific for the uptake of purines, pyrimidines and related analogues. The characterized mammalian NATs are specific for the uptake of L-ascorbic acid. In this work we identify in silico a group of fungal putative transporters, named UapD-like proteins, which represent a novel NAT subfamily. To understand the function and specificity of UapD proteins, we cloned and functionally characterized the two Aspergillus brasiliensis NAT members (named AbUapC and AbUapD) by heterologous expression in Aspergillus nidulans. AbUapC represents canonical NATs (UapC or UapA), while AbUapD represents the new subfamily. AbUapC is a high-affinity, high-capacity, H(+)/xanthine-uric acid transporter, which can also recognize other purines with very low affinity. No apparent transport function could be detected for AbUapD. GFP-tagging showed that, unlike AbUapC which is localized in the plasma membrane, AbUapD is ER-retained and degraded in the vacuoles, a characteristic of misfolded proteins. Chimeric UapA/AbUapD molecules are also turned-over in the vacuole, suggesting that UapD includes intrinsic peptidic sequences leading to misfolding. The possible evolutionary implication of such conserved, but inactive proteins is discussed.

  19. Sequence-independent Control of Peptide Conformation in Liposomal Vaccines for Targeting Protein Misfolding Diseases*

    PubMed Central

    Hickman, David T.; López-Deber, María Pilar; Ndao, Dorin Mlaki; Silva, Alberto B.; Nand, Deepak; Pihlgren, Maria; Giriens, Valérie; Madani, Rime; St-Pierre, Annie; Karastaneva, Hristina; Nagel-Steger, Luitgard; Willbold, Dieter; Riesner, Detlev; Nicolau, Claude; Baldus, Marc; Pfeifer, Andrea; Muhs, Andreas

    2011-01-01

    Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule β-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing β-sheet aggregated lipopeptide (Palm1–15) induced polyclonal IgG antibodies that specifically recognized β-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease. PMID:21343310

  20. HIV-1 matrix protein p17 misfolding forms toxic amyloidogenic assemblies that induce neurocognitive disorders.

    PubMed

    Zeinolabediny, Yasmin; Caccuri, Francesca; Colombo, Laura; Morelli, Federica; Romeo, Margherita; Rossi, Alessandro; Schiarea, Silvia; Ciaramelli, Carlotta; Airoldi, Cristina; Weston, Ria; Donghui, Liu; Krupinski, Jerzy; Corpas, Rubén; García-Lara, Elisa; Sarroca, Sara; Sanfeliu, Coral; Slevin, Mark; Caruso, Arnaldo; Salmona, Mario; Diomede, Luisa

    2017-09-04

    Human immunodeficiency virus type-1 (HIV-1)-associated neurocognitive disorder (HAND) remains an important neurological manifestation that adversely affects a patient's quality of life. HIV-1 matrix protein p17 (p17) has been detected in autoptic brain tissue of HAND individuals who presented early with severe AIDS encephalopathy. We hypothesised that the ability of p17 to misfold may result in the generation of toxic assemblies in the brain and may be relevant for HAND pathogenesis. A multidisciplinary integrated approach has been applied to determine the ability of p17 to form soluble amyloidogenic assemblies in vitro. To provide new information into the potential pathogenic role of soluble p17 species in HAND, their toxicological capability was evaluated in vivo. In C. elegans, capable of recognising toxic assemblies of amyloidogenic proteins, p17 induces a specific toxic effect which can be counteracted by tetracyclines, drugs able to hinder the formation of large oligomers and consequently amyloid fibrils. The intrahippocampal injection of p17 in mice reduces their cognitive function and induces behavioral deficiencies. These findings offer a new way of thinking about the possible cause of neurodegeneration in HIV-1-seropositive patients, which engages the ability of p17 to form soluble toxic assemblies.

  1. Potential candidate camelid antibodies for the treatment of protein-misfolding diseases.

    PubMed

    David, Monique Antoinette; Jones, Daryl Rhys; Tayebi, Mourad

    2014-07-15

    Protein-misfolding diseases (PMDs), including Alzheimer's disease would potentially reach epidemic proportion if effective ways to diagnose and treat them were not developed. The quest for effective therapy for PMDs has been ongoing for decades and some of the technologies developed so far show great promise. We report here the development of antibodies by immunization of camelids with prion (PrioV3) and Alzheimer's (PrioAD12, 13 & 120) disease-derived brain material. We show that anti-PrP antibody transmigration across the blood-brain barrier (BBB) was inhibited with phosphatidylinositol-specific phospholipase C (PIPLC). Our camelid anti-prion antibody was also shown to permanently abrogate prion replication in a prion-permissive cell line after crossing the artificial BBB. Furthermore, anti-Aβ/tau antibodies were able to bind their specific immunogens with ELISA and immunohistochemistry. Finally, both PrioV3 and PrioAD12 were shown to co-localize with Lamp-1, a marker of late endosomal/lysosomal compartments. These antibodies could prove to be a valuable tool for the neutralization/clearance of PrP(Sc), Aβ and tau proteins in cellular compartments of affected neurons and could potentially have wider applicability for the treatment of PMDs. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. In Vitro Amplification of Misfolded Prion Protein Using Lysate of Cultured Cells

    PubMed Central

    Mays, Charles E.; Yeom, Jihyun; Kang, Hae-Eun; Bian, Jifeng; Khaychuk, Vadim; Kim, Younghwan; Bartz, Jason C.; Telling, Glenn C.; Ryou, Chongsuk

    2011-01-01

    Protein misfolding cyclic amplification (PMCA) recapitulates the prion protein (PrP) conversion process under cell-free conditions. PMCA was initially established with brain material and then with further simplified constituents such as partially purified and recombinant PrP. However, availability of brain material from some species or brain material from animals with certain mutations or polymorphisms within the PrP gene is often limited. Moreover, preparation of native PrP from mammalian cells and tissues, as well as recombinant PrP from bacterial cells, involves time-consuming purification steps. To establish a convenient and versatile PMCA procedure unrestricted to the availability of substrate sources, we attempted to conduct PMCA with the lysate of cells that express cellular PrP (PrPC). PrPSc was efficiently amplified with lysate of rabbit kidney epithelial RK13 cells stably transfected with the mouse or Syrian hamster PrP gene. Furthermore, PMCA was also successful with lysate of other established cell lines of neuronal or non-neuronal origins. Together with the data showing that the abundance of PrPC in cell lysate was a critical factor to drive efficient PrPSc amplification, our results demonstrate that cell lysate in which PrPC is present abundantly serves as an excellent substrate source for PMCA. PMID:21464935

  3. Temperature-Induced Misfolding in Prion Protein: Evidence of Multiple Partially Disordered States Stabilized by Non-Native Hydrogen Bonds.

    PubMed

    Chamachi, Neharika G; Chakrabarty, Suman

    2017-02-14

    The structural basis of pathways of misfolding of a cellular prion (PrP(C)) into the toxic scrapie form (PrP(SC)) and identification of possible intermediates (e.g., PrP*) still eludes us. In this work, we have used a cumulative ∼65 μs of replica exchange molecular dynamics simulation data to construct the conformational free energy landscapes and capture the structural and thermodynamic characteristics associated with various stages of the thermal denaturation process in human prion protein. The temperature-dependent free energy surfaces consist of multiple metastable states stabilized by non-native contacts and hydrogen bonds, thus rendering the protein prone to misfolding. We have been able to identify metastable conformational states with high β-content (∼30-40%) and low α-content (∼10-20%) that might be precursors of PrP(SC) oligomer formation. These conformations also involve participation of the unstructured N-terminal domain, and its role in misfolding has been investigated. All the misfolded or partially unfolded states are quite compact in nature despite having large deviations from the native structure. Although the number of native contacts decreases dramatically at higher temperatures, the radius of gyration and number of intraprotein hydrogen bonds and contacts remain relatively unchanged, leading to stabilization of the misfolded conformations by non-native interactions. Our results are in good agreement with the established view that the C-terminal regions of the second and third helices (H2 and H3, respectively) of mammal prions might be the Achilles heels of their stability, while separation of B1-H1-B2 and H2-H3 domains seems to play a key role, as well.

  4. Cavitation during the protein misfolding cyclic amplification (PMCA) method – The trigger for de novo prion generation?

    SciTech Connect

    Haigh, Cathryn L.; Drew, Simon C.

    2015-06-05

    The protein misfolding cyclic amplification (PMCA) technique has become a widely-adopted method for amplifying minute amounts of the infectious conformer of the prion protein (PrP). PMCA involves repeated cycles of 20 kHz sonication and incubation, during which the infectious conformer seeds the conversion of normally folded protein by a templating interaction. Recently, it has proved possible to create an infectious PrP conformer without the need for an infectious seed, by including RNA and the phospholipid POPG as essential cofactors during PMCA. The mechanism underpinning this de novo prion formation remains unknown. In this study, we first establish by spin trapping methods that cavitation bubbles formed during PMCA provide a radical-rich environment. Using a substrate preparation comparable to that employed in studies of de novo prion formation, we demonstrate by immuno-spin trapping that PrP- and RNA-centered radicals are generated during sonication, in addition to PrP-RNA cross-links. We further show that serial PMCA produces protease-resistant PrP that is oxidatively modified. We suggest a unique confluence of structural (membrane-mimetic hydrophobic/hydrophilic bubble interface) and chemical (ROS) effects underlie the phenomenon of de novo prion formation by PMCA, and that these effects have meaningful biological counterparts of possible relevance to spontaneous prion formation in vivo. - Highlights: • Sonication during PMCA generates free radicals at the surface of cavitation bubbles. • PrP-centered and RNA-centered radicals are formed in addition to PrP-RNA adducts. • De novo prions may result from ROS and structural constraints during cavitation.

  5. Transthyretin suppresses the toxicity of oligomers formed by misfolded proteins in vitro.

    PubMed

    Cascella, Roberta; Conti, Simona; Mannini, Benedetta; Li, Xinyi; Buxbaum, Joel N; Tiribilli, Bruno; Chiti, Fabrizio; Cecchi, Cristina

    2013-12-01

    Although human transthyretin (TTR) is associated with systemic amyloidoses, an anti-amyloidogenic effect that prevents Aβ fibril formation in vitro and in animal models has been observed. Here we studied the ability of three different types of TTR, namely human tetramers (hTTR), mouse tetramers (muTTR) and an engineered monomer of the human protein (M-TTR), to suppress the toxicity of oligomers formed by two different amyloidogenic peptides/proteins (HypF-N and Aβ42). muTTR is the most stable homotetramer, hTTR can dissociate into partially unfolded monomers, whereas M-TTR maintains a monomeric state. Preformed toxic HypF-N and Aβ42 oligomers were incubated in the presence of each TTR then added to cell culture media. hTTR, and to a greater extent M-TTR, were found to protect human neuroblastoma cells and rat primary neurons against oligomer-induced toxicity, whereas muTTR had no protective effect. The thioflavin T assay and site-directed labeling experiments using pyrene ruled out disaggregation and structural reorganization within the discrete oligomers following incubation with TTRs, while confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and hTTR, particularly M-TTR. Moreover, atomic force microscopy (AFM), light scattering and turbidimetry analyses indicated that larger assemblies of oligomers are formed in the presence of M-TTR and, to a lesser extent, with hTTR. Overall, the data suggest a generic capacity of TTR to efficiently neutralize the toxicity of oligomers formed by misfolded proteins and reveal that such neutralization occurs through a mechanism of TTR-mediated assembly of protein oligomers into larger species, with an efficiency that correlates inversely with TTR tetramer stability. © 2013.

  6. Protein folding and misfolding: a paradigm of self-assembly and regulation in complex biological systems.

    PubMed

    Vendruscolo, Michele; Zurdo, Jesús; MacPhee, Cait E; Dobson, Christopher M

    2003-06-15

    Understanding biological complexity is one of the grand scientific challenges for the future. A living organism is a highly evolved system made up of a large number of interwoven molecular networks. These networks primarily involve proteins, the macromolecules that enable and control virtually every chemical process that takes place in the cell. Proteins are also key elements in the essential characteristic of living systems, their ability to function and replicate themselves through controlled molecular interactions. Recent progress in understanding the most fundamental aspect of polypeptide self-organization, the process by which proteins fold to attain their active conformations, provides a global platform to gain knowledge about the function of biological systems and the regulatory mechanisms that underpin their ability to adapt to changing conditions. In order to exploit such progress effectively, we are developing a variety of approaches, including procedures that use experimental data to restrain the properties of complex systems in computer simulations, to describe their behaviour under a wide variety of conditions. We believe that such approaches can lead to significant advances in understanding biological complexity, in general, and protein folding and misfolding in particular. These advances would contribute to: a more effective exploitation of the information from genome sequences; more rational therapeutic approaches to diseases, particularly those associated with ageing; the responsible control of our own evolution; and the development of new technologies based on mimicking the principles of biological self-assembly, for instance in nanotechnology. More fundamentally, we believe that this research will result in a more coherent understanding of the origin, evolution and functional properties of living systems.

  7. α1-antitrypsin Deficiency: A Misfolded Secretory Protein Variant with Unique Effects on the Endoplasmic Reticulum

    PubMed Central

    Perlmutter, David H

    2016-01-01

    In the classical form of α1-antitrypsin deficiency (ATD) a point mutation leads to accumulation of a misfolded secretory glycoprotein in the endoplasmic reticulum (ER) of liver cells and so ATD has come to be considered a prototypical ER storage disease. It is associated with two major types of clinical disorders, chronic obstructive pulmonary disease (COPD) by loss-of-function mechanisms and hepatic cirrhosis and carcinogenesis by gain-of-function mechanisms. The lung disease predominantly results from proteolytic damage to the pulmonary connective tissue matrix because of reduced levels of protease inhibitor activity of α1-anitrypsin (AT) in the circulating blood and body fluids. Cigarette smoking is a powerful disease-promoting modifier but other modifiers are known to exist because variation in the lung disease phenotype is still found in smoking and non-smoking homozygotes. The liver disease is highly likely to be caused by the proteotoxic effects of intracellular misfolded protein accumulation and a high degree of variation in the hepatic phenotype among affected homozygotes has been hypothetically attributed to genetic and environmental modifiers that alter proteostasis responses. Liver biopsies of homozygotes show intrahepatocytic inclusions with dilation and expansion of the ER and recent studies of iPS-derived hepatocyte-like cells from individuals with ATD indicate that the changes in the ER directly vary with the hepatic phenotype i.e there is much lesser alteration in the ER in cells derived from homozygotes that do not have clinically significant liver disease. From a signaling perspective, studies in mammalian cell line and animal models expressing the classical α1-antitrypsin Z variant (ATZ) have found that ER signaling is perturbed in a relatively unique way with powerful activation of autophagy and the NFκB pathway but relatively limited, if any, UPR signaling. It is still not known how much these unique structural and functional changes and

  8. Hsp70-GlcNAc-binding activity is released by stress, proteasome inhibition, and protein misfolding

    SciTech Connect

    Guinez, Celine; Mir, Anne-Marie; Leroy, Yves; Cacan, Rene; Michalski, Jean-Claude; Lefebvre, Tony . E-mail: tony.lefebvre@univ-lille1.fr

    2007-09-21

    Numerous recent works strengthen the idea that the nuclear and cytosolic-specific O-GlcNAc glycosylation protects cells against injuries. We have first investigated O-GlcNAc level and Hsp70-GlcNAc-binding activity (HGBA) behaviour after exposure of HeLa and HepG{sub 2} cells to a wide variety of stresses. O-GlcNAc and HGBA responses were different according to the stress and according to the cell. HGBA was released for almost all stresses, while O-GlcNAc level was modified either upwards or downwards, depending to the stress. Against all expectations, we demonstrated that energy charge did not significantly vary with stress whereas UDP-GlcNAc pools were more dramatically affected even if differences in UDP-GlcNAc contents were not correlated with O-GlcNAc variations suggesting that O-GlcNAc transferase is itself finely regulated during cell injury. Finally, HGBA could be triggered by proteasome inhibition and by L-azetidine-2-carboxylic acid (a proline analogue) incorporation demonstrating that protein misfolding is one of the key-activator of this Hsp70 property.

  9. In vitro strain adaptation of CWD prions by serial protein misfolding cyclic amplification.

    PubMed

    Meyerett, Crystal; Michel, Brady; Pulford, Bruce; Spraker, Terry R; Nichols, Traci A; Johnson, Theodore; Kurt, Timothy; Hoover, Edward A; Telling, Glenn C; Zabel, Mark D

    2008-12-20

    We used serial protein misfolding cyclic amplification (sPMCA) to amplify the D10 strain of CWD prions in a linear relationship over two logs of D10 dilutions. The resultant PMCA-amplified D10 induced terminal TSE disease in CWD-susceptible Tg(cerPrP)1536 mice with a survival time approximately 80 days shorter than the original D10 inoculum, similar to that produced by in vivo sub-passage of D10 in Tg(cerPrP)1536 mice. Both in vitro-amplified and mouse-passaged D10 produced brain lesion profiles, glycoform ratios and conformational stabilities significantly different than those produced by the original D10 inoculum in Tg(cerPrP)1536 mice. These findings demonstrate that sPMCA can amplify and adapt prion strains in vitro as effectively and much more quickly than in vivo strain adaptation by mouse passage. Thus sPMCA may represent a powerful tool to assess prion strain adaptation and species barriers in vitro.

  10. Larger aggregates of mutant seipin in Celia's Encephalopathy, a new protein misfolding neurodegenerative disease.

    PubMed

    Ruiz-Riquelme, Alejandro; Sánchez-Iglesias, Sofía; Rábano, Alberto; Guillén-Navarro, Encarna; Domingo-Jiménez, Rosario; Ramos, Adriana; Rosa, Isaac; Senra, Ana; Nilsson, Peter; García, Ángel; Araújo-Vilar, David; Requena, Jesús R

    2015-11-01

    Celia's Encephalopathy (MIM #615924) is a recently discovered fatal neurodegenerative syndrome associated with a new BSCL2 mutation (c.985C>T) that results in an aberrant isoform of seipin (Celia seipin). This mutation is lethal in both homozygosity and compounded heterozygosity with a lipodystrophic BSCL2 mutation, resulting in a progressive encephalopathy with fatal outcomes at ages 6-8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to this mutation. Here we report new key insights about the molecular pathogenic mechanism of this new syndrome. Intranuclear inclusions containing mutant seipin were found in brain tissue from a homozygous patient suggesting a pathogenic mechanism similar to other neurodegenerative diseases featuring brain accumulation of aggregated, misfolded proteins. Sucrose gradient distribution showed that mutant seipin forms much larger aggregates as compared with wild type (wt) seipin, indicating an impaired oligomerization. On the other hand, the interaction between wt and Celia seipin confirmed by coimmunoprecipitation (CoIP) assays, together with the identification of mixed oligomers in sucrose gradient fractionation experiments can explain the lack of symptoms in heterozygous carriers. We propose that the increased aggregation and subsequent impaired oligomerization of Celia seipin leads to cell death. In heterozygous carriers, wt seipin might prevent the damage caused by mutant seipin through its sequestration into harmless mixed oligomers. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Prion formation, but not clearance, is supported by protein misfolding cyclic amplification.

    PubMed

    Shikiya, Ronald A; Eckland, Thomas E; Young, Alan J; Bartz, Jason C

    2014-01-01

    Prion diseases are fatal transmissible neurodegenerative disorders that affect animals including humans. The kinetics of prion infectivity and PrP(Sc) accumulation can differ between prion strains and within a single strain in different tissues. The net accumulation of PrP(Sc) in animals is controlled by the relationship between the rate of PrP(Sc) formation and clearance. Protein misfolding cyclic amplification (PMCA) is a powerful technique that faithfully recapitulates PrP(Sc) formation and prion infectivity in a cell-free system. PMCA has been used as a surrogate for animal bioassay and can model species barriers, host range, strain co-factors and strain interference. In this study we investigated if degradation of PrP(Sc) and/or prion infectivity occurs during PMCA. To accomplish this we performed PMCA under conditions that do not support PrP(Sc) formation and did not observe either a reduction in PrP(Sc) abundance or an extension of prion incubation period, compared to untreated control samples. These results indicate that prion clearance does not occur during PMCA. These data have significant implications for the interpretation of PMCA based experiments such as prion amplification rate, adaptation to new species and strain interference where production and clearance of prions can affect the outcome.

  12. Hyperefficient PrP Sc amplification of mouse-adapted BSE and scrapie strain by protein misfolding cyclic amplification technique.

    PubMed

    Fujihara, Aiko; Atarashi, Ryuichiro; Fuse, Takayuki; Ubagai, Kaori; Nakagaki, Takehiro; Yamaguchi, Naohiro; Ishibashi, Daisuke; Katamine, Shigeru; Nishida, Noriyuki

    2009-05-01

    Abnormal forms of prion protein (PrP(Sc)) accumulate via structural conversion of normal PrP (PrP(C)) in the progression of transmissible spongiform encephalopathy. Under cell-free conditions, the process can be efficiently replicated using in vitro PrP(Sc) amplification methods, including protein misfolding cyclic amplification. These methods enable ultrasensitive detection of PrP(Sc); however, there remain difficulties in utilizing them in practice. For example, to date, several rounds of protein misfolding cyclic amplification have been necessary to reach maximal sensitivity, which not only take several weeks, but also result in an increased risk of contamination. In this study, we sought to further promote the rate of PrP(Sc) amplification in the protein misfolding cyclic amplification technique using mouse transmissible spongiform encephalopathy models infected with either mouse-adapted bovine spongiform encephalopathy or mouse-adapted scrapie, Chandler strain. Here, we demonstrate that appropriate regulation of sonication dramatically accelerates PrP(Sc) amplification in both strains. In fact, we reached maximum sensitivity, allowing the ultrasensitive detection of < 1 LD(50) of PrP(Sc) in the diluted brain homogenates, after only one or two reaction rounds, and in addition, we detected PrP(Sc) in the plasma of mouse-adapted bovine spongiform encephalopathy-infected mice. We believe that these results will advance the establishment of a fast, ultrasensitive diagnostic test for transmissible spongiform encephalopathies.

  13. The Pathogenic Mutation T182A Converts the Prion Protein into a Molten Globule-like Conformation Whose Misfolding to Oligomers but Not to Fibrils Is Drastically Accelerated.

    PubMed

    Singh, Jogender; Udgaonkar, Jayant B

    2016-01-26

    Delineation of the effects of pathogenic mutations linked with familial prion diseases on the structure and misfolding of prion protein (PrP) will be useful in understanding the molecular mechanism of PrP misfolding. Here, it has been shown that the pathogenic mutation T182A causes a drastic reduction in the apparent cooperativity and enthalpy of unfolding of the mouse prion protein (moPrP) under misfolding-prone conditions by converting the protein into a molten globule (MG)-like conformation. Hydrogen-deuterium exchange studies in conjunction with mass spectrometry indicate that the T182A mutation disrupts the core of the protein, thereby increasing overall structural dynamics. T182A moPrP is shown to misfold to oligomers very much faster than does wild-type (wt) moPrP but to misfold to fibrils at a rate similar to that of wt moPrP. This observation suggests that oligomers are unlikely to play a productive role in the direct pathway of aggregation from monomer to fibrils. The observation that fully folded T182A moPrP has a MG-like structure, and that it misfolds to oligomers much faster than does wt moPrP, suggests that a MG-like intermediate, whose structure resembles that of fully folded T182A moPrP, might be populated early on the pathway of misfolding of wt moPrP to oligomers.

  14. The Volumetric Diversity of Misfolded Prion Protein Oligomers Revealed by Pressure Dissociation.

    PubMed

    Torrent, Joan; Lange, Reinhard; Rezaei, Human

    2015-08-14

    Protein oligomerization has been associated with a wide range of diseases. High pressure approaches offer a powerful tool for deciphering the underlying molecular mechanisms by revealing volume changes associated with the misfolding and assembly reactions. We applied high pressure to induce conformational changes in three distinct β-sheet-rich oligomers of the prion protein PrP, a protein characterized by a variety of infectious quaternary structures that can propagate stably and faithfully and cause diseases with specific phenotypic traits. We show that pressure induces dissociation of the oligomers and leads to a lower volume monomeric PrP state that refolds into the native conformation after pressure release. By measuring the different pressure and temperature sensitivity of the tested PrP oligomers, we demonstrate significantly different void volumes in their quaternary structure. In addition, by focusing on the kinetic and energetic behavior of the pressure-induced dissociation of one specific PrP oligomer, we reveal a large negative activation volume and an increase in both apparent activation enthalpy and entropy. This suggests a transition state ensemble that is less structured and significantly more hydrated than the oligomeric state. Finally, we found that site-specific fluorescent labeling allows monitoring of the transient population of a kinetic intermediate in the dissociation reaction. Our results indicate that defects in atomic packing may deserve consideration as a new factor that influences differences between PrP assemblies and that could be relevant also for explaining the origin of prion strains. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Infrared microspectroscopy detects protein misfolding cyclic amplification (PMCA)-induced conformational alterations in hamster scrapie progeny seeds.

    PubMed

    Daus, Martin L; Wagenführ, Katja; Thomzig, Achim; Boerner, Susann; Hermann, Peter; Hermelink, Antje; Beekes, Michael; Lasch, Peter

    2013-12-06

    The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP(C)) into Proteinase K-resistant, infectious PrP particles (PrP(TSE)), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP(C) substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP(C) substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.

  16. The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases.

    PubMed

    Schmitz, Matthias; Cramm, Maria; Llorens, Franc; Müller-Cramm, Dominik; Collins, Steven; Atarashi, Ryuichiro; Satoh, Katsuya; Orrù, Christina D; Groveman, Bradley R; Zafar, Saima; Schulz-Schaeffer, Walter J; Caughey, Byron; Zerr, Inga

    2016-11-01

    The development and adaption of in vitro misfolded protein amplification systems has been a major innovation in the detection of abnormally folded prion protein scrapie (PrP(Sc)) in human brain and cerebrospinal fluid (CSF) samples. Herein, we describe a fast and efficient protein amplification technique, real-time quaking-induced conversion (RT-QuIC), for the detection of a PrP(Sc) seed in human brain and CSF. In contrast to other in vitro misfolded protein amplification assays-such as protein misfolding cyclic amplification (PMCA)-which are based on sonication, the RT-QuIC technique is based on prion seed-induced misfolding and aggregation of recombinant prion protein substrate, accelerated by alternating cycles of shaking and rest in fluorescence plate readers. A single RT-QuIC assay typically analyzes up to 32 samples in triplicate, using a 96-well-plate format. From sample preparation to analysis of results, the protocol takes ∼87 h to complete. In addition to diagnostics, this technique has substantial generic analytical applications, including drug screening, prion strain discrimination, biohazard screening (e.g., to reduce transmission risk related to prion diseases) and the study of protein misfolding; in addition, it can potentially be used for the investigation of other protein misfolding diseases such as Alzheimer's and Parkinson's disease.

  17. Quality control of inner nuclear membrane proteins by the Asi complex.

    PubMed

    Foresti, Ombretta; Rodriguez-Vaello, Victoria; Funaya, Charlotta; Carvalho, Pedro

    2014-11-07

    Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a quality control system called ER-associated protein degradation (ERAD). However, it is unknown how misfolded proteins in the inner nuclear membrane (INM), a specialized ER subdomain, are degraded. We used a quantitative proteomics approach to reveal an ERAD branch required for INM protein quality control in yeast. This branch involved the integral membrane proteins Asi1, Asi2, and Asi3, which assembled into an Asi complex. Besides INM misfolded proteins, the Asi complex promoted the degradation of functional regulators of sterol biosynthesis. Asi-mediated ERAD was required for ER homeostasis, which suggests that spatial segregation of protein quality control systems contributes to ER function. Copyright © 2014, American Association for the Advancement of Science.

  18. The Azoarcus Group I Intron Ribozyme Misfolds and Is Accelerated for Refolding by ATP-dependent RNA Chaperone Proteins*

    PubMed Central

    Sinan, Selma; Yuan, Xiaoyan; Russell, Rick

    2011-01-01

    Structured RNAs traverse complex energy landscapes that include valleys representing misfolded intermediates. In Neurospora crassa and Saccharomyces cerevisiae, efficient splicing of mitochondrial group I and II introns requires the DEAD box proteins CYT-19 and Mss116p, respectively, which promote folding transitions and function as general RNA chaperones. To test the generality of RNA misfolding and the activities of DEAD box proteins in vitro, here we measure native folding of a small group I intron ribozyme from the bacterium Azoarcus by monitoring its catalytic activity. To develop this assay, we first measure cleavage of an oligonucleotide substrate by the prefolded ribozyme. Substrate cleavage is rate-limited by binding and is readily reversible, with an internal equilibrium near unity, such that the amount of product observed is less than the amount of native ribozyme. We use this assay to show that approximately half of the ribozyme folds readily to the native state, whereas the other half forms an intermediate that transitions slowly to the native state. This folding transition is accelerated by urea and increased temperature and slowed by increased Mg2+ concentration, suggesting that the intermediate is misfolded and must undergo transient unfolding during refolding to the native state. CYT-19 and Mss116p accelerate refolding in an ATP-dependent manner, presumably by disrupting structure in the intermediate. These results highlight the tendency of RNAs to misfold, underscore the roles of CYT-19 and Mss116p as general RNA chaperones, and identify a refolding transition for further dissection of the roles of DEAD box proteins in RNA folding. PMID:21878649

  19. A novel mutation in PNLIP causes pancreatic triglyceride lipase deficiency through protein misfolding.

    PubMed

    Szabó, András; Xiao, Xunjun; Haughney, Margaret; Spector, Alyssa; Sahin-Tóth, Miklós; Lowe, Mark E

    2015-07-01

    Congenital pancreatic triglyceride lipase (PNLIP) deficiency is a rare disorder with uncertain genetic background as most cases were described before gene sequencing was readily available. Recently, two brothers with PNLIP deficiency were found to carry a homozygous missense mutation, c.662C>T (p.T221M) in the PNLIP gene (J. Lipid Res. 2014. 55:307-312). Molecular modeling suggested the substitution would change the orientation of residues in the catalytic site and disrupt the function of p.T221M PNLIP. To test the effect of the p.T221M mutation on PNLIP function, we expressed wild-type and p.T221M PNLIP in human embryonic kidney (HEK) 293A cells and dexamethasone-differentiated AR42J rat acinar cells. In both cellular models, wild-type PNLIP was secreted into the conditioned medium where it was readily detectable by protein staining, immunoblot or lipase activity assays. In contrast, mutant p.T221M was not secreted into the medium, but it was present in cell lysates where it accumulated in the insoluble fraction. Intracellular retention of mutant p.T221M resulted in endoplasmic reticulum (ER) stress as measured by elevated XBP1 splicing and increased levels of ER chaperones. Our results demonstrate that the presence of methionine at position 221 in the PNLIP protein sequence causes misfolding and aggregation of the p.T221M mutant inside the cell. The consequent loss of enzyme secretion adequately explains the clinical phenotype of PNLIP deficiency reported for homozygous carriers of p.T221M. Furthermore, the ability of mutant p.T221M to induce ER stress suggests that this form of PNLIP deficiency might cause acinar cell damage as well. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

    NASA Astrophysics Data System (ADS)

    Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A.; Iyer, Pooja; Lane, Fiona M.; Graham, James F.; Goldmann, Wilfred; Pinheiro, Teresa J. T.; Gill, Andrew C.

    2015-10-01

    The β2-α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2-α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2-α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

  1. Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein.

    PubMed

    Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A; Iyer, Pooja; Lane, Fiona M; Graham, James F; Goldmann, Wilfred; Pinheiro, Teresa J T; Gill, Andrew C

    2015-10-22

    The β2-α2 loop of PrP(C) is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrP(C) appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2-α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that 'rigidity' in the β2-α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

  2. A current pharmacologic agent versus the promise of next generation therapeutics to ameliorate protein misfolding and/or aggregation diseases.

    PubMed

    Baranczak, Aleksandra; Kelly, Jeffery W

    2016-06-01

    The list of protein aggregation-associated degenerative diseases is long and growing, while the portfolio of disease-modifying strategies is very small. In this review and perspective, we assess what has worked to slow the progression of an aggregation-associated degenerative disease, covering the underlying mechanism of pharmacologic action and what we have learned about the etiology of the transthyretin amyloid diseases and likely amyloidoses in general. Next, we introduce emerging therapies that should apply more generally to protein misfolding and/or aggregation diseases, approaches that rely on adapting the protein homeostasis or proteostasis network for disease amelioration. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Fluorescence spectroscopy of protein oligomerization in membranes.

    PubMed

    Gorbenko, Galyna P

    2011-05-01

    Fluorescence spectroscopy is one of the most powerful tools for characterization of a multitude of biological processes. Of these, the phenomenon of protein oligomerization attracts especial interest due to its crucial role in the formation of fibrillar protein aggregates (amyloid fibrils) involved in ethiology of so-called protein misfolding diseases. It is becoming increasingly substantiated that protein fibrillization in vivo can be initiated and modulated at membrane-water interface. All steps of membrane-assisted fibrillogenesis, viz., protein adsorption onto lipid bilayer, structural transition of polypeptide chain into a highly aggregation-prone partially folded conformation, assembly of oligomeric nucleus from membrane-bound monomeric species and fiber elongation can be monitored with a mighty family of fluorescence-based techniques. Furthermore, the mechanisms behind cytotoxicity of prefibrillar protein oligomers are highly amenable to fluorescence analysis. The applications of fluorescence spectroscopy to monitoring protein oligomerization in a membrane environment are exemplified and some problems encountered in such kinds of studies are highlighted.

  4. Protein folding, misfolding and aggregation: The importance of two-electron stabilizing interactions

    PubMed Central

    2017-01-01

    Proteins associated with neurodegenerative diseases are highly pleiomorphic and may adopt an all-α-helical fold in one environment, assemble into all-β-sheet or collapse into a coil in another, and rapidly polymerize in yet another one via divergent aggregation pathways that yield broad diversity of aggregates’ morphology. A thorough understanding of this behaviour may be necessary to develop a treatment for Alzheimer’s and related disorders. Unfortunately, our present comprehension of folding and misfolding is limited for want of a physicochemical theory of protein secondary and tertiary structure. Here we demonstrate that electronic configuration and hyperconjugation of the peptide amide bonds ought to be taken into account to advance such a theory. To capture the effect of polarization of peptide linkages on conformational and H-bonding propensity of the polypeptide backbone, we introduce a function of shielding tensors of the Cα atoms. Carrying no information about side chain-side chain interactions, this function nonetheless identifies basic features of the secondary and tertiary structure, establishes sequence correlates of the metamorphic and pH-driven equilibria, relates binding affinities and folding rate constants to secondary structure preferences, and manifests common patterns of backbone density distribution in amyloidogenic regions of Alzheimer’s amyloid β and tau, Parkinson’s α-synuclein and prions. Based on those findings, a split-intein like mechanism of molecular recognition is proposed to underlie dimerization of Aβ, tau, αS and PrPC, and divergent pathways for subsequent association of dimers are outlined; a related mechanism is proposed to underlie formation of PrPSc fibrils. The model does account for: (i) structural features of paranuclei, off-pathway oligomers, non-fibrillar aggregates and fibrils; (ii) effects of incubation conditions, point mutations, isoform lengths, small-molecule assembly modulators and chirality of solid

  5. Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment

    PubMed Central

    Zhou, Minghai; Ottenberg, Gregory; Sferrazza, Gian Franco; Hubbs, Christopher; Fallahi, Mohammad; Rumbaugh, Gavin; Brantley, Alicia F.

    2015-01-01

    The mechanisms of neuronal death in protein misfolding neurodegenerative diseases such as Alzheimer’s, Parkinson’s and prion diseases are poorly understood. We used a highly toxic misfolded prion protein (TPrP) model to understand neurotoxicity induced by prion protein misfolding. We show that abnormal autophagy activation and neuronal demise is due to severe, neuron-specific, nicotinamide adenine dinucleotide (NAD+) depletion. Toxic prion protein-exposed neuronal cells exhibit dramatic reductions of intracellular NAD+ followed by decreased ATP production, and are completely rescued by treatment with NAD+ or its precursor nicotinamide because of restoration of physiological NAD+ levels. Toxic prion protein-induced NAD+ depletion results from PARP1-independent excessive protein ADP-ribosylations. In vivo, toxic prion protein-induced degeneration of hippocampal neurons is prevented dose-dependently by intracerebral injection of NAD+. Intranasal NAD+ treatment of prion-infected sick mice significantly improves activity and delays motor impairment. Our study reveals NAD+ starvation as a novel mechanism of autophagy activation and neurodegeneration induced by a misfolded amyloidogenic protein. We propose the development of NAD+ replenishment strategies for neuroprotection in prion diseases and possibly other protein misfolding neurodegenerative diseases. PMID:25678560

  6. Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment.

    PubMed

    Zhou, Minghai; Ottenberg, Gregory; Sferrazza, Gian Franco; Hubbs, Christopher; Fallahi, Mohammad; Rumbaugh, Gavin; Brantley, Alicia F; Lasmézas, Corinne I

    2015-04-01

    The mechanisms of neuronal death in protein misfolding neurodegenerative diseases such as Alzheimer's, Parkinson's and prion diseases are poorly understood. We used a highly toxic misfolded prion protein (TPrP) model to understand neurotoxicity induced by prion protein misfolding. We show that abnormal autophagy activation and neuronal demise is due to severe, neuron-specific, nicotinamide adenine dinucleotide (NAD(+)) depletion. Toxic prion protein-exposed neuronal cells exhibit dramatic reductions of intracellular NAD(+) followed by decreased ATP production, and are completely rescued by treatment with NAD(+) or its precursor nicotinamide because of restoration of physiological NAD(+) levels. Toxic prion protein-induced NAD(+) depletion results from PARP1-independent excessive protein ADP-ribosylations. In vivo, toxic prion protein-induced degeneration of hippocampal neurons is prevented dose-dependently by intracerebral injection of NAD(+). Intranasal NAD(+) treatment of prion-infected sick mice significantly improves activity and delays motor impairment. Our study reveals NAD(+) starvation as a novel mechanism of autophagy activation and neurodegeneration induced by a misfolded amyloidogenic protein. We propose the development of NAD(+) replenishment strategies for neuroprotection in prion diseases and possibly other protein misfolding neurodegenerative diseases.

  7. Structures of membrane proteins

    PubMed Central

    Vinothkumar, Kutti R.; Henderson, Richard

    2010-01-01

    In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class. PMID:20667175

  8. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  9. Caenorhabditis elegans as a model system for studying non-cell-autonomous mechanisms in protein-misfolding diseases.

    PubMed

    Nussbaum-Krammer, Carmen I; Morimoto, Richard I

    2014-01-01

    Caenorhabditis elegans has a number of distinct advantages that are useful for understanding the basis for cellular and organismal dysfunction underlying age-associated diseases of protein misfolding. Although protein aggregation, a key feature of human neurodegenerative diseases, has been typically explored in vivo at the single-cell level using cells in culture, there is now increasing evidence that proteotoxicity has a non-cell-autonomous component and is communicated between cells and tissues in a multicellular organism. These discoveries have opened up new avenues for the use of C. elegans as an ideal animal model system to study non-cell-autonomous proteotoxicity, prion-like propagation of aggregation-prone proteins, and the organismal regulation of stress responses and proteostasis. This Review focuses on recent evidence that C. elegans has mechanisms to transmit certain classes of toxic proteins between tissues and a complex stress response that integrates and coordinates signals from single cells and tissues across the organism. These findings emphasize the potential of C. elegans to provide insights into non-cell-autonomous proteotoxic mechanisms underlying age-related protein-misfolding diseases.

  10. Polypeptide models to understand misfolding and amyloidogenesis and their relevance in protein design and therapeutics.

    PubMed

    Zurdo, Jesús

    2005-02-01

    The study of amyloid polypeptide models (polypeptides able to generate amyloid structures not necessarily connected with any pathology) provides an excellent tool to increase the understanding of the generic aspects of misfolding and aggregation as well as the details of the mechanism of polypeptide deposition in disease. This knowledge can be integrated and applied to different problems in therapy and biotechnology, and in particular to re-designing bio-active polypeptides (biopharmaceuticals) with improved properties.

  11. Protein misfolding and dysregulated protein homeostasis in autoinflammatory diseases and beyond.

    PubMed

    Agyemang, Amma F; Harrison, Stephanie R; Siegel, Richard M; McDermott, Michael F

    2015-07-01

    Cells have a number of mechanisms to maintain protein homeostasis, including proteasome-mediated degradation of ubiquitinated proteins and autophagy, a regulated process of "self-eating" where the contents of entire organelles can be recycled for other uses. The unfolded protein response prevents protein overload in the secretory pathway. In the past decade, it has become clear that these fundamental cellular processes also help contain inflammation though degrading pro-inflammatory protein complexes such as the NLRP3 inflammasome. Signaling pathways such as the UPR can also be co-opted by toll-like receptor and mitochondrial reactive oxygen species signaling to induce inflammatory responses. Mutations that alter key inflammatory proteins, such as NLRP3 or TNFR1, can overcome normal protein homeostasis mechanisms, resulting in autoinflammatory diseases. Conversely, Mendelian defects in the proteasome cause protein accumulation, which can trigger interferon-dependent autoinflammatory disease. In non-Mendelian inflammatory diseases, polymorphisms in genes affecting the UPR or autophagy pathways can contribute to disease, and in diseases not formerly considered inflammatory such as neurodegenerative conditions and type 2 diabetes, there is increasing evidence that cell intrinsic or environmental alterations in protein homeostasis may contribute to pathogenesis.

  12. Diabetes and pancreatic exocrine dysfunction due to mutations in the carboxyl ester lipase gene-maturity onset diabetes of the young (CEL-MODY): a protein misfolding disease.

    PubMed

    Johansson, Bente B; Torsvik, Janniche; Bjørkhaug, Lise; Vesterhus, Mette; Ragvin, Anja; Tjora, Erling; Fjeld, Karianne; Hoem, Dag; Johansson, Stefan; Ræder, Helge; Lindquist, Susanne; Hernell, Olle; Cnop, Miriam; Saraste, Jaakko; Flatmark, Torgeir; Molven, Anders; Njølstad, Pål R

    2011-10-07

    CEL-maturity onset diabetes of the young (MODY), diabetes with pancreatic lipomatosis and exocrine dysfunction, is due to dominant frameshift mutations in the acinar cell carboxyl ester lipase gene (CEL). As Cel knock-out mice do not express the phenotype and the mutant protein has an altered and intrinsically disordered tandem repeat domain, we hypothesized that the disease mechanism might involve a negative effect of the mutant protein. In silico analysis showed that the pI of the tandem repeat was markedly increased from pH 3.3 in wild-type (WT) to 11.8 in mutant (MUT) human CEL. By stably overexpressing CEL-WT and CEL-MUT in HEK293 cells, we found similar glycosylation, ubiquitination, constitutive secretion, and quality control of the two proteins. The CEL-MUT protein demonstrated, however, a high propensity to form aggregates found intracellularly and extracellularly. Different physicochemical properties of the intrinsically disordered tandem repeat domains of WT and MUT proteins may contribute to different short and long range interactions with the globular core domain and other macromolecules, including cell membranes. Thus, we propose that CEL-MODY is a protein misfolding disease caused by a negative gain-of-function effect of the mutant proteins in pancreatic tissues.

  13. Why are proteins with glutamine- and asparagine-rich regions associated with protein misfolding diseases?

    NASA Astrophysics Data System (ADS)

    Cruzeiro, Leonor

    2005-12-01

    The possibility that vibrational excited states (VESs) are the drivers of protein folding and function (the VES hypothesis) is explored to explain the reason why Gln- and Asn-rich proteins are associated with degenerative diseases. The Davydov/Scott model is extended to describe energy transfer from the water solution to the protein and vice versa. Computer simulations show that, on average, Gln and Asn residues lead to an initial larger absorption of energy from the environment to the protein, something that can explain the greater structural instability of prions. The sporadic, inherited and infectious character of prion diseases is discussed in the light of the VES hypothesis. An alternative treatment for prion diseases is suggested.

  14. Integrated Organotypic Slice Cultures and RT-QuIC (OSCAR) Assay: Implications for Translational Discovery in Protein Misfolding Diseases

    PubMed Central

    Kondru, Naveen; Manne, Sireesha; Greenlee, Justin; West Greenlee, Heather; Anantharam, Vellareddy; Halbur, Patrick; Kanthasamy, Arthi; Kanthasamy, Anumantha

    2017-01-01

    Protein misfolding is a key pathological event in neurodegenerative diseases like prion diseases, synucleinopathies, and tauopathies that are collectively termed protein misfolding disorders. Prions are a prototypic model to study protein aggregation biology and therapeutic development. Attempts to develop anti-prion therapeutics have been impeded by the lack of screening models that faithfully replicate prion diseases and the lack of rapid, sensitive biological screening systems. Therefore, a sensitive model encompassing prion replication and neurotoxicity would be indispensable to the pursuit of intervention strategies. We present an ultra-sensitive screening system coupled to an ex vivo prion organotypic slice culture model to rapidly advance rationale-based high-throughput therapeutic strategies. This hybrid Organotypic Slice Culture Assay coupled with RT-QuIC (OSCAR) permits sensitive, specific and quantitative detection of prions from an infectious slice culture model on a reduced time scale. We demonstrate that the anti-prion activity of test compounds can be readily resolved based on the power and kinetics of seeding activity in the OSCAR screening platform and that the prions generated in slice cultures are biologically active. Collectively, our results imply that OSCAR is a robust model of prion diseases that offers a promising platform for understanding prion proteinopathies and advancing anti-prion therapeutics. PMID:28233859

  15. Integrated Organotypic Slice Cultures and RT-QuIC (OSCAR) Assay: Implications for Translational Discovery in Protein Misfolding Diseases.

    PubMed

    Kondru, Naveen; Manne, Sireesha; Greenlee, Justin; West Greenlee, Heather; Anantharam, Vellareddy; Halbur, Patrick; Kanthasamy, Arthi; Kanthasamy, Anumantha

    2017-02-24

    Protein misfolding is a key pathological event in neurodegenerative diseases like prion diseases, synucleinopathies, and tauopathies that are collectively termed protein misfolding disorders. Prions are a prototypic model to study protein aggregation biology and therapeutic development. Attempts to develop anti-prion therapeutics have been impeded by the lack of screening models that faithfully replicate prion diseases and the lack of rapid, sensitive biological screening systems. Therefore, a sensitive model encompassing prion replication and neurotoxicity would be indispensable to the pursuit of intervention strategies. We present an ultra-sensitive screening system coupled to an ex vivo prion organotypic slice culture model to rapidly advance rationale-based high-throughput therapeutic strategies. This hybrid Organotypic Slice Culture Assay coupled with RT-QuIC (OSCAR) permits sensitive, specific and quantitative detection of prions from an infectious slice culture model on a reduced time scale. We demonstrate that the anti-prion activity of test compounds can be readily resolved based on the power and kinetics of seeding activity in the OSCAR screening platform and that the prions generated in slice cultures are biologically active. Collectively, our results imply that OSCAR is a robust model of prion diseases that offers a promising platform for understanding prion proteinopathies and advancing anti-prion therapeutics.

  16. Misfolding of a bacterial autotransporter.

    PubMed

    Mogensen, Jesper E; Kleinschmidt, Jörg H; Schmidt, M Alexander; Otzen, Daniel E

    2005-11-01

    The adhesin involved in diffuse adherence (AIDA) is an autotransporter protein that confers the diffuse adherence phenotype to certain diarrheagenic Escherichia coli strains. It consists of a 49 amino acid signal peptide, a 797 amino acid passenger domain, and a 440 amino acid beta-domain integrated into the outer membrane. The beta-domain consists of two parts: the beta(1)-domain, which is predicted to form two beta-strands on the bacterial cell surface, and the beta(2)-domain, which constitutes the transmembrane domain. We have previously shown that the beta-domain can be folded from the urea-denatured state when bound to a nickel column during purification. It has not been possible to achieve proper refolding of the beta-domain in solution; instead, a misfolded state C is formed. Here, we characterize this misfolded state in greater detail, showing that despite being misfolded, C can be analyzed as a conventional conformational state, with cooperative unfolding in urea and SDS as well as showing simple exponential kinetics during its formation in the presence of lipid vesicles and detergent micelles. The kinetics of formation of C is sensitive to the lipid composition in vesicles. We have also attempted to identify biological factors that might aid folding of the beta-domain to the properly folded state. However, no purified periplasmic or cytosolic chaperone was found to increase folding yields, and no factor in a periplasmic extract was identified that could bind to C. We conclude that it is the exposure to the unique spatial arrangement of the bacterial cell that leads to proper refolding of the beta-domain.

  17. The Unfolded Protein Response and Chemical Chaperones Reduce Protein Misfolding and Colitis in Mice

    PubMed Central

    CAO, STEWART SIYAN; ZIMMERMANN, ELLEN M.; CHUANG, BRANDY–MENGCHIEH; SONG, BENBO; NWOKOYE, ANOSIKE; WILKINSON, J. ERBY; EATON, KATHRYN A.; KAUFMAN, RANDAL J.

    2013-01-01

    BACKGROUND & AIMS Endoplasmic reticulum (ER) stress has been associated with development of inflammatory bowel disease. We examined the effects of ER stress–induced chaperone response and the orally active chemical chaperones tauroursodeoxycholate (TUDCA) and 4-phenylbutyrate (PBA), which facilitate protein folding and reduce ER stress, in mice with colitis. METHODS We used dextran sulfate sodium (DSS) to induce colitis in mice that do not express the transcription factor ATF6α or the protein chaperone P58IPK. We examined the effects of TUDCA and PBA in cultured intestinal epithelial cells (IECs); in wild-type, P58IPK−/−, and Atf6α−/− mice with colitis; and in Il10−/− mice. RESULTS P58IPK−/− and Atf6α−/− mice developed more severe colitis following administration of DSS than wild-type mice. IECs from P58IPK−/− mice had excessive ER stress, and apoptotic signaling was activated in IECs from Atf6α−/− mice. Inflammatory stimuli induced ER stress signals in cultured IECs, which were reduced by incubation with TUDCA or PBA. Oral administration of either PBA or TUDCA reduced features of DSS-induced acute and chronic colitis in wild-type mice, the colitis that develops in Il10−/− mice, and DSS-induced colitis in P58IPK−/− and Atf6α−/− mice. Reduced signs of colonic inflammation in these mice were associated with significantly decreased ER stress in colonic epithelial cells. CONCLUSIONS The unfolded protein response induces expression of genes that encode chaperones involved in ER protein folding; these factors prevent induction of colitis in mice. Chemical chaperones such as TUDCA and PBA alleviate different forms of colitis in mice and might be developed for treatment of inflammatory bowel diseases. PMID:23336977

  18. Protein misfolding in the late-onset neurodegenerative diseases: common themes and the unique case of amyotrophic lateral sclerosis.

    PubMed

    Mulligan, Vikram Khipple; Chakrabartty, Avijit

    2013-08-01

    Enormous strides have been made in the last 100 years to extend human life expectancy and to combat the major infectious diseases. Today, the major challenges for medical science are age-related diseases, including cancer, heart disease, lung disease, renal disease, and late-onset neurodegenerative disease. Of these, only the neurodegenerative diseases represent a class of disease so poorly understood that no general strategies for prevention or treatment exist. These diseases, which include Alzheimer's disease, Parkinson's disease, Huntington's disease, the transmissible spongiform encephalopathies, and amyotrophic lateral sclerosis (ALS), are generally fatal and incurable. The first section of this review summarizes the diversity and common features of the late-onset neurodegenerative diseases, with a particular focus on protein misfolding and aggregation-a recurring theme in the molecular pathology. The second section focuses on the particular case of ALS, a late-onset neurodegenerative disease characterized by the death of central nervous system motor neurons, leading to paralysis and patient death. Of the 10% of ALS cases that show familial inheritance (familial ALS), the largest subset is caused by mutations in the SOD1 gene, encoding the Cu, Zn superoxide dismutase (SOD1). The unusual kinetic stability of SOD1 has provided a unique opportunity for detailed structural characterization of conformational states potentially involved in SOD1-associated ALS. This review discusses past studies exploring the stability, folding, and misfolding behavior of SOD1, as well as the therapeutic possibilities of using detailed knowledge of misfolding pathways to target the molecular mechanisms underlying ALS and other neurodegenerative diseases. Copyright © 2013 Wiley Periodicals, Inc.

  19. Roles of methionine oxidation in E200K prion protein misfolding: Implications for the mechanism of pathogenesis in E200K linked familial Creutzfeldt-Jakob disease.

    PubMed

    Wang, Zonglin; Feng, Boya; Xiao, Gengfu; Zhou, Zheng

    2016-04-01

    Prion diseases are a group of neurodegenerative diseases caused by prion protein (PrP) conformational changes. More than 30 PRNP gene mutations have been associated with familial prion diseases. E200K-associated familial Creutzfeldt-Jakob disease (fCJD) is the most common inherited prion disease. One of the hallmarks of prion diseases is the accumulation of oxidative damage. The mechanism by which oxidative modification of methionine (Met) residues influence the E200K PrP misfolding remains unclear. Here, we examined the stability, structural change, oligomerization and proteinase K resistance of unoxidized/oxidized E200K PrP and Met-to-Leu mutants. We found that oxidation of surface-exposed Met109/112/129/134/154/166 residues significantly destabilized E200K PrP but had a limited impact on the protein's structure. The oxidation of Met213 was the initial step in the conformational conversion of E200K PrP and facilitated the further oxidation of Met205/206. The oxidation of Met213/205/206 led to the exposure of the inner hydrophobic core, disrupted the overall structure of E200K PrP and induced the formation of large soluble multimers at low pH. In addition, the aggregation behavior of oxidized E200K PrP at the cellular level was investigated using E200K PrP Met-to-Ser mutants. The results showed that M109/112/129/154S or M134/166S mutants were efficiently localized on the cell membrane, whereas the M213/205/206S mutant generated many of aggregated fluorescent dots in the cytoplasm. The present work provides important clues for understanding the special roles of methionine oxidation in E200K PrP misfolding and links oxidative stress and consequent misfolding of oxidative damaged E200K PrP with the pathogenic mechanism of E200K-associated fCJD. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Combination therapy utilizing shRNA knockdown and an optimized resistant transgene for rescue of diseases caused by misfolded proteins.

    PubMed

    Li, Chengwen; Xiao, Pingjie; Gray, Steven James; Weinberg, Marc Scott; Samulski, R Jude

    2011-08-23

    Molecular knockdown of disease proteins and restoration of wild-type activity represent a promising but challenging strategy for the treatment of diseases that result from the accumulation of misfolded proteins (i.e., Huntington disease, amyotrophic lateral sclerosis, and α-1 antitrypsin deficiency). In this study we used alpha-1 antitrypsin (AAT) deficiency with the piZZ mutant phenotype as a model system to evaluate the efficiency of gene-delivery approaches that both silence the piZZ transcript (e.g., shRNA) and restore circulating wild-type AAT expression from resistant codon-optimized AAT (AAT-opt) transgene cassette using adeno-associated virus (AAV) vector delivery. After systemic injection of a self-complimentary AAV serotype 8 (scAAV8) vector encoding shRNA in piZZ transgenic mice, both mutant AAT mRNA in the liver and defected serum protein level were inhibited by 95%, whereas liver pathology, as monitored by dPAS and fibrosis staining, reversed. To restore blood AAT levels in AAV8/shRNA-treated mice, several strategies to restore functional AAT levels were tested, including using AAV AAT-opt transgene cassettes targeted to muscle and liver, or combination vectors carrying piZZ shRNA and AAT-opt transgenes separately, or a single bicistronic AAV vector. With these molecular approaches, we observed over 90% knockdown of mutant AAT with a 13- to 30-fold increase of circulating wild-type AAT protein from the shRNA-resistant AAT-opt cassette. The molecular approaches applied in this study can simultaneously prevent liver pathology and restore blood AAT concentration in AAT deficiencies. Based on these observations, similar gene-therapy strategies could be considered for any diseases caused by accumulation of misfolded proteins.

  1. Probing Single Membrane Proteins by Atomic Force Microscopy

    NASA Astrophysics Data System (ADS)

    Scheuring, S.; Sapra, K. Tanuj; Müller, Daniel J.

    In this book chapter, we describe the working principle of the atomic force microscope (AFM), followed by the applications of AFM in high-resolution imaging and single-molecule force spectroscopy of membrane proteins. In the imaging mode, AFM allows observing the assembly of membrane proteins directly in native membranes approaching a resolution of ~0.5 nm with an outstanding signal-to-noise ratio. Conformational deviations of individual membrane proteins can be observed and their functional states directly imaged. Time-lapse AFM can image membrane proteins at work. In conjunction with high- resolution imaging, the use of the AFM as a single-molecule force spectroscope (SMFS) has gained tremendous importance in recent years. This combination allows to locate the inter- and intramolecular interactions of single membrane proteins. SMFS allows characterization of interactions that guide the folding of proteins and describe the parameters that lead to their destabilization, malfunction and misfolding. Moreover, it enables to measure the interactions established by ligand- and inhibitor-binding and in membrane protein assemblies. Because of its practical use in characterizing various parameters of membrane proteins in their native environment, AFM can be aptly described as a `lab on a tip' device.

  2. Proteins of Excitable Membranes

    PubMed Central

    Nachmansohn, David

    1969-01-01

    Excitable membranes have the special ability of changing rapidly and reversibly their permeability to ions, thereby controlling the ion movements that carry the electric currents propagating nerve impulses. Acetylcholine (ACh) is the specific signal which is released by excitation and is recognized by a specific protein, the ACh-receptor; it induces a conformational change, triggering off a sequence of reactions resulting in increased permeability. The hydrolysis of ACh by ACh-esterase restores the barrier to ions. The enzymes hydrolyzing and forming ACh and the receptor protein are present in the various types of excitable membranes. Properties of the two proteins directly associated with electrical activity, receptor and esterase, will be described in this and subsequent lectures. ACh-esterase has been shown to be located within the excitable membranes. Potent enzyme inhibitors block electrical activity demonstrating the essential role in this function. The enzyme has been recently crystallized and some protein properties will be described. The monocellular electroplax preparation offers a uniquely favorable material for analyzing the properties of the ACh-receptor and its relation to function. The essential role of the receptor in electrical activity has been demonstrated with specific receptor inhibitors. Recent data show the basically similar role of ACh in the axonal and junctional membranes; the differences of electrical events and pharmacological actions are due to variations of shape, structural organization, and environment. PMID:19873642

  3. Evolutionary origins of membrane proteins

    NASA Astrophysics Data System (ADS)

    Mulkidjanian, Armen Y.; Galperin, Michael Y.

    Although the genes that encode membrane proteins make about 30% of the sequenced genomes, the evolution of membrane proteins and their origins are still poorly understood. Here we address this topic by taking a closer look at those membrane proteins the ancestors of which were present in the Last Universal Common Ancestor, and in particular, the F/V-type rotating ATPases. Reconstruction of their evolutionary history provides hints for understanding not only the origin of membrane proteins, but also of membranes themselves. We argue that the evolution of biological membranes could occur as a process of coevolution of lipid bilayers and membrane proteins, where the increase in the ion-tightness of the membrane bilayer may have been accompanied by a transition from amphiphilic, pore-forming membrane proteins to highly hydrophobic integral membrane complexes.

  4. Temporal resolution of misfolded prion protein transport, accumulation, glial activation, and neuronal death in the retinas of mice inoculated with scrapie

    USDA-ARS?s Scientific Manuscript database

    Currently, there is a lack of pathologic landmarks to describe the progression of prion disease in vivo. The goal of this work was to determine the temporal relationship between the transport of misfolded prion protein from the brain to the retina, the accumulation of PrPSc in the retina, the respon...

  5. Amyloid misfolding, aggregation, and the early onset of protein deposition diseases: insights from AFM experiments and computational analyses.

    PubMed

    Lyubchenko, Yuri L

    2015-01-01

    The development of Alzheimer's disease is believed to be caused by the assembly of amyloid β proteins into aggregates and the formation of extracellular senile plaques. Similar models suggest that structural misfolding and aggregation of proteins are associated with the early onset of diseases such as Parkinson's, Huntington's, and other protein deposition diseases. Initially, the aggregates were structurally characterized by traditional techniques such as x-ray crystallography, NMR, electron microscopy, and AFM. However, data regarding the structures formed during the early stages of the aggregation process were unknown. Experimental models of protein deposition diseases have demonstrated that the small oligomeric species have significant neurotoxicity. This highlights the urgent need to discover the properties of these species, to enable the development of efficient diagnostic and therapeutic strategies. The oligomers exist transiently, making it impossible to use traditional structural techniques to study their characteristics. The recent implementation of single-molecule imaging and probing techniques that are capable of probing transient states have enabled the properties of these oligomers to be characterized. Additionally, powerful computational techniques capable of structurally analyzing oligomers at the atomic level advanced our understanding of the amyloid aggregation problem. This review outlines the progress in AFM experimental studies and computational analyses with a primary focus on understanding the very first stage of the aggregation process. Experimental approaches can aid in the development of novel sensitive diagnostic and preventive strategies for protein deposition diseases, and several examples of these approaches will be discussed.

  6. Amyloid misfolding, aggregation, and the early onset of protein deposition diseases: insights from AFM experiments and computational analyses

    PubMed Central

    Lyubchenko, Yuri L.

    2016-01-01

    The development of Alzheimer’s disease is believed to be caused by the assembly of amyloid β proteins into aggregates and the formation of extracellular senile plaques. Similar models suggest that structural misfolding and aggregation of proteins are associated with the early onset of diseases such as Parkinson’s, Huntington’s, and other protein deposition diseases. Initially, the aggregates were structurally characterized by traditional techniques such as x-ray crystallography, NMR, electron microscopy, and AFM. However, data regarding the structures formed during the early stages of the aggregation process were unknown. Experimental models of protein deposition diseases have demonstrated that the small oligomeric species have significant neurotoxicity. This highlights the urgent need to discover the properties of these species, to enable the development of efficient diagnostic and therapeutic strategies. The oligomers exist transiently, making it impossible to use traditional structural techniques to study their characteristics. The recent implementation of single-molecule imaging and probing techniques that are capable of probing transient states have enabled the properties of these oligomers to be characterized. Additionally, powerful computational techniques capable of structurally analyzing oligomers at the atomic level advanced our understanding of the amyloid aggregation problem. This review outlines the progress in AFM experimental studies and computational analyses with a primary focus on understanding the very first stage of the aggregation process. Experimental approaches can aid in the development of novel sensitive diagnostic and preventive strategies for protein deposition diseases, and several examples of these approaches will be discussed. PMID:27830177

  7. The Copper Metabolism MURR1 domain protein 1 (COMMD1) modulates the aggregation of misfolded protein species in a client-specific manner.

    PubMed

    Vonk, Willianne I M; Kakkar, Vaishali; Bartuzi, Paulina; Jaarsma, Dick; Berger, Ruud; Hofker, Marten H; Klomp, Leo W J; Wijmenga, Cisca; Kampinga, Harm H; van de Sluis, Bart

    2014-01-01

    The Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1α). Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1) and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS), cause misfolding and aggregation of the mutant SOD1 (mSOD1) protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson's disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington's disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies.

  8. Tracking membrane protein association in model membranes.

    PubMed

    Reffay, Myriam; Gambin, Yann; Benabdelhak, Houssain; Phan, Gilles; Taulier, Nicolas; Ducruix, Arnaud; Hodges, Robert S; Urbach, Wladimir

    2009-01-01

    Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue.We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well.After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 A, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We extract a

  9. Protein misfolding, amyotrophic lateral sclerosis and guanabenz: protocol for a phase II RCT with futility design (ProMISe trial).

    PubMed

    Bella, Eleonora Dalla; Tramacere, Irene; Antonini, Giovanni; Borghero, Giuseppe; Capasso, Margherita; Caponnetto, Claudia; Chiò, Adriano; Corbo, Massimo; Eleopra, Roberto; Filosto, Massimiliano; Giannini, Fabio; Granieri, Enrico; Bella, Vincenzo La; Lunetta, Christian; Mandrioli, Jessica; Mazzini, Letizia; Messina, Sonia; Monsurrò, Maria Rosaria; Mora, Gabriele; Riva, Nilo; Rizzi, Romana; Siciliano, Gabriele; Silani, Vincenzo; Simone, Isabella; Sorarù, Gianni; Volanti, Paolo; Lauria, Giuseppe

    2017-08-11

    Recent studies suggest that endoplasmic reticulum stress may play a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS) through an altered regulation of the proteostasis, the cellular pathway-balancing protein synthesis and degradation. A key mechanism is thought to be the dephosphorylation of eIF2α, a factor involved in the initiation of protein translation. Guanabenz is an alpha-2-adrenergic receptor agonist safely used in past to treat mild hypertension and is now an orphan drug. A pharmacological action recently discovered is its ability to modulate the synthesis of proteins by the activation of translational factors preventing misfolded protein accumulation and endoplasmic reticulum overload. Guanabenz proved to rescue motoneurons from misfolding protein stress both in in vitro and in vivo ALS models, making it a potential disease-modifying drug in patients. It is conceivable investigating whether its neuroprotective effects based on the inhibition of eIF2α dephosphorylation can change the progression of ALS. Protocolised Management In Sepsis is a multicentre, randomised, double-blind, placebo-controlled phase II clinical trial with futility design. We will investigate clinical outcomes, safety, tolerability and biomarkers of neurodegeneration in patients with ALS treated with guanabenz or riluzole alone for 6 months. The primary aim is to test if guanabenz can reduce the proportion of patients progressed to a higher stage of disease at 6 months compared with their baseline stage as measured by the ALS Milano-Torino Staging (ALS-MITOS) system and to the placebo group. Secondary aims are safety, tolerability and change in at least one biomarker of neurodegeneration in the guanabenz arm compared with the placebo group. Findings will provide reliable data on the likelihood that guanabenz can slow the course of ALS in a phase III trial. The study protocol was approved by the Ethics Committee of IRCCS 'Carlo Besta Foundation' of Milan

  10. A Two-step Protein Quality Control Pathway for a Misfolded DJ-1 Variant in Fission Yeast.

    PubMed

    Mathiassen, Søs G; Larsen, Ida B; Poulsen, Esben G; Madsen, Christian T; Papaleo, Elena; Lindorff-Larsen, Kresten; Kragelund, Birthe B; Nielsen, Michael L; Kriegenburg, Franziska; Hartmann-Petersen, Rasmus

    2015-08-21

    A mutation, L166P, in the cytosolic protein, PARK7/DJ-1, causes protein misfolding and is linked to Parkinson disease. Here, we identify the fission yeast protein Sdj1 as the orthologue of DJ-1 and calculate by in silico saturation mutagenesis the effects of point mutants on its structural stability. We also map the degradation pathways for Sdj1-L169P, the fission yeast orthologue of the disease-causing DJ-1 L166P protein. Sdj1-L169P forms inclusions, which are enriched for the Hsp104 disaggregase. Hsp104 and Hsp70-type chaperones are required for efficient degradation of Sdj1-L169P. This also depends on the ribosome-associated E3 ligase Ltn1 and its co-factor Rqc1. Although Hsp104 is absolutely required for proteasomal degradation of Sdj1-L169P aggregates, the degradation of already aggregated Sdj1-L169P occurs independently of Ltn1 and Rqc1. Thus, our data point to soluble Sdj1-L169P being targeted early by Ltn1 and Rqc1. The fraction of Sdj1-L169P that escapes this first inspection then forms aggregates that are subsequently cleared via an Hsp104- and proteasome-dependent pathway.

  11. Membrane fission by protein crowding.

    PubMed

    Snead, Wilton T; Hayden, Carl C; Gadok, Avinash K; Zhao, Chi; Lafer, Eileen M; Rangamani, Padmini; Stachowiak, Jeanne C

    2017-04-18

    Membrane fission, which facilitates compartmentalization of biological processes into discrete, membrane-bound volumes, is essential for cellular life. Proteins with specific structural features including constricting rings, helical scaffolds, and hydrophobic membrane insertions are thought to be the primary drivers of fission. In contrast, here we report a mechanism of fission that is independent of protein structure-steric pressure among membrane-bound proteins. In particular, random collisions among crowded proteins generate substantial pressure, which if unbalanced on the opposite membrane surface can dramatically increase membrane curvature, leading to fission. Using the endocytic protein epsin1 N-terminal homology domain (ENTH), previously thought to drive fission by hydrophobic insertion, our results show that membrane coverage correlates equally with fission regardless of the hydrophobicity of insertions. Specifically, combining FRET-based measurements of membrane coverage with multiple, independent measurements of membrane vesiculation revealed that fission became spontaneous as steric pressure increased. Further, fission efficiency remained equally potent when helices were replaced by synthetic membrane-binding motifs. These data challenge the view that hydrophobic insertions drive membrane fission, suggesting instead that the role of insertions is to anchor proteins strongly to membrane surfaces, amplifying steric pressure. In line with these conclusions, even green fluorescent protein (GFP) was able to drive fission efficiently when bound to the membrane at high coverage. Our conclusions are further strengthened by the finding that intrinsically disordered proteins, which have large hydrodynamic radii yet lack a defined structure, drove fission with substantially greater potency than smaller, structured proteins.

  12. The non-protein amino acid BMAA is misincorporated into human proteins in place of L-serine causing protein misfolding and aggregation.

    PubMed

    Dunlop, Rachael Anne; Cox, Paul Alan; Banack, Sandra Anne; Rodgers, Kenneth John

    2013-01-01

    Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNA(Ala) with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of L-serine into human proteins. We also report that this misincorporation can be inhibited by L-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS.

  13. The Non-Protein Amino Acid BMAA Is Misincorporated into Human Proteins in Place of l-Serine Causing Protein Misfolding and Aggregation

    PubMed Central

    Dunlop, Rachael Anne; Cox, Paul Alan; Banack, Sandra Anne; Rodgers, Kenneth John

    2013-01-01

    Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNAAla with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of l-serine into human proteins. We also report that this misincorporation can be inhibited by l-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. PMID:24086518

  14. Proteins causing membrane fouling in membrane bioreactors.

    PubMed

    Miyoshi, Taro; Nagai, Yuhei; Aizawa, Tomoyasu; Kimura, Katsuki; Watanabe, Yoshimasa

    2015-01-01

    In this study, the details of proteins causing membrane fouling in membrane bioreactors (MBRs) treating real municipal wastewater were investigated. Two separate pilot-scale MBRs were continuously operated under significantly different operating conditions; one MBR was a submerged type whereas the other was a side-stream type. The submerged and side-stream MBRs were operated for 20 and 10 days, respectively. At the end of continuous operation, the foulants were extracted from the fouled membranes. The proteins contained in the extracted foulants were enriched by using the combination of crude concentration with an ultrafiltration membrane and trichloroacetic acid precipitation, and then separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The N-terminal amino acid sequencing analysis of the proteins which formed intensive spots on the 2D-PAGE gels allowed us to partially identify one protein (OmpA family protein originated from genus Brevundimonas or Riemerella anatipestifer) from the foulant obtained from the submerged MBR, and two proteins (OprD and OprF originated from genus Pseudomonas) from that obtained from the side-stream MBR. Despite the significant difference in operating conditions of the two MBRs, all proteins identified in this study belong to β-barrel protein. These findings strongly suggest the importance of β-barrel proteins in developing membrane fouling in MBRs.

  15. Expression of three topologically distinct membrane proteins elicits unique stress response pathways in the yeast Saccharomyces cerevisiae

    PubMed Central

    Buck, Teresa M.; Jordan, Rick; Lyons-Weiler, James; Adelman, Joshua L.; Needham, Patrick G.; Kleyman, Thomas R.

    2015-01-01

    Misfolded membrane proteins are retained in the endoplasmic reticulum (ER) and are subject to ER-associated degradation, which clears the secretory pathway of potentially toxic species. While the transcriptional response to environmental stressors has been extensively studied, limited data exist describing the cellular response to misfolded membrane proteins. To this end, we expressed and then compared the transcriptional profiles elicited by the synthesis of three ER retained, misfolded ion channels: The α-subunit of the epithelial sodium channel, ENaC, the cystic fibrosis transmembrane conductance regulator, CFTR, and an inwardly rectifying potassium channel, Kir2.1, which vary in their mass, membrane topologies, and quaternary structures. To examine transcriptional profiles in a null background, the proteins were expressed in yeast, which was previously used to examine the degradation requirements for each substrate. Surprisingly, the proteins failed to induce a canonical unfolded protein response or heat shock response, although messages encoding several cytosolic and ER lumenal protein folding factors rose when αENaC or CFTR was expressed. In contrast, the levels of these genes were unaltered by Kir2.1 expression; instead, the yeast iron regulon was activated. Nevertheless, a significant number of genes that respond to various environmental stressors were upregulated by all three substrates, and compared with previous microarray data we deduced the existence of a group of genes that reflect a novel misfolded membrane protein response. These data indicate that aberrant proteins in the ER elicit profound yet unique cellular responses. PMID:25759377

  16. Unfolding story of inclusion-body myositis and myopathies: role of misfolded proteins, amyloid-beta, cholesterol, and aging.

    PubMed

    Askanas, Valerie; Engel, W King

    2003-03-01

    Sporadic inclusion-body myositis and hereditary inclusion-body myopathies are progressive muscle diseases leading to severe disability. We briefly summarize their clinical pictures and pathologic diagnostic criteria and discuss the latest advances in illuminating their pathogenic mechanism(s). We emphasize how different etiologies might lead to the strikingly similar pathology and possibly similar pathogenic cascade. On the basis of our research, several processes seem to be important in relation to the still speculative pathogenesis, including (a) increased transcription and accumulation of amyloid-beta precursor protein and accumulation of its proteolytic fragment amyloid-beta; (b) abnormal accumulation of components related to lipid metabolism, for example, cholesterol, accumulation of which is possibly owing to its abnormal trafficking; (c) oxidative stress; (d) accumulations of other Alzheimer's disease-related proteins; and (e) a milieu of muscle cellular aging in which these changes occur. We discuss a potentially very important role of unfolded and/or misfolded proteins as a possible mechanism in the formations of the inclusion bodies and other abnormalities.

  17. The b' domain provides the principal peptide-binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins.

    PubMed Central

    Klappa, P; Ruddock, L W; Darby, N J; Freedman, R B

    1998-01-01

    Protein disulfide isomerase (PDI) is a very efficient catalyst of folding of many disulfide-bonded proteins. A great deal is known about the catalytic functions of PDI, while little is known about its substrate binding. We recently demonstrated by cross-linking that PDI binds peptides and misfolded proteins, with high affinity but broad specificity. To characterize the substrate-binding site of PDI, we investigated the interactions of various recombinant fragments of human PDI, expressed in Escherichia coli, with different radiolabelled model peptides. We observed that the b' domain of human PDI is essential and sufficient for the binding of small peptides. In the case of larger peptides, specifically a 28 amino acid fragment derived from bovine pancreatic trypsin inhibitor, or misfolded proteins, the b' domain is essential but not sufficient for efficient binding, indicating that contributions from additional domains are required. Hence we propose that the different domains of PDI all contribute to the binding site, with the b' domain forming the essential core. PMID:9463371

  18. Conformation of Membrane Proteins: Bacteriorhodopsin

    DTIC Science & Technology

    1994-05-13

    membrane (PM) of Halobacterium halobium, was chemically modified with methoxypolyethylene glycol MINE (MW = 5000) succinimidyl carbonate. The...membrane protein (248 amino acids) which catalyzes the light-induced proton translocation across the membrane of Halobacterium halobium. Research was...purple membrane (PM) of Halobacterium halobium, was chemically modified with methoxypolyethylene glycol (MW = 5000) succinimidyl carbonate. The

  19. Bovine spongiform encephalopathy induces misfolding of alleged prion-resistant species cellular prion protein without altering its pathobiological features.

    PubMed

    Vidal, Enric; Fernández-Borges, Natalia; Pintado, Belén; Ordóñez, Montserrat; Márquez, Mercedes; Fondevila, Dolors; Torres, Juan María; Pumarola, Martí; Castilla, Joaquín

    2013-05-01

    Bovine spongiform encephalopathy (BSE) prions were responsible for an unforeseen epizootic in cattle which had a vast social, economic, and public health impact. This was primarily because BSE prions were found to be transmissible to humans. Other species were also susceptible to BSE either by natural infection (e.g., felids, caprids) or in experimental settings (e.g., sheep, mice). However, certain species closely related to humans, such as canids and leporids, were apparently resistant to BSE. In vitro prion amplification techniques (saPMCA) were used to successfully misfold the cellular prion protein (PrP(c)) of these allegedly resistant species into a BSE-type prion protein. The biochemical and biological properties of the new prions generated in vitro after seeding rabbit and dog brain homogenates with classical BSE were studied. Pathobiological features of the resultant prion strains were determined after their inoculation into transgenic mice expressing bovine and human PrP(C). Strain characteristics of the in vitro-adapted rabbit and dog BSE agent remained invariable with respect to the original cattle BSE prion, suggesting that the naturally low susceptibility of rabbits and dogs to prion infections should not alter their zoonotic potential if these animals became infected with BSE. This study provides a sound basis for risk assessment regarding prion diseases in purportedly resistant species.

  20. Protein Misfolding and Aggregation in Cataract Disease and Prospects for Prevention

    PubMed Central

    Moreau, Kate L.; King, Jonathan A.

    2012-01-01

    The transparency of the eye lens depends on maintaining the native tertiary structures and solubility of the lens crystallin proteins over a lifetime. Cataract, the leading cause of blindness worldwide, is caused by protein aggregation within the protected lens environment. With age, covalent protein damage accumulates through pathways thought to include UV radiation, oxidation, deamidation, and truncations. Experiments suggest that the resulting protein destabilization leads to partially unfolded, aggregation-prone intermediates and the formation of insoluble, light-scattering protein aggregates. These aggregates either include or overwhelm the protein chaperone content of the lens. Here we review the causes of cataracts and non-surgical methods being investigated to inhibit or delay cataract development, including natural product-based therapies, modulators of oxidation, and protein aggregation inhibitors. PMID:22520268

  1. SerpinB2 (PAI-2) Modulates Proteostasis via Binding Misfolded Proteins and Promotion of Cytoprotective Inclusion Formation

    PubMed Central

    Farrawell, Natalie; Shearer, Robert F.; Constantinescu, Patrick; Hatters, Danny M.; Schroder, Wayne A.; Suhrbier, Andreas; Wilson, Mark R.; Saunders, Darren N.; Ranson, Marie

    2015-01-01

    SerpinB2 (PAI-2), a member of the clade B family of serine protease inhibitors, is one of the most upregulated proteins following cellular stress. Originally described as an inhibitor of urokinase plasminogen activator, its predominant cytoplasmic localisation suggests an intracellular function. SerpinB2 has been reported to display cytoprotective properties in neurons and to interact with intracellular proteins including components of the ubiquitin-proteasome system (UPS). In the current study we explored the potential role of SerpinB2 as a modulator of proteotoxic stress. Initially, we transiently transfected wild-type SerpinB2 and SerpinB2-/- murine embryonic fibroblasts (MEFs) with Huntingtin exon1-polyglutamine (fused C-terminally to mCherry). Inclusion body formation as result of Huntingtin aggregation was evident in the SerpinB2 expressing cells but significantly impaired in the SerpinB2-/- cells, the latter concomitant with loss in cell viability. Importantly, recovery of the wild-type phenotype and cell viability was rescued by retroviral transduction of SerpinB2 expression. SerpinB2 modestly attenuated Huntingtin and amyloid beta fibril formation in vitro and was able to bind preferentially to misfolded proteins. Given the modest chaperone-like activity of SerpinB2 we tested the ability of SerpinB2 to modulate UPS and autophagy activity using a GFP reporter system and autophagy reporter, respectively. Activity of the UPS was reduced and autophagy was dysregulated in SerpinB2-/- compared to wild-type MEFs. Moreover, we observed a non-covalent interaction between ubiquitin and SerpinB2 in cells using GFP-pulldown assays and bimolecular fluorescence complementation. We conclude that SerpinB2 plays an important role in proteostasis as its loss leads to a proteotoxic phenotype associated with an inability to compartmentalize aggregating proteins and a reduced capacity of the UPS. PMID:26083412

  2. Proteins interacting with Membranes: Protein Sorting and Membrane Shaping

    NASA Astrophysics Data System (ADS)

    Callan-Jones, Andrew

    2015-03-01

    Membrane-bound transport in cells requires generating membrane curvature. In addition, transport is selective, in order to establish spatial gradients of membrane components in the cell. The mechanisms underlying cell membrane shaping by proteins and the influence of curvature on membrane composition are active areas of study in cell biophysics. In vitro approaches using Giant Unilamellar Vesicles (GUVs) are a useful tool to identify the physical mechanisms that drive sorting of membrane components and membrane shape change by proteins. I will present recent work on the curvature sensing and generation of IRSp53, a protein belonging to the BAR family, whose members, sharing a banana-shaped backbone, are involved in endocytosis. Pulling membrane tubes with 10-100 nm radii from GUVs containing encapsulated IRSp53 have, unexpectedly, revealed a non-monotonic dependence of the protein concentration on the tube as a function of curvature. Experiments also show that bound proteins alter the tube mechanics and that protein phase separation along the tube occurs at low tensions. I will present accompanying theoretical work that can explain these findings based on the competition between the protein's intrinsic curvature and the effective rigidity of a membrane-protein patch.

  3. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.

    PubMed

    Bucciantini, Monica; Giannoni, Elisa; Chiti, Fabrizio; Baroni, Fabiana; Formigli, Lucia; Zurdo, Jesús; Taddei, Niccolò; Ramponi, Giampietro; Dobson, Christopher M; Stefani, Massimo

    2002-04-04

    A range of human degenerative conditions, including Alzheimer's disease, light-chain amyloidosis and the spongiform encephalopathies, is associated with the deposition in tissue of proteinaceous aggregates known as amyloid fibrils or plaques. It has been shown previously that fibrillar aggregates that are closely similar to those associated with clinical amyloidoses can be formed in vitro from proteins not connected with these diseases, including the SH3 domain from bovine phosphatidyl-inositol-3'-kinase and the amino-terminal domain of the Escherichia coli HypF protein. Here we show that species formed early in the aggregation of these non-disease-associated proteins can be inherently highly cytotoxic. This finding provides added evidence that avoidance of protein aggregation is crucial for the preservation of biological function and suggests common features in the origins of this family of protein deposition diseases.

  4. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases

    NASA Astrophysics Data System (ADS)

    Bucciantini, Monica; Giannoni, Elisa; Chiti, Fabrizio; Baroni, Fabiana; Formigli, Lucia; Zurdo, Jesús; Taddei, Niccolò; Ramponi, Giampietro; Dobson, Christopher M.; Stefani, Massimo

    2002-04-01

    A range of human degenerative conditions, including Alzheimer's disease, light-chain amyloidosis and the spongiform encephalopathies, is associated with the deposition in tissue of proteinaceous aggregates known as amyloid fibrils or plaques. It has been shown previously that fibrillar aggregates that are closely similar to those associated with clinical amyloidoses can be formed in vitro from proteins not connected with these diseases, including the SH3 domain from bovine phosphatidyl-inositol-3'-kinase and the amino-terminal domain of the Escherichia coli HypF protein. Here we show that species formed early in the aggregation of these non-disease-associated proteins can be inherently highly cytotoxic. This finding provides added evidence that avoidance of protein aggregation is crucial for the preservation of biological function and suggests common features in the origins of this family of protein deposition diseases.

  5. Hsp110 Is a Bona Fide Chaperone Using ATP to Unfold Stable Misfolded Polypeptides and Reciprocally Collaborate with Hsp70 to Solubilize Protein Aggregates*

    PubMed Central

    Mattoo, Rayees U. H.; Sharma, Sandeep K.; Priya, Smriti; Finka, Andrija; Goloubinoff, Pierre

    2013-01-01

    Structurally and sequence-wise, the Hsp110s belong to a subfamily of the Hsp70 chaperones. Like the classical Hsp70s, members of the Hsp110 subfamily can bind misfolding polypeptides and hydrolyze ATP. However, they apparently act as a mere subordinate nucleotide exchange factors, regulating the ability of Hsp70 to hydrolyze ATP and convert stable protein aggregates into native proteins. Using stably misfolded and aggregated polypeptides as substrates in optimized in vitro chaperone assays, we show that the human cytosolic Hsp110s (HSPH1 and HSPH2) are bona fide chaperones on their own that collaborate with Hsp40 (DNAJA1 and DNAJB1) to hydrolyze ATP and unfold and thus convert stable misfolded polypeptides into natively refolded proteins. Moreover, equimolar Hsp70 (HSPA1A) and Hsp110 (HSPH1) formed a powerful molecular machinery that optimally reactivated stable luciferase aggregates in an ATP- and DNAJA1-dependent manner, in a disaggregation mechanism whereby the two paralogous chaperones alternatively activate the release of bound unfolded polypeptide substrates from one another, leading to native protein refolding. PMID:23737532

  6. Loss of Clcc1 Results in ER Stress, Misfolded Protein Accumulation, and Neurodegeneration

    PubMed Central

    Jia, Yichang; Jucius, Thomas J.; Cook, Susan A.

    2015-01-01

    Folding of transmembrane and secretory proteins occurs in the lumen of the endoplasmic reticulum (ER) before transportation to the cell surface and is monitored by the unfolded protein response (UPR) signaling pathway. The accumulation of unfolded proteins in the ER activates the UPR that restores ER homeostasis by regulating gene expression that leads to an increase in the protein-folding capacity of the ER and a decrease in the ER protein-folding load. However, prolonged UPR activity has been associated with cell death in multiple pathological conditions, including neurodegeneration. Here, we report a spontaneous recessive mouse mutation that causes progressive cerebellar granule cell death and peripheral motor axon degeneration. By positional cloning, we identify the mutation in this strain as a retrotransposon insertion in the Clcc1 gene, which encodes a putative chloride channel localized to the ER. Furthermore, we demonstrate that the C3H/HeSnJ inbred strain has late onset cerebellar degeneration due to this mutation. Interestingly, acute knockdown of Clcc1 expression in cultured cells increases sensitivity to ER stress. In agreement, GRP78, the major HSP70 family chaperone in the ER, is upregulated in Clcc1-deficient granule cells in vivo, and ubiquitinated proteins accumulate in these neurons before their degeneration. These data suggest that disruption of chloride homeostasis in the ER disrupts the protein-folding capacity of the ER, leading to eventual neuron death. PMID:25698737

  7. Highly sensitive detection of small ruminant bovine spongiform encephalopathy within transmissible spongiform encephalopathy mixes by serial protein misfolding cyclic amplification.

    PubMed

    Gough, Kevin C; Bishop, Keith; Maddison, Ben C

    2014-11-01

    It is assumed that sheep and goats consumed the same bovine spongiform encephalopathy (BSE)-contaminated meat and bone meal that was fed to cattle and precipitated the BSE epidemic in the United Kingdom that peaked more than 20 years ago. Despite intensive surveillance for cases of BSE within the small ruminant populations of the United Kingdom and European Union, no instances of BSE have been detected in sheep, and in only two instances has BSE been discovered in goats. If BSE is present within the small ruminant populations, it may be at subclinical levels, may manifest as scrapie, or may be masked by coinfection with scrapie. To determine whether BSE is potentially circulating at low levels within the European small ruminant populations, highly sensitive assays that can specifically detect BSE, even within the presence of scrapie prion protein, are required. Here, we present a novel assay based on the specific amplification of BSE PrP(Sc) using the serial protein misfolding cyclic amplification assay (sPMCA), which specifically amplified small amounts of ovine and caprine BSE agent which had been mixed into a range of scrapie-positive brain homogenates. We detected the BSE prion protein within a large excess of classical, atypical, and CH1641 scrapie isolates. In a blind trial, this sPMCA-based assay specifically amplified BSE PrP(Sc) within brain mixes with 100% specificity and 97% sensitivity when BSE agent was diluted into scrapie-infected brain homogenates at 1% (vol/vol). Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  8. Human Stefin B Role in Cell's Response to Misfolded Proteins and Autophagy

    PubMed Central

    Polajnar, Mira; Zavašnik-Bergant, Tina; Škerget, Katja; Vizovišek, Matej; Vidmar, Robert; Fonović, Marko; Kopitar-Jerala, Nataša; Petrovič, Uroš; Navarro, Susanna; Ventura, Salvador; Žerovnik, Eva

    2014-01-01

    Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates. PMID:25047918

  9. The synergistic effect of antiglycating agents (MB-92) on inhibition of protein glycation, misfolding and diabetic complications in diabetic-atherosclerotic rat.

    PubMed

    Mahdavifard, S; Bathaie, S Z; Nakhjavani, M; Taghikhani, M

    2016-10-04

    Protein glycation due to hyperglycemia resulting in misfolding and aggregation, which is known as one of the most important reasons of diabetes complications. We previously showed the beneficial effects of some antiglycating agents in diabetic rats. Here, the effect of MB-92, a combination of some amino acids and crocetin (Crt, a saffron carotenoid), was studied in the prevention of diabetic complications in diabetic-atherosclerotic rats. In addition, the inhibitory effect of these treatments on glycation intermediates, aggregation and misfolding of proteins was investigated both in vivo and in vitro. Thus, the streptozotocin-induced diabetic rats that underwent an atherogenic diet were treated with Crt, N-acetylcyctein and MB-92. Then, glycated products and markers of oxidation and inflammation, in addition to other markers of diabetes complications were studied. The results of the in vivo study indicated that the mentioned treatments prevented the atheromatos formation, reduced the increased blood glucose; inhibited the formation of various glycation products, induced glyoxalase system (I and II), diminished oxidation and inflammatory markers, and improved lipid profile and atherosclerotic index in the diabetic-atherosclerotic rats; but MB-92 was the most effective treatment. In vitro results also confirmed that MB-92 was the most effective treatment to inhibit protein glycation and misfolding in comparison with the other treatments. In conclusion, MB-92 showed the greatest potential for inhibition of glycation and oxidation products, atheromatose plaque formation and inflammation in diabetic-atherosclerotic rats, and to control protein glycation, misfolding and aggregation in high glucose concentration; thus, it can be suggested as a new drug to prevent diabetic complications.

  10. Random Amino Acid Mutations and Protein Misfolding Lead to Shannon Limit in Sequence-Structure Communication

    PubMed Central

    Lisewski, Andreas Martin

    2008-01-01

    The transmission of genomic information from coding sequence to protein structure during protein synthesis is subject to stochastic errors. To analyze transmission limits in the presence of spurious errors, Shannon's noisy channel theorem is applied to a communication channel between amino acid sequences and their structures established from a large-scale statistical analysis of protein atomic coordinates. While Shannon's theorem confirms that in close to native conformations information is transmitted with limited error probability, additional random errors in sequence (amino acid substitutions) and in structure (structural defects) trigger a decrease in communication capacity toward a Shannon limit at 0.010 bits per amino acid symbol at which communication breaks down. In several controls, simulated error rates above a critical threshold and models of unfolded structures always produce capacities below this limiting value. Thus an essential biological system can be realistically modeled as a digital communication channel that is (a) sensitive to random errors and (b) restricted by a Shannon error limit. This forms a novel basis for predictions consistent with observed rates of defective ribosomal products during protein synthesis, and with the estimated excess of mutual information in protein contact potentials. PMID:18769673

  11. Biophysical Insights into How Surfaces, Including Lipid Membranes, Modulate Protein Aggregation Related to Neurodegeneration

    PubMed Central

    Burke, Kathleen A.; Yates, Elizabeth A.; Legleiter, Justin

    2013-01-01

    There are a vast number of neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), associated with the rearrangement of specific proteins to non-native conformations that promotes aggregation and deposition within tissues and/or cellular compartments. These diseases are commonly classified as protein-misfolding or amyloid diseases. The interaction of these proteins with liquid/surface interfaces is a fundamental phenomenon with potential implications for protein-misfolding diseases. Kinetic and thermodynamic studies indicate that significant conformational changes can be induced in proteins encountering surfaces, which can play a critical role in nucleating aggregate formation or stabilizing specific aggregation states. Surfaces of particular interest in neurodegenerative diseases are cellular and subcellular membranes that are predominately comprised of lipid components. The two-dimensional liquid environments provided by lipid bilayers can profoundly alter protein structure and dynamics by both specific and non-specific interactions. Importantly for misfolding diseases, these bilayer properties can not only modulate protein conformation, but also exert influence on aggregation state. A detailed understanding of the influence of (sub)cellular surfaces in driving protein aggregation and/or stabilizing specific aggregate forms could provide new insights into toxic mechanisms associated with these diseases. Here, we review the influence of surfaces in driving and stabilizing protein aggregation with a specific emphasis on lipid membranes. PMID:23459674

  12. Protein misfolding and oxidative stress promote glial-mediated neurodegeneration in an Alexander disease model

    PubMed Central

    Wang, Liqun; Colodner, Kenneth J.; Feany, Mel B.

    2011-01-01

    Although alterations in glial structure and function commonly accompany death of neurons in neurodegenerative diseases, the role glia play in modulating neuronal loss is poorly understood. We have created a model of Alexander disease in Drosophila by expressing disease-linked mutant versions of glial fibrillary acidic protein (GFAP) in fly glia. We find aggregation of mutant human GFAP into inclusions bearing the hallmarks of authentic Rosenthal fibers. We also observe significant toxicity of mutant human GFAP to glia, which is mediated by protein aggregation and oxidative stress. Both protein aggregation and oxidative stress contribute to activation of a robust autophagic response in glia. Toxicity of mutant GFAP to glial cells induces a non-cell autonomous stress response and subsequent apoptosis in neurons, which is dependent on glial glutamate transport. Our findings thus establish a simple genetic model of Alexander disease and further identify cellular pathways critical for glial-induced neurodegeneration. PMID:21414908

  13. Iron in neurodegenerative disorders of protein misfolding: a case of prion disorders and Parkinson's disease.

    PubMed

    Singh, Neena; Haldar, Swati; Tripathi, Ajai K; McElwee, Matthew K; Horback, Katharine; Beserra, Amber

    2014-07-20

    Intracellular and extracellular aggregation of a specific protein or protein fragments is the principal pathological event in several neurodegenerative conditions. We describe two such conditions: sporadic Creutzfeldt-Jakob disease (sCJD), a rare but potentially infectious and invariably fatal human prion disorder, and Parkinson's disease (PD), a common neurodegenerative condition second only to Alzheimer's disease in prevalence. In sCJD, a cell surface glycoprotein known as the prion protein (PrP(C)) undergoes a conformational change to PrP-scrapie, a pathogenic and infectious isoform that accumulates in the brain parenchyma as insoluble aggregates. In PD, α-synuclein, a cytosolic protein, forms insoluble aggregates that accumulate in neurons of the substantia nigra and cause neurotoxicity. Although distinct processes are involved in the pathogenesis of sCJD and PD, both share brain iron dyshomeostasis as a common associated feature that is reflected in the cerebrospinal fluid in a disease-specific manner. Since PrP(C) and α-synuclein play a significant role in maintaining cellular iron homeostasis, it is important to understand whether the aggregation of these proteins and iron dyshomeostasis are causally related. Here, we discuss recent information on the normal function of PrP(C) and α-synuclein in cellular iron metabolism and the cellular and biochemical processes that contribute to iron imbalance in sCJD and PD. Improved understanding of the relationship between brain iron imbalance and protein aggregation is likely to help in the development of therapeutic strategies that can restore brain iron homeostasis and mitigate neurotoxicity.

  14. Protein Misfolding, Amyloid Formation, and Human Disease: A Summary of Progress Over the Last Decade.

    PubMed

    Chiti, Fabrizio; Dobson, Christopher M

    2017-06-20

    Peptides and proteins have been found to possess an inherent tendency to convert from their native functional states into intractable amyloid aggregates. This phenomenon is associated with a range of increasingly common human disorders, including Alzheimer and Parkinson diseases, type II diabetes, and a number of systemic amyloidoses. In this review, we describe this field of science with particular reference to the advances that have been made over the last decade in our understanding of its fundamental nature and consequences. We list the proteins that are known to be deposited as amyloid or other types of aggregates in human tissues and the disorders with which they are associated, as well as the proteins that exploit the amyloid motif to play specific functional roles in humans. In addition, we summarize the genetic factors that have provided insight into the mechanisms of disease onset. We describe recent advances in our knowledge of the structures of amyloid fibrils and their oligomeric precursors and of the mechanisms by which they are formed and proliferate to generate cellular dysfunction. We show evidence that a complex proteostasis network actively combats protein aggregation and that such an efficient system can fail in some circumstances and give rise to disease. Finally, we anticipate the development of novel therapeutic strategies with which to prevent or treat these highly debilitating and currently incurable conditions.

  15. Design of membrane proteins: toward functional systems.

    PubMed

    Ghirlanda, Giovanna

    2009-12-01

    Over the years, membrane-soluble peptides have provided a convenient model system to investigate the folding and assembly of integral membrane proteins. Recent advances in experimental and computational methods are now being translated into the design of functional membrane proteins. Applications include artificial modulators of membrane protein function, inhibitors of protein-protein interactions, and redox membrane proteins.

  16. The aqueous extract of Glycyrrhiza inflata can upregulate unfolded protein response-mediated chaperones to reduce tau misfolding in cell models of Alzheimer's disease.

    PubMed

    Chang, Kuo-Hsuan; Chen, I-Cheng; Lin, Hsuan-Yuan; Chen, Hsuan-Chiang; Lin, Chih-Hsin; Lin, Te-Hsien; Weng, Yu-Ting; Chao, Chih-Ying; Wu, Yih-Ru; Lin, Jung-Yaw; Lee-Chen, Guey-Jen; Chen, Chiung-Mei

    2016-01-01

    Alzheimer's disease (AD) and several neurodegenerative disorders known as tauopathies are characterized by misfolding and aggregation of tau protein. Although several studies have suggested the potential of traditional Chinese medicine (TCM) as treatment for neurodegenerative diseases, the role of TCM in treating AD and tauopathies have not been well explored. Tau protein was coupled to the DsRed fluorophore by fusing a pro-aggregation mutant of repeat domain of tau (ΔK280 tauRD) with DsRed. The ΔK280 tauRD-DsRed fusion gene was then used to generate Tet-On 293 and SH-SY5Y cell clones as platforms to test the efficacy of 39 aqueous extracts of TCM in reducing tau misfolding and in neuroprotection. Seven TCM extracts demonstrated a significant reduction in tau misfolding and reactive oxidative species with low cytotoxicity in the ΔK280 tauRD-DsRed 293 cell model. Glycyrrhiza inflata and Panax ginseng also demonstrated the potential to improve neurite outgrowth in the ΔK280 tauRD-DsRed SH-SY5Y neuronal cell model. G. inflata further rescued the upregulation of ERN2 (pro-apoptotic) and downregulation of unfolded-protein-response-mediated chaperones ERP44, DNAJC3, and SERP1 in ΔK280 tauRD-DsRed 293 cells. This in vitro study provides evidence that G. inflata may be a novel therapeutic for AD and tauopathies. Future applications of G. inflata on animal models of AD and tauopathies are warranted to corroborate its effect of reducing misfolding and potential disease modification.

  17. The aqueous extract of Glycyrrhiza inflata can upregulate unfolded protein response-mediated chaperones to reduce tau misfolding in cell models of Alzheimer’s disease

    PubMed Central

    Chang, Kuo-Hsuan; Chen, I-Cheng; Lin, Hsuan-Yuan; Chen, Hsuan-Chiang; Lin, Chih-Hsin; Lin, Te-Hsien; Weng, Yu-Ting; Chao, Chih-Ying; Wu, Yih-Ru; Lin, Jung-Yaw; Lee-Chen, Guey-Jen; Chen, Chiung-Mei

    2016-01-01

    Background Alzheimer’s disease (AD) and several neurodegenerative disorders known as tauopathies are characterized by misfolding and aggregation of tau protein. Although several studies have suggested the potential of traditional Chinese medicine (TCM) as treatment for neurodegenerative diseases, the role of TCM in treating AD and tauopathies have not been well explored. Materials and methods Tau protein was coupled to the DsRed fluorophore by fusing a pro-aggregation mutant of repeat domain of tau (ΔK280 tauRD) with DsRed. The ΔK280 tauRD-DsRed fusion gene was then used to generate Tet-On 293 and SH-SY5Y cell clones as platforms to test the efficacy of 39 aqueous extracts of TCM in reducing tau misfolding and in neuroprotection. Results Seven TCM extracts demonstrated a significant reduction in tau misfolding and reactive oxidative species with low cytotoxicity in the ΔK280 tauRD-DsRed 293 cell model. Glycyrrhiza inflata and Panax ginseng also demonstrated the potential to improve neurite outgrowth in the ΔK280 tauRD-DsRed SH-SY5Y neuronal cell model. G. inflata further rescued the upregulation of ERN2 (pro-apoptotic) and downregulation of unfolded-protein-response-mediated chaperones ERP44, DNAJC3, and SERP1 in ΔK280 tauRD-DsRed 293 cells. Conclusion This in vitro study provides evidence that G. inflata may be a novel therapeutic for AD and tauopathies. Future applications of G. inflata on animal models of AD and tauopathies are warranted to corroborate its effect of reducing misfolding and potential disease modification. PMID:27013866

  18. Deduction of the evaluation limit and termination timing of multi-round protein misfolding cyclic amplification from a titration curve.

    PubMed

    Takeuchi, Atsuko; Komiya, Mayumi; Kitamoto, Tetsuyuki; Morita, Masanori

    2011-07-01

    In this study, the efficacy of disinfectants in reducing the partially protease-resistant isoform of prion protein was evaluated by a multi-round protein misfolding cyclic amplification (PMCA) technique. Hamster brains infected with scrapie-derived strain 263K were homogenized, treated under inactivating or mock conditions, and subjected to multi-round PMCA. Four sets of serial 10-fold dilutions of mock-treated samples were analyzed. Although considerable variability was observed in the signal patterns, between the second and sixth rounds the number of the PMCA round correlated in a linear fashion with the mean dilution factor of mock-treated, infected brains, corresponding to a log reduction factor (LRF) of 3.8-7.3 log. No signals were observed in the PMCA products amplified from normal hamster brain homogenates. The mean numbers of rounds at the first appearance of the signal for 1 M and 2 M NaOH-treated samples were 4.33 and 4, respectively. Using the linear regression line as the titration curve, the LRFs of these disinfectants were found to be 6.1 and 5.8 log, respectively; these values were not significantly different. The mean number of rounds for the alkaline cleaner and sodium dodecyl sulfate were 9 and 10.33, respectively, and were outside the range of both the linear regression line and evaluation limit. The disinfectants were considered very effective because their LRFs were ≥7.3 log. These estimations were concordant with previous bioassay-based reports. Thus, the evaluation limit seems to be valuable in some applications of multi-round PMCA, such as disinfectant assessment and process validation.

  19. Asymmetric folding pathways and transient misfolding in a coarse-grained model of proteins

    NASA Astrophysics Data System (ADS)

    Wolff, K.; Vendruscolo, M.; Porto, M.

    2011-05-01

    Coarse-grained approaches to study the protein folding process provide the possibility to explore timescales longer than those accessible to all-atom models and thus provide access, albeit in less detail, to larger regions of the conformational space. Here, we investigate the behaviour of a coarse-grained model whose two primary characteristics are a tube-like geometry to describe the self-avoidance effects of the polypeptide chain, and an energy function based on a one-dimensional structural representation that specifies the sequence's connectivity in a given conformation. Such an energy function, rather than favouring the formation of specific native pairwise contacts, promotes the establishment of a specific target connectivity for each amino acid. We illustrate the use of this model by showing that it enables to follow the complete process of folding and to efficiently determine the free energy landscapes of two small α-helical proteins, the villin headpiece domain and the ubiquitin associated domain, providing results that closely resemble those found in extensive molecular dynamics studies. These results support the idea that the use of coarse-grained models that capture the self-avoidance and the connectivity of a polypeptide chain represents a promising approach for obtaining effective descriptions of many aspects of the behaviour of proteins.

  20. Structural elucidation of the interaction between neurodegenerative disease-related tau protein with model lipid membranes

    NASA Astrophysics Data System (ADS)

    Jones, Emmalee M.

    A protein's sequence of amino acids determines how it folds. That folded structure is linked to protein function, and misfolding to dysfunction. Protein misfolding and aggregation into beta-sheet rich fibrillar aggregates is connected with over 20 neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized in part by misfolding, aggregation and deposition of the microtubule associated tau protein into neurofibrillary tangles (NFTs). However, two questions remain: What is tau's fibrillization mechanism, and what is tau's cytotoxicity mechanism? Tau is prone to heterogeneous interactions, including with lipid membranes. Lipids have been found in NFTs, anionic lipid vesicles induced aggregation of the microtubule binding domain of tau, and other protein aggregates induced ion permeability in cells. This evidence prompted our investigation of tau's interaction with model lipid membranes to elucidate the structural perturbations those interactions induced in tau protein and in the membrane. We show that although tau is highly charged and soluble, it is highly surface active and preferentially interacts with anionic membranes. To resolve molecular-scale structural details of tau and model membranes, we utilized X-ray and neutron scattering techniques. X-ray reflectivity indicated tau aggregated at air/water and anionic lipid membrane interfaces and penetrated into membranes. More significantly, membrane interfaces induced tau protein to partially adopt a more compact conformation with density similar to folded protein and ordered structure characteristic of beta-sheet formation. This suggests possible membrane-based mechanisms of tau aggregation. Membrane morphological changes were seen using fluorescence microscopy, and X-ray scattering techniques showed tau completely disrupts anionic membranes, suggesting an aggregate-based cytotoxicity mechanism. Further investigation of protein constructs and a "hyperphosphorylation" disease mimic helped

  1. Robust Yet Fragile: Expression Noise, Protein Misfolding, and Gene Dosage in the Evolution of Genomes.

    PubMed

    Pires, J Chris; Conant, Gavin C

    2016-11-23

    The complex manner in which organisms respond to changes in their gene dosage has long fascinated geneticists. Oddly, although the existence of dominance implies that dosage reductions often have mild phenotypes, extra copies of whole chromosomes (aneuploidy) are generally strongly deleterious. Even more paradoxically, an extra copy of the genome is better tolerated than is aneuploidy. We review the resolution of this paradox, highlighting the roles of biochemistry, protein aggregation, and disruption of cellular microstructure in that explanation. Returning to life's curious combination of robustness and sensitivity to dosage changes, we argue that understanding how biological robustness evolved makes these observations less inexplicable. We propose that noise in gene expression and evolutionary strategies for its suppression play a role in generating dosage phenotypes. Finally, we outline an unappreciated mechanism for the preservation of duplicate genes, namely preservation to limit expression noise, arguing that it is particularly relevant in polyploid organisms.

  2. Glycosaminoglycan Sulphation Affects the Seeded Misfolding of a Mutant Prion Protein

    PubMed Central

    Lawson, Victoria A.; Lumicisi, Brooke; Welton, Jeremy; Machalek, Dorothy; Gouramanis, Katrina; Klemm, Helen M.; Stewart, James D.; Masters, Colin L.; Hoke, David E.; Collins, Steven J.; Hill, Andrew F.

    2010-01-01

    Background The accumulation of protease resistant conformers of the prion protein (PrPres) is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific. Methodology/Principal Finding In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrPres formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrPC substrate was found to specifically prevent PrPres formation seeded by mouse derived PrPSc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrPres formation, while having no effect on PrPres formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans. Conclusions/Significance Cofactor requirements for PrPres formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains. PMID:20808809

  3. The extent of protease resistance of misfolded prion protein is highly dependent on the salt concentration.

    PubMed

    Concha-Marambio, Luis; Diaz-Espinoza, Rodrigo; Soto, Claudio

    2014-01-31

    Transmissible spongiform encephalopathies are neurodegenerative diseases caused by prions in mammals. An aberrantly folded protein (PrP(Sc)) is the main component of these proteinaceous infectious particles. Prions exhibit strong resistance to protease digestion, which is typically exploited for biochemical discrimination from its native cellular form (PrP(C)). This classical feature has been partially challenged by the isolation of sizeable amounts of protease-sensitive PrP(Sc) isoforms that self-propagate in vivo. Here, we report that the degree of PrP(Sc) protease resistance is highly dependent on the concentration of salt in the solution. Similar changes were observed in PrP(Sc) obtained from different strains and species. Strikingly, the effect of salt is reversible and is associated with changes on the size of PrP(Sc) particles, but surprisingly, the more protease-sensitive species consists of a larger size. These findings shed light on the mechanistic aspects of prion proteolysis and should be considered when assessing samples of biomedical relevance.

  4. Two misfolding routes for the prion protein around pH 4.5.

    PubMed

    Garrec, Julian; Tavernelli, Ivano; Rothlisberger, Ursula

    2013-01-01

    Using molecular dynamics simulations, we show that the prion protein (PrP) exhibits a dual behavior, with two possible transition routes, upon protonation of H187 around pH 4.5, which mimics specific conditions encountered in endosomes. Our results suggest a picture in which the protonated imidazole ring of H187 experiences an electrostatic repulsion with the nearby guanidinium group of R136, to which the system responds by pushing either H187 or R136 sidechains away from their native cavities. The regions to which H187 and R136 are linked, namely the C-terminal part of H2 and the loop connecting S1 to H1, respectively, are affected in a different manner depending on which pathway is taken. Specific in vivo or in vitro conditions, such as the presence of molecular chaperones or a particular experimental setup, may favor one transition pathway over the other, which can result in very different [Formula: see text] monomers. This has some possible connections with the observation of various fibril morphologies and the outcome of prion strains. In addition, the finding that the interaction of H187 with R136 is a weak point in mammalian PrP is supported by the absence of the [Formula: see text] residue pair in non-mammalian species that are known to be resistant to prion diseases.

  5. Nanotechnology drives a paradigm shift on protein misfolding diseases and amyloidosis

    NASA Astrophysics Data System (ADS)

    Bellotti, Vittorio; Stoppini, Monica

    2012-06-01

    In almost a century of scientific work on the mechanism of amyloid diseases much of the attention has been focused on the amyloid fibrils, which still represent the diagnostic hallmark of the disease and are easily identified in affected organs for their peculiar tinctorial properties and the fibrillar shape. However, it has been lately discovered that the seeds of the pathogenesis are deeply hidden in the structure and folding dynamics of proteins at the monomeric state which almost indistinguishable from the normal counterpart through classical biochemical approaches. In the recent years soluble oligomeric/prefibrillar species, putatively cytotoxic, were discovered and even more recently polymorphisms of shape and structure of fibrils was emerging as a property that could dictate the bioactivity of amyloid as well as the specificity of its tissue localization. Nanotechnology through the biophysical analysis of the single molecules (monomers or oligomers or fibrils) is the propulsive disciplines in the transformation of our knowledge on the molecular mechanism of this disease. It will provide, in the forthcoming years, precious analytical devices mimicking the biological microenvironment where the molecular events causing the amyloid formation will be monitored and possibly modulated in a real time frame.

  6. Nitrosative stress mediated misfolded protein aggregation mitigated by Na-D-{beta}-hydroxybutyrate intervention

    SciTech Connect

    Kabiraj, Parijat; Pal, Rituraj; Varela-Ramirez, Armando; Miranda, Manuel; Narayan, Mahesh

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Rotenone is a model for inducing apoptosis and synphilin-1 accumulation in Parkinson Prime s studies. Black-Right-Pointing-Pointer The metabolite sodium betahydroxybutryate mitigates these effects in SHSY5Y cell lines. Black-Right-Pointing-Pointer Results reveal a novel and innate mechanism to prevent neurodegeneration/cell death. -- Abstract: Mitochondrial dysfunction, leading to elevated levels of reactive oxygen species, is associated with the pathogenesis of neurodegenerative disorders. Rotenone, a mitochondrial stressor induces caspase-9 and caspase-3 activation leading proteolytic cleavage of substrate nuclear poly(ADP-ribose) polymerase (PARP). PARP cleavage is directly related to apoptotic cell death. In this study, we have monitored the aggregation of green-fluorescent protein (GFP)-tagged synphilin-1, as a rotenone-induced Parkinsonia-onset biomarker. We report that the innate ketone body, Na-D-{beta}-hydroxybutyrate (Na{beta}HB) reduces markedly the incidence of synphilin-1 aggregation. Furthermore, our data reveal that the metabolic byproduct also prevents rotenone-induced caspase-activated apoptotic cell death in dopaminergic SH-SY5Y cells. Together, these results suggest that Na{beta}HB is neuroprotective; it attenuates effects originating from mitochondrial insult and can serve as a scaffold for the design and development of sporadic neuropathies.

  7. Lipids and topological rules governing membrane protein assembly☆

    PubMed Central

    Bogdanov, Mikhail; Dowhan, William; Vitrac, Heidi

    2014-01-01

    Membrane protein folding and topogenesis are tuned to a given lipid profile since lipids and proteins have co-evolved to follow a set of interdependent rules governing final protein topological organization. Transmembrane domain (TMD) topology is determined via a dynamic process in which topogenic signals in the nascent protein are recognized and interpreted initially by the translocon followed by a given lipid profile in accordance with the Positive Inside Rule. The net zero charged phospholipid phosphatidylethanolamine and other neutral lipids dampen the translocation potential of negatively charged residues in favor of the cytoplasmic retention potential of positively charged residues (Charge Balance Rule). This explains why positively charged residues are more potent topological signals than negatively charged residues. Dynamic changes in orientation of TMDs during or after membrane insertion are attributed to non-sequential cooperative and collective lipid–protein charge interactions as well as long-term interactions within a protein. The proportion of dual topological conformers of a membrane protein varies in a dose responsive manner with changes in the membrane lipid composition not only in vivo but also in vitro and therefore is determined by the membrane lipid composition. Switching between two opposite TMD topologies can occur in either direction in vivo and also in liposomes (designated as fliposomes) independent of any other cellular factors. Such lipid-dependent post-insertional reversibility of TMD orientation indicates a thermodynamically driven process that can occur at any time and in any cell membrane driven by changes in the lipid composition. This dynamic view of protein topological organization influenced by the lipid environment reveals previously unrecognized possibilities for cellular regulation and understanding of disease states resulting from mis-folded proteins. This article is part of a Special Issue entitled: Protein Trafficking

  8. The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells

    PubMed Central

    Johansson, Ida; Monsen, Vivi Talstad; Pettersen, Kristine; Mildenberger, Jennifer; Misund, Kristine; Kaarniranta, Kai; Schønberg, Svanhild; Bjørkøy, Geir

    2015-01-01

    Accumulation and aggregation of misfolded proteins is a hallmark of several diseases collectively known as proteinopathies. Autophagy has a cytoprotective role in diseases associated with protein aggregates. Age-related macular degeneration (AMD) is the most common neurodegenerative eye disease that evokes blindness in elderly. AMD is characterized by degeneration of retinal pigment epithelial (RPE) cells and leads to loss of photoreceptor cells and central vision. The initial phase associates with accumulation of intracellular lipofuscin and extracellular deposits called drusen. Epidemiological studies have suggested an inverse correlation between dietary intake of marine n-3 polyunsaturated fatty acids (PUFAs) and the risk of developing neurodegenerative diseases, including AMD. However, the disease-preventive mechanism(s) mobilized by n-3 PUFAs is not completely understood. In human retinal pigment epithelial cells we find that physiologically relevant doses of the n-3 PUFA docosahexaenoic acid (DHA) induce a transient increase in cellular reactive oxygen species (ROS) levels that activates the oxidative stress response regulator NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2). Simultaneously, there is a transient increase in intracellular protein aggregates containing SQSTM1/p62 (sequestosome 1) and an increase in autophagy. Pretreatment with DHA rescues the cells from cell cycle arrest induced by misfolded proteins or oxidative stress. Cells with a downregulated oxidative stress response, or autophagy, respond with reduced cell growth and survival after DHA supplementation. These results suggest that DHA both induces endogenous antioxidants and mobilizes selective autophagy of misfolded proteins. Both mechanisms could be relevant to reduce the risk of developing aggregate-associate diseases such as AMD. PMID:26237736

  9. Interaction of tau protein with model lipid membranes induces tau structural compaction and membrane disruption

    PubMed Central

    Jones, Emmalee M.; Dubey, Manish; Camp, Phillip J.; Vernon, Briana C.; Biernat, Jacek; Mandelkow, Eckhard; Majewski, Jaroslaw; Chi, Eva Y.

    2012-01-01

    The misfolding and aggregation of the intrinsically disordered, microtubule-associated tau protein into neurofibrillary tangles is implicated in the pathogenesis of Alzheimer's disease. However, the mechanisms of tau aggregation and toxicity remain unknown. Recent work has shown that lipid membrane can induce tau aggregation and that membrane permeabilization may serve as a pathway by which protein aggregates exert toxicity, suggesting that the plasma membrane may play dual roles in tau pathology. This prompted our investigation to assess tau's propensity to interact with membranes and to elucidate the mutually disruptive structural perturbations the interactions induce in both tau and the membrane. We show that although highly charged and soluble, the full-length tau (hTau40) is also highly surface active, selectively inserts into anionic DMPG lipid monolayers and induces membrane morphological changes. To resolve molecular-scale structural details of hTau40 associated with lipid membranes, X-ray and neutron scattering techniques are utilized. X-ray reflectivity indicates hTau40's presence underneath a DMPG monolayer and penetration into the lipid headgroups and tailgroups, whereas grazing incidence X-ray diffraction shows that hTau40 insertion disrupts lipid packing. Moreover, both air/water and DMPG lipid membrane interfaces induce the disordered hTau40 to partially adopt a more compact conformation with density similar to that of a folded protein. Neutron reflectivity shows that tau completely disrupts supported DMPG bilayers while leaving the neutral DPPC bilayer intact. Our results show that hTau40's strong interaction with anionic lipids induces tau structural compaction and membrane disruption, suggesting possible membrane-based mechanisms of tau aggregation and toxicity in neurodegenerative diseases. PMID:22401494

  10. Molecular dynamics of membrane proteins.

    SciTech Connect

    Woolf, Thomas B.; Crozier, Paul Stewart; Stevens, Mark Jackson

    2004-10-01

    Understanding the dynamics of the membrane protein rhodopsin will have broad implications for other membrane proteins and cellular signaling processes. Rhodopsin (Rho) is a light activated G-protein coupled receptor (GPCR). When activated by ligands, GPCRs bind and activate G-proteins residing within the cell and begin a signaling cascade that results in the cell's response to external stimuli. More than 50% of all current drugs are targeted toward G-proteins. Rho is the prototypical member of the class A GPCR superfamily. Understanding the activation of Rho and its interaction with its Gprotein can therefore lead to a wider understanding of the mechanisms of GPCR activation and G-protein activation. Understanding the dark to light transition of Rho is fully analogous to the general ligand binding and activation problem for GPCRs. This transition is dependent on the lipid environment. The effect of lipids on membrane protein activity in general has had little attention, but evidence is beginning to show a significant role for lipids in membrane protein activity. Using the LAMMPS program and simulation methods benchmarked under the IBIG program, we perform a variety of allatom molecular dynamics simulations of membrane proteins.

  11. Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena.

    PubMed

    Pain, Coralie; Dumont, Janice; Dumoulin, Mireille

    2015-04-01

    The deposition of misfolded peptides and proteins in the form of amyloid fibrils is the hallmark of nearly fifty medical disorders, including Alzheimer's disease, Parkinson's disease, prion diseases and type II diabetes. These disorders, referred to as amyloidoses, generally become apparent late in life. Their psycho-sociological and economic incidence in western societies will be therefore considerable in the coming decades due to the ageing of the population. Neither preventing nor curative treatments are available yet. These disorders constitute therefore a medical challenge of great importance. Thus, an extensive research is being carried out to understand, at the molecular level, (i) how amyloidogenic proteins misfold and convert from their soluble form into amyloid fibrils, and (ii) how these aggregates or some of their oligomeric precursor species are toxic. The formation of amyloid fibrils proceeds through a complex nucleation/polymerisation mechanism with the formation of various species, including small oligomers. In this review, we focus on how VHHs or nanobodies, the antigen-binding domains of camelid heavy-chain antibodies, are being increasingly used to characterise each of the species formed on the pathway of fibril formation in terms of structure, stability, kinetics of formation and toxicity. We first introduce the characteristic features of nanobodies compared to those of conventional antibody fragments. Thereafter, we discuss how nanobodies, due to their unique properties, are used as probes to dissect the molecular mechanisms of misfolding and aggregation of six proteins associated with diseases, i.e. human lysozyme, β2-microglobulin, α-synuclein, prion, polyadenylate binding protein nuclear 1 and amyloid β-peptide. A brief general presentation of each disease and the associated peptide/protein is also provided. In addition, we discuss how nanobodies could be used as early diagnostic tools and as novel strategies to treat diseases associated

  12. Neuronal cells but not muscle cells are resistant to oxidative stress mediated protein misfolding and cell death: role of molecular chaperones.

    PubMed

    Bhattacharya, Arunabh; Wei, Rochelle; Hamilton, Ryan T; Chaudhuri, Asish R

    2014-04-18

    Our recent study in a mouse model of familial-Amyotrophic Lateral Sclerosis (f-ALS) revealed that muscle proteins are equally sensitive to misfolding as spinal cord proteins despite the presence of low mutant CuZn-superoxide dismutase, which is considered to be the key toxic element for initiation and progression of f-ALS. More importantly, we observed differential level of heat shock proteins (Hsp's) between skeletal muscle and spinal cord tissues prior to the onset and during disease progression; spinal cord maintains significantly higher level of Hsp's compared to skeletal muscle. In this study, we report two important observations; (i) muscle cells (but not neuronal cells) are extremely vulnerable to protein misfolding and cell death during challenge with oxidative stress and (ii) muscle cells fail to mount Hsp's during challenge unlike neuronal cells. These two findings can possibly explain why muscle atrophy precedes the death of motor neurons in f-ALS mice. Copyright © 2014. Published by Elsevier Inc.

  13. Structural Symmetry in Membrane Proteins.

    PubMed

    Forrest, Lucy R

    2015-01-01

    Symmetry is a common feature among natural systems, including protein structures. A strong propensity toward symmetric architectures has long been recognized for water-soluble proteins, and this propensity has been rationalized from an evolutionary standpoint. Proteins residing in cellular membranes, however, have traditionally been less amenable to structural studies, and thus the prevalence and significance of symmetry in this important class of molecules is not as well understood. In the past two decades, researchers have made great strides in this area, and these advances have provided exciting insights into the range of architectures adopted by membrane proteins. These structural studies have revealed a similarly strong bias toward symmetric arrangements, which were often unexpected and which occurred despite the restrictions imposed by the membrane environment on the possible symmetry groups. Moreover, membrane proteins disproportionately contain internal structural repeats resulting from duplication and fusion of smaller segments. This article discusses the types and origins of symmetry in membrane proteins and the implications of symmetry for protein function.

  14. Misfolding of luciferase at the single-molecule level.

    PubMed

    Mashaghi, Alireza; Mashaghi, Samaneh; Tans, Sander J

    2014-09-22

    The folding of complex proteins can be dramatically affected by misfolding transitions. Directly observing misfolding and distinguishing it from aggregation is challenging. Experiments with optical tweezers revealed transitions between the folded states of a single protein in the absence of mechanical tension. Nonfolded chains of the multidomain protein luciferase folded within seconds to different partially folded states, one of which was stable over several minutes and was more resistant to forced unfolding than other partially folded states. Luciferase monomers can thus adopt a stable misfolded state and can do so without interacting with aggregation partners. This result supports the notion that luciferase misfolding is the cause of the low refolding yields and aggregation observed with this protein. This approach could be used to study misfolding transitions in other large proteins, as well as the factors that affect misfolding. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Structure and function in rhodopsin: Mass spectrometric identification of the abnormal intradiscal disulfide bond in misfolded retinitis pigmentosa mutants

    PubMed Central

    Hwa, John; Klein-Seetharaman, Judith; Khorana, H. Gobind

    2001-01-01

    Retinitis pigmentosa (RP) point mutations in both the intradiscal (ID) and transmembrane domains of rhodopsin cause partial or complete misfolding of rhodopsin, resulting in loss of 11-cis-retinal binding. Previous work has shown that misfolding is caused by the formation of a disulfide bond in the ID domain different from the native Cys-110–Cys-187 disulfide bond in native rhodopsin. Here we report on direct identification of the abnormal disulfide bond in misfolded RP mutants in the transmembrane domain by mass spectrometric analysis. This disulfide bond is between Cys-185 and Cys-187, the same as previously identified in misfolded RP mutations in the ID domain. The strategy described here should be generally applicable to identification of disulfide bonds in other integral membrane proteins. PMID:11320236

  16. Kinetic stability of membrane proteins.

    PubMed

    González Flecha, F Luis

    2017-09-18

    Although membrane proteins constitute an important class of biomolecules involved in key cellular processes, study of the thermodynamic and kinetic stability of their structures is far behind that of soluble proteins. It is known that many membrane proteins become unstable when removed by detergent extraction from the lipid environment. In addition, most of them undergo irreversible denaturation, even under mild experimental conditions. This process was found to be associated with partial unfolding of the polypeptide chain exposing hydrophobic regions to water, and it was proposed that the formation of kinetically trapped conformations could be involved. In this review, we will describe some of the efforts toward understanding the irreversible inactivation of membrane proteins. Furthermore, its modulation by phospholipids, ligands, and temperature will be herein discussed.

  17. Dangerous liaisons between detergents and membrane proteins. The case of mitochondrial uncoupling protein 2.

    PubMed

    Zoonens, Manuela; Comer, Jeffrey; Masscheleyn, Sandrine; Pebay-Peyroula, Eva; Chipot, Christophe; Miroux, Bruno; Dehez, François

    2013-10-09

    The extraction of membrane proteins from their native environment by detergents is central to their biophysical characterization. Recent studies have emphasized that detergents may perturb the structure locally and modify the dynamics of membrane proteins. However, it remains challenging to determine whether these perturbations are negligible or could be responsible for misfolded conformations, altering the protein's function. In this work, we propose an original strategy combining functional studies and molecular simulations to address the physiological relevance of membrane protein structures obtained in the presence of detergents. We apply our strategy to a structure of isoform 2 of an uncoupling protein (UCP2) binding an inhibitor recently obtained in dodecylphosphocholine detergent micelles. Although this structure shares common traits with the ADP/ATP carrier, a member of the same protein family, its functional and biological significance remains to be addressed. In the present investigation, we demonstrate how dodecylphosphocholine severely alters the structure as well as the function of UCPs. The proposed original strategy opens new vistas for probing the physiological relevance of three-dimensional structures of membrane proteins obtained in non-native environments.

  18. The interactions of peripheral membrane proteins with biological membranes

    SciTech Connect

    Johs, Alexander; Whited, A. M.

    2015-01-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approaches continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.

  19. The interactions of peripheral membrane proteins with biological membranes

    DOE PAGES

    Johs, Alexander; Whited, A. M.

    2015-01-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

  20. YidC assists the stepwise and stochastic folding of membrane proteins

    PubMed Central

    Serdiuk, Tetiana; Balasubramaniam, Dhandayuthapani; Sugihara, Junichi; Mari, Stefania A.; Kaback, H. Ronald; Müller, Daniel J.

    2016-01-01

    How chaperones, insertases and translocases facilitate insertion and folding of complex cytoplasmic proteins into cellular membranes is not fully understood. Here, we utilize single-molecule force spectroscopy to observe YidC, a transmembrane chaperone/insertase, sculpting the folding trajectory of the polytopic α-helical membrane protein lactose permease (LacY). In the absence of YidC, unfolded LacY inserts individual structural segments into the membrane; however, misfolding dominates the process so that folding cannot be completed. YidC prevents LacY from misfolding by stabilizing the unfolded state from which LacY inserts structural segments stepwise into the membrane until folding is completed. During stepwise insertion, YidC and membrane together stabilize the transient folds. Remarkably, the order of insertion of structural segments is stochastic, thereby indicating that LacY can fold along variable pathways towards the native structure. Since YidC is essential in membrane protein biogenesis and LacY a paradigm for the major facilitator superfamily, our observations have general relevance. PMID:27595331

  1. Thermodynamic competition between membrane protein oligomeric states

    NASA Astrophysics Data System (ADS)

    Kahraman, Osman; Haselwandter, Christoph A.

    2016-10-01

    Self-assembly of protein monomers into distinct membrane protein oligomers provides a general mechanism for diversity in the molecular architectures, and resulting biological functions, of membrane proteins. We develop a general physical framework describing the thermodynamic competition between different oligomeric states of membrane proteins. Using the mechanosensitive channel of large conductance as a model system, we show how the dominant oligomeric states of membrane proteins emerge from the interplay of protein concentration in the cell membrane, protein-induced lipid bilayer deformations, and direct monomer-monomer interactions. Our results suggest general physical mechanisms and principles underlying regulation of protein function via control of membrane protein oligomeric state.

  2. Autophagy-linked FYVE protein (Alfy) promotes autophagic removal of misfolded proteins involved in amyotrophic lateral sclerosis (ALS).

    PubMed

    Han, Huihui; Wei, Wanyi; Duan, Weisong; Guo, Yansu; Li, Yi; Wang, Jie; Bi, Yue; Li, Chunyan

    2015-03-01

    Autophagy-linked FYVE (Alfy) is a protein implicated in the selective degradation of aggregated proteins. In our present study, we found that Alfy was recruited into the aggregated G93A-SOD1 in transgenic mice with amyotrophic lateral sclerosis (ALS). We demonstrated that Alfy overexpression could decrease the expression of mutant proteins via the autophagosome-lysosome pathway, and thereby, the toxicity of mutant proteins was reduced. The clearance of the mutant proteins in NSC34 cells was significantly inhibited in an Alfy knockdown cellular model. We therefore deduced that Alfy translocalization likely is involved in the pathogenesis of ALS. Alfy may be developed into a useful target for ALS therapy.

  3. Elevated protein carbonylation, and misfolding in sciatic nerve from db/db and Sod1(-/-) mice: plausible link between oxidative stress and demyelination.

    PubMed

    Hamilton, Ryan T; Bhattacharya, Arunabh; Walsh, Michael E; Shi, Yun; Wei, Rochelle; Zhang, Yiqiang; Rodriguez, Karl A; Buffenstein, Rochelle; Chaudhuri, Asish R; Van Remmen, Holly

    2013-01-01

    Diabetic peripheral polyneuropathy is associated with decrements in motor/sensory neuron myelination, nerve conduction and muscle function; however, the mechanisms of reduced myelination in diabetes are poorly understood. Chronic elevation of oxidative stress may be one of the potential determinants for demyelination as lipids and proteins are important structural constituents of myelin and highly susceptible to oxidation. The goal of the current study was to determine whether there is a link between protein oxidation/misfolding and demyelination. We chose two distinct models to test our hypothesis: 1) the leptin receptor deficient mouse (dbdb) model of diabetic polyneuropathy and 2) superoxide dismutase 1 knockout (Sod1(-/-) ) mouse model of in vivo oxidative stress. Both experimental models displayed a significant decrement in nerve conduction, increase in tail distal motor latency as well as reduced myelin thickness and fiber/axon diameter. Further biochemical studies demonstrated that oxidative stress is likely to be a potential key player in the demyelination process as both models exhibited significant elevation in protein carbonylation and alterations in protein conformation. Since peripheral myelin protein 22 (PMP22) is a key component of myelin sheath and has been found mutated and aggregated in several peripheral neuropathies, we predicted that an increase in carbonylation and aggregation of PMP22 may be associated with demyelination in dbdb mice. Indeed, PMP22 was found to be carbonylated and aggregated in sciatic nerves of dbdb mice. Sequence-driven hydropathy plot analysis and in vitro oxidation-induced aggregation of purified PMP22 protein supported the premise for oxidation-dependent aggregation of PMP22 in dbdb mice. Collectively, these data strongly suggest for the first time that oxidation-mediated protein misfolding and aggregation of key myelin proteins may be linked to demyelination and reduced nerve conduction in peripheral neuropathies.

  4. SNARE proteins and 'membrane rafts'.

    PubMed

    Lang, Thorsten

    2007-12-15

    The original 'lipid raft' hypothesis proposed that lipid-platforms/rafts form in the exoplasmic plasmalemmal leaflet by tight clustering of sphingolipids and cholesterol. Their physical state, presumably similar to liquid-ordered phases in model membranes, would confer detergent resistance to rafts and enriched proteins therein. Based on this concept, detergent resistant membranes (DRMs) from solubilized cells were considered to reflect pre-existing 'lipid rafts' in live cells. To date, more than 200 proteins were found in DRMs including also members of the SNARE superfamily, which are small membrane proteins involved in intracellular fusion steps. Their raft association indicates that they are not uniformly distributed, and, indeed, microscopic studies revealed that SNAREs concentrate in submicrometre-sized, cholesterol-dependent clusters at which vesicles fuse. However, the idea that SNARE clusters are 'lipid rafts' was challenged, as they do not colocalize with raft markers, and SNAREs are excluded from liquid-ordered phases in model membranes. Independent from this disagreement, in recent years the solubilization criterion has been criticized for several reasons, calling for a more exact definition of rafts. At a recent consensus on a revised raft model, the term 'lipid rafts' was replaced by 'membrane rafts' that were defined as 'small (10-200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes'. As a result, after dismissing the terms 'detergent resistant' and 'liquid-ordered', it now appears that SNARE clusters are bona fide 'membrane rafts'.

  5. Glycoprotein misfolding in the endoplasmic reticulum: identification of released oligosaccharides reveals a second ER-associated degradation pathway for Golgi-retrieved proteins.

    PubMed

    Alonzi, Dominic S; Kukushkin, Nikolay V; Allman, Sarah A; Hakki, Zalihe; Williams, Spencer J; Pierce, Lorna; Dwek, Raymond A; Butters, Terry D

    2013-08-01

    Endoplasmic reticulum-associated degradation (ERAD) is a key cellular process whereby misfolded proteins are removed from the endoplasmic reticulum (ER) for subsequent degradation by the ubiquitin/proteasome system. In the present work, analysis of the released, free oligosaccharides (FOS) derived from all glycoproteins undergoing ERAD, has allowed a global estimation of the mechanisms of this pathway rather than following model proteins through degradative routes. Examining the FOS produced in endomannosidase-compromised cells following α-glucosidase inhibition has revealed a mechanism for clearing Golgi-retrieved glycoproteins that have failed to enter the ER quality control cycle. The Glc3Man7GlcNAc2 FOS species has been shown to be produced in the ER lumen by a mechanism involving a peptide: N-glycanase-like activity, and its production was sensitive to disruption of Golgi-ER trafficking. The detection of this oligosaccharide was unaffected by the overexpression of EDEM1 or cytosolic mannosidase, both of which increased the production of previously characterised cytosolically localised FOS. The lumenal FOS identified are therefore distinct in their production and regulation compared to FOS produced by the conventional route of misfolded glycoproteins directly removed from the ER. The production of such lumenal FOS is indicative of a novel degradative route for cellular glycoproteins that may exist under certain conditions.

  6. Folding, Binding, Misfolding and Aggregation with AWSEM

    NASA Astrophysics Data System (ADS)

    Schafer, Nicholas P.

    This thesis discusses our recent results using the Associative-memory, Water-mediated, Structure and Energy Model (AWSEM), an optimized, coarse-grained molecular dynamics protein folding model, to fold, bind, and predict the misfolding behavior of proteins. AWSEM is capable of performing de novo structure prediction on small alpha-helical protein domains and predict the binding interfaces of homo- and hetero-dimers. More recent work demonstrates how the misfolding behavior of tandem constructs in AWSEM is consistent with crucial aspects of ensemble and single molecule experiments on the aggregation and misfolding of these constructs. The first chapter is a review of the energy landscape theory of protein folding as it applies to the problem of protein structure prediction, and more specifically how energy landscape theory and the principle of minimal frustration can be used to optimize parameters of coarse-grained protein folding simulation models. The subsequent four chapters are reports of novel research performed with one such model.

  7. Misfolding of mutant adenine nucleotide translocase in yeast supports a novel mechanism of Ant1-induced muscle diseases.

    PubMed

    Liu, Yaxin; Wang, Xiaowen; Chen, Xin Jie

    2015-06-01

    Approximately one-third of proteins in the cell reside in the membrane. Mutations in membrane proteins can induce conformational changes and expose nonnative polar domains/residues to the lipid environment. The molecular effect of the resulting membrane stress is poorly defined. Adenine nucleotide translocase 1 (Ant1) is a mitochondrial inner membrane protein involved in ATP/ADP exchange. Missense mutations in the Ant1 isoform cause autosomal dominant progressive external ophthalmoplegia (adPEO), cardiomyopathy, and myopathy. The mechanism of the Ant1-induced pathologies is highly debated. Here we show that equivalent mutations in the yeast Aac2 protein cause protein misfolding. Misfolded Aac2 drastically affects the assembly and stability of multiple protein complexes in the membrane, which ultimately inhibits cell growth. Despite causing similar proteostatic damages, the adPEO- but not the cardiomyopathy/myopathy-type Aac2 proteins form large aggregates. The data suggest that the Ant1-induced diseases belong to protein misfolding disorders. Protein homeostasis is subtly maintained on the mitochondrial inner membrane and can be derailed by the misfolding of one single protein with or without aggregate formation. This finding could have broad implications for understanding other dominant diseases (e.g., retinitis pigmentosa) caused by missense mutations in membrane proteins. © 2015 Liu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  8. Heparan Sulfate and Heparin Promote Faithful Prion Replication in Vitro by Binding to Normal and Abnormal Prion Proteins in Protein Misfolding Cyclic Amplification.

    PubMed

    Imamura, Morikazu; Tabeta, Naoko; Kato, Nobuko; Matsuura, Yuichi; Iwamaru, Yoshifumi; Yokoyama, Takashi; Murayama, Yuichi

    2016-12-16

    The precise mechanism underlying the conversion of normal prion protein (PrP(C)) into abnormal prion protein (PrP(Sc)) remains unclear. Protein misfolding cyclic amplification (PMCA), an in vitro technique used for amplifying PrP(Sc), results in PrP(Sc) replication that preserves the strain-specific characteristics of the input PrP(Sc); thus, PMCA mimics the process of in vivo PrP(Sc) replication. Previous work has demonstrated that in PMCA, nucleic acids are critical for PrP(Sc) amplification, but little information has been reported on glycosaminoglycan (GAG) participation in PrP(Sc) replication in vitro Here, we investigated whether GAGs play a role in the faithful replication of PrP(Sc) by using a modified PMCA performed with baculovirus-derived recombinant PrP (Bac-PrP) as a substrate. The addition of heparan sulfate (HS) or its analog heparin (HP) restored the conversion efficiency in PMCA that was inhibited through nucleic acid depletion. Moreover, the PMCA products obtained under these conditions were infectious and preserved the properties of the input PrP(Sc) These data suggest that HS and HP play the same role as nucleic acids in facilitating faithful replication of prions in PMCA. Furthermore, we showed that HP binds to both Bac-PrP and Bac-PrP(Sc) through the sulfated groups present on HP and that the N-terminal domain of Bac-PrP(Sc) might potentially not be involved in the binding to HP. These results suggest that the interaction of GAGs such as HS and HP with PrP(C) and/or PrP(Sc) through their sulfate groups is critical for the faithful replication of prions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Strategies for the purification of membrane proteins.

    PubMed

    Smith, Sinead Marian

    2011-01-01

    Although membrane proteins account for 20-30% of the coding regions of all sequenced genomes and play crucial roles in many fundamental cell processes, there are relatively few membranes proteins with known 3D structure. This is likely due to technical challenges associated with membrane protein extraction, solubilisation, and purification. Membrane proteins are classified based on the level of interaction with membrane lipid bilayers, with peripheral membrane proteins associating non-covalently with the membrane, and integral membrane proteins associating more strongly by means of hydrophobic interactions. Generally speaking, peripheral membrane proteins can be purified by milder techniques than integral membrane proteins, whose extraction requires phospholipid bilayer disruption by detergents. Here, important criteria for strategies of membrane protein purification are addressed, with a focus on the initial stages of membrane protein solublilisation, where problems are most frequently encountered. Protocols are outlined for the successful extraction of peripheral membrane proteins, solubilisation of integral membrane proteins, and detergent removal which is important not only for retaining native protein stability and biological functions, but also for the efficiency of later purification techniques.

  10. Rapid and Highly Sensitive Detection of Variant Creutzfeldt - Jakob Disease Abnormal Prion Protein on Steel Surfaces by Protein Misfolding Cyclic Amplification: Application to Prion Decontamination Studies

    PubMed Central

    Belondrade, Maxime; Nicot, Simon; Béringue, Vincent; Coste, Joliette; Lehmann, Sylvain; Bougard, Daisy

    2016-01-01

    The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10−8 brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions. PMID:26800081

  11. The domain-specific and temperature-dependent protein misfolding phenotype of variant medium-chain acyl-CoA dehydrogenase.

    PubMed

    Jank, Johanna M; Maier, Esther M; Reiβ, Dunja D; Haslbeck, Martin; Kemter, Kristina F; Truger, Marietta S; Sommerhoff, Christian P; Ferdinandusse, Sacha; Wanders, Ronald J; Gersting, Søren W; Muntau, Ania C

    2014-01-01

    The implementation of expanded newborn screening programs reduced mortality and morbidity in medium-chain acyl-CoA dehydrogenase deficiency (MCADD) caused by mutations in the ACADM gene. However, the disease is still potentially fatal. Missense induced MCADD is a protein misfolding disease with a molecular loss-of-function phenotype. Here we established a comprehensive experimental setup to analyze the structural consequences of eight ACADM missense mutations (p.Ala52Val, p.Tyr67His, p.Tyr158His, p.Arg206Cys, p.Asp266Gly, p.Lys329Glu, p.Arg334Lys, p.Arg413Ser) identified after newborn screening and linked the corresponding protein misfolding phenotype to the site of side-chain replacement with respect to the domain. With fever being the crucial risk factor for metabolic decompensation of patients with MCADD, special emphasis was put on the analysis of structural and functional derangements related to thermal stress. Based on protein conformation, thermal stability and kinetic stability, the molecular phenotype in MCADD depends on the structural region that is affected by missense-induced conformational changes with the central β-domain being particularly prone to structural derangement and destabilization. Since systematic classification of conformational derangements induced by ACADM mutations may be a helpful tool in assessing the clinical risk of patients, we scored the misfolding phenotype of the variants in comparison to p.Lys329Glu (K304E), the classical severe mutation, and p.Tyr67His (Y42H), discussed to be mild. Experiments assessing the impact of thermal stress revealed that mutations in the ACADM gene lower the temperature threshold at which MCAD loss-of-function occurs. Consequently, increased temperature as it occurs during intercurrent infections, significantly increases the risk of further conformational derangement and loss of function of the MCAD enzyme explaining the life-threatening clinical courses observed during fever episodes. Early and

  12. Degradation-mediated protein quality control at the inner nuclear membrane

    PubMed Central

    Boban, Mirta; Foisner, Roland

    2016-01-01

    abstract An intricate machinery protects cells from the accumulation of misfolded, non-functional proteins and protein aggregates. Protein quality control pathways have been best described in the cytoplasm and the endoplasmic reticulum, however, recent findings indicate that the nucleus is also an important compartment for protein quality control. Several nuclear ubiquitinylation pathways target soluble and membrane proteins in the nucleus and mediate their degradation through nuclear proteasomes. In addition, emerging data suggest that nuclear envelope components are also degraded by autophagy, although the mechanisms by which cytoplasmic autophagy machineries get access to nuclear targets remain unclear. In this minireview we summarize the nuclear ubiquitin-proteasome pathways in yeast, focusing on pathways involved in the protein degradation at the inner nuclear membrane. In addition, we discuss potential mechanisms how nuclear targets at the nuclear envelope may be delivered to the cytoplasmic autophagy pathways in yeast and mammals. PMID:26760377

  13. Physiological Response to Membrane Protein Overexpression in E. coli*

    PubMed Central

    Gubellini, Francesca; Verdon, Grégory; Karpowich, Nathan K.; Luff, Jon D.; Boël, Grégory; Gauthier, Nils; Handelman, Samuel K.; Ades, Sarah E.; Hunt, John F.

    2011-01-01

    Overexpression represents a principal bottleneck in structural and functional studies of integral membrane proteins (IMPs). Although E. coli remains the leading organism for convenient and economical protein overexpression, many IMPs exhibit toxicity on induction in this host and give low yields of properly folded protein. Different mechanisms related to membrane biogenesis and IMP folding have been proposed to contribute to these problems, but there is limited understanding of the physical and physiological constraints on IMP overexpression and folding in vivo. Therefore, we used a variety of genetic, genomic, and microscopy techniques to characterize the physiological responses of Escherichia coli MG1655 cells to overexpression of a set of soluble proteins and IMPs, including constructs exhibiting different levels of toxicity and producing different levels of properly folded versus misfolded product on induction. Genetic marker studies coupled with transcriptomic results indicate only minor perturbations in many of the physiological systems implicated in previous studies of IMP biogenesis. Overexpression of either IMPs or soluble proteins tends to block execution of the standard stationary-phase transcriptional program, although these effects are consistently stronger for the IMPs included in our study. However, these perturbations are not an impediment to successful protein overexpression. We present evidence that, at least for the target proteins included in our study, there is no inherent obstacle to IMP overexpression in E. coli at moderate levels suitable for structural studies and that the biochemical and conformational properties of the proteins themselves are the major obstacles to success. Toxicity associated with target protein activity produces selective pressure leading to preferential growth of cells harboring expression-reducing and inactivating mutations, which can produce chemical heterogeneity in the target protein population, potentially

  14. Proteomic analysis of integral plasma membrane proteins.

    PubMed

    Zhao, Yingxin; Zhang, Wei; Kho, Yoonjung; Zhao, Yingming

    2004-04-01

    Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 microg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.

  15. Membrane stiffness is modified by integral membrane proteins.

    PubMed

    Fowler, Philip W; Hélie, Jean; Duncan, Anna; Chavent, Matthieu; Koldsø, Heidi; Sansom, Mark S P

    2016-09-20

    The ease with which a cell membrane can bend and deform is important for a wide range of biological functions. Peripheral proteins that induce curvature in membranes (e.g. BAR domains) have been studied for a number of years. Little is known, however, about the effect of integral membrane proteins on the stiffness of a membrane (characterised by the bending rigidity, Kc). We demonstrate by computer simulation that adding integral membrane proteins at physiological densities alters the stiffness of the membrane. First we establish that the coarse-grained MARTINI forcefield is able to accurately reproduce the bending rigidity of a small patch of 1500 phosphatidyl choline lipids by comparing the calculated value to both experiment and an atomistic simulation of the same system. This enables us to simulate the dynamics of large (ca. 50 000 lipids) patches of membrane using the MARTINI coarse-grained description. We find that altering the lipid composition changes the bending rigidity. Adding integral membrane proteins to lipid bilayers also changes the bending rigidity, whilst adding a simple peripheral membrane protein has no effect. Our results suggest that integral membrane proteins can have different effects, and in the case of the bacterial outer membrane protein, BtuB, the greater the density of protein, the larger the reduction in stiffness.

  16. Yeast mutants affecting possible quality control of plasma membrane proteins.

    PubMed

    Li, Y; Kane, T; Tipper, C; Spatrick, P; Jenness, D D

    1999-05-01

    Mutations gef1, stp22, STP26, and STP27 in Saccharomyces cerevisiae were identified as suppressors of the temperature-sensitive alpha-factor receptor (mutation ste2-3) and arginine permease (mutation can1(ts)). These suppressors inhibited the elimination of misfolded receptors (synthesized at 34 degrees C) as well as damaged surface receptors (shifted from 22 to 34 degrees C). The stp22 mutation (allelic to vps23 [M. Babst and S. Emr, personal communication] and the STP26 mutation also caused missorting of carboxypeptidase Y, and ste2-3 was suppressed by mutations vps1, vps8, vps10, and vps28 but not by mutation vps3. In the stp22 mutant, both the mutant and the wild-type receptors (tagged with green fluorescent protein [GFP]) accumulated within an endosome-like compartment and were excluded from the vacuole. GFP-tagged Stp22p also accumulated in this compartment. Upon reaching the vacuole, cytoplasmic domains of both mutant and wild-type receptors appeared within the vacuolar lumen. Stp22p and Gef1p are similar to tumor susceptibility protein TSG101 and voltage-gated chloride channel, respectively. These results identify potential elements of plasma membrane quality control and indicate that cytoplasmic domains of membrane proteins are translocated into the vacuolar lumen.

  17. Biopores/membrane proteins in synthetic polymer membranes.

    PubMed

    Garni, Martina; Thamboo, Sagana; Schoenenberger, Cora-Ann; Palivan, Cornelia G

    2017-04-01

    Mimicking cell membranes by simple models based on the reconstitution of membrane proteins in lipid bilayers represents a straightforward approach to understand biological function of these proteins. This biomimetic strategy has been extended to synthetic membranes that have advantages in terms of chemical and mechanical stability, thus providing more robust hybrid membranes. We present here how membrane proteins and biopores have been inserted both in the membrane of nanosized and microsized compartments, and in planar membranes under various conditions. Such bio-hybrid membranes have new properties (as for example, permeability to ions/molecules), and functionality depending on the specificity of the inserted biomolecules. Interestingly, membrane proteins can be functionally inserted in synthetic membranes provided these have appropriate properties to overcome the high hydrophobic mismatch between the size of the biomolecule and the membrane thickness. Functional insertion of membrane proteins and biopores in synthetic membranes of compartments or in planar membranes is possible by an appropriate selection of the amphiphilic copolymers, and conditions of the self-assembly process. These hybrid membranes have new properties and functionality based on the specificity of the biomolecules and the nature of the synthetic membranes. Bio-hybrid membranes represent new solutions for the development of nanoreactors, artificial organelles or active surfaces/membranes that, by further gaining in complexity and functionality, will promote translational applications. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider. Copyright © 2016. Published by Elsevier B.V.

  18. The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria.

    PubMed

    Jin, Seok Min; Youle, Richard J

    2013-11-01

    Defective mitochondria exert deleterious effects on host cells. To manage this risk, mitochondria display several lines of quality control mechanisms: mitochondria-specific chaperones and proteases protect against misfolded proteins at the molecular level, and fission/fusion and mitophagy segregate and eliminate damage at the organelle level. An increase in unfolded proteins in mitochondria activates a mitochondrial unfolded protein response (UPR(mt)) to increase chaperone production, while the mitochondrial kinase PINK1 and the E3 ubiquitin ligase PARK2/Parkin, whose mutations cause familial Parkinson disease, remove depolarized mitochondria through mitophagy. It is unclear, however, if there is a connection between those different levels of quality control (QC). Here, we show that the expression of unfolded proteins in the matrix causes the accumulation of PINK1 on energetically healthy mitochondria, resulting in mitochondrial translocation of PARK2, mitophagy and subsequent reduction of unfolded protein load. Also, PINK1 accumulation is greatly enhanced by the knockdown of the LONP1 protease. We suggest that the accumulation of unfolded proteins in mitochondria is a physiological trigger of mitophagy.

  19. Computational modeling of membrane proteins

    PubMed Central

    Leman, Julia Koehler; Ulmschneider, Martin B.; Gray, Jeffrey J.

    2014-01-01

    The determination of membrane protein (MP) structures has always trailed that of soluble proteins due to difficulties in their overexpression, reconstitution into membrane mimetics, and subsequent structure determination. The percentage of MP structures in the protein databank (PDB) has been at a constant 1-2% for the last decade. In contrast, over half of all drugs target MPs, only highlighting how little we understand about drug-specific effects in the human body. To reduce this gap, researchers have attempted to predict structural features of MPs even before the first structure was experimentally elucidated. In this review, we present current computational methods to predict MP structure, starting with secondary structure prediction, prediction of trans-membrane spans, and topology. Even though these methods generate reliable predictions, challenges such as predicting kinks or precise beginnings and ends of secondary structure elements are still waiting to be addressed. We describe recent developments in the prediction of 3D structures of both α-helical MPs as well as β-barrels using comparative modeling techniques, de novo methods, and molecular dynamics (MD) simulations. The increase of MP structures has (1) facilitated comparative modeling due to availability of more and better templates, and (2) improved the statistics for knowledge-based scoring functions. Moreover, de novo methods have benefitted from the use of correlated mutations as restraints. Finally, we outline current advances that will likely shape the field in the forthcoming decade. PMID:25355688

  20. Protein roles in group I intron RNA folding: The tyrosyl-tRNA synthetase CYT-18 stabilizes the native state relative to a long-lived misfolded structure without compromising folding kinetics

    PubMed Central

    Chadee, Amanda B.; Bhaskaran, Hari; Russell, Rick

    2009-01-01

    The Neurospora crassa CYT-18 protein is a mitochondrial tyrosyl-tRNA synthetase that also promotes self-splicing of group I intron RNAs by stabilizing functional structure in the conserved core. CYT-18 binds the core along the same surface as a common peripheral element, P5abc, suggesting that CYT-18 can replace P5abc functionally. In addition to stabilizing structure generally, P5abc stabilizes the native conformation of the Tetrahymena group I intron relative to a globally-similar misfolded conformation, which has only local differences within the core and is populated significantly at equilibrium by a ribozyme variant lacking P5abc (EΔP5abc). Here we show that CYT-18 specifically promotes formation of the native group I intron core from this misfolded conformation. Catalytic activity assays demonstrate that CYT-18 shifts the equilibrium of EΔP5abc toward the native state by at least 35-fold, and binding assays suggest an even larger effect. Thus, like P5abc, CYT-18 preferentially recognizes the native core, despite the global similarity of the misfolded core and despite forming crudely similar complexes, as revealed by DMS footprinting. Interestingly, the effects of CYT-18 and P5abc on folding kinetics differ. Whereas P5abc inhibits refolding of the misfolded conformation by forming peripheral contacts that must break during refolding, CYT-18 does not display analogous inhibition, most likely because it relies to a greater extent on direct interactions with the core. Although CYT-18 does not encounter this RNA in vivo, our results suggest that it stabilizes its cognate group I introns relative to analogous misfolded intermediates. By specifically recognizing native structure, CYT-18 may also interact with earlier folding intermediates to avoid RNA misfolding or to trap native structure as it forms. More generally, our results highlight the ability of a protein cofactor to stabilize a functional RNA structure specifically without incurring associated costs in

  1. The role of the cytosolic HSP70 chaperone system in diseases caused by misfolding and aberrant trafficking of ion channels.

    PubMed

    Young, Jason C

    2014-03-01

    Protein-folding diseases are an ongoing medical challenge. Many diseases within this group are genetically determined, and have no known cure. Among the examples in which the underlying cellular and molecular mechanisms are well understood are diseases driven by misfolding of transmembrane proteins that normally function as cell-surface ion channels. Wild-type forms are synthesized and integrated into the endoplasmic reticulum (ER) membrane system and, upon correct folding, are trafficked by the secretory pathway to the cell surface. Misfolded mutant forms traffic poorly, if at all, and are instead degraded by the ER-associated proteasomal degradation (ERAD) system. Molecular chaperones can assist the folding of the cytosolic domains of these transmembrane proteins; however, these chaperones are also involved in selecting misfolded forms for ERAD. Given this dual role of chaperones, diseases caused by the misfolding and aberrant trafficking of ion channels (referred to here as ion-channel-misfolding diseases) can be regarded as a consequence of insufficiency of the pro-folding chaperone activity and/or overefficiency of the chaperone ERAD role. An attractive idea is that manipulation of the chaperones might allow increased folding and trafficking of the mutant proteins, and thereby partial restoration of function. This Review outlines the roles of the cytosolic HSP70 chaperone system in the best-studied paradigms of ion-channel-misfolding disease--the CFTR chloride channel in cystic fibrosis and the hERG potassium channel in cardiac long QT syndrome type 2. In addition, other ion channels implicated in ion-channel-misfolding diseases are discussed.

  2. Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

    DOEpatents

    Laible, Philip D; Hanson, Deborah K

    2013-06-04

    The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

  3. Bcl-2 Decreases the Affinity of SQSTM1/p62 to Poly-Ubiquitin Chains and Suppresses the Aggregation of Misfolded Protein in Neurodegenerative Disease.

    PubMed

    Zhou, Liang; Wang, Hongfeng; Ren, Haigang; Hu, Qingsong; Ying, Zheng; Wang, Guanghui

    2015-12-01

    Poly-ubiquitinated protein aggregate formation is the most striking hallmark of various neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, and prion disease. Mutations of many ubiquitin-associated proteins involved in the regulation of protein aggregation, such as SQSTM1/p62 (p62), parkin, and VCP, are closely linked to neurodegeneration. B-cell lymphoma 2 (Bcl-2) is a key regulator in autophagy, apoptosis, and mitochondria quality control in many cell types including neurons, and it plays important roles in the pathogenesis of neurodegenerative diseases mentioned above. Our previous work showed that Bcl-2 can directly bind to p62, and here we report that Bcl-2 directly interacts with the N-terminus of p62, but not the C-terminus (UBA domain). Interestingly and importantly, Bcl-2 affects the affinity of p62 to poly-ubiquitin chains and suppresses the aggregation of poly-ubiquitinated proteins such as mutant huntingtin associated with Huntington's disease. Our study reveals a role of Bcl-2 that involves in the regulation of misfolded proteins.

  4. Membrane shape modulates transmembrane protein distribution

    PubMed Central

    Aimon, Sophie; Callan-Jones, Andrew; Berthaud, Alice; Pinot, Mathieu; Toombes, Gilman E. S.; Bassereau, Patricia

    2014-01-01

    Summary Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown due to the numerous sorting mechanisms taking place concurrently in cells. To isolate the effect of membrane shape, cellsized Giant Unilamellar Vesicles (GUVs) containing either the potassium channel, KvAP, or water channel, AQP0, were used to form membrane nanotubes with controlled radii. While the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. FRAP measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins, and provides a method for determining the effective shape and flexibility of membrane proteins. PMID:24480645

  5. Cystamine-mediated inhibition of protein disulfide isomerase triggers aggregation of misfolded orexin-A in the Golgi apparatus and prevents extracellular secretion of orexin-A.

    PubMed

    Fujita, Issei; Nobunaga, Mizuki; Seki, Takahiro; Kurauchi, Yuki; Hisatsune, Akinori; Katsuki, Hiroshi

    2017-07-22

    Orexins (orexin-A and orexin-B) are neuropeptides that are reduced in narcolepsy, a sleep disorder that is characterized by excessive daytime sleepiness, sudden sleep attacks and cataplexy. However, it remains unclear how orexins in the brain and orexin neurons are reduced in narcolepsy. Orexin-A has two closely located intramolecular disulfide bonds and is prone to misfolding due to the formation of incorrect disulfide bonds. Protein disulfide isomerase (PDI) possesses disulfide interchange activity. PDI can modify misfolded orexin-A to its native form by rearrangement of two disulfide bonds. We have previously demonstrated that sleep deprivation and a high fat diet increase nitric oxide in the brain. This increase triggers S-nitrosation and inactivation of PDI, leading to aggregation of orexin-A and reduction of orexin neurons. However, the relationship between PDI inactivation and loss of orexin neurons has not yet been fully elucidated. In the present study, we used a PDI inhibitor, cystamine, to elucidate the precise molecular mechanism by which PDI inhibition reduces the number of orexin neurons. In rat hypothalamic slice cultures, cystamine induced selective depletion of orexin-A, but not orexin-B and melanin-concentrating hormone. Moreover, cystamine triggered aggregation of orexin-A, but not orexin-B in the Golgi apparatus of hypothalamic slice cultures and in vivo mouse brains. However, cystamine did not induce endoplasmic reticulum (ER) stress, and an ER stress inducer did not trigger aggregation of orexin-A in slice cultures. Finally, we demonstrated that cystamine significantly decreased extracellular secretion of orexin-A in AD293 cells overexpressing prepro-orexin. These findings suggest that cystamine-induced PDI inhibition induces selective depletion, aggregation in the Golgi apparatus and impaired secretion of orexin-A. These effects may represent an initial step in the pathogenesis of narcolepsy. Copyright © 2017. Published by Elsevier Inc.

  6. Enhanced membrane protein expression by engineering increased intracellular membrane production

    PubMed Central

    2013-01-01

    Background Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of

  7. Membrane Structure: Lipid-Protein Interactions in Microsomal Membranes*

    PubMed Central

    Trump, Benjamin F.; Duttera, Sue M.; Byrne, William L.; Arstila, Antti U.

    1970-01-01

    The relationships of phospholipid to membrane structure and function were examined in hepatic microsomes. Findings indicate that normal microsomal membrane structure is dependent on lipid-protein interactions and that it correlates closely with glucose-6-phosphatase activity. Modification of most phospholipid with phospholipase-C is associated with widening of the membrane which can be reversed following readdition of phospholipid. Images PMID:4317915

  8. Membrane proteins: always an insoluble problem?

    PubMed Central

    Rawlings, Andrea E.

    2016-01-01

    Membrane proteins play crucial roles in cellular processes and are often important pharmacological drug targets. The hydrophobic properties of these proteins make full structural and functional characterization challenging because of the need to use detergents or other solubilizing agents when extracting them from their native lipid membranes. To aid membrane protein research, new methodologies are required to allow these proteins to be expressed and purified cheaply, easily, in high yield and to provide water soluble proteins for subsequent study. This mini review focuses on the relatively new area of water soluble membrane proteins and in particular two innovative approaches: the redesign of membrane proteins to yield water soluble variants and how adding solubilizing fusion proteins can help to overcome these challenges. This review also looks at naturally occurring membrane proteins, which are able to exist as stable, functional, water soluble assemblies with no alteration to their native sequence. PMID:27284043

  9. Membrane tension and peripheral protein density mediate membrane shape transitions

    NASA Astrophysics Data System (ADS)

    Shi, Zheng; Baumgart, Tobias

    2015-01-01

    Endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. The mechanism of the coupling between these two processes to enable homeostasis is not well understood. Recently, an ultrafast endocytosis (UFE) pathway was revealed with a speed significantly exceeding classical clathrin-mediated endocytosis (CME). Membrane tension reduction is a potential mechanism by which endocytosis can be rapidly activated at remote sites. Here, we provide experimental evidence for a mechanism whereby membrane tension reduction initiates membrane budding and tubulation mediated by endocytic proteins, such as endophilin A1. We find that shape instabilities occur at well-defined membrane tensions and surface densities of endophilin. From our data, we obtain a membrane shape stability diagram that shows remarkable consistency with a quantitative model. This model applies to all laterally diffusive curvature-coupling proteins and therefore a wide range of endocytic proteins.

  10. Detection of Proteins on Blot Membranes.

    PubMed

    Goldman, Aaron; Harper, Sandra; Speicher, David W

    2016-11-01

    Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc.

  11. Capillary electrophoresis analysis of different variants of the amyloidogenic protein β2 -microglobulin as a simple tool for misfolding and stability studies.

    PubMed

    Bertoletti, Laura; Bisceglia, Federica; Colombo, Raffaella; Giorgetti, Sofia; Raimondi, Sara; Mangione, P Patrizia; De Lorenzi, Ersilia

    2015-10-01

    Free solution capillary electrophoresis with UV detection is here used to retrieve information on the conformational changes of wild-type β2 -microglobulin and a series of naturally and artificially created variants known to have different stability and amyloidogenic potential. Under nondenaturing conditions, the resolution of at least two folding conformers at equilibrium is obtained and a third species is detected for the less stable isoforms. Partial denaturation by using chaotropic agents such as acetonitrile or trifluoroethanol reveals that the separated peaks are at equilibrium, as the presence of less structured species is either enhanced or induced at the expenses of the native form. Reproducible CE data allow to obtain an interesting semiquantitative correlation between the peak areas observed and the protein stability. Thermal unfolding over the range 25-42°C is induced inside the capillary for the two pathogenic proteins (wtβ2 -microglobulin and D76N variant): the large differences observed upon small temperature variation draw attention on the robustness of analytical methods when dealing with proteins prone to misfolding and aggregation.

  12. Lipids in the assembly of membrane proteins and organization of protein supercomplexes: implications for lipid-linked disorders.

    PubMed

    Bogdanov, Mikhail; Mileykovskaya, Eugenia; Dowhan, William

    2008-01-01

    Lipids play important roles in cellular dysfunction leading to disease. Although a major role for phospholipids is in defining the membrane permeability barrier, phospholipids play a central role in a diverse range of cellular processes and therefore are important factors in cellular dysfunction and disease. This review is focused on the role of phospholipids in normal assembly and organization of the membrane proteins, multimeric protein complexes, and higher order supercomplexes. Since lipids have no catalytic activity, it is difficult to determine their function at the molecular level. Lipid function has generally been defined by affects on protein function or cellular processes. Molecular details derived from genetic, biochemical, and structural approaches are presented for involvement of phosphatidylethanolamine and cardiolipin in protein organization. Experimental evidence is presented that changes in phosphatidylethanolamine levels results in misfolding and topological misorientation of membrane proteins leading to dysfunctional proteins. Examples are presented for diseases in which proper protein folding or topological organization is not attained due to either demonstrated or proposed involvement of a lipid. Similar changes in cardiolipin levels affects the structure and function of individual components of the mitochondrial electron transport chain and their organization into supercomplexes resulting in reduced mitochondrial oxidative phosphorylation efficiency and apoptosis. Diseases in which mitochondrial dysfunction has been linked to reduced cardiolipin levels are described. Therefore, understanding the principles governing lipid-dependent assembly and organization of membrane proteins and protein complexes will be useful in developing novel therapeutic approaches for disorders in which lipids play an important role.

  13. Malate synthase a membrane protein

    SciTech Connect

    Chapman, K.D.; Turley, R.B.; Hermerath, C.A.; Carrapico, F.; Trelease, R.N.

    1987-04-01

    Malate synthase (MS) is generally regarded as a peripheral membrane protein, and believed by some to be ontogenetically associated with ER. However, immuno- and cyto-chemical in situ localizations show MS throughout the matrix of cotton (and cucumber) glyoxysomes, not specifically near their boundary membranes, nor in ER. Only a maximum of 50% MS can be solubilized from cotton glyoxysomes with 1% Triton X-100, 2mM Zwittergen 14, or 10mM DOC +/- salts. Cotton MS does not incorporate /sup 3/H-glucosamine in vivo, nor does it react with Con A on columns or blots. Cotton MS banded with ER in sucrose gradients (20-40%) in Tricine after 3h, but not after 22h in Tricine or Hepes, or after 3h in Hepes or K-phosphate. Collectively the authors data are inconsistent with physiologically meaningful MS-membrane associations in ER or glyoxysomes. It appears that experimentally-induced aggregates of MS migrate in ER gradients and occur in isolated glyoxysomes. These data indicate that ER is not involved in synthesis or modification of cottonseed MS prior to its import into the glyoxysomal matrix.

  14. Protein-Induced Membrane Curvature Alters Local Membrane Tension

    PubMed Central

    Rangamani, Padmini; Mandadap, Kranthi K.; Oster, George

    2014-01-01

    Adsorption of proteins onto membranes can alter the local membrane curvature. This phenomenon has been observed in biological processes such as endocytosis, tubulation, and vesiculation. However, it is not clear how the local surface properties of the membrane, such as membrane tension, change in response to protein adsorption. In this article, we show that the partial differential equations arising from classical elastic model of lipid membranes, which account for simultaneous changes in shape and membrane tension due to protein adsorption in a local region, cannot be solved for nonaxisymmetric geometries using straightforward numerical techniques; instead, a viscous-elastic formulation is necessary to fully describe the system. Therefore, we develop a viscous-elastic model for inhomogeneous membranes of the Helfrich type. Using the newly available viscous-elastic model, we find that the lipids flow to accommodate changes in membrane curvature during protein adsorption. We show that, at the end of protein adsorption process, the system sustains a residual local tension to balance the difference between the actual mean curvature and the imposed spontaneous curvature. We also show that this change in membrane tension can have a functional impact such as altered response to pulling forces in the presence of proteins. PMID:25099814

  15. Internal packing of helical membrane proteins

    PubMed Central

    Eilers, Markus; Shekar, Srinivasan C.; Shieh, Ted; Smith, Steven O.; Fleming, Patrick J.

    2000-01-01

    Helix packing is important in the folding, stability, and association of membrane proteins. Packing analysis of the helical portions of 7 integral membrane proteins and 37 soluble proteins show that the helices in membrane proteins have higher packing values (0.431) than in soluble proteins (0.405). The highest packing values in integral membrane proteins originate from small hydrophobic (G and A) and small hydroxyl-containing (S and T) amino acids, whereas in soluble proteins large hydrophobic and aromatic residues have the highest packing values. The highest packing values for membrane proteins are found in the transmembrane helix–helix interfaces. Glycine and alanine have the highest occurrence among the buried amino acids in membrane proteins, whereas leucine and alanine are the most common buried residue in soluble proteins. These observations are consistent with a shorter axial separation between helices in membrane proteins. The tight helix packing revealed in this analysis contributes to membrane protein stability and likely compensates for the lack of the hydrophobic effect as a driving force for helix–helix association in membranes. PMID:10823938

  16. Artificial membranes for membrane protein purification, functionality and structure studies.

    PubMed

    Parmar, Mayuriben J; Lousa, Carine De Marcos; Muench, Stephen P; Goldman, Adrian; Postis, Vincent L G

    2016-06-15

    Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  17. Solid State NMR and Protein-Protein Interactions in Membranes

    PubMed Central

    Miao, Yimin; Cross, Timothy A.

    2013-01-01

    Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water soluble proteins and other membrane proteins. PMID:24034903

  18. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion.

    PubMed

    Dong, Jinlan; Bruening, Merlin L

    2015-01-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  19. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

    NASA Astrophysics Data System (ADS)

    Dong, Jinlan; Bruening, Merlin L.

    2015-07-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  20. Protein Homeostasis at the Plasma Membrane

    PubMed Central

    2014-01-01

    The plasma membrane (PM) and endocytic protein quality control (QC) in conjunction with the endosomal sorting machinery either repairs or targets conformationally damaged membrane proteins for lysosomal/vacuolar degradation. Here, we provide an overview of emerging aspects of the underlying mechanisms of PM QC that fulfill a critical role in preserving cellular protein homeostasis in health and diseases. PMID:24985330

  1. Tuning microbial hosts for membrane protein production

    PubMed Central

    2009-01-01

    The last four years have brought exciting progress in membrane protein research. Finally those many efforts that have been put into expression of eukaryotic membrane proteins are coming to fruition and enable to solve an ever-growing number of high resolution structures. In the past, many skilful optimization steps were required to achieve sufficient expression of functional membrane proteins. Optimization was performed individually for every membrane protein, but provided insight about commonly encountered bottlenecks and, more importantly, general guidelines how to alleviate cellular limitations during microbial membrane protein expression. Lately, system-wide analyses are emerging as powerful means to decipher cellular bottlenecks during heterologous protein production and their use in microbial membrane protein expression has grown in popularity during the past months. This review covers the most prominent solutions and pitfalls in expression of eukaryotic membrane proteins using microbial hosts (prokaryotes, yeasts), highlights skilful applications of our basic understanding to improve membrane protein production. Omics technologies provide new concepts to engineer microbial hosts for membrane protein production. PMID:20040113

  2. Folding or misfolding: the choice of β-hairpin.

    PubMed

    Shao, Qiang

    2015-03-12

    Proteins fold through complex inter-residue interactions which are mutually supportive and cooperatively lead to thermodynamically favorable native structures. Competing (misfolded) structures, however, could exist, which might affect the thermodynamic and kinetic properties of folded structure. Running long-time REMD simulations on two β-structured polypeptides, the present study identifies the folded and (less populated) competing misfolded states of β-hairpins. Of particular interest is a one-residue shifted misfolded state which has been often seen in previous reports. The folding and misfolding pathways are then energetically characterized by free energy landscape analysis, indicating that the folding and misfolding of β-hairpin are parallel pathways and a protein's selection of following which pathway is a consequence of the competition between the formation of alterable turn configurations and cross-strand hydrophobic interactions. Proteins possessing high percentage of hydrophobic residues introduce strong cross-strand hydrophobic interactions which stabilize the native structural elements in the folding pathway, leading to low possibility of misfolding. The present study provides novel insights into the origin of sequence-dependent β-hairpin misfolding "hidden" behind experimentally detectable β-hairpin folding, suggesting the direction for the structure design of β-structured protein.

  3. Circumnavigating misfolding traps in the energy landscape through protein engineering: suppression of molten globule and aggregation in carbonic anhydrase.

    PubMed

    Karlsson, Martin; Mårtensson, Lars-Göran; Olofsson, Patrik; Carlsson, Uno

    2004-06-01

    The native state of the enzyme human carbonic anhydrase (HCA II) has been stabilized by the introduction of a disulfide bond, the oxidized A23C/L203C mutant. This stabilized protein variant undergoes an apparent two-state unfolding process with suppression of the otherwise stable equilibrium, molten-globule intermediate, which is normally very prone to aggregation. Stopped-flow measurements also showed that lower amounts of the transiently occurring molten globule were formed during refolding. This led to a markedly lowered tendency for aggregation during equilibrium denaturing conditions and, more importantly, to significantly higher reactivation yields upon refolding of the fully denatured protein. Thus, a general strategy to circumvent aggregation during the refolding of proteins could be to stabilize the native state of a protein at the expense of partially folded intermediates, thereby shifting the unfolding behavior from a three-state process to a two-state one.

  4. [Membrane protein characterization by photoactivatable localization microscopy].

    PubMed

    Huang, Li; Fang, Weihuan; Yu, Ying; Song, Houhui

    2012-11-01

    The on-site labeling and localization tracking of membrane proteins in pathogenic bacteria are tedious work. In order to develop a novel protein labeling technology at super resolution level (nanometer scale) using the photoactivatable localization microscopy (PALM), the chimeric protein of the outer membrane protein A (OmpA) of Mycobacterium tuberculosis and the photoactivatable mEos2m protein were expressed in the non-pathogenic Mycobacterium smegmatis. The recombinant bacteria were fixed on slide, activated by 405 nm laser and subject to PALM imaging to capture photons released by the fusion protein. Meanwhile, colony and cell morphology were visualized under regular fluorescent stereomicroscope and upright fluorescent microscope to characterize fluorescence conversion and protein localization. The fusion proteins formed a "belt"-like structure on cell membrane of M. smegmatis under PALM, providing direct evidence of on-site imaging of membrane proteins. Expression of fusion protein did not compromise the localization properties of OmpA. Thus, mEos2m could be used as a labeling probe to track localizations of non-oligomer oriented membrane proteins. This indicates non-pathogenic M. smegmatis could be served as a model strain to characterize the function and localization of the proteins derived from pathogenic M. tuberculosis. This is the first report using PALM to characterize localization of membrane proteins.

  5. Membrane topology of transmembrane proteins: determinants and experimental tools.

    PubMed

    Lee, Hunsang; Kim, Hyun

    2014-10-17

    Membrane topology refers to the two-dimensional structural information of a membrane protein that indicates the number of transmembrane (TM) segments and the orientation of soluble domains relative to the plane of the membrane. Since membrane proteins are co-translationally translocated across and inserted into the membrane, the TM segments orient themselves properly in an early stage of membrane protein biogenesis. Each membrane protein must contain some topogenic signals, but the translocation components and the membrane environment also influence the membrane topology of proteins. We discuss the factors that affect membrane protein orientation and have listed available experimental tools that can be used in determining membrane protein topology.

  6. Insight into PreImplantation Factor (PIF*) Mechanism for Embryo Protection and Development: Target Oxidative Stress and Protein Misfolding (PDI and HSP) through Essential RIPK Binding Site

    PubMed Central

    Barnea, Eytan R.; Lubman, David M.; Liu, Yan-Hui; Absalon-Medina, Victor; Hayrabedyan, Soren; Todorova, Krassimira; Gilbert, Robert O.; Guingab, Joy; Barder, Timothy J.

    2014-01-01

    Background Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised. Methods FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). In silico evaluation examined binding of PIF to critical targets, using mutation analysis. Results PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIPK site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented. Conclusion Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF

  7. Insight into PreImplantation Factor (PIF*) mechanism for embryo protection and development: target oxidative stress and protein misfolding (PDI and HSP) through essential RIKP [corrected] binding site.

    PubMed

    Barnea, Eytan R; Lubman, David M; Liu, Yan-Hui; Absalon-Medina, Victor; Hayrabedyan, Soren; Todorova, Krassimira; Gilbert, Robert O; Guingab, Joy; Barder, Timothy J

    2014-01-01

    Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised. FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). In silico evaluation examined binding of PIF to critical targets, using mutation analysis. PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIKP [corrected] site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented. Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a novel

  8. Pharmacological correction of misfolding of ABC proteins☆

    PubMed Central

    Rudashevskaya, Elena L.; Stockner, Thomas; Trauner, Michael; Freissmuth, Michael; Chiba, Peter

    2014-01-01

    The endoplasmic reticulum (ER) quality control system distinguishes between correctly and incorrectly folded proteins to prevent processing of aberrantly folded conformations along the secretory pathway. Non-synonymous mutations can lead to misfolding of ABC proteins and associated disease phenotypes. Specific phenotypes may at least partially be corrected by small molecules, so-called pharmacological chaperones. Screening for folding correctors is expected to open an avenue for treatment of diseases such as cystic fibrosis and intrahepatic cholestasis. PMID:25027379

  9. Predictions of Protein-Protein Interfaces within Membrane Protein Complexes

    PubMed Central

    Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz

    2013-01-01

    Background Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. Methods In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. Results The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Conclusion Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues. PMID:23919118

  10. The unfolded protein response in melanocytes: activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding.

    PubMed

    Manga, Prashiela; Bis, Sabina; Knoll, Kristen; Perez, Beremis; Orlow, Seth J

    2010-10-01

    Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways.

  11. Membrane Protein Structure Determination in Membrana

    PubMed Central

    DING, YI; YAO, YONG; MARASSI, FRANCESCA M.

    2014-01-01

    CONSPECTUS The two principal components of biological membranes, the lipid bilayer and the proteins integrated within it, have coevolved for specific functions that mediate the interactions of cells with their environment. Molecular structures can provide very significant insights about protein function. In the case of membrane proteins, the physical and chemical properties of lipids and proteins are highly interdependent; therefore structure determination should include the membrane environment. Considering the membrane alongside the protein eliminates the possibility that crystal contacts or detergent molecules could distort protein structure, dynamics, and function and enables ligand binding studies to be performed in a natural setting. Solid-state NMR spectroscopy is compatible with three-dimensional structure determination of membrane proteins in phospholipid bilayer membranes under physiological conditions and has played an important role in elucidating the physical and chemical properties of biological membranes, providing key information about the structure and dynamics of the phospholipid components. Recently, developments in the recombinant expression of membrane proteins, sample preparation, pulse sequences for high-resolution spectroscopy, radio frequency probes, high-field magnets, and computational methods have enabled a number of membrane protein structures to be determined in lipid bilayer membranes. In this Account, we illustrate solid-state NMR methods with examples from two bacterial outer membrane proteins (OmpX and Ail) that form integral membrane β-barrels. The ability to measure orientation-dependent frequencies in the solid-state NMR spectra of membrane-embedded proteins provides the foundation for a powerful approach to structure determination based primarily on orientation restraints. Orientation restraints are particularly useful for NMR structural studies of membrane proteins because they provide information about both three

  12. Membrane protein structure determination in membrana.

    PubMed

    Ding, Yi; Yao, Yong; Marassi, Francesca M

    2013-09-17

    The two principal components of biological membranes, the lipid bilayer and the proteins integrated within it, have coevolved for specific functions that mediate the interactions of cells with their environment. Molecular structures can provide very significant insights about protein function. In the case of membrane proteins, the physical and chemical properties of lipids and proteins are highly interdependent; therefore structure determination should include the membrane environment. Considering the membrane alongside the protein eliminates the possibility that crystal contacts or detergent molecules could distort protein structure, dynamics, and function and enables ligand binding studies to be performed in a natural setting. Solid-state NMR spectroscopy is compatible with three-dimensional structure determination of membrane proteins in phospholipid bilayer membranes under physiological conditions and has played an important role in elucidating the physical and chemical properties of biological membranes, providing key information about the structure and dynamics of the phospholipid components. Recently, developments in the recombinant expression of membrane proteins, sample preparation, pulse sequences for high-resolution spectroscopy, radio frequency probes, high-field magnets, and computational methods have enabled a number of membrane protein structures to be determined in lipid bilayer membranes. In this Account, we illustrate solid-state NMR methods with examples from two bacterial outer membrane proteins (OmpX and Ail) that form integral membrane β-barrels. The ability to measure orientation-dependent frequencies in the solid-state NMR spectra of membrane-embedded proteins provides the foundation for a powerful approach to structure determination based primarily on orientation restraints. Orientation restraints are particularly useful for NMR structural studies of membrane proteins because they provide information about both three-dimensional structure

  13. Protein Solvation in Membranes and at Water-Membrane Interfaces

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Chipot, Christophe; Wilson, Michael A.

    2002-01-01

    Different salvation properties of water and membranes mediate a host of biologically important processes, such as folding, insertion into a lipid bilayer, associations and functions of membrane proteins. These processes will be discussed in several examples involving synthetic and natural peptides. In particular, a mechanism by which a helical peptide becomes inserted into a model membrane will be described. Further, the molecular mechanism of recognition and association of protein helical segments in membranes will be discussed. These processes are crucial for proper functioning of a cell. A membrane-spanning domain of glycophorin A, which exists as a helical dimer, serves as the model system. For this system, the free energy of dissociation of the helices is being determined for both the wild type and a mutant, in which dimerization is disrupted.

  14. Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

    PubMed Central

    Findlay, Heather E; McClafferty, Heather; Ashley, Richard H

    2005-01-01

    Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP) with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells) after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded) β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS) domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP's topology, could provide

  15. Assessment of the genetic susceptibility of sheep to scrapie by protein misfolding cyclic amplification and comparison with experimental scrapie transmission studies.

    PubMed

    Bucalossi, Cecilia; Cosseddu, Gianmario; D'Agostino, Claudia; Di Bari, Michele Angelo; Chiappini, Barbara; Conte, Michela; Rosone, Francesca; De Grossi, Luigi; Scavia, Gaia; Agrimi, Umberto; Nonno, Romolo; Vaccari, Gabriele

    2011-08-01

    The susceptibility of sheep to scrapie is influenced mainly by the prion protein polymorphisms A136V, R154H, and Q171R/H. Here we analyzed the ability of protein misfolding cyclic amplification (PMCA) to model the genetic susceptibility of sheep to scrapie. For this purpose, we studied the efficiency of brain homogenates from sheep with different PrP genotypes to support PrP(Sc) amplification by PMCA using an ARQ/ARQ scrapie inoculum. The results were then compared with those obtained in vivo using the same sheep breed, genotypes, and scrapie inoculum. Genotypes associated with susceptibility (ARQ/ARQ, ARQ/AHQ, and AHQ/ARH) were able to sustain PrP(Sc) amplification in PMCA reactions, while genotypes associated with resistance to scrapie (ARQ/ARR and ARR/ARR) were unable to support the in vitro conversion. The incubation times of the experimental infection were then compared with the in vitro amplification factors. Linear regression analysis showed that the efficiency of in vitro PrP(Sc) amplification of the different genotypes was indeed inversely proportional to their incubation times. Finally, the rare ARQK₁₇₆/ARQK₁₇₆ genotype, for which no in vivo data are available, was studied by PMCA. No amplification was obtained, suggesting ARQK₁₇₆/ARQK₁₇₆ as an additional genotype associated with resistance, at least to the isolate tested. Our results indicate a direct correlation between the ability of different PrP genotypes to undergo PrP(C)-to-PrP(Sc) conversion by PMCA and their in vivo susceptibility and point to PMCA as an alternative to transmission studies and a potential tool to test the susceptibility of numerous sheep PrP genotypes to a variety of prion sources.

  16. Detection of PrPCWD in feces from naturally exposed Rocky Mountain elk (Cervus elaphus nelsoni) using protein misfolding cyclic amplification.

    PubMed

    Pulford, Bruce; Spraker, Terry R; Wyckoff, A Christy; Meyerett, Crystal; Bender, Heather; Ferguson, Adam; Wyatt, Brittney; Lockwood, Krista; Powers, Jenny; Telling, Glenn C; Wild, Margaret A; Zabel, Mark D

    2012-04-01

    Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting captive and free-ranging cervids. Currently, tests for CWD in live animals involve relatively invasive procedures to collect lymphoid tissue biopsies and examine them for CWD-associated, protease-resistant cervid prion protein (PrP(CWD)) detected by immunohistochemistry (IHC). We adapted an ultrasensitive prion detection system, protein misfolding cyclic amplification (PMCA), to detect PrP(CWD) in Rocky Mountain elk (Cervus elaphus nelsoni) feces. Our PMCA reproducibly detected a 1.2 × 10(7) dilution of PrP(CWD) (a 10% infected brain homogenate diluted 1.2 × 10(6)-fold into 10% fecal homogenates), equivalent to approximately 100 pg of PrP(CWD)/g of feces. We developed a semiquantitative scoring system based on the first PMCA round at which PrP(CWD) was detected and fit a nonlinear regression curve to our serial dilutions to correlate PMCA scores with known PrP(CWD) concentrations. We used this PMCA scoring system to detect PrP(CWD) and estimate its concentration in feces from free-ranging elk from Rocky Mountain National Park, Colorado. We compared our results to PrP(CWD) IHC of rectoanal mucosa-associated lymphoid tissue and obex from the same animals. The PMCA successfully detected PrP(CWD) in feces from elk that were positive by IHC, with estimated prion loads from 100 to 5,000 pg PrP(CWD)/g of feces. These data show for the first time PrP(CWD) in feces from naturally exposed free-ranging elk and demonstrate the potential of PMCA as a new, noninvasive CWD diagnostic tool to complement IHC.

  17. Assessment of the Genetic Susceptibility of Sheep to Scrapie by Protein Misfolding Cyclic Amplification and Comparison with Experimental Scrapie Transmission Studies ▿

    PubMed Central

    Bucalossi, Cecilia; Cosseddu, GianMario; D'Agostino, Claudia; Di Bari, Michele Angelo; Chiappini, Barbara; Conte, Michela; Rosone, Francesca; De Grossi, Luigi; Scavia, Gaia; Agrimi, Umberto; Nonno, Romolo; Vaccari, Gabriele

    2011-01-01

    The susceptibility of sheep to scrapie is influenced mainly by the prion protein polymorphisms A136V, R154H, and Q171R/H. Here we analyzed the ability of protein misfolding cyclic amplification (PMCA) to model the genetic susceptibility of sheep to scrapie. For this purpose, we studied the efficiency of brain homogenates from sheep with different PrP genotypes to support PrPSc amplification by PMCA using an ARQ/ARQ scrapie inoculum. The results were then compared with those obtained in vivo using the same sheep breed, genotypes, and scrapie inoculum. Genotypes associated with susceptibility (ARQ/ARQ, ARQ/AHQ, and AHQ/ARH) were able to sustain PrPSc amplification in PMCA reactions, while genotypes associated with resistance to scrapie (ARQ/ARR and ARR/ARR) were unable to support the in vitro conversion. The incubation times of the experimental infection were then compared with the in vitro amplification factors. Linear regression analysis showed that the efficiency of in vitro PrPSc amplification of the different genotypes was indeed inversely proportional to their incubation times. Finally, the rare ARQK176/ARQK176 genotype, for which no in vivo data are available, was studied by PMCA. No amplification was obtained, suggesting ARQK176/ARQK176 as an additional genotype associated with resistance, at least to the isolate tested. Our results indicate a direct correlation between the ability of different PrP genotypes to undergo PrPC-to-PrPSc conversion by PMCA and their in vivo susceptibility and point to PMCA as an alternative to transmission studies and a potential tool to test the susceptibility of numerous sheep PrP genotypes to a variety of prion sources. PMID:21680531

  18. Crystallization of Membrane protein under Microgravity

    NASA Astrophysics Data System (ADS)

    Henning, C.; Frank, J.; Laubender, G.; Fromme, P.

    2002-01-01

    Proteins are biological molecules which catalyse all essential reactions of cells. The knowledge on the structure of these molecular machines is necessary for the understanding of their function. Many diseases are caused by defects of membrane proteins. In order to develop new medical therapies the construction principle of the proteins must be known. The main difficulty in the determination of the structure of these membrane protein complexes is the crystallisation. Membrane proteins are normally not soluble in water and have therefore to be solubilised from the membranes by use of detergents. The whole protein-detergent micelle must be crystallised to maintain the functional integrity of the protein complexes. These difficulties are the reasons for the fact that crystals of membrane proteins are difficult to grow and most of them are badly ordered, being not appropriate for X-ray structure analysis. The crystallisation of proteins under microgravity leads to the growth of better-ordered crystals by reduction of nucleation rate and the undisturbed growth of the hovering seeds by the absence of sedimentation and convection. The successful crystallistation of a membrane protein under microgravity has been performed during the space shuttle missions USML2 and STS95 in the Space Shuttle with Photosystem I as model protein. Photosystem I is a large membrane protein complex which catalyses one of the first and fundamental steps in oxygen photosynthesis. The crystals of Photosystem I, grown under microgravity were twenty times larger than all Photosystem I crystals which have been grown on earth. They were the basis for the determination of an improved X-ray structure of Photo- system I. These experiments opened the way for the structure enlightenment of more membrane proteins on the basis of microgravity experiments. On board of the International Space Station ideal conditions for the crystallisation of proteins under zero gravity are existing.

  19. On the Design of Broad Based Screening Assays to Identify Potential Pharmacological Chaperones of Protein Misfolding Diseases†

    PubMed Central

    Naik, Subhashchandra; Zhang, Na; Gao, Phillip; Fisher, Mark T.

    2013-01-01

    Correcting aberrant folds that develop during protein folding disease states is now an active research endeavor that is attracting increasing attention from both academic and industrial circles. One particular approach focuses on developing or identifying small molecule correctors or pharmacological chaperones that specifically stabilize the native fold. Unfortunately, the limited screening platforms available to rapidly identify or validate potential drug candidates are usually inadequate or slow because the folding disease proteins in question are often transiently folded and/or aggregation-prone, complicating and/or interfering with the assay outcomes. In this review, we outline and discuss the numerous platform options currently being employed to identify small molecule therapeutics for folding diseases. Finally, we describe a new stability screening approach that is broad based and is easily applicable toward a very large number of both common and rare protein folding diseases. The label free screening method described herein couples the promiscuity of the GroEL binding to transient aggregation-prone hydrophobic folds with surface plasmon resonance enabling one to rapidly identify potential small molecule pharmacological chaperones. PMID:23339304

  20. IFITM Proteins Restrict Viral Membrane Hemifusion

    PubMed Central

    Golfetto, Ottavia; Bungart, Brittani; Li, Minghua; Ding, Shilei; He, Yuxian; Liang, Chen; Lee, James C.; Gratton, Enrico; Cohen, Fredric S.; Liu, Shan-Lu

    2013-01-01

    The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous

  1. Disulfide Bonding in Neurodegenerative Misfolding Diseases

    PubMed Central

    2013-01-01

    In recent years an increasing number of neurodegenerative diseases has been linked to the misfolding of a specific protein and its subsequent accumulation into aggregated species, often toxic to the cell. Of all the factors that affect the behavior of these proteins, disulfide bonds are likely to be important, being very conserved in protein sequences and being the enzymes devoted to their formation among the most conserved machineries in mammals. Their crucial role in the folding and in the function of a big fraction of the human proteome is well established. The role of disulfide bonding in preventing and managing protein misfolding and aggregation is currently under investigation. New insights into their involvement in neurodegenerative diseases, their effect on the process of protein misfolding and aggregation, and into the role of the cellular machineries devoted to disulfide bond formation in neurodegenerative diseases are emerging. These studies mark a step forward in the comprehension of the biological base of neurodegenerative disorders and highlight the numerous questions that still remain open. PMID:23983694

  2. A predictor of membrane class: Discriminating alpha-helical and beta-barrel membrane proteins from non-membranous proteins.

    PubMed

    Taylor, Paul D; Toseland, Christopher P; Attwood, Teresa K; Flower, Darren R

    2006-10-07

    Accurate protein structure prediction remains an active objective of research in bioinformatics. Membrane proteins comprise approximately 20% of most genomes. They are, however, poorly tractable targets of experimental structure determination. Their analysis using bioinformatics thus makes an important contribution to their on-going study. Using a method based on Bayesian Networks, which provides a flexible and powerful framework for statistical inference, we have addressed the alignment-free discrimination of membrane from non-membrane proteins. The method successfully identifies prokaryotic and eukaryotic alpha-helical membrane proteins at 94.4% accuracy, beta-barrel proteins at 72.4% accuracy, and distinguishes assorted non-membranous proteins with 85.9% accuracy. The method here is an important potential advance in the computational analysis of membrane protein structure. It represents a useful tool for the characterisation of membrane proteins with a wide variety of potential applications.

  3. Membrane Protein Crystallization Using Laser Irradiation

    NASA Astrophysics Data System (ADS)

    Adachi, Hiroaki; Murakami, Satoshi; Niino, Ai; Matsumura, Hiroyoshi; Takano, Kazufumi; Inoue, Tsuyoshi; Mori, Yusuke; Yamaguchi, Akihito; Sasaki, Takatomo

    2004-10-01

    We demonstrate the crystallization of a membrane protein using femtosecond laser irradiation. This method, which we call the laser irradiated growth technique (LIGHT), is useful for producing AcrB crystals in a solution of low supersaturation range. LIGHT is characterized by reduced nucleation times. This feature is important for crystallizing membrane proteins because of their labile properties when solubilized as protein-detergent micelles. Using LIGHT, high-quality crystals of a membrane transporter protein, AcrB, were obtained. The resulting crystals were found to be of sufficiently high resolution for X-ray diffraction. The results reported here indicate that LIGHT is a powerful tool for membrane protein crystallization, as well as for the growth of soluble proteins.

  4. alpha-Synuclein misfolding: single molecule AFM force spectroscopy study.

    PubMed

    Yu, Junping; Malkova, Sarka; Lyubchenko, Yuri L

    2008-12-26

    Protein misfolding and aggregation are the very first and critical steps in development of various neurodegenerative disorders, including Parkinson's disease, induced by misfolding of alpha-synuclein. Thus, elucidating properties of proteins in misfolded states and understanding the mechanisms of their assembly into the disease prone aggregates are critical for the development of rational approaches to prevent protein misfolding-mediated pathologies. To accomplish this goal and as a first step to elucidate the mechanism of alpha-synuclein misfolding, we applied single-molecule force spectroscopy capable of detecting protein misfolding. We immobilized alpha-synuclein molecules at their C-termini at the atomic force microscope tips and substrate surfaces, and measured the interaction between the proteins by probing the microscope tip at various locations on the surface. Using this approach, we detected alpha-synuclein misfolded states by enhanced interprotein interaction. We used a dynamics force spectroscopy approach to measure such an important characteristic of dimers of misfolded alpha-synuclein as their lifetimes. We found that the dimer lifetimes are in the range of seconds and these values are much higher than the characteristics for the dynamics of the protein in monomeric state. These data show that compared to highly dynamic monomeric forms, alpha-synuclein dimers are much more stable and thus can serve as stable nuclei for the formation of multimeric and aggregated forms of alpha-synuclein. Importantly, two different lifetimes were observed for the dimers, suggesting that aggregation can follow different pathways that may lead to different aggregated morphologies of alpha-synuclein.

  5. Functional dynamics of cell surface membrane proteins

    NASA Astrophysics Data System (ADS)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

  6. Lateral proton transfer between the membrane and a membrane protein.

    PubMed

    Ojemyr, Linda; Sandén, Tor; Widengren, Jerker; Brzezinski, Peter

    2009-03-17

    Proton transport across biological membranes is a key step of the energy conservation machinery in living organisms, and it has been proposed that the membrane itself plays an important role in this process. In the present study we have investigated the effect of incorporation of a proton transporter, cytochrome c oxidase, into a membrane on the protonation kinetics of a fluorescent pH-sensitive probe attached at the surface of the protein. The results show that proton transfer to the probe was slightly accelerated upon attachment at the protein surface (approximately 7 x 1010 s(-1) M(-1), compared to the expected value of (1-2) x 10(10) s(-1) M(-1)), which is presumably due to the presence of acidic/His groups in the vicinity. Upon incorporation of the protein into small unilamellar phospholipid vesicles the rate increased by more than a factor of 400 to approximately 3 x 10(13) s(-1) M(-1), which indicates that the protein-attached probe is in rapid protonic contact with the membrane surface. The results indicate that the membrane acts to accelerate proton uptake by the membrane-bound proton transporter.

  7. Inherently tunable electrostatic assembly of membrane proteins.

    PubMed

    Liang, Hongjun; Whited, Gregg; Nguyen, Chi; Okerlund, Adam; Stucky, Galen D

    2008-01-01

    Membrane proteins are a class of nanoscopic entities that control the matter, energy, and information transport across cellular boundaries. Electrostatic interactions are shown to direct the rapid co-assembly of proteorhodopsin (PR) and lipids into long-range crystalline arrays. The roles of inherent charge variations on lipid membranes and PR variants with different compositions are examined by tuning recombinant PR variants with different extramembrane domain sizes and charged amino acid substitutions, lipid membrane compositions, and lipid-to-PR stoichiometric ratios. Rational control of this predominantly electrostatic assembly for PR crystallization is demonstrated, and the same principles should be applicable to the assembly and crystallization of other integral membrane proteins.

  8. Membrane injury by pore-forming proteins.

    PubMed

    Bischofberger, Mirko; Gonzalez, Manuel R; van der Goot, F Gisou

    2009-08-01

    The plasma membrane defines the boundary of every living cell, and its integrity is essential for life. The plasma membrane may, however, be challenged by mechanical stress or pore-forming proteins produced by the organism itself or invading pathogens. We will here review recent findings about pore-forming proteins from different organisms, highlighting their structural and functional similarities, and describe the mechanisms that lead to membrane repair, since remarkably, cells can repair breaches in their plasma membrane of up to 10,000 microm(2).

  9. Active Nuclear Import of Membrane Proteins Revisited

    PubMed Central

    Laba, Justyna K.; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M.

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

  10. Intermolecular transmission of superoxide dismutase 1 misfolding in living cells

    PubMed Central

    Grad, Leslie I.; Guest, Will C.; Yanai, Anat; Pokrishevsky, Edward; O'Neill, Megan A.; Gibbs, Ebrima; Semenchenko, Valentyna; Yousefi, Masoud; Wishart, David S.; Plotkin, Steven S.; Cashman, Neil R.

    2011-01-01

    Human wild-type superoxide dismutase-1 (wtSOD1) is known to coaggregate with mutant SOD1 in familial amyotrophic lateral sclerosis (FALS), in double transgenic models of FALS, and in cell culture systems, but the structural determinants of this process are unclear. Here we molecularly dissect the effects of intracellular and cell-free obligately misfolded SOD1 mutant proteins on natively structured wild-type SOD1. Expression of the enzymatically inactive, natural familial ALS SOD1 mutations G127X and G85R in human mesenchymal and neural cell lines induces misfolding of wild-type natively structured SOD1, as indicated by: acquisition of immunoreactivity with SOD1 misfolding-specific monoclonal antibodies; markedly enhanced protease sensitivity suggestive of structural loosening; and nonnative disulfide-linked oligomer and multimer formation. Expression of G127X and G85R in mouse cell lines did not induce misfolding of murine wtSOD1, and a species restriction element for human wtSOD1 conversion was mapped to a region of sequence divergence in loop II and β-strand 3 of the SOD1 β-barrel (residues 24–36), then further refined surprisingly to a single tryptophan residue at codon 32 (W32) in human SOD1. Time course experiments enabled by W32 restriction revealed that G127X and misfolded wtSOD1 can induce misfolding of cell-endogenous wtSOD1. Finally, aggregated recombinant G127X is capable of inducing misfolding and protease sensitivity of recombinant human wtSOD1 in a cell-free system containing reducing and chelating agents; cell-free wtSOD1 conversion was also restricted by W32. These observations demonstrate that misfolded SOD1 can induce misfolding of natively structured wtSOD1 in a physiological intracellular milieu, consistent with a direct protein–protein interaction. PMID:21930926

  11. Protein misfolding cyclic amplification corroborates the absence of PrP(Sc) accumulation in placenta from foetuses with the ARR/ARQ genotype in natural scrapie.

    PubMed

    Garza, María Carmen; Eraña, Hasier; Castilla, Joaquín; Acín, Cristina; Vargas, Antonia; Badiola, Juan José; Monleón, Eva

    2017-05-01

    Ovine scrapie is a worldwide spread prion disease that is transmitted horizontally under field conditions. Placenta from scrapie-infected ewes is an important source of infection, since this tissue can accumulate high amounts of PrP(Sc) depending on the foetal genotype. Therefore, placentas carrying susceptible foetuses can accumulate PrP(Sc) but there is not PrP(Sc) accumulation in presence of foetuses with at least one ARR haplotype. In scrapie eradication programs, ARR/ARR males are used for breeding to increase the resistant progeny and reduce the horizontal transmission of the disease through the placenta. The development of highly sensitive techniques, that allow the detection of minimal amounts of PrP(Sc), has caused many secretions/excretions and tissues that had previously been deemed negative to be relabeled as positive for PrP(Sc). This has raised concerns about the possible presence of minimal amounts of PrP(Sc) in placentas from ARR foetuses that conventional techniques had indicated were negative. In the present study we examined 30 placentas from a total of 23 gestations; 15 gestations resulted from naturally ARQ/ARQ scrapie-infected ewes mated with ARR/ARR rams. The absence of PrP(Sc) in placentas carrying the foetal ARR haplotype (n=19) was determined by IDEXX HerdChek scrapie/BSE Antigen EIA Test, Prionics(®)-Check WESTERN and corroborated by the highly sensitive Protein Misfolding Cyclic Amplification technique (PMCA). By immunohistochemistry, several unspecific stainings that might mislead a diagnosis were observed. The results of the present study support that using ARR/ARR males in scrapie eradication programs efficiently decreases the spreading of the agent in the environment via shed placentas. Copyright © 2017. Published by Elsevier B.V.

  12. Membrane protein architects: the role of the BAM complex in outer membrane protein assembly.

    PubMed

    Knowles, Timothy J; Scott-Tucker, Anthony; Overduin, Michael; Henderson, Ian R

    2009-03-01

    The folding of transmembrane proteins into the outer membrane presents formidable challenges to Gram-negative bacteria. These proteins must migrate from the cytoplasm, through the inner membrane and into the periplasm, before being recognized by the beta-barrel assembly machinery, which mediates efficient insertion of folded beta-barrels into the outer membrane. Recent discoveries of component structures and accessory interactions of this complex are yielding insights into how cells fold membrane proteins. Here, we discuss how these structures illuminate the mechanisms responsible for the biogenesis of outer membrane proteins.

  13. MECHANISM OF ALPHA-SYNUCLEIN OLIGOMERIZATION AND MEMBRANE INTERACTION: THEORETICAL APPROACH TO UNSTRUCTURED PROTEINS STUDIES

    PubMed Central

    Tsigelny, Igor F.; Sharikov, Yuriy; Miller, Mark A.; Masliah, Eliezer

    2008-01-01

    Misfolding and oligomerization of unstructured proteins is involved in the pathogenesis of Parkinson’s (PD), Alzheimer’s (AD), Huntington’s, and other neurodegenerative disorders. Elucidation of possible conformations of these proteins and their interactions with the membrane is necessary to understand the molecular mechanisms of neurodegeneration. We developed a strategy that makes it possible to elucidate the molecular mechanisms of of alpha-synuclein aggregation- a key molecular event in the pathogenesis of PD. This strategy can be also useful for the study of other unstructured proteins involved in neurodegeneration. The results of these theoretical studies have been confirmed with biochemical and electrophysiological studies. Our studies provide insights into the molecular mechanism for PD initiation and progression, and provide a useful paradigm for identifying possible therapeutic interventions through computational modeling. PMID:18640077

  14. Helical Membrane Protein Conformations and their Environment

    PubMed Central

    Cross, Timothy A.; Murray, Dylan T.; Watts, Anthony

    2013-01-01

    Evidence that membrane proteins respond conformationally and functionally to their environment is gaining pace. Structural models, by necessity, have been characterized in preparations where the protein has been removed from its native environment. Different structural methods have used various membrane mimetics that have recently included lipid bilayers as a more native-like environment. Structural tools applied to lipid bilayer-embedded integral proteins are informing us about important generic characteristics of how membrane proteins respond to the lipid environment as compared with their response to other non-lipid environments. Here, we review the current status of the field, with specific reference to observations of some well-studied α-helical membrane proteins, as a starting point to aid the development of possible generic principals for model refinement. PMID:23996195

  15. Thermostabilisation of membrane proteins for structural studies

    PubMed Central

    Magnani, Francesca; Serrano-Vega, Maria J.; Shibata, Yoko; Abdul-Hussein, Saba; Lebon, Guillaume; Miller-Gallacher, Jennifer; Singhal, Ankita; Strege, Annette; Thomas, Jennifer A.; Tate, Christopher G.

    2017-01-01

    The thermostability of an integral membrane protein in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals suitable for structure determination. However, many mammalian membrane proteins are too unstable for crystallisation. We developed a thermostabilisation strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes approximately 6-12 months to thermostabilise a G protein-coupled receptor (GPCR) containing 300 amino acid residues. The resulting thermostabilised membrane proteins are more easily crystallised and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs, because it is possible to determine multiple structures of the thermostabilised receptors bound to low affinity ligands. Protocols and advice are given on how to develop thermostability assays for membrane proteins and how to combine mutations to make an optimally stable mutant suitable for structural studies. PMID:27466713

  16. Polyene antibiotic that inhibits membrane transport proteins.

    PubMed

    te Welscher, Yvonne Maria; van Leeuwen, Martin Richard; de Kruijff, Ben; Dijksterhuis, Jan; Breukink, Eefjan

    2012-07-10

    The limited therapeutic arsenal and the increase in reports of fungal resistance to multiple antifungal agents have made fungal infections a major therapeutic challenge. The polyene antibiotics are the only group of antifungal antibiotics that directly target the plasma membrane via a specific interaction with the main fungal sterol, ergosterol, often resulting in membrane permeabilization. In contrast to other polyene antibiotics that form pores in the membrane, the mode of action of natamycin has remained obscure but is not related to membrane permeabilization. Here, we demonstrate that natamycin inhibits growth of yeasts and fungi via the immediate inhibition of amino acid and glucose transport across the plasma membrane. This is attributable to ergosterol-specific and reversible inhibition of membrane transport proteins. It is proposed that ergosterol-dependent inhibition of membrane proteins is a general mode of action of all the polyene antibiotics, of which some have been shown additionally to permeabilize the plasma membrane. Our results imply that sterol-protein interactions are fundamentally important for protein function even for those proteins that are not known to reside in sterol-rich domains.

  17. Solid-state NMR and Membrane Proteins

    PubMed Central

    Opella, Stanley J.

    2015-01-01

    The native environment for a membrane protein is a phospholipid bilayer. Because the protein is immobilized on NMR timescales by the interactions within a bilayer membrane, solid-state NMR methods are essential to obtain high-resolution spectra. Approaches have been developed for both unoriented and oriented samples, however, they all rest on the foundation of the most fundamental aspects solid-state NMR, and the chemical shift and homo- and hetero-nuclear dipole-dipole interactions. Solid-state NMR has advanced sufficiently to enable the structures of membrane proteins to be determined under near-native conditions in phospholipid bilayers. PMID:25681966

  18. Solid-state NMR and membrane proteins

    NASA Astrophysics Data System (ADS)

    Opella, Stanley J.

    2015-04-01

    The native environment for a membrane protein is a phospholipid bilayer. Because the protein is immobilized on NMR timescales by the interactions within a bilayer membrane, solid-state NMR methods are essential to obtain high-resolution spectra. Approaches have been developed for both unoriented and oriented samples, however, they all rest on the foundation of the most fundamental aspects of solid-state NMR, and the chemical shift and homo- and hetero-nuclear dipole-dipole interactions. Solid-state NMR has advanced sufficiently to enable the structures of membrane proteins to be determined under near-native conditions in phospholipid bilayers.

  19. Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR

    NASA Astrophysics Data System (ADS)

    Varkey, Jobin; Langen, Ralf

    2017-07-01

    The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single

  20. Dissection of the Dislocation Pathway for Type I Membrane Proteins with a New Small Molecule Inhibitor, Eeyarestatin

    PubMed Central

    Fiebiger, Edda; Hirsch, Christian; Vyas, Jatin M.; Gordon, Eva; Ploegh, Hidde L.; Tortorella, Domenico

    2004-01-01

    The mammalian endoplasmic reticulum (ER)-to-cytosol degradation pathway for disposal of misfolded proteins is an attractive target for therapeutic intervention in diseases that are characterized by impaired protein degradation. The ability to do so is hampered by the small number of specific inhibitors available and by our limited understanding of the individual steps involved in this pathway. Cells that express a class I major histocompatibility complex (MHC) heavy chain-enhanced green fluorescent protein (EGFP) fusion protein and the human cytomegalovirus protein US11, which catalyzes dislocation of the class I MHC EGFP reporter, show only little fluorescence. Treatment with proteasome inhibitors increases their fluorescence by stabilizing EGFP-tagged MHC class I molecules. We used this change in signal intensity as a readout to screen a chemical library of 16,320 compounds and identified two structurally related compounds (eeyarestatin I and II) that interfered with the degradation of both EGFP-heavy chain and its endogenous unmodified class I MHC heavy chain counterpart. Eeyarestatin I also inhibited degradation of a second misfolded type I membrane protein, T-cell receptor α. Both compounds stabilize these dislocation substrates in the ER membrane, without preventing proteasomal turnover of cytosolic substrates. The new inhibitors must therefore interfere with a step that precedes proteasomal degradation. The use of eeyarestatin I thus allows the definition of a new intermediate in dislocation. PMID:14767067

  1. A Hydrophobic Gold Surface Triggers Misfolding and Aggregation of the Amyloidogenic Josephin Domain in Monomeric Form, While Leaving the Oligomers Unaffected

    PubMed Central

    Apicella, Alessandra; Soncini, Monica; Deriu, Marco Agostino; Natalello, Antonino; Bonanomi, Marcella; Dellasega, David; Tortora, Paolo; Regonesi, Maria Elena; Casari, Carlo Spartaco

    2013-01-01

    Protein misfolding and aggregation in intracellular and extracellular spaces is regarded as a main marker of the presence of degenerative disorders such as amyloidoses. To elucidate the mechanisms of protein misfolding, the interaction of proteins with inorganic surfaces is of particular relevance, since surfaces displaying different wettability properties may represent model systems of the cell membrane. Here, we unveil the role of surface hydrophobicity/hydrophilicity in the misfolding of the Josephin domain (JD), a globular-shaped domain of ataxin-3, the protein responsible for the spinocerebellar ataxia type 3. By means of a combined experimental and theoretical approach based on atomic force microscopy, Fourier transform infrared spectroscopy and molecular dynamics simulations, we reveal changes in JD morphology and secondary structure elicited by the interaction with the hydrophobic gold substrate, but not by the hydrophilic mica. Our results demonstrate that the interaction with the gold surface triggers misfolding of the JD when it is in native-like configuration, while no structural modification is observed after the protein has undergone oligomerization. This raises the possibility that biological membranes would be unable to affect amyloid oligomeric structures and toxicity. PMID:23527026

  2. Expression and purification of membrane proteins.

    PubMed

    Kubicek, Jan; Block, Helena; Maertens, Barbara; Spriestersbach, Anne; Labahn, Jörg

    2014-01-01

    Approximately 30% of a genome encodes for membrane proteins. They are one of the most important classes of proteins in that they can receive, differentiate, and transmit intra- and intercellular signals. Some examples of classes of membrane proteins include cell-adhesion molecules, translocases, and receptors in signaling pathways. Defects in membrane proteins may be involved in a number of serious disorders such as neurodegenerative diseases (e.g., Alzheimer's) and diabetes. Furthermore, membrane proteins provide natural entry and anchoring points for the molecular agents of infectious diseases. Thus, membrane proteins constitute ~50% of known and novel drug targets. Progress in this area is slowed by the requirement to develop methods and procedures for expression and isolation that are tailored to characteristic properties of membrane proteins. A set of standard protocols for the isolation of the targets in quantities that allow for the characterization of their individual properties for further optimization is required. The standard protocols given below represent a workable starting point. If optimization of yields is desired, a variation of conditions as outlined in the theory section is recommended.

  3. Protein profiles of hatchery egg shell membrane

    USDA-ARS?s Scientific Manuscript database

    Background: Eggshells, which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of m...

  4. Detergents in Membrane Protein Purification and Crystallisation.

    PubMed

    Anandan, Anandhi; Vrielink, Alice

    2016-01-01

    Detergents play a significant role in structural and functional characterisation of integral membrane proteins (IMPs). IMPs reside in the biological membranes and exhibit a great variation in their structural and physical properties. For in vitro biophysical studies, structural and functional analyses, IMPs need to be extracted from the membrane lipid bilayer environment in which they are found and purified to homogeneity while maintaining a folded and functionally active state. Detergents are capable of successfully solubilising and extracting the IMPs from the membrane bilayers. A number of detergents with varying structure and physicochemical properties are commercially available and can be applied for this purpose. Nevertheless, it is important to choose a detergent that is not only able to extract the membrane protein but also provide an optimal environment while retaining the correct structural and physical properties of the protein molecule. Choosing the best detergent for this task can be made possible by understanding the physical and chemical properties of the different detergents and their interaction with the IMPs. In addition, understanding the mechanism of membrane solubilisation and protein extraction along with crystallisation requirements, if crystallographic studies are going to be undertaken, can help in choosing the best detergent for the purpose. This chapter aims to present the fundamental properties of detergents and highlight information relevant to IMP crystallisation. The first section of the chapter reviews the physicochemical properties of detergents and parameters essential for predicting their behaviour in solution. The second section covers the interaction of detergents with the biologic membranes and proteins followed by their role in membrane protein crystallisation. The last section will briefly cover the types of detergent and their properties focusing on custom designed detergents for membrane protein studies.

  5. The secretory carrier membrane protein family: structure and membrane topology.

    PubMed

    Hubbard, C; Singleton, D; Rauch, M; Jayasinghe, S; Cafiso, D; Castle, D

    2000-09-01

    Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane

  6. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

    Laganowsky, Arthur; Reading, Eamonn; Hopper, Jonathan T.S.; Robinson, Carol V.

    2014-01-01

    Mass spectrometry of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here, we describe a protocol for mass spectrometry of membrane protein complexes. The protocol begins with preparation of the membrane protein complex enabling not only the direct assessment of stoichiometry, delipidation, and quality of the target complex, but also evaluation of the purification strategy. A detailed list of compatible non-ionic detergents is included, along with a protocol for screening detergents to find an optimal one for mass spectrometry, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a Q-ToF mass spectrometer after introduction of complexes from gold-coated nanoflow capillaries. PMID:23471109

  7. Protein engineering methods applied to membrane protein targets.

    PubMed

    Lluis, M W; Godfroy, J I; Yin, H

    2013-02-01

    Genes encoding membrane proteins have been estimated to comprise as much as 30% of the human genome. Among these membrane, proteins are a large number of signaling receptors, transporters, ion channels and enzymes that are vital to cellular regulation, metabolism and homeostasis. While many membrane proteins are considered high-priority targets for drug design, there is a dearth of structural and biochemical information on them. This lack of information stems from the inherent insolubility and instability of transmembrane domains, which prevents easy obtainment of high-resolution crystals to specifically study structure-function relationships. In part, this lack of structures has greatly impeded our understanding in the field of membrane proteins. One method that can be used to enhance our understanding is directed evolution, a molecular biology method that mimics natural selection to engineer proteins that have specific phenotypes. It is a powerful technique that has considerable success with globular proteins, notably the engineering of protein therapeutics. With respect to transmembrane protein targets, this tool may be underutilized. Another powerful tool to investigate membrane protein structure-function relationships is computational modeling. This review will discuss these protein engineering methods and their tremendous potential in the study of membrane proteins.

  8. Protein profiles of hatchery egg shell membrane.

    PubMed

    Rath, N C; Liyanage, R; Makkar, S K; Lay, J O

    2016-01-01

    Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM). Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins. Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic. The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

  9. Tuning Escherichia coli for membrane protein overexpression.

    PubMed

    Wagner, Samuel; Klepsch, Mirjam M; Schlegel, Susan; Appel, Ansgar; Draheim, Roger; Tarry, Michael; Högbom, Martin; van Wijk, Klaas J; Slotboom, Dirk J; Persson, Jan O; de Gier, Jan-Willem

    2008-09-23

    A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.

  10. Tuning Escherichia coli for membrane protein overexpression

    PubMed Central

    Wagner, Samuel; Klepsch, Mirjam M.; Schlegel, Susan; Appel, Ansgar; Draheim, Roger; Tarry, Michael; Högbom, Martin; van Wijk, Klaas J.; Slotboom, Dirk J.; Persson, Jan O.; de Gier, Jan-Willem

    2008-01-01

    A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used “Walker strains” C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications. PMID:18796603

  11. Ponticulin is an atypical membrane protein

    PubMed Central

    1994-01-01

    We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha- helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s). PMID:8089175

  12. Overexpression of membrane proteins using Pichia pastoris.

    PubMed

    Bornert, Olivier; Alkhalfioui, Fatima; Logez, Christel; Wagner, Renaud

    2012-02-01

    Among the small number of expression systems validated for the mass production of eukaryotic membrane proteins (EMPs), the methylotrophic yeast Pichia pastoris stands as one of the most efficient hosts. This system has been used to produce crystallization-grade proteins for a variety of EMPs, from which high-resolution 3D structures have been determined. This unit describes a set of guidelines and instructions to overexpress membrane proteins using the P. pastoris system. Using a G protein-coupled receptor (GPCR) as a model EMP, these protocols illustrate the necessary steps, starting with the design of the DNA sequence to be expressed, through the preparation and analysis of samples containing the corresponding membrane protein of interest. In addition, recommendations are given on a series of experimental parameters that can be optimized to substantially improve the amount and/or the functionality of the expressed EMPs.

  13. Protein quality control at the plasma membrane

    PubMed Central

    Okiyoneda, Tsukasa; Apaja, Pirjo M.; Lukacs, Gergely L.

    2011-01-01

    Cellular proteostasis (or protein homeostasis) depends on the timely folding and disposal of conformationally damaged polypeptides during their life span at all subcellular locations. This process is particularly important for membrane proteins confined to the cell surface with critical regulatory role in cellular hom