Sample records for collagen chaperone protein
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Structural mechanisms of chaperone mediated protein disaggregation
Sousa, Rui
2014-01-01
The ClpB/Hsp104 and Hsp70 classes of molecular chaperones use ATP hydrolysis to dissociate protein aggregates and complexes, and to move proteins through membranes. ClpB/Hsp104 are members of the AAA+ family of proteins which form ring-shaped hexamers. Loops lining the pore in the ring engage substrate proteins as extended polypeptides. Interdomain rotations and conformational changes in these loops coupled to ATP hydrolysis unfold and pull proteins through the pore. This provides a mechanism that progressively disrupts local secondary and tertiary structure in substrates, allowing these chaperones to dissociate stable aggregates such as β-sheet rich prions or coiled coil SNARE complexes. While the ClpB/Hsp104 mechanism appears to embody a true power-stroke in which an ATP powered conformational change in one protein is directly coupled to movement or structural change in another, the mechanism of force generation by Hsp70s is distinct and less well understood. Both active power-stroke and purely passive mechanisms in which Hsp70 captures spontaneous fluctuations in a substrate have been proposed, while a third proposed mechanism—entropic pulling—may be able to generate forces larger than seen in ATP-driven molecular motors without the conformational coupling required for a power-stroke. The disaggregase activity of these chaperones is required for thermotolerance, but unrestrained protein complex/aggregate dissociation is potentially detrimental. Disaggregating chaperones are strongly auto-repressed, and are regulated by co-chaperones which recruit them to protein substrates and activate the disaggregases via mechanisms involving either sequential transfer of substrate from one chaperone to another and/or simultaneous interaction of substrate with multiple chaperones. By effectively subjecting substrates to multiple levels of selection by multiple chaperones, this may insure that these potent disaggregases are only activated in the appropriate context. PMID
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Kitamura, Akira; Ishida, Yoshihito; Kubota, Hiroshi; Pack, Chan-Gi; Homma, Takayuki; Ito, Shinya; Araki, Kazutaka; Kinjo, Masataka; Nagata, Kazuhiro
2018-02-26
Heat shock protein 47 kDa (HSP47), an ER-resident and collagen-specific molecular chaperone, recognizes collagenous hydrophobic amino acid sequences (Gly-Pro-Hyp) and assists in secretion of correctly folded collagen. Elevated collagen production is correlated with HSP47 expression in various diseases, including fibrosis and keloid. HSP47 knockdown ameliorates liver fibrosis by inhibiting collagen secretion, and inhibition of the interaction of HSP47 with procollagen also prevents collagen secretion. Therefore, a high-throughput system for screening of drugs capable of inhibiting the interaction between HSP47 and collagen would aid the development of novel therapies for fibrotic diseases. In this study, we established a straightforward method for rapidly and quantitatively measuring the interaction between HSP47 and collagen in solution using fluorescence correlation spectroscopy (FCS). The diffusion rate of HSP47 labeled with Alexa Fluor 488 (HSP47-AF), a green fluorescent dye, decreased upon addition of type I or III collagen, whereas that of dye-labeled protein disulfide isomerase (PDI) or bovine serum albumin (BSA) did not, indicating that specific binding of HSP47 to collagen could be detected using FCS. Using this method, we calculated the dissociation constant of the interaction between HSP47 and collagen. The binding ratio between HSP47-AF and collagen did not change in the presence of sodium chloride, confirming that the interaction was hydrophobic in nature. In addition, we observed dissociation of collagen from HSP47 at low pH and re-association after recovery to neutral pH. These observations indicate that this system is appropriate for detecting the interaction between HSP47 and collagen, and could be applied to high-throughput screening for drugs capable of suppressing and/or curing fibrosis. Copyright © 2018 Elsevier Inc. All rights reserved.
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Protein Quality Control by Molecular Chaperones in Neurodegeneration
Ciechanover, Aaron; Kwon, Yong Tae
2017-01-01
Protein homeostasis (proteostasis) requires the timely degradation of misfolded proteins and their aggregates by protein quality control (PQC), of which molecular chaperones are an essential component. Compared with other cell types, PQC in neurons is particularly challenging because they have a unique cellular structure with long extensions. Making it worse, neurons are postmitotic, i.e., cannot dilute toxic substances by division, and, thus, are highly sensitive to misfolded proteins, especially as they age. Failure in PQC is often associated with neurodegenerative diseases, such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), and prion disease. In fact, many neurodegenerative diseases are considered to be protein misfolding disorders. To prevent the accumulation of disease-causing aggregates, neurons utilize a repertoire of chaperones that recognize misfolded proteins through exposed hydrophobic surfaces and assist their refolding. If such an effort fails, chaperones can facilitate the degradation of terminally misfolded proteins through either the ubiquitin (Ub)-proteasome system (UPS) or the autophagy-lysosome system (hereafter autophagy). If soluble, the substrates associated with chaperones, such as Hsp70, are ubiquitinated by Ub ligases and degraded through the proteasome complex. Some misfolded proteins carrying the KFERQ motif are recognized by the chaperone Hsc70 and delivered to the lysosomal lumen through a process called, chaperone-mediated autophagy (CMA). Aggregation-prone misfolded proteins that remain unprocessed are directed to macroautophagy in which cargoes are collected by adaptors, such as p62/SQSTM-1/Sequestosome-1, and delivered to the autophagosome for lysosomal degradation. The aggregates that have survived all these refolding/degradative processes can still be directly dissolved, i.e., disaggregated by chaperones. Studies have shown that molecular chaperones alleviate the pathogenic symptoms by
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Visualizing chaperone-assisted protein folding
Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp; ...
2016-05-30
We present that challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone–substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperonemore » Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.« less
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NASA Technical Reports Server (NTRS)
Kumei, Yasuhiro; Morita, Sadao; Shimokawa, Hitoyata; Ohya, Kei'ichi; Akiyama, Hideo; Hirano, Masahiko; Sams, Clarence F.; Whitson, Peggy A.
2003-01-01
Rat osteoblasts were cultured aboard a space shuttle for 4 or 5 days. Cells were exposed to 1alpha, 25 dihydroxyvitamin D(3) during the last 20 h and then solubilized by guanidine solution. The mRNA levels for molecular chaperones were analyzed by semi-quantitative RT-PCR. ELISA was used to quantify TGF-beta1 in the conditioned medium. The HSP70 mRNA levels in the flight cultures were almost completely suppressed, as compared to the ground (1 x g) controls. The inducible HSP70 is known as the major heat shock protein that prevents stress-induced apoptosis. The mean mRNA levels for the constitutive HSC73 in the flight cultures were reduced to 69%, approximately 60% of the ground controls. HSC73 is reported to prevent the pathological state that is induced by disruption of microtubule network. The mean HSP47 mRNA levels in the flight cultures were decreased to 50% and 19% of the ground controls on the 4th and 5th days. Concomitantly, the concentration of TGF-beta1 in the conditioned medium of the flight cultures was reduced to 37% and 19% of the ground controls on the 4th and 5th days. HSP47 is the collagen-specific molecular chaperone that controls collagen processing and quality and is regulated by TGF-beta1. Microgravity differentially modulated the expression of molecular chaperones in osteoblasts, which might be involved in induction and/or prevention of osteopenia in space.
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Chaperone activity of human small heat shock protein-GST fusion proteins.
Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A
2017-07-01
Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST
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Yeast prions are useful for studying protein chaperones and protein quality control.
Masison, Daniel C; Reidy, Michael
2015-01-01
Protein chaperones help proteins adopt and maintain native conformations and play vital roles in cellular processes where proteins are partially folded. They comprise a major part of the cellular protein quality control system that protects the integrity of the proteome. Many disorders are caused when proteins misfold despite this protection. Yeast prions are fibrous amyloid aggregates of misfolded proteins. The normal action of chaperones on yeast prions breaks the fibers into pieces, which results in prion replication. Because this process is necessary for propagation of yeast prions, even small differences in activity of many chaperones noticeably affect prion phenotypes. Several other factors involved in protein processing also influence formation, propagation or elimination of prions in yeast. Thus, in much the same way that the dependency of viruses on cellular functions has allowed us to learn much about cell biology, the dependency of yeast prions on chaperones presents a unique and sensitive way to monitor the functions and interactions of many components of the cell's protein quality control system. Our recent work illustrates the utility of this system for identifying and defining chaperone machinery interactions.
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Allostery in the Hsp70 chaperone proteins
Zuiderweg, Erik R.P.; Bertelsen, Eric B.; Rousaki, Aikaterini; Mayer, Matthias P.; Gestwicki, Jason E.; Ahmad, Atta
2013-01-01
Heat shock 70 kDa (Hsp70) chaperones are essential to in-vivo protein folding, protein transport and protein re-folding. They carry out these activities using repeated cycles of binding and release of client proteins. This process is under allosteric control of nucleotide binding and hydrolysis. X-ray crystallography, NMR spectroscopy and other biophysical techniques have contributed much to the understanding of the allosteric mechanism linking these activities and the effect of co-chaperones on this mechanism. In this chapter, these findings are critically reviewed. Studies on the allosteric mechanisms of Hsp70 have gained enhanced urgency, as recent studies have implicated this chaperone as a potential drug target in diseases such as Alzheimer's and cancer. Recent approaches to combat these diseases through interference with the Hsp70 allosteric mechanism are discussed. PMID:22576356
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Martínez-Alonso, Mónica; Villaverde, Antonio
2010-01-01
Main Escherichia coli cytosolic chaperones such as DnaK are key components of the control quality network designed to minimize the prevalence of polypeptides with aberrant conformations. This is achieved by both favoring refolding activities but also stimulating proteolytic degradation of folding reluctant species. This last activity is responsible for the decrease of the proteolytic stability of recombinant proteins when co-produced along with DnaK, where an increase in solubility might be associated to a decrease in protein yield. However, when DnaK and its co-chaperone DnaJ are co-produced in cultured insect cells or whole insect larvae (and expectedly, in other heterologous hosts), only positive, folding-related effects of these chaperones are observed, in absence of proteolysis-mediated reduction of recombinant protein yield. PMID:21326941
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Mamipour, Mina; Yousefi, Mohammadreza; Hasanzadeh, Mohammad
2017-09-01
The majority of research topics declared that most of the recombinant proteins have been expressed by Escherichia coli in basic investigations. But the majority of high expressed proteins formed as inactive recombinant proteins that are called inclusion body. To overcome this problem, several methods have been used including suitable promoter, environmental factors, ladder tag to secretion of proteins into the periplasm, gene protein optimization, chemical chaperones and molecular chaperones sets. Co-expression of the interest protein with molecular chaperones is one of the common methods The chaperones are a group of proteins, which are involved in making correct folding of recombinant proteins. Chaperones are divided two groups including; cytoplasmic and periplasmic chaperones. Moreover, periplasmic chaperones and proteases can be manipulated to increase the yields of secreted proteins. In this article, we attempted to review cytoplasmic chaperones such as Hsp families and periplasmic chaperones including; generic chaperones, specialized chaperones, PPIases, and proteins involved in disulfide bond formation. Copyright © 2017 Elsevier B.V. All rights reserved.
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Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan
2014-01-01
Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of co-factors (co-chaperones) that regulate their specificity and function. However, how these co-chaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We have combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone/co-chaperone/client interaction network in human cells. We uncover hundreds of novel chaperone clients, delineate their participation in specific co-chaperone complexes, and establish a surprisingly distinct network of protein/protein interactions for co-chaperones. As a salient example of the power of such analysis, we establish that NUDC family co-chaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network, its regulation in development and disease, and expand the use of chaperones as sensors for drug/target engagement. PMID:25036637
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Douglas, Peter M; Summers, Daniel W
2009-01-01
The self-association of misfolded or damaged proteins into ordered amyloid-like aggregates characterizes numerous neurodegenerative disorders. Insoluble amyloid plaques are diagnostic of many disease states. Yet soluble, oligomeric intermediates in the aggregation pathway appear to represent the toxic culprit. Molecular chaperones regulate the fate of misfolded proteins and thereby influence their aggregation state. Chaperones conventionally antagonize aggregation of misfolded, disease proteins and assist in refolding or degradation pathways. Recent work suggests that chaperones may also suppress neurotoxicity by converting toxic, soluble oligomers into benign aggregates. Chaperones can therefore suppress or promote aggregation of disease proteins to ameliorate the proteotoxic accumulation of soluble, assembly intermediates. PMID:19421006
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Interplay between Chaperones and Protein Disorder Promotes the Evolution of Protein Networks
Pechmann, Sebastian; Frydman, Judith
2014-01-01
Evolution is driven by mutations, which lead to new protein functions but come at a cost to protein stability. Non-conservative substitutions are of interest in this regard because they may most profoundly affect both function and stability. Accordingly, organisms must balance the benefit of accepting advantageous substitutions with the possible cost of deleterious effects on protein folding and stability. We here examine factors that systematically promote non-conservative mutations at the proteome level. Intrinsically disordered regions in proteins play pivotal roles in protein interactions, but many questions regarding their evolution remain unanswered. Similarly, whether and how molecular chaperones, which have been shown to buffer destabilizing mutations in individual proteins, generally provide robustness during proteome evolution remains unclear. To this end, we introduce an evolutionary parameter λ that directly estimates the rate of non-conservative substitutions. Our analysis of λ in Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens sequences reveals how co- and post-translationally acting chaperones differentially promote non-conservative substitutions in their substrates, likely through buffering of their destabilizing effects. We further find that λ serves well to quantify the evolution of intrinsically disordered proteins even though the unstructured, thus generally variable regions in proteins are often flanked by very conserved sequences. Crucially, we show that both intrinsically disordered proteins and highly re-wired proteins in protein interaction networks, which have evolved new interactions and functions, exhibit a higher λ at the expense of enhanced chaperone assistance. Our findings thus highlight an intricate interplay of molecular chaperones and protein disorder in the evolvability of protein networks. Our results illuminate the role of chaperones in enabling protein evolution, and underline the importance of the cellular
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RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae.
Ivanyi-Nagy, Roland; Lavergne, Jean-Pierre; Gabus, Caroline; Ficheux, Damien; Darlix, Jean-Luc
2008-02-01
RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning-possibly mediated by intrinsically disordered protein segments-is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication.
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Ebrahimi-Fakhari, Darius; Saidi, Laiq-Jan; Wahlster, Lara
2013-12-05
Changes in protein metabolism are key to disease onset and progression in many neurodegenerative diseases. As a prime example, in Parkinson's disease, folding, post-translational modification and recycling of the synaptic protein α-synuclein are clearly altered, leading to a progressive accumulation of pathogenic protein species and the formation of intracellular inclusion bodies. Altered protein folding is one of the first steps of an increasingly understood cascade in which α-synuclein forms complex oligomers and finally distinct protein aggregates, termed Lewy bodies and Lewy neurites. In neurons, an elaborated network of chaperone and co-chaperone proteins is instrumental in mediating protein folding and re-folding. In addition to their direct influence on client proteins, chaperones interact with protein degradation pathways such as the ubiquitin-proteasome-system or autophagy in order to ensure the effective removal of irreversibly misfolded and potentially pathogenic proteins. Because of the vital role of proper protein folding for protein homeostasis, a growing number of studies have evaluated the contribution of chaperone proteins to neurodegeneration. We herein review our current understanding of the involvement of chaperones, co-chaperones and chaperone-mediated autophagy in synucleinopathies with a focus on the Hsp90 and Hsp70 chaperone system. We discuss genetic and pathological studies in Parkinson's disease as well as experimental studies in models of synucleinopathies that explore molecular chaperones and protein degradation pathways as a novel therapeutic target. To this end, we examine the capacity of chaperones to prevent or modulate neurodegeneration and summarize the current progress in models of Parkinson's disease and related neurodegenerative disorders.
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Immunodominant protein MIP_05962 from Mycobacterium indicus pranii displays chaperone activity.
Sharma, Ashish; Equbal, Md Javed; Pandey, Saurabh; Sheikh, Javaid A; Ehtesham, Nasreen Z; Hasnain, Seyed E; Chaudhuri, Tapan K
2017-05-01
Tuberculosis, a contagious disease of infectious origin is currently a major cause of deaths worldwide. Mycobacterium indicus pranii (MIP), a saprophytic nonpathogen and a potent immunomodulator is currently being investigated as an intervention against tuberculosis along with many other diseases with positive outcome. The apparent paradox of multiple chaperones in mycobacterial species and enigma about the cellular functions of the client proteins of these chaperones need to be explored. Chaperones are the known immunomodulators; thus, there is need to exploit the proteome of MIP for identification and characterization of putative chaperones. One of the immunogenic proteins, MIP_05962 is a member of heat shock protein (HSP) 20 family due to the presence of α-crystallin domain, and has amino acid similarity with Mycobacterium lepraeHSP18 protein. The diverse functions of M. lepraeHSP18 in stress conditions implicate MIP_05962 as an important protein that needs to be explored. Biophysical and biochemical characterization of the said protein proved it to be a chaperone. The observations of aggregation prevention and refolding of substrate proteins in the presence of MIP_05962 along with interaction with non-native proteins, surface hydrophobicity, formation of large oligomers, in-vivo thermal rescue of Escherichia coli expressing MIP_05962, enhancing solubility of insoluble protein maltodextrin glucosidase (MalZ) under in-vivo conditions, and thermal stability and reversibility confirmed MIP_05962 as a molecular chaperone. © 2017 Federation of European Biochemical Societies.
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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.
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RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae
Ivanyi-Nagy, Roland; Lavergne, Jean-Pierre; Gabus, Caroline; Ficheux, Damien; Darlix, Jean-Luc
2008-01-01
RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning—possibly mediated by intrinsically disordered protein segments—is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication. PMID:18033802
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Alcantara, Marice B; Nemazannikova, Natalie; Elahy, Mina; Dass, Crispin R
2014-11-01
Pigment epithelium-derived factor (PEDF) has proven anti-osteosarcoma activity. However, the mechanism(s) underpinning its ability to reduce primary bone tumour (osteosarcoma) metastasis is unknown. Adult and fetal murine bone were immunostained for PEDF, collagen I (major protein in bone) and its processing proteins, heat shock protein 47 (HSP47, a chaperone protein for collagen I), membrane type I matrix metalloproteinase (MT1-MMP, a collagenase), and matrix metalloproteinase 2 (MMP-2, which is activated by MT1-MMP). Immunoblotting and immunocytochemistry were used to observe levels of the above biomarkers when human osteosarcoma cells were treated with PEDF. Immunohistochemical staining in adult and fetal bone mirrors collagen I. PEDF localised to ridges of trabecular bone in tibial cortex and to megakaryocytes within bone marrow. Second, we observed that PEDF upregulates collagen I, HSP47 and MT1-MMP, while downregulating MMP-2 in osteosarcoma cells in vitro. PEDF is a promising antagonist to osteosarcoma cell metastasis via downregulation of MMP-2, and can induce tumour cells to further adopt differentiative properties, thereby possibly reducing their aggressive growth in vitro and in vivo. © 2014 Royal Pharmaceutical Society.
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Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones
Pausch, Patrick; Singh, Ujjwala; Ahmed, Yasar Luqman; Pillet, Benjamin; Murat, Guillaume; Altegoer, Florian; Stier, Gunter; Thoms, Matthias; Hurt, Ed; Sinning, Irmgard; Bange, Gert; Kressler, Dieter
2015-01-01
Exponentially growing yeast cells produce every minute >160,000 ribosomal proteins. Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge. Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site. Here we explore the intuitive possibility that ribosomal proteins are captured by dedicated chaperones in a co-translational manner. Affinity purification of four chaperones (Rrb1, Syo1, Sqt1 and Yar1) selectively enriched the mRNAs encoding their specific ribosomal protein clients (Rpl3, Rpl5, Rpl10 and Rps3). X-ray crystallography reveals how the N-terminal, rRNA-binding residues of Rpl10 are shielded by Sqt1's WD-repeat β-propeller, providing mechanistic insight into the incorporation of Rpl10 into pre-60S subunits. Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation. PMID:26112308
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Analysis of molecular chaperones using a Xenopus oocyte protein refolding assay.
Heikkila, John J; Kaldis, Angelo; Abdulle, Rashid
2006-01-01
Heat shock proteins (Hsps) are molecular chaperones that aid in the folding and translocation of protein under normal conditions and protect cellular proteins during stressful situations. A family of Hsps, the small Hsps, can maintain denatured target proteins in a folding-competent state such that they can be refolded and regain biological activity in the presence of other molecular chaperones. Previous assays have employed cellular lysates as a source of molecular chaperones involved in folding. In this chapter, we describe the production and purification of a Xenopus laevis recombinant small Hsp, Hsp30C, and an in vivo luciferase (LUC) refolding assay employing microinjected Xenopus oocytes. This assay tests whether LUC can be maintained in a folding-competent state when heat denatured in the presence of a small Hsp or other molecular chaperone. For example, micro-injection of heat-denatured LUC alone into oocytes resulted in minimal reactivation of enzyme activity. However, LUC heat denatured in the presence of Hsp30C resulted in 100% recovery of enzyme activity after microinjection. The in vivo oocyte refolding system is more sensitive and requires less molecular chaperone than in vitro refolding assays. Also, this protocol is not limited to testing Xenopus molecular chaperones because small Hsps from other organisms have been used successfully.
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Fe-S Cluster Hsp70 Chaperones: The ATPase Cycle and Protein Interactions.
Dutkiewicz, Rafal; Nowak, Malgorzata; Craig, Elizabeth A; Marszalek, Jaroslaw
2017-01-01
Hsp70 chaperones and their obligatory J-protein cochaperones function together in many cellular processes. Via cycles of binding to short stretches of exposed amino acids on substrate proteins, Hsp70/J-protein chaperones not only facilitate protein folding but also drive intracellular protein transport, biogenesis of cellular structures, and disassembly of protein complexes. The biogenesis of iron-sulfur (Fe-S) clusters is one of the critical cellular processes that require Hsp70/J-protein action. Fe-S clusters are ubiquitous cofactors critical for activity of proteins performing diverse functions in, for example, metabolism, RNA/DNA transactions, and environmental sensing. This biogenesis process can be divided into two sequential steps: first, the assembly of an Fe-S cluster on a conserved scaffold protein, and second, the transfer of the cluster from the scaffold to a recipient protein. The second step involves Hsp70/J-protein chaperones. Via binding to the scaffold, chaperones enable cluster transfer to recipient proteins. In eukaryotic cells mitochondria have a key role in Fe-S cluster biogenesis. In this review, we focus on methods that enabled us to dissect protein interactions critical for the function of Hsp70/J-protein chaperones in the mitochondrial process of Fe-S cluster biogenesis in the yeast Saccharomyces cerevisiae. © 2017 Elsevier Inc. All rights reserved.
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Engineering and Evolution of Molecular Chaperones and Protein Disaggregases with Enhanced Activity
Mack, Korrie L.; Shorter, James
2016-01-01
Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson's disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector. PMID:27014702
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Regulation of Effector Delivery by Type III Secretion Chaperone Proteins in Erwinia amylovora.
Castiblanco, Luisa F; Triplett, Lindsay R; Sundin, George W
2018-01-01
Type III secretion (TTS) chaperones are critical for the delivery of many effector proteins from Gram-negative bacterial pathogens into host cells, functioning in the stabilization and hierarchical delivery of the effectors to the type III secretion system (TTSS). The plant pathogen Erwinia amylovora secretes at least four TTS effector proteins: DspE, Eop1, Eop3, and Eop4. DspE specifically interacts with the TTS chaperone protein DspF, which stabilizes the effector protein in the cytoplasm and promotes its efficient translocation through the TTSS. However, the role of E. amylovora chaperones in regulating the delivery of other secreted effectors is unknown. In this study, we identified functional interactions between the effector proteins DspE, Eop1, and Eop3 with the TTS chaperones DspF, Esc1 and Esc3 in yeast. Using site-directed mutagenesis, secretion, and translocation assays, we demonstrated that the three TTS chaperones have additive roles for the secretion and translocation of DspE into plant cells whereas DspF negatively affects the translocation of Eop1 and Eop3. Collectively, these results indicate that TTS chaperone proteins exhibit a cooperative behavior to orchestrate the effector secretion and translocation dynamics in E. amylovora .
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Singh, Bhanu Pratap; Saha, Ishita; Nandi, Indrani; Swamy, Musti J
2017-12-02
The major bovine seminal plasma protein, PDC-109, binds to choline phospholipids of the sperm plasma membrane and induces an efflux of cholesterol and choline phospholipids (cholesterol efflux), which is crucial for sperm capacitation. PDC-109 also exhibits chaperone-like activity and protects target proteins against various kinds of stress. Here we show that the polyamines spermine and spermidine, present in high concentration in the seminal plasma of various mammals, increase the ability of PDC-109 to perturb membrane structure as well as its chaperone-like activity. Interestingly, spermine/spermidine alone did not perturb membrane structure but exhibited chaperone-like activity by protecting target proteins against thermal and oxidative stress. When spermine/spermidine was used along with PDC-109, the observed chaperone-like activity was considerably higher than that expected for a simple additive effect, suggesting that PDC-109 and the polyamines act in a synergistic fashion. These results indicate that at the high concentrations present in the seminal plasma spermine/spermidine exhibit a positive modulatory effect on the chaperone-like activity of PDC-109 and may also function as chemical chaperones and protect other seminal plasma proteins from various kinds of stress. Copyright © 2017 Elsevier Inc. All rights reserved.
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Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone
Góral, Agnieszka; Bieganowski, Paweł; Prus, Wiktor; Krzemień-Ojak, Łucja; Kądziołka, Beata; Fabczak, Hanna; Filipek, Anna
2016-01-01
The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery. PMID:27249023
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Ito, Shinya; Ogawa, Koji; Takeuchi, Koh; Takagi, Motoki; Yoshida, Masahito; Hirokawa, Takatsugu; Hirayama, Shoshiro; Shin-Ya, Kazuo; Shimada, Ichio; Doi, Takayuki; Goshima, Naoki; Natsume, Tohru; Nagata, Kazuhiro
2017-12-08
Fibrosis can disrupt tissue structure and integrity and impair organ function. Fibrosis is characterized by abnormal collagen accumulation in the extracellular matrix. Pharmacological inhibition of collagen secretion therefore represents a promising strategy for the management of fibrotic disorders, such as liver and lung fibrosis. Hsp47 is an endoplasmic reticulum (ER)-resident collagen-specific molecular chaperone essential for correct folding of procollagen in the ER. Genetic deletion of Hsp47 or inhibition of its interaction with procollagen interferes with procollagen triple helix production, which vastly reduces procollagen secretion from fibroblasts. Thus, Hsp47 could be a potential and promising target for the management of fibrosis. In this study, we screened small-molecule compounds that inhibit the interaction of Hsp47 with collagen from chemical libraries using surface plasmon resonance (BIAcore), and we found a molecule AK778 and its cleavage product Col003 competitively inhibited the interaction and caused the inhibition of collagen secretion by destabilizing the collagen triple helix. Structural information obtained with NMR analysis revealed that Col003 competitively binds to the collagen-binding site on Hsp47. We propose that these structural insights could provide a basis for designing more effective therapeutic drugs for managing fibrosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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The major protein of bovine seminal plasma, PDC-109, is a molecular chaperone.
Sankhala, Rajeshwer Singh; Swamy, Musti J
2010-05-11
The major protein of bovine seminal plasma, PDC-109, binds to choline phospholipids on the sperm plasma membrane and induces the efflux of cholesterol and choline phospholipids, which is an important step in sperm capacitation. The high abundance, polydisperse nature and reversibility of thermal unfolding of PDC-109 suggest significant similarities to chaperone-like proteins such as spectrin, alpha-crystallin, and alpha-synuclein. In the present study, biochemical and biophysical approaches were employed to investigate the chaperone-like activity of PDC-109. The effect of various stress factors such as high temperature, chemical denaturant (urea), and acidic pH on target proteins such as lactate dehydrogenase, alcohol dehydrogenase, and insulin were studied in both the presence and absence of PDC-109. The results obtained indicate that PDC-109 exhibits chaperone-like activity, as evidenced by its ability to suppress the nonspecific aggregation of target proteins and direct them into productive folding. Atomic force microscopic studies demonstrate that PDC-109 effectively prevents the fibrillation of insulin, which is of considerable significance since amyloidogenesis has been reported to be a serious problem during sperm maturation in certain species. Binding of phosphorylcholine or high ionic strength in the medium inhibited the chaperone-like activity of PDC-109, suggesting that most likely the aggregation state of the protein is important for the chaperone function. These observations show that PDC-109 functions as a molecular chaperone in vitro, suggesting that it may assist the proper folding of proteins involved in the bovine sperm capacitation pathway. To the best of our knowledge, this is the first study reporting chaperone-like activity of a seminal plasma protein.
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Peroxisomal Proteostasis Involves a Lon Family Protein That Functions as Protease and Chaperone*
Bartoszewska, Magdalena; Williams, Chris; Kikhney, Alexey; Opaliński, Łukasz; van Roermund, Carlo W. T.; de Boer, Rinse; Veenhuis, Marten; van der Klei, Ida J.
2012-01-01
Proteins are subject to continuous quality control for optimal proteostasis. The knowledge of peroxisome quality control systems is still in its infancy. Here we show that peroxisomes contain a member of the Lon family of proteases (Pln). We show that Pln is a heptameric protein and acts as an ATP-fueled protease and chaperone. Hence, Pln is the first chaperone identified in fungal peroxisomes. In cells of a PLN deletion strain peroxisomes contain protein aggregates, a major component of which is catalase-peroxidase. We show that this enzyme is sensitive to oxidative damage. The oxidatively damaged, but not the native protein, is a substrate of the Pln protease. Cells of the pln strain contain enhanced levels of catalase-peroxidase protein but reduced catalase-peroxidase enzyme activities. Together with the observation that Pln has chaperone activity in vitro, our data suggest that catalase-peroxidase aggregates accumulate in peroxisomes of pln cells due to the combined absence of Pln protease and chaperone activities. PMID:22733816
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Chaperones and protein folding in the archaea.
Large, Andrew T; Goldberg, Martin D; Lund, Peter A
2009-02-01
A survey of archaeal genomes for the presence of homologues of bacterial and eukaryotic chaperones reveals several interesting features. All archaea contain chaperonins, also known as Hsp60s (where Hsp is heat-shock protein). These are more similar to the type II chaperonins found in the eukaryotic cytosol than to the type I chaperonins found in bacteria, mitochondria and chloroplasts, although some archaea also contain type I chaperonin homologues, presumably acquired by horizontal gene transfer. Most archaea contain several genes for these proteins. Our studies on the type II chaperonins of the genetically tractable archaeon Haloferax volcanii have shown that only one of the three genes has to be present for the organisms to grow, but that there is some evidence for functional specialization between the different chaperonin proteins. All archaea also possess genes for prefoldin proteins and for small heat-shock proteins, but they generally lack genes for Hsp90 and Hsp100 homologues. Genes for Hsp70 (DnaK) and Hsp40 (DnaJ) homologues are only found in a subset of archaea. Thus chaperone-assisted protein folding in archaea is likely to display some unique features when compared with that in eukaryotes and bacteria, and there may be important differences in the process between euryarchaea and crenarchaea.
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Molecular chaperones and photoreceptor function
Kosmaoglou, Maria; Schwarz, Nele; Bett, John S.; Cheetham, Michael E.
2008-01-01
Molecular chaperones facilitate and regulate protein conformational change within cells. This encompasses many fundamental cellular processes: including the correct folding of nascent chains; protein transport and translocation; signal transduction and protein quality control. Chaperones are, therefore, important in several forms of human disease, including neurodegeneration. Within the retina, the highly specialized photoreceptor cell presents a fascinating paradigm to investigate the specialization of molecular chaperone function and reveals unique chaperone requirements essential to photoreceptor function. Mutations in several photoreceptor proteins lead to protein misfolding mediated neurodegeneration. The best characterized of these are mutations in the molecular light sensor, rhodopsin, which cause autosomal dominant retinitis pigmentosa. Rhodopsin biogenesis is likely to require chaperones, while rhodopsin misfolding involves molecular chaperones in quality control and the cellular response to protein aggregation. Furthermore, the specialization of components of the chaperone machinery to photoreceptor specific roles has been revealed by the identification of mutations in molecular chaperones that cause inherited retinal dysfunction and degeneration. These chaperones are involved in several important cellular pathways and further illuminate the essential and diverse roles of molecular chaperones. PMID:18490186
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The heat-shock protein/chaperone network and multiple stress resistance.
Jacob, Pierre; Hirt, Heribert; Bendahmane, Abdelhafid
2017-04-01
Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multistress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat-shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone 'client proteins', many are primary metabolism enzymes and signal transduction components with essential roles for the proper functioning of a cell. HSPs/chaperones are controlled by the action of diverse heat-shock factors, which are recruited under stress conditions. In this review, we give an overview of the regulation of the HSP/chaperone network with a focus on Arabidopsis thaliana. We illustrate the role of HSPs/chaperones in regulating diverse signalling pathways and discuss several basic principles that should be considered for engineering multiple stress resistance in crops through the HSP/chaperone network. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
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Valaviciute, Monika; Norkiene, Milda; Goda, Karolis; Slibinskas, Rimantas; Gedvilaite, Alma
2016-07-01
A number of viruses utilize molecular chaperones during various stages of their life cycle. It has been shown that members of the heat-shock protein 70 (Hsp70) chaperone family assist polyomavirus capsids during infection. However, the molecular chaperones that assist the formation of recombinant capsid viral protein 1 (VP1)-derived virus-like particles (VLPs) in yeast remain unclear. A panel of yeast strains with single chaperone gene deletions were used to evaluate the chaperones required for biosynthesis of recombinant hamster polyomavirus capsid protein VP1. The impact of deletion or mild overexpression of chaperone genes was determined in live cells by flow cytometry using enhanced green fluorescent protein (EGFP) fused with VP1. Targeted genetic analysis demonstrated that VP1-EGFP fusion protein levels were significantly higher in yeast strains in which the SSZ1 or ZUO1 genes encoding ribosome-associated complex components were deleted. The results confirmed the participation of cytosolic Hsp70 chaperones and suggested the potential involvement of the Ydj1 and Caj1 co-chaperones and the endoplasmic reticulum chaperones in the biosynthesis of VP1 VLPs in yeast. Likewise, the markedly reduced levels of VP1-EGFP in Δhsc82 and Δhsp82 yeast strains indicated that both Hsp70 and Hsp90 chaperones might assist VP1 VLPs during protein biosynthesis.
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Barnes, Aileen M.; Cabral, Wayne A.; Weis, MaryAnn; Makareeva, Elena; Mertz, Edward L.; Leikin, Sergey; Eyre, David; Trujillo, Carlos; Marini, Joan C.
2012-01-01
Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% overmodification. Normal chain incorporation, helix folding, and collagen Tm support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix. PMID:22718341
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Mammalian Fe-S proteins: definition of a consensus motif recognized by the co-chaperone HSC20.
Maio, N; Rouault, T A
2016-10-01
Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery.
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Differential effects of collagen prolyl 3-hydroxylation on skeletal tissues.
Homan, Erica P; Lietman, Caressa; Grafe, Ingo; Lennington, Jennifer; Morello, Roy; Napierala, Dobrawa; Jiang, Ming-Ming; Munivez, Elda M; Dawson, Brian; Bertin, Terry K; Chen, Yuqing; Lua, Rhonald; Lichtarge, Olivier; Hicks, John; Weis, Mary Ann; Eyre, David; Lee, Brendan H L
2014-01-01
Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1(H662A) ). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity
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Bone Collagen: New Clues to its Mineralization Mechanism From Recessive Osteogenesis Imperfecta
Eyre, David R.; Ann Weis, Mary
2013-01-01
Until 2006 the only mutations known to cause osteogenesis imperfecta (OI) were in the two genes coding for type I collagen chains. These dominant mutations affecting the expression or primary sequence of collagen α1(I) and α2(I) chains account for over 90% of OI cases. Since then a growing list of mutant genes causing the 5–10% of recessive cases has rapidly emerged. They include CRTAP, LEPRE1 and PPIB, which encode three proteins forming the prolyl 3-hydroxylase complex; PLOD2 and FKBP10, which encode respectively lysyl hydroxylase 2 and a foldase required for its activity in forming mature cross-links in bone collagen; SERPIN H1, which encodes the collagen chaperone HSP47; SERPIN F1, which encodes pigment epithelium-derived factor required for osteoid mineralization; and BMP1, which encodes the type I procollagen C-propeptidase. All cause fragile bone in infancy, which can include over-mineralization or under-mineralization defects as well as abnormal collagen post-translational modifications. Consistently both dominant and recessive variants lead to abnormal cross-linking chemistry in bone collagen. These recent discoveries strengthen the potential for a common pathogenic mechanism of misassembled collagen fibrils. Of the new genes identified, eight encode proteins required for collagen post-translational modification, chaperoning of newly synthesized collagen chains into native molecules or transport through the endoplasmic reticulum and Golgi for polymerization, cross-linking and mineralization. In reviewing these findings, we conclude that a common theme is emerging in the pathogenesis of brittle bone disease of mishandled collagen assembly with important insights on post-translational features of bone collagen that have evolved to optimize it as a biomineral template. PMID:23508630
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The radical SAM protein HemW is a heme chaperone.
Haskamp, Vera; Karrie, Simone; Mingers, Toni; Barthels, Stefan; Alberge, François; Magalon, Axel; Müller, Katrin; Bill, Eckhard; Lubitz, Wolfgang; Kleeberg, Kirstin; Schweyen, Peter; Bröring, Martin; Jahn, Martina; Jahn, Dieter
2018-02-16
Radical S -adenosylmethionine (SAM) enzymes exist in organisms from all kingdoms of life, and all of these proteins generate an adenosyl radical via the homolytic cleavage of the S-C(5') bond of SAM. Of particular interest are radical SAM enzymes, such as heme chaperones, that insert heme into respiratory enzymes. For example, heme chaperones insert heme into target proteins but have been studied only for the formation of cytochrome c -type hemoproteins. Here, we report that a radical SAM protein, the heme chaperone HemW from bacteria, is required for the insertion of heme b into respiratory chain enzymes. As other radical SAM proteins, HemW contains three cysteines and one SAM coordinating an [4Fe-4S] cluster, and we observed one heme per subunit of HemW. We found that an intact iron-sulfur cluster was required for HemW dimerization and HemW-catalyzed heme transfer but not for stable heme binding. A bacterial two-hybrid system screen identified bacterioferritins and the heme-containing subunit NarI of the respiratory nitrate reductase NarGHI as proteins that interact with HemW. We also noted that the bacterioferritins potentially serve as heme donors for HemW. Of note, heme that was covalently bound to HemW was actively transferred to a heme-depleted, catalytically inactive nitrate reductase, restoring its nitrate-reducing enzyme activity. Finally, the human HemW orthologue radical SAM domain-containing 1 (RSAD1) stably bound heme. In conclusion, our findings indicate that the radical SAM protein family HemW/RSAD1 is a heme chaperone catalyzing the insertion of heme into hemoproteins. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
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Mammalian Fe-S proteins: definition of a consensus motif recognized by the co-chaperone HSC20
Maio, N.; Rouault, T. A.
2017-01-01
Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery. PMID:27714045
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Heat Shock Proteins: A Review of the Molecular Chaperones for Plant Immunity.
Park, Chang-Jin; Seo, Young-Su
2015-12-01
As sessile organisms, plants are exposed to persistently changing stresses and have to be able to interpret and respond to them. The stresses, drought, salinity, chemicals, cold and hot temperatures, and various pathogen attacks have interconnected effects on plants, resulting in the disruption of protein homeostasis. Maintenance of proteins in their functional native conformations and preventing aggregation of non-native proteins are important for cell survival under stress. Heat shock proteins (HSPs) functioning as molecular chaperones are the key components responsible for protein folding, assembly, translocation, and degradation under stress conditions and in many normal cellular processes. Plants respond to pathogen invasion using two different innate immune responses mediated by pattern recognition receptors (PRRs) or resistance (R) proteins. HSPs play an indispensable role as molecular chaperones in the quality control of plasma membrane-resident PRRs and intracellular R proteins against potential invaders. Here, we specifically discuss the functional involvement of cytosolic and endoplasmic reticulum (ER) HSPs/chaperones in plant immunity to obtain an integrated understanding of the immune responses in plant cells.
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Cooperative Subunit Refolding of a Light-Harvesting Protein through a Self-Chaperone Mechanism.
Laos, Alistair J; Dean, Jacob C; Toa, Zi S D; Wilk, Krystyna E; Scholes, Gregory D; Curmi, Paul M G; Thordarson, Pall
2017-07-10
The fold of a protein is encoded by its amino acid sequence, but how complex multimeric proteins fold and assemble into functional quaternary structures remains unclear. Here we show that two structurally different phycobiliproteins refold and reassemble in a cooperative manner from their unfolded polypeptide subunits, without biological chaperones. Refolding was confirmed by ultrafast broadband transient absorption and two-dimensional electronic spectroscopy to probe internal chromophores as a marker of quaternary structure. Our results demonstrate a cooperative, self-chaperone refolding mechanism, whereby the β-subunits independently refold, thereby templating the folding of the α-subunits, which then chaperone the assembly of the native complex, quantitatively returning all coherences. Our results indicate that subunit self-chaperoning is a robust mechanism for heteromeric protein folding and assembly that could also be applied in self-assembled synthetic hierarchical systems. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1.
Derrington, Edmund; Gabus, Caroline; Leblanc, Pascal; Chnaidermann, Jonas; Grave, Linda; Dormont, Dominique; Swietnicki, Wieslaw; Morillas, Manuel; Marck, Daniel; Nandi, Pradip; Darlix, Jean-Luc
2002-01-01
The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.
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The fragile X mental retardation protein has nucleic acid chaperone properties
Gabus, Caroline; Mazroui, Rachid; Tremblay, Sandra; Khandjian, Edouard W.; Darlix, Jean-Luc
2004-01-01
The fragile X syndrome is the most common cause of inherited mental retardation resulting from the absence of the fragile X mental retardation protein (FMRP). FMRP contains two K-homology (KH) domains and one RGG box that are landmarks characteristic of RNA-binding proteins. In agreement with this, FMRP associates with messenger ribonucleoparticles (mRNPs) within actively translating ribosomes, and is thought to regulate translation of target mRNAs, including its own transcript. To investigate whether FMRP might chaperone nucleic acid folding and hybridization, we analysed the annealing and strand exchange activities of DNA oligonucleotides and the enhancement of ribozyme-directed RNA substrate cleavage by FMRP and deleted variants relative to canonical nucleic acid chaperones, such as the cellular YB-1/p50 protein and the retroviral nucleocapsid protein HIV-1 NCp7. FMRP was found to possess all the properties of a potent nucleic acid chaperone, requiring the KH motifs and RGG box for optimal activity. These findings suggest that FMRP may regulate translation by acting on RNA–RNA interactions and thus on the structural status of mRNAs. PMID:15096575
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The fragile X mental retardation protein has nucleic acid chaperone properties.
Gabus, Caroline; Mazroui, Rachid; Tremblay, Sandra; Khandjian, Edouard W; Darlix, Jean-Luc
2004-01-01
The fragile X syndrome is the most common cause of inherited mental retardation resulting from the absence of the fragile X mental retardation protein (FMRP). FMRP contains two K-homology (KH) domains and one RGG box that are landmarks characteristic of RNA-binding proteins. In agreement with this, FMRP associates with messenger ribonucleoparticles (mRNPs) within actively translating ribosomes, and is thought to regulate translation of target mRNAs, including its own transcript. To investigate whether FMRP might chaperone nucleic acid folding and hybridization, we analysed the annealing and strand exchange activities of DNA oligonucleotides and the enhancement of ribozyme-directed RNA substrate cleavage by FMRP and deleted variants relative to canonical nucleic acid chaperones, such as the cellular YB-1/p50 protein and the retroviral nucleocapsid protein HIV-1 NCp7. FMRP was found to possess all the properties of a potent nucleic acid chaperone, requiring the KH motifs and RGG box for optimal activity. These findings suggest that FMRP may regulate translation by acting on RNA-RNA interactions and thus on the structural status of mRNAs.
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Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon
Chou, Ming-Lun; Fitzpatrick, Lynda M.; Tu, Shuh-Long; Budziszewski, Gregory; Potter-Lewis, Sharon; Akita, Mitsuru; Levin, Joshua Z.; Keegstra, Kenneth; Li, Hsou-min
2003-01-01
The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal. Isolated mutant chloroplasts imported precursor proteins at a lower rate than wild-type chloroplasts. Mutant chloroplasts were normal in allowing binding of precursor proteins. However, during subsequent translocation across the inner membrane, fewer precursors were translocated and more precursors were released from the mutant chloroplasts. Cross-linking experiments demonstrated that Tic40 was part of the translocon complex and functioned at the same stage of import as Tic110 and Hsp93, a member of the Hsp100 family of molecular chaperones. Tertiary structure prediction and immunological studies indicated that the C-terminal portion of Tic40 contains a TPR domain followed by a domain with sequence similarity to co-chaperones Sti1p/Hop and Hip. We propose that Tic40 functions as a co-chaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane. PMID:12805212
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Lee, Won Jai; Lee, Ju Hee; Ahn, Hyo Min; Song, Seung Yong; Kim, Yong Oock; Lew, Dae Hyun; Yun, Chae-Ok
2015-09-01
The 90-kDa heat-shock protein (heat-shock protein 90) is an abundant cytosolic chaperone, and inhibition of heat-shock protein 90 by 17-allylamino-17-demethoxygeldanamycin (17-AAG) compromises transforming growth factor (TGF)-β-mediated transcriptional responses by enhancing TGF-β receptor I and II degradation, thus preventing Smad2/3 activation. In this study, the authors evaluated whether heat-shock protein 90 regulates TGF-β signaling in the pathogenesis and treatment of keloids. Keloid fibroblasts were treated with 17-AAG (10 μM), and mRNA levels of collagen types I and III were determined by real-time reverse- transcriptase polymerase chain reaction. Also, secreted TGF-β1 was assessed by enzyme-linked immunosorbent assay. The effect of 17-AAG on protein levels of Smad2/3 complex was determined by Western blot analysis. In addition, in 17-AAG-treated keloid spheroids, the collagen deposition and expression of major extracellular matrix proteins were investigated by means of Masson trichrome staining and immunohistochemistry. The authors found that heat-shock protein 90 is overexpressed in human keloid tissue compared with adjacent normal tissue, and 17-AAG decreased mRNA levels of type I collagen, secreted TGF-ß1, and Smad2/3 complex protein expression in keloid fibroblasts. Masson trichrome staining revealed that collagen deposition was decreased in 17-AAG-treated keloid spheroids, and immunohistochemical analysis showed that expression of collagen types I and III, elastin, and fibronectin was markedly decreased in 17-AAG-treated keloid spheroids. These results suggest that the antifibrotic action of heat-shock protein 90 inhibitors such as 17-AAG may have therapeutic effects on keloids.
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Barnes, Aileen M; Cabral, Wayne A; Weis, MaryAnn; Makareeva, Elena; Mertz, Edward L; Leikin, Sergey; Eyre, David; Trujillo, Carlos; Marini, Joan C
2012-11-01
Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% over-modification. Normal chain incorporation, helix folding, and collagen T(m) support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix. Published 2012 Wiley Periodicals, Inc.*This article is a US Government work and, as such, is in the public domain of the United States of America.
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Kumsta, Caroline; Jakob, Ursula
2009-06-09
Redox regulation of stress proteins, such as molecular chaperones, guarantees an immediate response to oxidative stress conditions. This review focuses on the two major classes of redox-regulated chaperones, Hsp33 in bacteria and typical 2-Cys peroxiredoxins in eukaryotes. Both proteins employ redox-sensitive cysteines, whose oxidation status directly controls their affinity for unfolding proteins and therefore their chaperone function. We will first discuss Hsp33, whose oxidative stress-induced disulfide bond formation triggers the partial unfolding of the chaperone, which, in turn, leads to the exposure of a high-affinity binding site for unfolded proteins. This rapid mode of activation makes Hsp33 essential for protecting bacteria against severe oxidative stress conditions, such as hypochlorite (i.e., bleach) treatment, which leads to widespread protein unfolding and aggregation. We will compare Hsp33 to the highly abundant eukaryotic typical 2-Cys peroxiredoxin, whose oxidative stress-induced sulfinic acid formation turns the peroxidase into a molecular chaperone in vitro and presumably in vivo. These examples illustrate how proteins use reversible cysteine modifications to rapidly adjust to oxidative stress conditions and demonstrate that redox regulation plays a vital role in protecting organisms against reactive oxygen species-mediated cell death.
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Chaperoning G Protein-Coupled Receptors: From Cell Biology to Therapeutics
Conn, P. Michael
2014-01-01
G protein-coupled receptors (GPCRs) are membrane proteins that traverse the plasma membrane seven times (hence, are also called 7TM receptors). The polytopic structure of GPCRs makes the folding of GPCRs difficult and complex. Indeed, many wild-type GPCRs are not folded optimally, and defects in folding are the most common cause of genetic diseases due to GPCR mutations. Both general and receptor-specific molecular chaperones aid the folding of GPCRs. Chemical chaperones have been shown to be able to correct the misfolding in mutant GPCRs, proving to be important tools for studying the structure-function relationship of GPCRs. However, their potential therapeutic value is very limited. Pharmacological chaperones (pharmacoperones) are potentially important novel therapeutics for treating genetic diseases caused by mutations in GPCR genes that resulted in misfolded mutant proteins. Pharmacoperones also increase cell surface expression of wild-type GPCRs; therefore, they could be used to treat diseases that do not harbor mutations in GPCRs. Recent studies have shown that indeed pharmacoperones work in both experimental animals and patients. High-throughput assays have been developed to identify new pharmacoperones that could be used as therapeutics for a number of endocrine and other genetic diseases. PMID:24661201
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Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A; Nagata, Kazuhiro; Bächinger, Hans Peter
2009-06-26
The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the alpha chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1.CRTAP.CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1.CRTAP.CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1.CRTAP.CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone.
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Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides
Guichard, Cécile; Ivanyi-Nagy, Roland; Sharma, Kamal Kant; Gabus, Caroline; Marc, Daniel; Mély, Yves; Darlix, Jean-Luc
2011-01-01
Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrPC) into the aggregated misfolded scrapie isoform, named PrPSc. Recent studies on the physiological role of PrPC revealed that this protein has probably multiple functions, notably in cell–cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5′-GACACAAGCCGA-3′ was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities. PMID:21737432
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Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides.
Guichard, Cécile; Ivanyi-Nagy, Roland; Sharma, Kamal Kant; Gabus, Caroline; Marc, Daniel; Mély, Yves; Darlix, Jean-Luc
2011-10-01
Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrP(C)) into the aggregated misfolded scrapie isoform, named PrP(Sc). Recent studies on the physiological role of PrP(C) revealed that this protein has probably multiple functions, notably in cell-cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5'-GACACAAGCCGA-3' was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities.
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2012-01-01
Background Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro. Methods The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining. Results TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1. Conclusion We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition. PMID:22694981
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Forces Driving Chaperone Action
Koldewey, Philipp; Stull, Frederick; Horowitz, Scott; Martin, Raoul; Bardwell, James C. A.
2016-01-01
SUMMARY It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the four key steps of chaperone-client interaction: initial binding, complex stabilization, folding, and release. Contrary to the common belief that chaperones recognize unfolding intermediates by their hydrophobic nature, we discover that the model chaperone Spy uses long-range electrostatic interactions to rapidly bind to its unfolded client protein Im7. Short-range hydrophobic interactions follow, which serve to stabilize the complex. Hydrophobic collapse of the client protein then drives its folding. By burying hydrophobic residues in its core, the client’s affinity to Spy decreases, which causes client release. By allowing the client to fold itself, Spy circumvents the need for client-specific folding instructions. This mechanism might help explain how chaperones can facilitate the folding of various unrelated proteins. PMID:27293188
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Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A.; Nagata, Kazuhiro; Bächinger, Hans Peter
2009-01-01
The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the α chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1·CRTAP·CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1·CRTAP·CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1·CRTAP·CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone. PMID:19419969
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RNA chaperone activity of human La protein is mediated by variant RNA recognition motif.
Naeeni, Amir R; Conte, Maria R; Bayfield, Mark A
2012-02-17
La proteins are conserved factors in eukaryotes that bind and protect the 3' trailers of pre-tRNAs from exonuclease digestion via sequence-specific recognition of UUU-3'OH. La has also been hypothesized to assist pre-tRNAs in attaining their native fold through RNA chaperone activity. In addition to binding polymerase III transcripts, human La has also been shown to enhance the translation of several internal ribosome entry sites and upstream ORF-containing mRNA targets, also potentially through RNA chaperone activity. Using in vitro FRET-based assays, we show that human and Schizosaccharomyces pombe La proteins harbor RNA chaperone activity by enhancing RNA strand annealing and strand dissociation. We use various RNA substrates and La mutants to show that UUU-3'OH-dependent La-RNA binding is not required for this function, and we map RNA chaperone activity to its RRM1 motif including a noncanonical α3-helix. We validate the importance of this α3-helix by appending it to the RRM of the unrelated U1A protein and show that this fusion protein acquires significant strand annealing activity. Finally, we show that residues required for La-mediated RNA chaperone activity in vitro are required for La-dependent rescue of tRNA-mediated suppression via a mutated suppressor tRNA in vivo. This work delineates the structural elements required for La-mediated RNA chaperone activity and provides a basis for understanding how La can enhance the folding of its various RNA targets.
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RNA Chaperone Activity of Human La Protein Is Mediated by Variant RNA Recognition Motif*
Naeeni, Amir R.; Conte, Maria R.; Bayfield, Mark A.
2012-01-01
La proteins are conserved factors in eukaryotes that bind and protect the 3′ trailers of pre-tRNAs from exonuclease digestion via sequence-specific recognition of UUU-3′OH. La has also been hypothesized to assist pre-tRNAs in attaining their native fold through RNA chaperone activity. In addition to binding polymerase III transcripts, human La has also been shown to enhance the translation of several internal ribosome entry sites and upstream ORF-containing mRNA targets, also potentially through RNA chaperone activity. Using in vitro FRET-based assays, we show that human and Schizosaccharomyces pombe La proteins harbor RNA chaperone activity by enhancing RNA strand annealing and strand dissociation. We use various RNA substrates and La mutants to show that UUU-3′OH-dependent La-RNA binding is not required for this function, and we map RNA chaperone activity to its RRM1 motif including a noncanonical α3-helix. We validate the importance of this α3-helix by appending it to the RRM of the unrelated U1A protein and show that this fusion protein acquires significant strand annealing activity. Finally, we show that residues required for La-mediated RNA chaperone activity in vitro are required for La-dependent rescue of tRNA-mediated suppression via a mutated suppressor tRNA in vivo. This work delineates the structural elements required for La-mediated RNA chaperone activity and provides a basis for understanding how La can enhance the folding of its various RNA targets. PMID:22203678
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Stress Chaperone GRP-78 Functions in Mineralized Matrix Formation*
Ravindran, Sriram; Gao, Qi; Ramachandran, Amsaveni; Blond, Sylvie; Predescu, Sanda A.; George, Anne
2011-01-01
Mineralized matrix formation is a well orchestrated event requiring several players. Glucose-regulated protein-78 (GRP-78) is an endoplasmic reticulum chaperone protein that has been implicated in functional roles ranging from involvement in cancer biology to serving as a receptor for viruses. In the present study we explored the role of GRP-78 in mineralized matrix formation. Differential expression of GRP-78 mRNA and protein was observed upon in vitro differentiation of primary mouse calvarial cells. An interesting observation was that GRP-78 was identified in the secretome of these cells and in the bone matrix, suggesting an extracellular function during matrix formation. In vitro nucleation experiments under physiological concentrations of calcium and phosphate ions indicated that GRP-78 can induce the formation of calcium phosphate polymorphs by itself, when bound to immobilized type I collagen and on demineralized collagen wafers. We provide evidence that GRP-78 can bind to DMP1 and type I collagen independent of each other in a simulated extracellular environment. Furthermore, we demonstrate the cell surface localization of GRP-78 and provide evidence that it functions as a receptor for DMP1 endocytosis in pre-osteoblasts and primary calvarial cells. Overall, this study represents a paradigm shift in the biological function of GRP-78. PMID:21239500
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Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.
Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju
2017-08-04
Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Modulation of intracellular protein degradation by SSB1-SIS1 chaperon system in yeast S. cerevisiae.
Ohba, M
1997-06-09
In prokaryotes, DnaK-DnaJ chaperon is involved in the protein degradation catalyzed by proteases La and ClpA/B complex as shown in E. coli. To extend this into eukaryotic cells, we examined the effects of hsp70 genes, SSA1 and SSB1, and DnaJ genes, SIS1 and YDJ1, on the growth of proteasome subunit mutants of the yeast S. cerevisiae. The results identified SSB1 and SIS1 as a pair of chaperon genes specifically involved in efficient protein turnover in the yeast, whose overexpression suppressed the growth defects caused by the proteasome mutations. Moreover, a single amino acid substitution in the putative peptide-binding site of SSB1 protein profoundly enhanced the suppression activity, indicating that the activity is mediated by the peptide-binding activity of this chaperon. Thus SSB1, with its partner DnaJ, SIS1, modulates the efficiency of protein turnover through its chaperon activity.
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Crucial HSP70 co–chaperone complex unlocks metazoan protein disaggregation
Nillegoda, Nadinath B.; Kirstein, Janine; Szlachcic, Anna; Berynskyy, Mykhaylo; Stank, Antonia; Stengel, Florian; Arnsburg, Kristin; Gao, Xuechao; Scior, Annika; Aebersold, Ruedi; Guilbride, D. Lys; Wade, Rebecca C.; Morimoto, Richard I.; Mayer, Matthias P.; Bukau, Bernd
2016-01-01
Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1,2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3,4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5,6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4,7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control. PMID:26245380
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Wang, Chao; Yu, Jiang; Huo, Lin; Wang, Lei; Feng, Wei; Wang, Chih-chen
2012-01-01
Protein-disulfide isomerase (PDI), with domains arranged as abb′xa′c, is a key enzyme and chaperone localized in the endoplasmic reticulum (ER) catalyzing oxidative folding and preventing misfolding/aggregation of proteins. It has been controversial whether the chaperone activity of PDI is redox-regulated, and the molecular basis is unclear. Here, we show that both the chaperone activity and the overall conformation of human PDI are redox-regulated. We further demonstrate that the conformational changes are triggered by the active site of domain a′, and the minimum redox-regulated cassette is located in b′xa′. The structure of the reduced bb′xa′ reveals for the first time that domain a′ packs tightly with both domain b′ and linker x to form one compact structural module. Oxidation of domain a′ releases the compact conformation and exposes the shielded hydrophobic areas to facilitate its high chaperone activity. Thus, the study unequivocally provides mechanistic insights into the redox-regulated chaperone activity of human PDI. PMID:22090031
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Collagen-Like Proteins in Pathogenic E. coli Strains
Ghosh, Neelanjana; McKillop, Thomas J.; Jowitt, Thomas A.; Howard, Marjorie; Davies, Heather; Holmes, David F.; Roberts, Ian S.; Bella, Jordi
2012-01-01
The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages. PMID:22701585
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Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi
2015-01-01
Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central "hubs". Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.
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Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB.
Malki, Abderrahim; Le, Hai-Tuong; Milles, Sigrid; Kern, Renée; Caldas, Teresa; Abdallah, Jad; Richarme, Gilbert
2008-05-16
The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100-5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1'-bis(4-anilino)naphtalene-5,5'-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.
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The G Protein α Chaperone Ric-8 as a Potential Therapeutic Target
Papasergi, Makaía M.; Patel, Bharti R.
2015-01-01
Resistance to inhibitors of cholinesterase (Ric-8)A and Ric-8B are essential genes that encode positive regulators of heterotrimeric G protein α subunits. Controversy persists surrounding the precise way(s) that Ric-8 proteins affect G protein biology and signaling. Ric-8 proteins chaperone nucleotide-free Gα-subunit states during biosynthetic protein folding prior to G protein heterotrimer assembly. In organisms spanning the evolutionary window of Ric-8 expression, experimental perturbation of Ric-8 genes results in reduced functional abundances of G proteins because G protein α subunits are misfolded and degraded rapidly. Ric-8 proteins also act as Gα-subunit guanine nucleotide exchange factors (GEFs) in vitro. However, Ric-8 GEF activity could strictly be an in vitro phenomenon stemming from the ability of Ric-8 to induce partial Gα unfolding, thereby enhancing GDP release. Ric-8 GEF activity clearly differs from the GEF activity of G protein–coupled receptors (GPCRs). G protein βγ is inhibitory to Ric-8 action but obligate for receptors. It remains an open question whether Ric-8 has dual functions in cells and regulates G proteins as both a molecular chaperone and GEF. Clearly, Ric-8 has a profound influence on heterotrimeric G protein function. For this reason, we propose that Ric-8 proteins are as yet untested therapeutic targets in which pharmacological inhibition of the Ric-8/Gα protein–protein interface could serve to attenuate the effects of disease-causing G proteins (constitutively active mutants) and/or GPCR signaling. This minireview will chronicle the understanding of Ric-8 function, provide a comparative discussion of the Ric-8 molecular chaperoning and GEF activities, and support the case for why Ric-8 proteins should be considered potential targets for development of new therapies. PMID:25319541
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N-glycan based ER molecular chaperone and protein quality control system: the calnexin binding cycle
Lamriben, Lydia; Graham, Jill B.; Adams, Benjamin M.; Hebert, Daniel N.
2015-01-01
Helenius and colleagues proposed over twenty-years ago a paradigm-shifting model for how chaperone binding in the endoplasmic reticulum was mediated and controlled for a new type of molecular chaperone- the carbohydrate binding chaperones, calnexin and calreticulin. While the originally established basics for this lectin chaperone binding cycle holds true today, there has been a number of important advances that have expanded our understanding of its mechanisms of action, role in protein homeostasis, and its connection to disease states that are highlighted in this review. PMID:26676362
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Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man
2015-11-01
Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.
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Visualization of a radical B 12 enzyme with its G-protein chaperone
Jost, Marco; Cracan, Valentin; Hubbard, Paul A.; ...
2015-02-09
G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. In this paper, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms ofmore » IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Finally and notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.« less
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Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi
2015-01-01
Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates. PMID:26394388
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Action of the Hsp70 chaperone system observed with single proteins
NASA Astrophysics Data System (ADS)
Nunes, João M.; Mayer-Hartl, Manajit; Hartl, F. Ulrich; Müller, Daniel J.
2015-02-01
In Escherichia coli, the binding of non-native protein substrates to the Hsp70 chaperone DnaK is mediated by the co-chaperone DnaJ. DnaJ accelerates ATP hydrolysis on DnaK, by closing the peptide-binding cleft of DnaK. GrpE catalysed nucleotide exchange and ATP re-binding then lead to substrate release from DnaK, allowing folding. Here we refold immunoglobulin 27 (I27) to better understand how DnaJ-DnaK-GrpE chaperones cooperate. When DnaJ is present, I27 is less likely to misfold and more likely to fold, whereas the unfolded state remains unaffected. Thus, the ‘holdase’ DnaJ shows foldase behaviour. Misfolding of I27 is fully abrogated when DnaJ cooperates with DnaK, which stabilizes the unfolded state and increases the probability of folding. Addition of GrpE shifts the unfolded fraction of I27 to pre-chaperone levels. These insights reveal synergistic mechanisms within the evolutionary highly conserved Hsp70 system that prevent substrates from misfolding and promote their productive transition to the native state.
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cpSRP43 Is a Novel Chaperone Specific for Light-harvesting Chlorophyll a,b-binding Proteins*
Falk, Sebastian; Sinning, Irmgard
2010-01-01
The biosynthesis of most membrane proteins is directly coupled to membrane insertion, and therefore, molecular chaperones are not required. The light-harvesting chlorophyll a,b-binding proteins (LHCPs) present a prominent exception as they are synthesized in the cytoplasm, and after import into the chloroplast, they are targeted and inserted into the thylakoid membrane. Upon arrival in the stroma, LHCPs form a soluble transit complex with the chloroplast signal recognition particle (cpSRP) consisting of an SRP54 homolog and the unique cpSRP43 composed of three chromodomains and four ankyrin repeats. Here we describe that cpSRP43 alone prevents aggregation of LHCP by formation of a complex with nanomolar affinity, whereas cpSRP54 is not required for this chaperone activity. Other stromal chaperones like trigger factor cannot replace cpSRP43, which implies that LHCPs require a specific chaperone. Although cpSRP43 does not have an ATPase activity, it can dissolve aggregates of LHCPs similar to chaperones of the Hsp104/ClpB family. We show that the LHCP-cpSRP43 interaction is predominantly hydrophobic but strictly depends on an intact DPLG motif between the second and third transmembrane region. The cpSRP43 ankyrin repeats that provide the binding site for the DPLG motif are sufficient for the chaperone function, whereas the chromodomains are dispensable. Taken together, we define cpSRP43 as a highly specific chaperone for LHCPs in addition to its established function as a targeting factor for this family of membrane proteins. PMID:20498370
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Mechanism of Nucleic Acid Chaperone Function of Retroviral Nuceleocapsid (NC) Proteins
NASA Astrophysics Data System (ADS)
Rouzina, Ioulia; Vo, My-Nuong; Stewart, Kristen; Musier-Forsyth, Karin; Cruceanu, Margareta; Williams, Mark
2006-03-01
Recent studies have highlighted two main activities of HIV-1 NC protein contributing to its function as a universal nucleic acid chaperone. Firstly, it is the ability of NC to weakly destabilize all nucleic acid,(NA), secondary structures, thus resolving the kinetic traps for NA refolding, while leaving the annealed state stable. Secondly, it is the ability of NC to aggregate NA, facilitating the nucleation step of bi-molecular annealing by increasing the local NA concentration. In this work we use single molecule DNA stretching and gel-based annealing assays to characterize these two chaperone activities of NC by using various HIV-1 NC mutants and several other retroviral NC proteins. Our results suggest that two NC functions are associated with its zinc fingers and cationic residues, respectively. NC proteins from other retroviruses have similar activities, although expressed to a different degree. Thus, NA aggregating ability improves, and NA duplex destabilizing activity decreases in the sequence: MLV NC, HIV NC, RSV NC. In contrast, HTLV NC protein works very differently from other NC proteins, and similarly to typical single stranded NA binding proteins. These features of retroviral NCs co-evolved with the structure of their genomes.
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Hartl, F. Ulrich
2017-01-01
Protein folding in the cell was originally assumed to be a spontaneous process, based on Anfinsen’s discovery that purified proteins can fold on their own after removal from denaturant. Consequently cell biologists showed little interest in the protein folding process. This changed only in the mid and late 1980s, when the chaperone story began to unfold. As a result, we now know that in vivo, protein folding requires assistance by a complex machinery of molecular chaperones. To ensure efficient folding, members of different chaperone classes receive the nascent protein chain emerging from the ribosome and guide it along an ordered pathway toward the native state. I was fortunate to contribute to these developments early on. In this short essay, I will describe some of the critical steps leading to the current concept of protein folding as a highly organized cellular process. PMID:29084909
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Comparison of intra-organellar chaperone capacity for dealing with stress-induced protein unfolding.
Hageman, Jurre; Vos, Michel J; van Waarde, Maria A W H; Kampinga, Harm H
2007-11-23
Molecular chaperones are essential for cells to prevent that partially unfolded proteins form non-functional, toxic aggregates. This requirement is increased when cells experience protein unfolding stresses and such could affect all compartments in the eukaryotic cell. Whether all organelles are equipped with comparable chaperone capacities is largely unknown, mainly due to the lack of suitable reporters that allow such a comparison. Here we describe the development of fluorescent luciferase reporters that are sorted to various cellular locations (nucleus, cytoplasm, endoplasmic reticulum, and peroxisomes) and that differ minimally in their intrinsic thermal stability properties. When heating living cells, the rate of inactivation was most rapid for the nuclear-targeted luciferase, indicating that the nucleus is the most sensitive organelle toward heat-induced denaturing stress. Post-heat re-activation, however, occurred at equal kinetics irrespective of luciferase localization. Also, induction of thermotolerance by a priming heat treatment, that coordinately up-regulates all heat-inducible chaperones, resulted in a transient heat resistance of the luciferase in all organelles in a comparable manner. Overexpression of the main heat-inducible Hsp70 family member, HspA1A, protected only the cytosolic and nuclear, but not the other luciferases. Together, our data suggest that in each compartment investigated, including the peroxisome in which so far no chaperones could be detected, chaperone machines are present and can be induced with activities similar to those present in the cytosolic/nuclear compartment.
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Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.
Schilke, Brenda A; Ciesielski, Szymon J; Ziegelhoffer, Thomas; Kamiya, Erina; Tonelli, Marco; Lee, Woonghee; Cornilescu, Gabriel; Hines, Justin K; Markley, John L; Craig, Elizabeth A
2017-10-01
By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.
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Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process*
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
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Verghese, Jacob; Abrams, Jennifer; Wang, Yanyu
2012-01-01
Summary: The eukaryotic heat shock response is an ancient and highly conserved transcriptional program that results in the immediate synthesis of a battery of cytoprotective genes in the presence of thermal and other environmental stresses. Many of these genes encode molecular chaperones, powerful protein remodelers with the capacity to shield, fold, or unfold substrates in a context-dependent manner. The budding yeast Saccharomyces cerevisiae continues to be an invaluable model for driving the discovery of regulatory features of this fundamental stress response. In addition, budding yeast has been an outstanding model system to elucidate the cell biology of protein chaperones and their organization into functional networks. In this review, we evaluate our understanding of the multifaceted response to heat shock. In addition, the chaperone complement of the cytosol is compared to those of mitochondria and the endoplasmic reticulum, organelles with their own unique protein homeostasis milieus. Finally, we examine recent advances in the understanding of the roles of protein chaperones and the heat shock response in pathogenic fungi, which is being accelerated by the wealth of information gained for budding yeast. PMID:22688810
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Molecular chaperones in Parkinson's disease--present and future.
Ebrahimi-Fakhari, Darius; Wahlster, Lara; McLean, Pamela J
2011-01-01
Parkinson's disease, like many other neurodegenerative disorders, is characterized by the progressive accumulation of pathogenic protein species and the formation of intracellular inclusion bodies. The cascade by which the small synaptic protein α-synuclein misfolds to form distinctive protein aggregates, termed Lewy bodies and Lewy neurites, has been the subject of intensive research for more than a decade. Genetic and pathological studies in Parkinson's disease patients as well as experimental studies in disease models have clearly established altered protein metabolism as a key element in the pathogenesis of Parkinson's disease. Alterations in protein metabolism include misfolding and aggregation, post-translational modification and dysfunctional degradation of cytotoxic protein species. Protein folding and re-folding are both mediated by a highly conserved network of molecules, called molecular chaperones and co-chaperones. In addition to the regulatory role in protein folding, molecular chaperone function is intimately associated with pathways of protein degradation, such as the ubiquitin-proteasome system and the autophagy-lysosomal pathway, to effectively remove irreversibly misfolded proteins. Because of the central role of molecular chaperones in maintaining protein homeostasis, we herein review our current knowledge on the involvement of molecular chaperones and co-chaperones in Parkinson's disease. We further discuss the capacity of molecular chaperones to prevent or modulate neurodegeneration, an important concept for future neuroprotective strategies and summarize the current progress in preclinical studies in models of Parkinson's disease and other neurodegenerative disorders. Finally we include a discussion on the future potential of using molecular chaperones as a disease modifying therapy.
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Pachulska-Wieczorek, Katarzyna; Stefaniak, Agnieszka K; Purzycka, Katarzyna J
2014-07-03
The nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein. We have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (-) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity. Our data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5'UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may
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Fujimori, Tsutomu; Suno, Ryoji; Iemura, Shun-Ichiro; Natsume, Tohru; Wada, Ikuo; Hosokawa, Nobuko
2017-08-01
The folding of newly synthesized proteins in the endoplasmic reticulum (ER) is assisted by ER-resident chaperone proteins. BiP (immunoglobulin heavy-chain-binding protein), a member of the HSP70 family, plays a central role in protein quality control. The chaperone function of BiP is regulated by its intrinsic ATPase activity, which is stimulated by ER-resident proteins of the HSP40/DnaJ family, including ERdj3. Here, we report that two closely related proteins, SDF2 and SDF2L1, regulate the BiP chaperone cycle. Both are ER-resident, but SDF2 is constitutively expressed, whereas SDF2L1 expression is induced by ER stress. Both luminal proteins formed a stable complex with ERdj3 and potently inhibited the aggregation of different types of misfolded ER cargo. These proteins associated with non-native proteins, thus promoting the BiP-substrate interaction cycle. A dominant-negative ERdj3 mutant that inhibits the interaction between ERdj3 and BiP prevented the dissociation of misfolded cargo from the ERdj3-SDF2L1 complex. Our findings indicate that SDF2 and SDF2L1 associate with ERdj3 and act as components in the BiP chaperone cycle to prevent the aggregation of misfolded proteins, partly explaining the broad folding capabilities of the ER under various physiological conditions. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.
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Colorful protein-based fluorescent probes for collagen imaging.
Aper, Stijn J A; van Spreeuwel, Ariane C C; van Turnhout, Mark C; van der Linden, Ardjan J; Pieters, Pascal A; van der Zon, Nick L L; de la Rambelje, Sander L; Bouten, Carlijn V C; Merkx, Maarten
2014-01-01
Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.
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Bacterial collagen-like proteins that form triple-helical structures
Yu, Zhuoxin; An, Bo; Ramshaw, John A.M.; Brodsky, Barbara
2014-01-01
A large number of collagen-like proteins have been identified in bacteria during the past ten years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in E. coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35–39 °C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions. PMID:24434612
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Mezzina, Mariela P; Wetzler, Diana E; de Almeida, Alejandra; Dinjaski, Nina; Prieto, M Auxiliadora; Pettinari, Maria Julia
2015-05-01
Phasins are proteins associated to intracellular polyhydroxyalkanoate granules that affect polymer accumulation and the number and size of the granules. Previous work demonstrated that a phasin from Azotobacter sp FA-8 (PhaPAz ) had an unexpected growth-promoting and stress-protecting effect in Escherichia coli, suggesting it could have chaperone-like activities. In this work, in vitro and in vivo experiments were performed in order to investigate this possibility. PhaPAz was shown to prevent in vitro thermal aggregation of the model protein citrate synthase and to facilitate the refolding process of this enzyme after chemical denaturation. Microscopy techniques were used to analyse the subcellular localization of PhaPAz in E. coli strains and to study the role of PhaPAz in in vivo protein folding and aggregation. PhaPAz was shown to colocalize with inclusion bodies of PD, a protein that aggregates when overexpressed. A reduction in the number of inclusion bodies of PD was observed when it was coexpressed with PhaPAz or with the known chaperone GroELS. These results demonstrate that PhaPAz has chaperone-like functions both in vitro and in vivo in E. coli recombinants, and suggests that phasins could have a general protective role in natural polyhydroxyalkanoate producers. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.
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Quantitative analysis of chaperone network throughput in budding yeast
Brownridge, Philip; Lawless, Craig; Payapilly, Aishwarya B; Lanthaler, Karin; Holman, Stephen W; Harman, Victoria M; Grant, Christopher M; Beynon, Robert J; Hubbard, Simon J
2013-01-01
The network of molecular chaperones mediates the folding and translocation of the many proteins encoded in the genome of eukaryotic organisms, as well as a response to stress. It has been particularly well characterised in the budding yeast, Saccharomyces cerevisiae, where 63 known chaperones have been annotated and recent affinity purification and MS/MS experiments have helped characterise the attendant network of chaperone targets to a high degree. In this study, we apply our QconCAT methodology to directly quantify the set of yeast chaperones in absolute terms (copies per cell) via SRM MS. Firstly, we compare these to existing quantitative estimates of these yeast proteins, highlighting differences between approaches. Secondly, we cast the results into the context of the chaperone target network and show a distinct relationship between abundance of individual chaperones and their targets. This allows us to characterise the ‘throughput’ of protein molecules passing through individual chaperones and their groups on a proteome-wide scale in an unstressed model eukaryote for the first time. The results demonstrate specialisations of the chaperone classes, which display different overall workloads, efficiencies and preference for the sub-cellular localisation of their targets. The novel integration of the interactome data with quantification supports re-estimates of the level of protein throughout going through molecular chaperones. Additionally, although chaperones target fewer than 40% of annotated proteins we show that they mediate the folding of the majority of protein molecules (∼62% of the total protein flux in the cell), highlighting their importance. PMID:23420633
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Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins?
Nakanishi, Kotaro
2016-09-01
RNA silencing is a eukaryote-specific phenomenon in which microRNAs and small interfering RNAs degrade messenger RNAs containing a complementary sequence. To this end, these small RNAs need to be loaded onto an Argonaute protein (AGO protein) to form the effector complex referred to as RNA-induced silencing complex (RISC). RISC assembly undergoes multiple and sequential steps with the aid of Hsc70/Hsp90 chaperone machinery. The molecular mechanisms for this assembly process remain unclear, despite their significance for the development of gene silencing techniques and RNA interference-based therapeutics. This review dissects the currently available structures of AGO proteins and proposes models and hypotheses for RISC assembly, covering the conformation of unloaded AGO proteins, the chaperone-assisted duplex loading, and the slicer-dependent and slicer-independent duplex separation. The differences in the properties of RISC between prokaryotes and eukaryotes will also be clarified. WIREs RNA 2016, 7:637-660. doi: 10.1002/wrna.1356 For further resources related to this article, please visit the WIREs website. © 2016 The Authors. WIREs RNA published by Wiley Periodicals, Inc.
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Chaperone-assisted protein aggregate reactivation: Different solutions for the same problem.
Aguado, Alejandra; Fernández-Higuero, José Angel; Moro, Fernando; Muga, Arturo
2015-08-15
The oligomeric AAA+ chaperones Hsp104 in yeast and ClpB in bacteria are responsible for the reactivation of aggregated proteins, an activity essential for cell survival during severe stress. The protein disaggregase activity of these members of the Hsp100 family is linked to the activity of chaperones from the Hsp70 and Hsp40 families. The precise mechanism by which these proteins untangle protein aggregates remains unclear. Strikingly, Hsp100 proteins are not present in metazoans. This does not mean that animal cells do not have a disaggregase activity, but that this activity is performed by the Hsp70 system and a representative of the Hsp110 family instead of a Hsp100 protein. This review describes the actual view of Hsp100-mediated aggregate reactivation, including the ATP-induced conformational changes associated with their disaggregase activity, the dynamics of the oligomeric assembly that is regulated by its ATPase cycle and the DnaK system, and the tight allosteric coupling between the ATPase domains within the hexameric ring complexes. The lack of homologs of these disaggregases in metazoans has suggested that they might be used as potential targets to develop antimicrobials. The current knowledge of the human disaggregase machinery and the role of Hsp110 are also discussed. Copyright © 2015 Elsevier Inc. All rights reserved.
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Chaperones in hepatitis C virus infection
Khachatoorian, Ronik; French, Samuel W
2016-01-01
The hepatitis C virus (HCV) infects approximately 3% of the world population or more than 185 million people worldwide. Each year, an estimated 350000-500000 deaths occur worldwide due to HCV-associated diseases including cirrhosis and hepatocellular carcinoma. HCV is the most common indication for liver transplantation in patients with cirrhosis worldwide. HCV is an enveloped RNA virus classified in the genus Hepacivirus in the Flaviviridae family. The HCV viral life cycle in a cell can be divided into six phases: (1) binding and internalization; (2) cytoplasmic release and uncoating; (3) viral polyprotein translation and processing; (4) RNA genome replication; (5) encapsidation (packaging) and assembly; and (6) virus morphogenesis (maturation) and secretion. Many host factors are involved in the HCV life cycle. Chaperones are an important group of host cytoprotective molecules that coordinate numerous cellular processes including protein folding, multimeric protein assembly, protein trafficking, and protein degradation. All phases of the viral life cycle require chaperone activity and the interaction of viral proteins with chaperones. This review will present our current knowledge and understanding of the role of chaperones in the HCV life cycle. Analysis of chaperones in HCV infection will provide further insights into viral/host interactions and potential therapeutic targets for both HCV and other viruses. PMID:26783419
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Structural basis for the antifolding activity of a molecular chaperone
NASA Astrophysics Data System (ADS)
Huang, Chengdong; Rossi, Paolo; Saio, Tomohide; Kalodimos, Charalampos G.
2016-09-01
Molecular chaperones act on non-native proteins in the cell to prevent their aggregation, premature folding or misfolding. Different chaperones often exert distinct effects, such as acceleration or delay of folding, on client proteins via mechanisms that are poorly understood. Here we report the solution structure of SecB, a chaperone that exhibits strong antifolding activity, in complex with alkaline phosphatase and maltose-binding protein captured in their unfolded states. SecB uses long hydrophobic grooves that run around its disk-like shape to recognize and bind to multiple hydrophobic segments across the length of non-native proteins. The multivalent binding mode results in proteins wrapping around SecB. This unique complex architecture alters the kinetics of protein binding to SecB and confers strong antifolding activity on the chaperone. The data show how the different architectures of chaperones result in distinct binding modes with non-native proteins that ultimately define the activity of the chaperone.
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Stefanovic, Branko
2013-01-01
Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments. Copyright © 2013 John Wiley & Sons, Ltd.
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Unfolding the chaperone story.
Hartl, F Ulrich
2017-11-01
Protein folding in the cell was originally assumed to be a spontaneous process, based on Anfinsen's discovery that purified proteins can fold on their own after removal from denaturant. Consequently cell biologists showed little interest in the protein folding process. This changed only in the mid and late 1980s, when the chaperone story began to unfold. As a result, we now know that in vivo, protein folding requires assistance by a complex machinery of molecular chaperones. To ensure efficient folding, members of different chaperone classes receive the nascent protein chain emerging from the ribosome and guide it along an ordered pathway toward the native state. I was fortunate to contribute to these developments early on. In this short essay, I will describe some of the critical steps leading to the current concept of protein folding as a highly organized cellular process. © 2017 Hartl. 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).
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Yuan, Bangqing; Xian, Ronghua; Wu, Xianqu; Jing, Junjie; Chen, Kangning; Liu, Guojun; Zhou, Zhenhua
2012-07-01
Previous evidence suggested that the stress protein grp170 can function as a highly efficient molecular chaperone, binding to large protein substrates and acting as a potent vaccine against specific tumors when purified from the same tumor. In addition, Pokemon can be found in almost all malignant tumor cells and is regarded to be a promising candidate for the treatment of tumors. However, the potential of the grp170-Pokemon chaperone complex has not been well described. In the present study, the natural chaperone complex between grp170 and the Pokemon was formed by heat shock, and its immunogenicity was detected by ELISPOT and (51)Cr-release assays in vitro and by tumor bearing models in vivo. Our results demonstrated that the grp170-Pokemon chaperone complex could elicit T cell responses as determined by ELISPOT and (51)Cr-release assays. In addition, immunized C57BL/6 mice were challenged with subcutaneous (s.c.) injection of Lewis cancer cells to induce primary tumors. Treatment of mice with the grp170-Pokemon chaperone complex also significantly inhibited tumor growth and prolonged the life span of tumor-bearing mice. Our results indicated that the grp170-Pokemon chaperone complex might represent a powerful approach to tumor immunotherapy and have significant potential for clinical application. Copyright © 2012 Elsevier GmbH. All rights reserved.
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Nakamoto, Hitoshi; Fujita, Kensaku; Ohtaki, Aguru; Watanabe, Satoru; Narumi, Shoichi; Maruyama, Takahiro; Suenaga, Emi; Misono, Tomoko S; Kumar, Penmetcha K R; Goloubinoff, Pierre; Yoshikawa, Hirofumi
2014-02-28
In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.
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Ahmed, Ali Abdurehim; Pedersen, Carsten; Schultz-Larsen, Torsten; Kwaaitaal, Mark; Jørgensen, Hans Jørgen Lyngs; Thordal-Christensen, Hans
2015-01-01
Pathogens secrete effector proteins to establish a successful interaction with their host. Here, we describe two barley (Hordeum vulgare) powdery mildew candidate secreted effector proteins, CSEP0105 and CSEP0162, which contribute to pathogen success and appear to be required during or after haustorial formation. Silencing of either CSEP using host-induced gene silencing significantly reduced the fungal haustorial formation rate. Interestingly, both CSEPs interact with the barley small heat shock proteins, Hsp16.9 and Hsp17.5, in a yeast two-hybrid assay. Small heat shock proteins are known to stabilize several intracellular proteins, including defense-related signaling components, through their chaperone activity. CSEP0105 and CSEP0162 localized to the cytosol and the nucleus of barley epidermal cells, whereas Hsp16.9 and Hsp17.5 are cytosolic. Intriguingly, only those specific CSEPs changed localization and became restricted to the cytosol when coexpressed with Hsp16.9 and Hsp17.5, confirming the CSEP-small heat shock protein interaction. As predicted, Hsp16.9 showed chaperone activity, as it could prevent the aggregation of Escherichia coli proteins during thermal stress. Remarkably, CSEP0105 compromised this activity. These data suggest that CSEP0105 promotes virulence by interfering with the chaperone activity of a barley small heat shock protein essential for defense and stress responses. PMID:25770154
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Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin.
Vainberg, I E; Lewis, S A; Rommelaere, H; Ampe, C; Vandekerckhove, J; Klein, H L; Cowan, N J
1998-05-29
We describe the discovery of a heterohexameric chaperone protein, prefoldin, based on its ability to capture unfolded actin. Prefoldin binds specifically to cytosolic chaperonin (c-cpn) and transfers target proteins to it. Deletion of the gene encoding a prefoldin subunit in S. cerevisiae results in a phenotype similar to those found when c-cpn is mutated, namely impaired functions of the actin and tubulin-based cytoskeleton. Consistent with prefoldin having a general role in chaperonin-mediated folding, we identify homologs in archaea, which have a class II chaperonin but contain neither actin nor tubulin. We show that by directing target proteins to chaperonin, prefoldin promotes folding in an environment in which there are many competing pathways for nonnative proteins.
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Conservation of RNA chaperone activity of the human La-related proteins 4, 6 and 7.
Hussain, Rawaa H; Zawawi, Mariam; Bayfield, Mark A
2013-10-01
The La module is a conserved tandem arrangement of a La motif and RNA recognition motif whose function has been best characterized in genuine La proteins. The best-characterized substrates of La proteins are pre-tRNAs, and previous work using tRNA mediated suppression in Schizosaccharomyces pombe has demonstrated that yeast and human La enhance the maturation of these using two distinguishable activities: UUU-3'OH-dependent trailer binding/protection and a UUU-3'OH independent activity related to RNA chaperone function. The La module has also been identified in several conserved families of La-related proteins (LARPs) that engage other RNAs, but their mode of RNA binding and function(s) are not well understood. We demonstrate that the La modules of the human LARPs 4, 6 and 7 are also active in tRNA-mediated suppression, even in the absence of stable UUU-3'OH trailer protection. Rather, the capacity of these to enhance pre-tRNA maturation is associated with RNA chaperone function, which we demonstrate to be a conserved activity for each hLARP in vitro. Our work reveals insight into the mechanisms by which La module containing proteins discriminate RNA targets and demonstrates that RNA chaperone activity is a conserved function across representative members of the La motif-containing superfamily.
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A molecular mechanism of chaperone-client recognition
He, Lichun; Sharpe, Timothy; Mazur, Adam; Hiller, Sebastian
2016-01-01
Molecular chaperones are essential in aiding client proteins to fold into their native structure and in maintaining cellular protein homeostasis. However, mechanistic aspects of chaperone function are still not well understood at the atomic level. We use nuclear magnetic resonance spectroscopy to elucidate the mechanism underlying client recognition by the adenosine triphosphate-independent chaperone Spy at the atomic level and derive a structural model for the chaperone-client complex. Spy interacts with its partially folded client Im7 by selective recognition of flexible, locally frustrated regions in a dynamic fashion. The interaction with Spy destabilizes a partially folded client but spatially compacts an unfolded client conformational ensemble. By increasing client backbone dynamics, the chaperone facilitates the search for the native structure. A comparison of the interaction of Im7 with two other chaperones suggests that the underlying principle of recognizing frustrated segments is of a fundamental nature. PMID:28138538
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Redox Aspects of Chaperones in Cardiac Function
Penna, Claudia; Sorge, Matteo; Femminò, Saveria; Pagliaro, Pasquale; Brancaccio, Mara
2018-01-01
Molecular chaperones are stress proteins that allow the correct folding or unfolding as well as the assembly or disassembly of macromolecular cellular components. Changes in expression and post-translational modifications of chaperones have been linked to a number of age- and stress-related diseases including cancer, neurodegeneration, and cardiovascular diseases. Redox sensible post-translational modifications, such as S-nitrosylation, glutathionylation and phosphorylation of chaperone proteins have been reported. Redox-dependent regulation of chaperones is likely to be a phenomenon involved in metabolic processes and may represent an adaptive response to several stress conditions, especially within mitochondria, where it impacts cellular bioenergetics. These post-translational modifications might underlie the mechanisms leading to cardioprotection by conditioning maneuvers as well as to ischemia/reperfusion injury. In this review, we discuss this topic and focus on two important aspects of redox-regulated chaperones, namely redox regulation of mitochondrial chaperone function and cardiac protection against ischemia/reperfusion injury. PMID:29615920
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Cho, Younhee; Zhang, Xin; Pobre, Kristine Faye R; Liu, Yu; Powers, David L; Kelly, Jeffery W; Gierasch, Lila M; Powers, Evan T
2015-04-14
The folding fate of a protein in vivo is determined by the interplay between a protein's folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding versus aggregation versus degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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Conservation of RNA chaperone activity of the human La-related proteins 4, 6 and 7
Hussain, Rawaa H.; Zawawi, Mariam; Bayfield, Mark A.
2013-01-01
The La module is a conserved tandem arrangement of a La motif and RNA recognition motif whose function has been best characterized in genuine La proteins. The best-characterized substrates of La proteins are pre-tRNAs, and previous work using tRNA mediated suppression in Schizosaccharomyces pombe has demonstrated that yeast and human La enhance the maturation of these using two distinguishable activities: UUU-3′OH-dependent trailer binding/protection and a UUU-3′OH independent activity related to RNA chaperone function. The La module has also been identified in several conserved families of La-related proteins (LARPs) that engage other RNAs, but their mode of RNA binding and function(s) are not well understood. We demonstrate that the La modules of the human LARPs 4, 6 and 7 are also active in tRNA-mediated suppression, even in the absence of stable UUU-3′OH trailer protection. Rather, the capacity of these to enhance pre-tRNA maturation is associated with RNA chaperone function, which we demonstrate to be a conserved activity for each hLARP in vitro. Our work reveals insight into the mechanisms by which La module containing proteins discriminate RNA targets and demonstrates that RNA chaperone activity is a conserved function across representative members of the La motif-containing superfamily. PMID:23887937
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Molecular chaperones: functional mechanisms and nanotechnological applications
NASA Astrophysics Data System (ADS)
Rosario Fernández-Fernández, M.; Sot, Begoña; María Valpuesta, José
2016-08-01
Molecular chaperones are a group of proteins that assist in protein homeostasis. They not only prevent protein misfolding and aggregation, but also target misfolded proteins for degradation. Despite differences in structure, all types of chaperones share a common general feature, a surface that recognizes and interacts with the misfolded protein. This and other, more specialized properties can be adapted for various nanotechnological purposes, by modification of the original biomolecules or by de novo design based on artificial structures.
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Marini, Joan C; Reich, Adi; Smith, Simone M
2014-08-01
Osteogenesis imperfecta or 'brittle bone disease' has mainly been considered a bone disorder caused by collagen mutations. Within the last decade, however, a surge of genetic discoveries has created a new paradigm for osteogenesis imperfecta as a collagen-related disorder, where most cases are due to autosomal dominant type I collagen defects, while rare, mostly recessive, forms are due to defects in genes whose protein products interact with collagen protein. This review is both timely and relevant in outlining the genesis, development, and future of this paradigm shift in the understanding of osteogenesis imperfecta. Bone-restricted interferon-induced transmembrane (IFITM)-like protein (BRIL) and pigment epithelium-derived factor (PEDF) defects cause types V and VI osteogenesis imperfecta via defective bone mineralization, while defects in cartilage-associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1), and cyclophilin B (CYPB) cause types VII-IX osteogenesis imperfecta via defective collagen post-translational modification. Heat shock protein 47 (HSP47) and FK506-binding protein-65 (FKBP65) defects cause types X and XI osteogenesis imperfecta via aberrant collagen crosslinking, folding, and chaperoning, while defects in SP7 transcription factor, wingless-type MMTV integration site family member 1 (WNT1), trimeric intracellular cation channel type b (TRIC-B), and old astrocyte specifically induced substance (OASIS) disrupt osteoblast development. Finally, absence of the type I collagen C-propeptidase bone morphogenetic protein 1 (BMP1) causes type XII osteogenesis imperfecta due to altered collagen maturation/processing. Identification of these multiple causative defects has provided crucial information for accurate genetic counseling, inspired a recently proposed functional grouping of osteogenesis imperfecta types by shared mechanism to simplify current nosology, and has prodded investigations into common pathways in osteogenesis imperfecta. Such
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Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine
2016-01-01
Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725
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Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; ...
2015-02-13
Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activitymore » with Hsp70ΔEEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interaction(s) between the J-domain and glycine-rich region controls co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. Yet, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD-binding adaptor proteins. Finally, these interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy
2015-04-01
Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at heir C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways, Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activitymore » with Hsp70ΔEEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interactions between the J-domain and glycine-rich region control co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. However, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.« less
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Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; Ciesielski, Szymon J.; Baranowski, Maciej; Zhou, Min; Joachimiak, Andrzej; Craig, Elizabeth A.
2015-01-01
Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70ΔEEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interaction(s) between the J-domain and glycine-rich region controls co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. Yet, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD-binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively. PMID:25687964
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Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis.
Finka, Andrija; Goloubinoff, Pierre
2013-09-01
In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here "the chaperome," which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10% of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.
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Collagen-binding proteins of Streptococcus mutans and related streptococci.
Avilés-Reyes, A; Miller, J H; Lemos, J A; Abranches, J
2017-04-01
The ability of Streptococcus mutans to interact with collagen through the expression of collagen-binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose-dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra-oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms used by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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Liang, Jingjing; Sagum, Cari A.; Bedford, Mark T.; Sudol, Marius; Han, Ziying
2017-01-01
Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles. PMID:28076420
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Liang, Jingjing; Sagum, Cari A; Bedford, Mark T; Sidhu, Sachdev S; Sudol, Marius; Han, Ziying; Harty, Ronald N
2017-01-01
Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles.
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Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc
2004-01-01
The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3′ untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication. PMID:15141033
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Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc
2004-01-01
The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication.
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DOE R&D Accomplishments Database
Trent, J. D.
1996-02-09
Acquired thermotolerance, the associated synthesis of heat-shock proteins (HSPs) under stress conditions, and the role of HSPs as molecular chaperones under normal growth conditions have been studied extensively in eukaryotes and bacteria, whereas research in these areas in archaea is only beginning. All organisms have evolved a variety of strategies for coping with high-temperature stress, and among these strategies is the increased synthesis of HSPs. The facts that both high temperatures and chemical stresses induce the HSPs and that some of the HSPs recognize and bind to unfolded proteins in vitro have led to the theory that the function of HSPs is to prevent protein aggregation in vivo. The facts that some HSPs are abundant under normal growth conditions and that they assist in protein folding in vitro have led to the theory that they assist protein folding in vivo; in this role, they are referred to as molecular chaperones. The limited research on acquired thermotolerance, HSPs, and molecular chaperones in archaea, particularly the hyperthermophilic archaea, suggests that these extremophiles provide a new perspective in these areas of research, both because they are members of a separate phylogenetic domain and because they have evolved to live under extreme conditions.
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Zhang, Huaqun; Amick, Joseph; Chakravarti, Ritu; ...
2015-02-12
The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 Å resolution structure of the tetratricopeptide repeat (TPR) domain of CHIP in complex with the α-helical lid subdomain and unstructured tail of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting an atypical mode of interaction between chaperones and TPR domains. Here, we demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required formore » proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Posttranslational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a bipartite mode of interaction between TPR domains and their binding partners.« less
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Mitochondrial chaperones may be targets for anti-cancer drugs
Scientists at NCI have found that a mitochondrial chaperone protein, TRAP1, may act indirectly as a tumor suppressor as well as a novel target for developing anti-cancer drugs. Chaperone proteins, such as TRAP1, help other proteins adapt to stress, but sc
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Howard, Jeffrey C.; Heinemann, Christine; Thatcher, Bradley J.; Martin, Brian; Gan, Bing Siang; Reid, Gregor
2000-01-01
Biosurfactants produced by Lactobacillus fermentum RC-14, L. rhamnosus GR-1 and 36, and L. casei Shirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)–time of flight mass spectrometry. Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins. Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form). Although biosurfactants isolated from L. casei Shirota and L. rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein. Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions. PMID:11010889
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Preventing α-synuclein aggregation: the role of the small heat-shock molecular chaperone proteins.
Cox, Dezerae; Carver, John A; Ecroyd, Heath
2014-09-01
Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.
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Mouradian, Michael; Ma, Irvin V; Vicente, Erika D; Kikawa, Keith D; Pardini, Ronald S
2017-01-01
The molecular chaperone, heat shock protein 90 (Hsp90), is a critical regulator for the proper folding and stabilization of several client proteins, and is a major contributor to carcinogenesis. Specific Hsp90 inhibitors have been designed to target the ATP-binding site in order to prevent Hsp90 chaperone maturation. The current study investigated the effects of docosahexaenoic acid (DHA; C22:6 n-3) on Hsp90 function and downstream client protein expression. In vitro analyses of BT-474 human breast carcinoma and A549 human lung adenocarcinoma cell lines revealed dose-dependent decreases in intracellular ATP levels by DHA treatment, resulting in a significant reduction of Hsp90 and p23 association in both cell lines. Attenuation of the Hsp90-p23 complex led to the inhibition of Hsp90 client proteins, epidermal growth factor receptor 2 (ErbB2), and hypoxia-inducible factor 1α (HIF-1α). Similar results were observed when employing 2-deoxyglucose (2-DG), confirming that DHA and 2-DG, both independently and combined, can disturb Hsp90 molecular chaperone function. In vivo A549 xenograft analysis also demonstrated decreased expression levels of Hsp90-p23 association and diminished protein levels of ErbB2 and HIF-1α in mice supplemented with dietary DHA. These data support a role for dietary intervention to improve cancer therapy in tumors overexpressing Hsp90 and its client proteins.
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Hsp100/ClpB Chaperone Function and Mechanism
DOE Office of Scientific and Technical Information (OSTI.GOV)
Vierling, Elizabeth
2015-01-27
The supported research investigated the mechanism of action of a unique class of molecular chaperones in higher plants, the Hsp100/ClpB proteins, with the ultimate goal of defining how these chaperones influence plant growth, development, stress tolerance and productivity. Molecular chaperones are essential effectors of cellular “protein quality control”, which comprises processes that ensure the proper folding, localization, activation and turnover of proteins. Hsp100/ClpB proteins are required for temperature acclimation in plants, optimal seed yield, and proper chloroplast development. The model plant Arabidopsis thaliana and genetic and molecular approaches were used to investigate two of the three members of the Hsp100/ClpBmore » proteins in plants, cytosolic AtHsp101 and chloroplast-localized AtClpB-p. Investigating the chaperone activity of the Hsp100/ClpB proteins addresses DOE goals in that this activity impacts how “plants generate and assemble components” as well as “allowing for their self repair”. Additionally, Hsp100/ClpB protein function in plants is directly required for optimal “utilization of biological energy” and is involved in “mechanisms that control the architecture of energy transduction systems”.« less
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Super Spy variants implicate flexibility in chaperone action.
Quan, Shu; Wang, Lili; Petrotchenko, Evgeniy V; Makepeace, Karl At; Horowitz, Scott; Yang, Jianyi; Zhang, Yang; Borchers, Christoph H; Bardwell, James Ca
2014-01-01
Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These "Super Spy" variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001.
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Super Spy variants implicate flexibility in chaperone action
Quan, Shu; Wang, Lili; Petrotchenko, Evgeniy V; Makepeace, Karl AT; Horowitz, Scott; Yang, Jianyi; Zhang, Yang; Borchers, Christoph H; Bardwell, James CA
2014-01-01
Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001 PMID:24497545
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Chaperone-client complexes: A dynamic liaison
NASA Astrophysics Data System (ADS)
Hiller, Sebastian; Burmann, Björn M.
2018-04-01
Living cells contain molecular chaperones that are organized in intricate networks to surveil protein homeostasis by avoiding polypeptide misfolding, aggregation, and the generation of toxic species. In addition, cellular chaperones also fulfill a multitude of alternative functionalities: transport of clients towards a target location, help them fold, unfold misfolded species, resolve aggregates, or deliver clients towards proteolysis machineries. Until recently, the only available source of atomic resolution information for virtually all chaperones were crystal structures of their client-free, apo-forms. These structures were unable to explain details of the functional mechanisms underlying chaperone-client interactions. The difficulties to crystallize chaperones in complexes with clients arise from their highly dynamic nature, making solution NMR spectroscopy the method of choice for their study. With the advent of advanced solution NMR techniques, in the past few years a substantial number of structural and functional studies on chaperone-client complexes have been resolved, allowing unique insight into the chaperone-client interaction. This review summarizes the recent insights provided by advanced high-resolution NMR-spectroscopy to understand chaperone-client interaction mechanisms at the atomic scale.
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The Collagen Binding Proteins of Streptococcus mutans and Related Streptococci
Avilés-Reyes, Alejandro; Miller, James H.; Lemos, José A.; Abranches, Jacqueline
2016-01-01
Summary The ability of Streptococcus mutans to interact with collagen through the expression of collagen-binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose-dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra-oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms utilized by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host. PMID:26991416
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Histone chaperones: an escort network regulating histone traffic.
De Koning, Leanne; Corpet, Armelle; Haber, James E; Almouzni, Geneviève
2007-11-01
In eukaryotes, DNA is organized into chromatin in a dynamic manner that enables it to be accessed for processes such as transcription and repair. Histones, the chief protein component of chromatin, must be assembled, replaced or exchanged to preserve or change this organization according to cellular needs. Histone chaperones are key actors during histone metabolism. Here we classify known histone chaperones and discuss how they build a network to escort histone proteins. Molecular interactions with histones and their potential specificity or redundancy are also discussed in light of chaperone structural properties. The multiplicity of histone chaperone partners, including histone modifiers, nucleosome remodelers and cell-cycle regulators, is relevant to their coordination with key cellular processes. Given the current interest in chromatin as a source of epigenetic marks, we address the potential contributions of histone chaperones to epigenetic memory and genome stability.
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Zmijewski, Michał A; Skórko-Glonek, Joanna; Tanfani, Fabio; Banecki, Bogdan; Kotlarz, Agnieszka; Macario, Alberto J L; Lipińska, Barbara
2007-01-01
Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.
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Cloning, expression and crystallisation of SGT1 co-chaperone protein from Glaciozyma antarctica
NASA Astrophysics Data System (ADS)
Yusof, Nur Athirah; Bakar, Farah Diba Abu; Beddoe, Travis; Murad, Abdul Munir Abdul
2013-11-01
Studies on psycrophiles are now in the limelight of today's post genomic era as they fascinate the research and development industries. The discovery from Glaciozyma antarctica, an extreme cold adapted yeast from Antarctica shows promising future to provide cost effective natural sustainable energy and create wider understanding of the property that permits this organisms to adapt to extreme temperature downshift. In plants and yeast, studies show the interaction between SGT1 and HSP90 are essential for disease resistance and heat stress by activating a number of resistance proteins. Here we report for the first time cloning, expression and crystallization of the recombinant SGT1 protein of G. antarctica (rGa_SGT1), a highly conserved eukaryotic protein that interacts with the molecular chaperones HSP90 (heat shock protein 90) apparently associated in a role of co-chaperone that may play important role in cold adaptation. The sequence analysis of rGa_SGT1 revealed the presence of all the characteristic features of SGT1 protein. In this study, we present the outlines and results of protein structural study of G. antarctica SGT1 protein. We validate this approach by starting with cloning the target insert into Ligation Independent Cloning system proceeded with expression using E. coli system, and crystallisation of the target rGA_SGT1 protein. The work is still on going with the target subunit of the complex proteins yielded crystals. These results, still ongoing, open a platform for better understanding of the uniqueness of this crucial molecular machine function in cold adaptation.
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The Structure and Function of Non-Collagenous Bone Proteins
NASA Technical Reports Server (NTRS)
Hook, Magnus; McQuillan, David J.
1997-01-01
The research done under the cooperative research agreement for the project titled 'The structure and function of non-collagenous bone proteins' represented the first phase of an ongoing program to define the structural and functional relationships of the principal noncollagenous proteins in bone. An ultimate goal of this research is to enable design and execution of useful pharmacological compounds that will have a beneficial effect in treatment of osteoporosis, both land-based and induced by long-duration space travel. The goals of the now complete first phase were as follows: 1. Establish and/or develop powerful recombinant protein expression systems; 2. Develop and refine isolation and purification of recombinant proteins; 3. Express wild-type non-collagenous bone proteins; 4. Express site-specific mutant proteins and domains of wild-type proteins to enhance likelihood of crystal formation for subsequent solution of structure.
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Han, Jaeseok; Song, Benbo; Kim, Jiun; Kodali, Vamsi K.; Pottekat, Anita; Wang, Miao; Hassler, Justin; Wang, Shiyu; Pennathur, Subramaniam; Back, Sung Hoon; Katze, Michael G.
2015-01-01
Proinsulin misfolding in the endoplasmic reticulum (ER) initiates a cell death response, although the mechanism(s) remains unknown. To provide insight into how protein misfolding may cause β-cell failure, we analyzed mice with the deletion of P58IPK/DnajC3, an ER luminal co-chaperone. P58IPK−/− mice become diabetic as a result of decreased β-cell function and mass accompanied by induction of oxidative stress and cell death. Treatment with a chemical chaperone, as well as deletion of Chop, improved β-cell function and ameliorated the diabetic phenotype in P58IPK−/− mice, suggesting P58IPK deletion causes β-cell death through ER stress. Significantly, a diet of chow supplemented with antioxidant dramatically and rapidly restored β-cell function in P58IPK−/− mice and corrected abnormal localization of MafA, a critical transcription factor for β-cell function. Antioxidant feeding also preserved β-cell function in Akita mice that express mutant misfolded proinsulin. Therefore defective protein folding in the β-cell causes oxidative stress as an essential proximal signal required for apoptosis in response to ER stress. Remarkably, these findings demonstrate that antioxidant feeding restores cell function upon deletion of an ER molecular chaperone. Therefore antioxidant or chemical chaperone treatment may be a promising therapeutic approach for type 2 diabetes. PMID:25795214
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Information encoded in non-native states drives substrate-chaperone pairing.
Mapa, Koyeli; Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik
2012-09-05
Many proteins refold in vitro through kinetic folding intermediates that are believed to be by-products of native-state centric evolution. These intermediates are postulated to play only minor roles, if any, in vivo because they lack any information related to translation-associated vectorial folding. We demonstrate that refolding intermediate of a test protein, generated in vitro, is able to find its cognate chaperone, from the whole complement of Escherichia coli soluble chaperones. Cognate chaperone-binding uniquely alters the conformation of non-native substrate. Importantly, precise chaperone targeting of substrates are maintained as long as physiological molar ratios of chaperones remain unaltered. Using a library of different chaperone substrates, we demonstrate that kinetically trapped refolding intermediates contain sufficient structural features for precise targeting to cognate chaperones. We posit that evolution favors sequences that, in addition to coding for a functional native state, encode folding intermediates with higher affinity for cognate chaperones than noncognate ones. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Three reasons protein disorder analysis makes more sense in the light of collagen
Oates, Matt E.; Tompa, Peter; Gough, Julian
2016-01-01
Abstract We have identified that the collagen helix has the potential to be disruptive to analyses of intrinsically disordered proteins. The collagen helix is an extended fibrous structure that is both promiscuous and repetitive. Whilst its sequence is predicted to be disordered, this type of protein structure is not typically considered as intrinsic disorder. Here, we show that collagen‐encoding proteins skew the distribution of exon lengths in genes. We find that previous results, demonstrating that exons encoding disordered regions are more likely to be symmetric, are due to the abundance of the collagen helix. Other related results, showing increased levels of alternative splicing in disorder‐encoding exons, still hold after considering collagen‐containing proteins. Aside from analyses of exons, we find that the set of proteins that contain collagen significantly alters the amino acid composition of regions predicted as disordered. We conclude that research in this area should be conducted in the light of the collagen helix. PMID:26941008
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Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; Ciesielski, Szymon J; Baranowski, Maciej; Zhou, Min; Joachimiak, Andrzej; Craig, Elizabeth A
2015-04-10
Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways, Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70∆EEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interactions between the J-domain and glycine-rich region control co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. However, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Powis, Katie; Schrul, Bianca; Tienson, Heather; Gostimskaya, Irina; Breker, Michal; High, Stephen; Schuldiner, Maya; Jakob, Ursula; Schwappach, Blanche
2013-01-01
Summary The endomembrane system of yeast contains different tail-anchored proteins that are post-translationally targeted to membranes via their C-terminal transmembrane domain. This hydrophobic segment could be hazardous in the cytosol if membrane insertion fails, resulting in the need for energy-dependent chaperoning and the degradation of aggregated tail-anchored proteins. A cascade of GET proteins cooperates in a conserved pathway to accept newly synthesized tail-anchored proteins from ribosomes and guide them to a receptor at the endoplasmic reticulum, where membrane integration takes place. It is, however, unclear how the GET system reacts to conditions of energy depletion that might prevent membrane insertion and hence lead to the accumulation of hydrophobic proteins in the cytosol. Here we show that the ATPase Get3, which accommodates the hydrophobic tail anchor of clients, has a dual function: promoting tail-anchored protein insertion when glucose is abundant and serving as an ATP-independent holdase chaperone during energy depletion. Like the generic chaperones Hsp42, Ssa2, Sis1 and Hsp104, we found that Get3 moves reversibly to deposition sites for protein aggregates, hence supporting the sequestration of tail-anchored proteins under conditions that prevent tail-anchored protein insertion. Our findings support a ubiquitous role for the cytosolic GET complex as a triaging platform involved in cellular proteostasis. PMID:23203805
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Meimaridou, Eirini; Gooljar, Sakina B; Chapple, J Paul
2009-01-01
Molecular chaperones are best recognized for their roles in de novo protein folding and the cellular response to stress. However, many molecular chaperones, and in particular the Hsp70 chaperone machinery, have multiple diverse cellular functions. At the molecular level, chaperones are mediators of protein conformational change. To facilitate conformational change of client/substrate proteins, in manifold contexts, chaperone power must be closely regulated and harnessed to specific cellular locales--this is controlled by cochaperones. This review considers specialized functions of the Hsp70 chaperone machinery mediated by its cochaperones. We focus on vesicular trafficking, protein degradation and a potential role in G protein-coupled receptor processing.
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Chaperone-like properties of tobacco plastid thioredoxins f and m
Sanz-Barrio, Ruth; Fernández-San Millán, Alicia; Carballeda, Jon; Corral-Martínez, Patricia; Seguí-Simarro, José M.; Farran, Inmaculada
2012-01-01
Thioredoxins (Trxs) are ubiquitous disulphide reductases that play important roles in the redox regulation of many cellular processes. However, some redox-independent functions, such as chaperone activity, have also been attributed to Trxs in recent years. The focus of our study is on the putative chaperone function of the well-described plastid Trxs f and m. To that end, the cDNA of both Trxs, designated as NtTrxf and NtTrxm, was isolated from Nicotiana tabacum plants. It was found that bacterially expressed tobacco Trx f and Trx m, in addition to their disulphide reductase activity, possessed chaperone-like properties. In vitro, Trx f and Trx m could both facilitate the reactivation of the cysteine-free form of chemically denatured glucose-6 phosphate dehydrogenase (foldase chaperone activity) and prevent heat-induced malate dehydrogenase aggregation (holdase chaperone activity). Our results led us to infer that the disulphide reductase and foldase chaperone functions prevail when the proteins occur as monomers and the well-conserved non-active cysteine present in Trx f is critical for both functions. By contrast, the holdase chaperone activity of both Trxs depended on their oligomeric status: the proteins were functional only when they were associated with high molecular mass protein complexes. Because the oligomeric status of both Trxs was induced by salt and temperature, our data suggest that plastid Trxs could operate as molecular holdase chaperones upon oxidative stress, acting as a type of small stress protein. PMID:21948853
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Zhadobov, M; Sauleau, R; Le Coq, L; Debure, L; Thouroude, D; Michel, D; Le Dréan, Y
2007-04-01
This article reports experimental results on the influence of low-power millimeter wave (MMW) radiation at 60 GHz on a set of stress-sensitive gene expression of molecular chaperones, namely clusterin (CLU) and HSP70, in a human brain cell line. Selection of the exposure frequency is determined by its near-future applications for the new broadband civil wireless communication systems including wireless local area networks (WLAN) for domestic and professional uses. Frequencies around 60 GHz are strongly attenuated in the earth's atmosphere and such radiations represent a new environmental factor. An exposure system operating in V-band (50-75 GHz) was developed for cell exposure. U-251 MG glial cell line was sham-exposed or exposed to MMW radiation for different durations (1-33 h) and two different power densities (5.4 microW/cm(2) or 0.54 mW/cm(2)). As gene expression is a multiple-step process, we analyzed chaperone proteins induction at different levels. First, using luciferase reporter gene, we investigated potential effect of MMWs on the activation of transcription factors (TFs) and gene promoter activity. Next, using RT-PCR and Western blot assays, we verified whether MMW exposure could alter RNA accumulation, translation, or protein stability. Experimental data demonstrated the absence of significant modifications in gene transcription, mRNA, and protein amount for the considered stress-sensitive genes for the exposure durations and power densities investigated. The main results of this study suggest that low-power 60 GHz radiation does not modify stress-sensitive gene expression of chaperone proteins. (c) 2006 Wiley-Liss, Inc.
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Yang, Zi; Hong, Joo Y.; Derkatch, Irina L.; Liebman, Susan W.
2013-01-01
Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q)/asparagine (N)-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller “seeds.” We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI +] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI +] or [URE3] prions. We explore in detail the events leading to the loss (curing) of [PSI+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI +]. PMID:23358669
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Sankhala, Rajeshwer S; Damai, Rajani S; Swamy, Musti J
2011-03-08
The major protein of bovine seminal plasma, PDC-109 binds to choline phospholipids present on the sperm plasma membrane upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. PDC-109 also shares significant similarities with small heat shock proteins and exhibits chaperone-like activity (CLA). Although the polydisperse nature of this protein has been shown to be important for its CLA, knowledge of other factors responsible for such an activity is scarce. Since surface exposure of hydrophobic residues is known to be an important factor which modulates the CLA of chaperone proteins, in the present study we have probed the surface hydrophobicity of PDC-109 using bisANS and ANS. Further, effect of phospholipids on the structure and chaperone-like activity of PDC-109 was studied. Presence of DMPC was found to increase the CLA of PDC-109 significantly, which could be due to the considerable exposure of hydrophobic regions on the lipid-protein recombinants, which can interact productively with the nonnative structures of target proteins, resulting in their protection. However, inclusion of DMPG instead of DMPC did not significantly alter the CLA of PDC-109, which could be due to the lower specificity of PDC-109 for DMPG as compared to DMPC. Cholesterol incorporation into DMPC membranes led to a decrease in the CLA of PDC-109-lipid recombinants, which could be attributed to reduced accessibility of hydrophobic surfaces to the substrate protein(s). These results underscore the relevance of phospholipid binding and hydrophobicity to the chaperone-like activity of PDC-109.
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Sankhala, Rajeshwer S.; Damai, Rajani S.; Swamy, Musti J.
2011-01-01
The major protein of bovine seminal plasma, PDC-109 binds to choline phospholipids present on the sperm plasma membrane upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. PDC-109 also shares significant similarities with small heat shock proteins and exhibits chaperone-like activity (CLA). Although the polydisperse nature of this protein has been shown to be important for its CLA, knowledge of other factors responsible for such an activity is scarce. Since surface exposure of hydrophobic residues is known to be an important factor which modulates the CLA of chaperone proteins, in the present study we have probed the surface hydrophobicity of PDC-109 using bisANS and ANS. Further, effect of phospholipids on the structure and chaperone-like activity of PDC-109 was studied. Presence of DMPC was found to increase the CLA of PDC-109 significantly, which could be due to the considerable exposure of hydrophobic regions on the lipid-protein recombinants, which can interact productively with the nonnative structures of target proteins, resulting in their protection. However, inclusion of DMPG instead of DMPC did not significantly alter the CLA of PDC-109, which could be due to the lower specificity of PDC-109 for DMPG as compared to DMPC. Cholesterol incorporation into DMPC membranes led to a decrease in the CLA of PDC-109-lipid recombinants, which could be attributed to reduced accessibility of hydrophobic surfaces to the substrate protein(s). These results underscore the relevance of phospholipid binding and hydrophobicity to the chaperone-like activity of PDC-109. PMID:21408153
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Blacklock, Kristin; Verkhivker, Gennady M.
2014-01-01
A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This
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Blacklock, Kristin; Verkhivker, Gennady M
2014-06-01
A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This
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Marcu, M G; Chadli, A; Bouhouche, I; Catelli, M; Neckers, L M
2000-11-24
Heat shock protein 90 (Hsp90), one of the most abundant chaperones in eukaryotes, participates in folding and stabilization of signal-transducing molecules including steroid hormone receptors and protein kinases. The amino terminus of Hsp90 contains a non-conventional nucleotide-binding site, related to the ATP-binding motif of bacterial DNA gyrase. The anti-tumor agents geldanamycin and radicicol bind specifically at this site and induce destabilization of Hsp90-dependent client proteins. We recently demonstrated that the gyrase inhibitor novobiocin also interacts with Hsp90, altering the affinity of the chaperone for geldanamycin and radicicol and causing in vitro and in vivo depletion of key regulatory Hsp90-dependent kinases including v-Src, Raf-1, and p185(ErbB2). In the present study we used deletion/mutation analysis to identify the site of interaction of novobiocin with Hsp90, and we demonstrate that the novobiocin-binding site resides in the carboxyl terminus of the chaperone. Surprisingly, this motif also recognizes ATP, and ATP and novobiocin efficiently compete with each other for binding to this region of Hsp90. Novobiocin interferes with association of the co-chaperones Hsc70 and p23 with Hsp90. These results identify a second site on Hsp90 where the binding of small molecule inhibitors can significantly impact the function of this chaperone, and they support the hypothesis that both amino- and carboxyl-terminal domains of Hsp90 interact to modulate chaperone activity.
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NASA Astrophysics Data System (ADS)
Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.
2016-06-01
The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.
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The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site
Pillet, Benjamin; García-Gómez, Juan J.; Pausch, Patrick; Falquet, Laurent; Bange, Gert; de la Cruz, Jesús; Kressler, Dieter
2015-01-01
Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S
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Conformational dynamics of the molecular chaperone Hsp90
Krukenberg, Kristin A.; Street, Timothy O.; Lavery, Laura A.; Agard, David A.
2016-01-01
The molecular chaperone Hsp90 is an essential eukaryotic protein that makes up 1–2% of all cytosolic proteins. Hsp90 is vital for the maturation and maintenance of a wide variety of substrate proteins largely involved in signaling and regulatory processes. Many of these substrates have also been implicated in cancer and other diseases making Hsp90 an attractive target for therapeutics. Hsp90 is a highly dynamic and flexible molecule that can adapt its conformation to the wide variety of substrate proteins with which it acts. Large conformational rearrangements are also required for the activation of these client proteins. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shifts the equilibrium between a pre-existing set of conformational states in an organism-dependent manner. In vivo Hsp90 functions as part of larger heterocomplexes. The binding partners of Hsp90, co-chaperones, assist in the recruitment and activation of substrates, and many co-chaperones further regulate the conformational dynamics of Hsp90 by shifting the conformational equilibrium towards a particular state. Studies have also suggested alternative mechanisms for the regulation of Hsp90’s conformation. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90 and the role that nucleotide, co-chaperones, post-translational modification and clients play in regulating Hsp90’s conformation. We also discuss the effects of current Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics. PMID:21414251
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Mohammadkhah, Melika; Simms, Ciaran K; Murphy, Paula
2017-02-01
Detection and visualisation of Collagen structure are important to understand the relationship between mechanical behaviour and microstructure in skeletal muscle since Collagen is the main structural protein in animal connective tissues, and is primarily responsible for their passive load-bearing properties. In the current study, the direct detection and visualization of Collagen using fluorescently tagged CNA35 binding protein (fused to EGFP or tdTomato) is reported for the first time on fixed skeletal muscle tissue. This Technical Note also establishes a working protocol by examining tissue preparation, dilution factor, exposure time etc. for sensitivity and specificity. Penetration of the binding protein into intact mature skeletal muscle was found to be very limited, but detection works well on tissue sections with higher sensitivity on wax embedded sections compared to frozen sections. CNA35 fused to tdTomato has a higher sensitivity than CNA35 fused to EGFP but both show specific detection. Best results were obtained with 15μm wax embedded sections, with blocking of non-specific binding in 1% BSA and antigen retrieval in Sodium Citrate. There was a play-off between dilution of the binding protein and time of incubation but both CNA35-tdTomato and CNA35-EGFP worked well with approximately 100μg/ml of purified protein with overnight incubation, while CNA35-tdTomato could be utilized at 5 fold less concentration. This approach can be applied to study the relationship between skeletal muscle micro-structure and to observe mechanical response to applied deformation. It can be used more broadly to detect Collagen in a variety of fixed tissues, useful for structure-functions studies, constitutive modelling, tissue engineering and assessment of muscle tissue pathologies. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Salomone, Federico; Li Volti, Giovanni; Vitaglione, Paola; Morisco, Filomena; Fogliano, Vincenzo; Zappalà, Agata; Palmigiano, Angelo; Garozzo, Domenico; Caporaso, Nicola; D'Argenio, Giuseppe; Galvano, Fabio
2014-06-01
Coffee consumption is inversely related to the degree of liver injury in patients with nonalcoholic fatty liver disease (NAFLD). Molecular mediators contributing to coffee's beneficial effects in NAFLD remain to be elucidated. In this study, we administrated decaffeinated espresso coffee or vehicle to rats fed an high-fat diet (HFD) for 12 weeks and examined the effects of coffee on liver injury by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) proteomic analysis combined with mass spectrometry. Rats fed an HFD and water developed panacinar steatosis, lobular inflammation, and mild fibrosis, whereas rats fed an HFD and coffee exhibited only mild steatosis. Coffee consumption increased liver expression of the endoplasmic reticulum chaperones glucose-related protein 78 and protein disulfide-isomerase A3; similarly, coffee drinking enhanced the expression of the mitochondrial chaperones heat stress protein 70 and DJ-1. Furthermore, in agreement with reduced hepatic levels of 8-isoprostanes and 8-hydroxy-2'-deoxyguanosine, proteomic analysis showed that coffee consumption induces the expression of master regulators of redox status (i.e., peroxiredoxin 1, glutathione S-transferase α2, and D-dopachrome tautomerase). Last, proteomics revealed an association of coffee intake with decreased expression of electron transfer flavoprotein subunit α, a component of the mitochondrial respiratory chain, involved in de novo lipogenesis. In this study, we were able to identify by proteomic analysis the stress proteins mediating the antioxidant effects of coffee; moreover, we establish for the first time the contribution of specific coffee-induced endoplasmic reticulum and mitochondrial chaperones ensuring correct protein folding and degradation in the liver. Copyright © 2014 Mosby, Inc. All rights reserved.
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Rout, Ashok K.; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico
2014-01-01
Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation. PMID:25342746
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Crystal structure of human proteasome assembly chaperone PAC4 involved in proteasome formation.
Kurimoto, Eiji; Satoh, Tadashi; Ito, Yuri; Ishihara, Eri; Okamoto, Kenta; Yagi-Utsumi, Maho; Tanaka, Keiji; Kato, Koichi
2017-05-01
The 26S proteasome is a large protein complex, responsible for degradation of ubiquinated proteins in eukaryotic cells. Eukaryotic proteasome formation is a highly ordered process that is assisted by several assembly chaperones. The assembly of its catalytic 20S core particle depends on at least five proteasome-specific chaperones, i.e., proteasome-assembling chaperons 1-4 (PAC1-4) and proteasome maturation protein (POMP). The orthologues of yeast assembly chaperones have been structurally characterized, whereas most mammalian assembly chaperones are not. In the present study, we determined a crystal structure of human PAC4 at 1.90-Å resolution. Our crystallographic data identify a hydrophobic surface that is surrounded by charged residues. The hydrophobic surface is complementary to that of its binding partner, PAC3. The surface also exhibits charge complementarity with the proteasomal α4-5 subunits. This will provide insights into human proteasome-assembling chaperones as potential anticancer drug targets. © 2017 The Protein Society.
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Contribution of chaperones to STAT pathway signaling
Bocchini, Claire E; Kasembeli, Moses M; Roh, Soung-Hun; Tweardy, David J
2014-01-01
Aberrant STAT signaling is associated with the development and progression of many cancers and immune related diseases. Recent findings demonstrate that proteostasis modulators under clinical investigation for cancer therapy have a significant impact on STAT signaling, which may be critical for mediating their anti-cancer effects. Chaperones are critical for protein folding, stability and function and, thus, play an essential role in the maintenance of proteostasis. In this review we discuss the role of chaperones in STAT and tyrosine kinase (TK) protein folding, modulation of STAT and TK activity, and degradation of TKs. We highlight the important role of chaperones in STAT signaling, and how this knowledge has provided a framework for the development of new therapeutic avenues of targeting STAT signaling related pathologies. PMID:26413421
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Czemeres, Josh; Buse, Kurt
2017-01-01
A fundamental role of the Hsp90 and Cdc37 chaperones in mediating conformational development and activation of diverse protein kinase clients is essential in signal transduction. There has been increasing evidence that the Hsp90-Cdc37 system executes its chaperoning duties by recognizing conformational instability of kinase clients and modulating their folding landscapes. The recent cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex has provided a framework for dissecting regulatory principles underlying differentiation and recruitment of protein kinase clients to the chaperone machinery. In this work, we have combined atomistic simulations with protein stability and network-based rigidity decomposition analyses to characterize dynamic factors underlying allosteric mechanism of the chaperone-kinase cycle and identify regulatory hotspots that control client recognition. Through comprehensive characterization of conformational dynamics and systematic identification of stabilization centers in the unbound and client- bound Hsp90 forms, we have simulated key stages of the allosteric mechanism, in which Hsp90 binding can induce instability and partial unfolding of Cdk4 client. Conformational landscapes of the Hsp90 and Cdk4 structures suggested that client binding can trigger coordinated dynamic changes and induce global rigidification of the Hsp90 inter-domain regions that is coupled with a concomitant increase in conformational flexibility of the kinase client. This process is allosteric in nature and can involve reciprocal dynamic exchanges that exert global effect on stability of the Hsp90 dimer, while promoting client instability. The network-based rigidity analysis and emulation of thermal unfolding of the Cdk4-cyclin D complex and Hsp90-Cdc37-Cdk4 complex revealed weak spots of kinase instability that are present in the native Cdk4 structure and are targeted by the chaperone during client recruitment. Our findings suggested that this
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The Role of Co-chaperones in Synaptic Proteostasis and Neurodegenerative Disease
Gorenberg, Erica L.; Chandra, Sreeganga S.
2017-01-01
Synapses must be preserved throughout an organism's lifespan to allow for normal brain function and behavior. Synapse maintenance is challenging given the long distances between the termini and the cell body, reliance on axonal transport for delivery of newly synthesized presynaptic proteins, and high rates of synaptic vesicle exo- and endocytosis. Hence, synapses rely on efficient proteostasis mechanisms to preserve their structure and function. To this end, the synaptic compartment has specific chaperones to support its functions. Without proper synaptic chaperone activity, local proteostasis imbalances lead to neurotransmission deficits, dismantling of synapses, and neurodegeneration. In this review, we address the roles of four synaptic chaperones in the maintenance of the nerve terminal, as well as their genetic links to neurodegenerative disease. Three of these are Hsp40 co-chaperones (DNAJs): Cysteine String Protein alpha (CSPα; DNAJC5), auxilin (DNAJC6), and Receptor-Mediated Endocytosis 8 (RME-8; DNAJC13). These co-chaperones contain a conserved J domain through which they form a complex with heat shock cognate 70 (Hsc70), enhancing the chaperone's ATPase activity. CSPα is a synaptic vesicle protein known to chaperone the t-SNARE SNAP-25 and the endocytic GTPase dynamin-1, thereby regulating synaptic vesicle exocytosis and endocytosis. Auxilin binds assembled clathrin cages, and through its interactions with Hsc70 leads to the uncoating of clathrin-coated vesicles, a process necessary for the regeneration of synaptic vesicles. RME-8 is a co-chaperone on endosomes and may have a role in clathrin-coated vesicle endocytosis on this organelle. These three co-chaperones maintain client function by preserving folding and assembly to prevent client aggregation, but they do not break down aggregates that have already formed. The fourth synaptic chaperone we will discuss is Heat shock protein 110 (Hsp110), which interacts with Hsc70, DNAJAs, and DNAJBs to
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Chaperoning the Cancer: The Proteostatic Functions of the Heat Shock Proteins in Cancer.
Vahid, Sepideh; Thaper, Daksh; Zoubeidi, Amina
2017-01-01
Protein homeostasis (proteostasis) is vital for the survival of cells in physiological and pathological conditions. Particularly, cancer cells are in constant state of cellular stress due to rapid proliferation and decreased quality control in proteosynthesis and therefore, are exceedingly dependent on the homeostasis pathways. Among the complex biological mechanisms regulating proteostasis are the highly conserved molecular chaperones, heat shock proteins (HSPs). HSPs assist cell survival by catalysing the proper folding of proteins, modulation of the apoptotic machinery and finally regulating the protein degradation machinery, providing either the stability or the degradation of selected proteins under stress conditions. Inevitably, HSPs are upregulated in malignancies and participate in different hallmarks of cancer, with indispensable roles in the onset and progression of the disease. Moreover, high levels of HSPs contribute to poor prognosis and treatment resistance in various cancers. Therefore these molecular chaperones present as attractive targets for anti-cancer therapy. This review describes how HSPs regulate different hallmarks of cancer and provides an overview on the most relevant patents which have recently appeared in the literature. The patents were extracted from Google Patents (2012-2016) while the clinical trial results were mined from www.clinicaltrial.gov. Review of literature shows that the proteostatic functions of HSPs can modify different hallmarks of cancer. Moreover, targeting HSPs (notably HSP27, HSP70 and HSP90) exhibited positive results in clinical trials so far. However, more studies should be designed to optimize the efficacy of mono or combination therapy in various malignancies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
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Verma, Amit K; Diwan, Danish; Raut, Sandeep; Dobriyal, Neha; Brown, Rebecca E; Gowda, Vinita; Hines, Justin K; Sahi, Chandan
2017-06-07
Heat shock proteins of 70 kDa (Hsp70s) partner with structurally diverse Hsp40s (J proteins), generating distinct chaperone networks in various cellular compartments that perform myriad housekeeping and stress-associated functions in all organisms. Plants, being sessile, need to constantly maintain their cellular proteostasis in response to external environmental cues. In these situations, the Hsp70:J protein machines may play an important role in fine-tuning cellular protein quality control. Although ubiquitous, the functional specificity and complexity of the plant Hsp70:J protein network has not been studied. Here, we analyzed the J protein network in the cytosol of Arabidopsis thaliana and, using yeast genetics, show that the functional specificities of most plant J proteins in fundamental chaperone functions are conserved across long evolutionary timescales. Detailed phylogenetic and functional analysis revealed that increased number, regulatory differences, and neofunctionalization in J proteins together contribute to the emerging functional diversity and complexity in the Hsp70:J protein network in higher plants. Based on the data presented, we propose that higher plants have orchestrated their "chaperome," especially their J protein complement, according to their specialized cellular and physiological stipulations. Copyright © 2017 Verma et al.
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Raut, Sandeep; Mallik, Bhagaban; Parichha, Arpan; Amrutha, Valsakumar; Sahi, Chandan; Kumar, Vimlesh
2017-07-05
Accumulation of toxic proteins in neurons has been linked with the onset of neurodegenerative diseases, which in many cases are characterized by altered neuronal function and synapse loss. Molecular chaperones help protein folding and the resolubilization of unfolded proteins, thereby reducing the protein aggregation stress. While most of the chaperones are expressed in neurons, their functional relevance remains largely unknown. Here, using bioinformatics analysis, we identified 95 Drosophila chaperones and classified them into seven different classes. Ubiquitous actin5C -Gal4-mediated RNAi knockdown revealed that ∼50% of the chaperones are essential in Drosophila Knocking down these genes in eyes revealed that ∼30% of the essential chaperones are crucial for eye development. Using neuron-specific knockdown, immunocytochemistry, and robust behavioral assays, we identified a new set of chaperones that play critical roles in the regulation of Drosophila NMJ structural organization. Together, our data present the first classification and comprehensive analysis of Drosophila chaperones. Our screen identified a new set of chaperones that regulate eye and NMJ morphogenesis. The outcome of the screen reported here provides a useful resource for further elucidating the role of individual chaperones in Drosophila eye morphogenesis and synaptic development. Copyright © 2017 Raut et al.
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Rout, Ashok K; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico
2014-12-19
Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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Dual inhibition of chaperoning process by taxifolin: molecular dynamics simulation study.
Verma, Sharad; Singh, Amit; Mishra, Abha
2012-07-01
Hsp90 (heat shock protein 90), a molecular chaperone, stabilizes more than 200 mutated and over expressed oncogenic proteins in cancer development. Cdc37 (cell division cycle protein 37), a co-chaperone of Hsp90, has been found to facilitate the maturation of protein kinases by acting as an adaptor and load these kinases onto the Hsp90 complex. Taxifolin (a natural phytochemical) was found to bind at ATP-binding site of Hsp90 and stabilized the inactive "open" or "lid-up" conformation as evidenced by molecular dynamic simulation. Furthermore, taxifolin was found to bind to interface of Hsp90 and Cdc37 complex and disrupt the interaction of residues of both proteins which were essential for the formation of active super-chaperone complex. Thus, taxifolin was found to act as an inhibitor of chaperoning process and may play a potential role in the cancer chemotherapeutics. Copyright © 2012 Elsevier Inc. All rights reserved.
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Marini, Joan C.; Reich, Adi; Smith, Simone M.
2014-01-01
Purpose of Review Osteogenesis imperfecta (OI), or “brittle bone disease”, has mainly been considered a bone disorder caused by collagen mutations. Within the last decade, however, a surge of genetic discoveries has created a new paradigm for OI as a collagen-related disorder, where autosomal dominant type I collagen defects cause most cases, while rare, mostly recessive forms are due to defects in genes whose protein products interact with collagen protein. This review is both timely and relevant in outlining the genesis, development and future of this paradigm shift in the understanding of OI. Recent Findings BRIL and PEDF defects cause types V and VI OI via defective bone mineralization, while defects in CRTAP, P3H1 and CyPB cause types VII-IX via defective collagen post-translational modification. Hsp47 and FKBP65 defects cause types X and XI OI via aberrant collagen crosslinking, folding and chaperoning, while defects in SP7, WNT1, TRIC-B and OASIS disrupt osteoblast development. Finally, absence of the type I collagen C-propeptidase BMP1 causes type XII OI due to altered collagen maturation/processing. Summary Identification of these multiple causative defects has provided crucial information for accurate genetic counseling, inspired a recently proposed functional grouping of OI types by shared mechanism to simplify current nosology, and should prod investigations into common pathways in OI. Such investigations could yield critical information on cellular and bone tissue mechanisms and translate to new mechanistic insight into clinical therapies for patients. PMID:25007323
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Sakono, Masafumi; Motomura, Konomi; Maruyama, Tatsuo; Kamiya, Noriho; Goto, Masahiro
2011-01-07
Casein micelles are a major component of milk proteins. It is well known that casein micelles show chaperone-like activity such as inhibition of protein aggregation and stabilization of proteins. In this study, it was revealed that casein micelles also possess a high refolding activity for denatured proteins. A buffer containing caseins exhibited higher refolding activity for denatured bovine carbonic anhydrase than buffers including other proteins. In particular, a buffer containing α-casein showed about a twofold higher refolding activity compared with absence of α-casein. Casein properties of surface hydrophobicity, a flexible structure and assembly formation are thought to contribute to this high refolding activity. Our results indicate that casein micelles stabilize milk proteins by both chaperone-like activity and refolding properties. Copyright © 2010 Elsevier Inc. All rights reserved.
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Kita, Kazuko; Jin, Yuan-Hu; Sun, Zhuo; Chen, Shi-Ping; Sumiya, Yoko; Hongo, Toshio; Suzuki, Nobuo
2009-06-01
We examined changes in the levels of chaperone proteins to evaluate the toxic effects of environmental chemicals in human cells in vitro. Some chaperones are up-regulated by estrogenic chemicals, but the effect is not necessarily dependent on the receptor. Thus we also investigated whether a chemical-induced change in chaperone protein expression is human estrogen receptor (hER)-dependent or not, using cultured human cell lines transfected with hERalpha cDNA or an empty vector. In the hERalpha-expressed cells, the protein levels of the heat shock protein 27 (HSP27), the glucose-regulated protein 78 (GRP78/BiP), and GRP94 increased after exposure to beta-estradiol (E(2)) (from 10(-9)M to 10(-6)M) and bisphenol A (BPA) (from 10(-6)M to 10(-5)M). On the other hand, the increase was not observed in the cells without hERalpha expression. These results suggest that the E(2)- and BPA-induced increase in the protein levels were hERalpha dependent. We next examined the effect of four phenolic chemicals similar in structure to BPA, and found that among them, 4-methoxyphenol (from 10(-6)M to 10(-5)M) increased the levels of the chaperone proteins with hERalpha dependency. Thus the human cultured cells would be suitable for evaluating whether an increase in chaperone proteins occurs upon exposure to environmental chemicals and whether the effect is ER-dependent.
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Oligomerization of a molecular chaperone modulates its activity
Kawagoe, Soichiro; Ishimori, Koichiro
2018-01-01
Molecular chaperones alter the folding properties of cellular proteins via mechanisms that are not well understood. Here, we show that Trigger Factor (TF), an ATP-independent chaperone, exerts strikingly contrasting effects on the folding of non-native proteins as it transitions between a monomeric and a dimeric state. We used NMR spectroscopy to determine the atomic resolution structure of the 100 kDa dimeric TF. The structural data show that some of the substrate-binding sites are buried in the dimeric interface, explaining the lower affinity for protein substrates of the dimeric compared to the monomeric TF. Surprisingly, the dimeric TF associates faster with proteins and it exhibits stronger anti-aggregation and holdase activity than the monomeric TF. The structural data show that the dimer assembles in a way that substrate-binding sites in the two subunits form a large contiguous surface inside a cavity, thus accounting for the observed accelerated association with unfolded proteins. Our results demonstrate how the activity of a chaperone can be modulated to provide distinct functional outcomes in the cell. PMID:29714686
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Caldas, Thérèse; Malki, Abderrahim; Kern, Renée; Abdallah, Jad; Richarme, Gilbert
2006-05-12
Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN functions with two redox cysteines. Accordingly, YbbN catalyzes the oxidation of reduced RNase and the isomerization of scrambled RNase. Moreover, upon oxidation, its oligomeric state changes from dimers to tetramers and higher oligomers. YbbN also possesses chaperone properties, promoting protein folding after urea denaturation and forming complexes with unfolded proteins. This is the first biochemical characterization of a member of the YbbN class of bacterial thioredoxin-like proteins, and in vivo experiments will allow to determine the importance of its redox and chaperone properties in the cellular physiology.
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Identification of a polymorphic collagen-like protein in the crustacean bacteria Pasteuria ramosa.
Mouton, Laurence; Traunecker, Emmanuel; McElroy, Kerensa; Du Pasquier, Louis; Ebert, Dieter
2009-12-01
Pasteuria ramosa is a spore-forming bacterium that infects Daphnia species. Previous results demonstrated a high specificity of host clone/parasite genotype interactions. Surface proteins of bacteria often play an important role in attachment to host cells prior to infection. We analyzed surface proteins of P. ramosa spores by two-dimensional gel electrophoresis. For the first time, we prove that two isolates selected for their differences in infectivity reveal few but clear-cut differences in protein patterns. Using internal sequencing and LC/MS/MS, we identified a collagen-like protein named Pcl1a (Pasteuria collagen-like protein 1a). This protein, reconstructed with the help of Pasteuria genome sequences, contains three domains: a 75-amino-acid amino-terminal domain with a potential transmembrane helix domain, a central collagen-like region (CLR) containing Gly-Xaa-Yaa (GXY) repeats, and a 7-amino-acid carboxy-terminal domain. The CLR region is polymorphic among the two isolates with amino-acid substitutions and a variable number of GXY triplets. Collagen-like proteins are rare in prokaryotes, although they have been described in several pathogenic bacteria, including Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis, closely related to Pasteuria species, in which they could be involved in the adherence of bacteria to host cells.
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Interaction of the Disordered Yersinia Effector Protein YopE with Its Cognate Chaperone SycE
2009-01-01
structures of YopECBD were molten globules with a hydrophobic core. Molecular dynamics (MD) simulations indi- cated that the structure remained compact at...ensembles of unfolded conformations of the Yersinia effector YopE using REMD simulations and docked them to the chaper- one SycE using a multistep protein...disordered state but transitions into an ordered state upon binding to its cognate chaperone (7). The dynamics of the disordered effector protein and
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Chemical Endoplasmic Reticulum Chaperone Alleviates Doxorubicin-Induced Cardiac Dysfunction.
Fu, Hai Ying; Sanada, Shoji; Matsuzaki, Takashi; Liao, Yulin; Okuda, Keiji; Yamato, Masaki; Tsuchida, Shota; Araki, Ryo; Asano, Yoshihiro; Asanuma, Hiroshi; Asakura, Masanori; French, Brent A; Sakata, Yasushi; Kitakaze, Masafumi; Minamino, Tetsuo
2016-03-04
Doxorubicin is an effective chemotherapeutic agent for cancer, but its use is often limited by cardiotoxicity. Doxorubicin causes endoplasmic reticulum (ER) dilation in cardiomyocytes, and we have demonstrated that ER stress plays important roles in the pathophysiology of heart failure. We evaluated the role of ER stress in doxorubicin-induced cardiotoxicity and examined whether the chemical ER chaperone could prevent doxorubicin-induced cardiac dysfunction. We confirmed that doxorubicin caused ER dilation in mouse hearts, indicating that doxorubicin may affect ER function. Doxorubicin activated an ER transmembrane stress sensor, activating transcription factor 6, in cultured cardiomyocytes and mouse hearts. However, doxorubicin suppressed the expression of genes downstream of activating transcription factor 6, including X-box binding protein 1. The decreased levels of X-box binding protein 1 resulted in a failure to induce the expression of the ER chaperone glucose-regulated protein 78 which plays a major role in adaptive responses to ER stress. In addition, doxorubicin activated caspase-12, an ER membrane-resident apoptotic molecule, which can lead to cardiomyocyte apoptosis and cardiac dysfunction. Cardiac-specific overexpression of glucose-regulated protein 78 by adeno-associated virus 9 or the administration of the chemical ER chaperone 4-phenylbutyrate attenuated caspase-12 cleavage, and alleviated cardiac apoptosis and dysfunction induced by doxorubicin. Doxorubicin activated the ER stress-initiated apoptotic response without inducing the ER chaperone glucose-regulated protein 78, further augmenting ER stress in mouse hearts. Cardiac-specific overexpression of glucose-regulated protein 78 or the administration of the chemical ER chaperone alleviated the cardiac dysfunction induced by doxorubicin and may facilitate the safe use of doxorubicin for cancer treatment. © 2016 American Heart Association, Inc.
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Mitochondrial carrier protein biogenesis: role of the chaperones Hsc70 and Hsp90
Zara, Vincenzo; Ferramosca, Alessandra; Robitaille-Foucher, Philippe; Palmieri, Ferdinando; Young, Jason C.
2016-01-01
Metabolite carrier proteins of the mitochondrial inner membrane share homology in their transmembrane domains, which also carries their targeting information. In addition, some carriers have cleavable presequences which are not essential for targeting, but have some other function before import. The cytosolic chaperones Hsc70 (heat-shock cognate 70) and Hsp90 (heat-shock protein 90) complex with carrier precursors and interact specifically with the Tom (translocase of the mitochondrial outer membrane) 70 import receptor to promote import. We analysed how the presequences of the PiC (phosphate carrier) and CIC (citrate carrier) relate to the mechanisms of chaperone-mediated import. Deletion of the PiC presequence reduced the efficiency of import but, notably, not by causing aggregation. Instead, binding of the protein to Hsc70 was reduced, as well as the dependence on Hsc70 for import. Hsp90 binding and function in import was not greatly affected, but it could not entirely compensate for the lack of Hsc70 interaction. Deletion of the presequence from CIC was shown to cause its aggregation, but had little effect on the contribution to import of either Hsc70 or Hsp90. The presequence of PiC, but not that of CIC, conferred Hsc70 binding to dihydrofolate reductase fusion proteins. In comparison, OGC (oxoglutarate carrier) lacks a presequence and was more soluble, though it is still dependent on both Hsc70 and Hsp90. We propose that carrier presequences evolved to improve targeting competence by different mechanisms, depending on physical properties of the precursors in the cytosolic targeting environment. PMID:19143589
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Mitochondrial carrier protein biogenesis: role of the chaperones Hsc70 and Hsp90.
Zara, Vincenzo; Ferramosca, Alessandra; Robitaille-Foucher, Philippe; Palmieri, Ferdinando; Young, Jason C
2009-04-15
Metabolite carrier proteins of the mitochondrial inner membrane share homology in their transmembrane domains, which also carries their targeting information. In addition, some carriers have cleavable presequences which are not essential for targeting, but have some other function before import. The cytosolic chaperones Hsc70 (heat-shock cognate 70) and Hsp90 (heat-shock protein 90) complex with carrier precursors and interact specifically with the Tom (translocase of the mitochondrial outer membrane) 70 import receptor to promote import. We analysed how the presequences of the PiC (phosphate carrier) and CIC (citrate carrier) relate to the mechanisms of chaperone-mediated import. Deletion of the PiC presequence reduced the efficiency of import but, notably, not by causing aggregation. Instead, binding of the protein to Hsc70 was reduced, as well as the dependence on Hsc70 for import. Hsp90 binding and function in import was not greatly affected, but it could not entirely compensate for the lack of Hsc70 interaction. Deletion of the presequence from CIC was shown to cause its aggregation, but had little effect on the contribution to import of either Hsc70 or Hsp90. The presequence of PiC, but not that of CIC, conferred Hsc70 binding to dihydrofolate reductase fusion proteins. In comparison, OGC (oxoglutarate carrier) lacks a presequence and was more soluble, though it is still dependent on both Hsc70 and Hsp90. We propose that carrier presequences evolved to improve targeting competence by different mechanisms, depending on physical properties of the precursors in the cytosolic targeting environment.
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Botha, M; Pesce, E-R; Blatch, G L
2007-01-01
Extensive structural and functional remodelling of Plasmodium falciparum (malaria)-infected erythrocytes follows the export of a range of proteins of parasite origin (exportome) across the parasitophorous vacuole into the host erythrocyte. The genome of P. falciparum encodes a diverse chaperone complement including at least 43 members of the heat shock protein 40kDa (Hsp40) family, and six members of the heat shock protein 70kDa (Hsp70) family. Nearly half of the Hsp40 proteins of P. falciparum are predicted to contain a PEXEL/HT (Plasmodium export element/host targeting signal) sequence motif, and hence are likely to be part of the exportome. In this review we critically evaluate the classification, sequence similarity and clustering, and possible interactors of the P. falciparum Hsp40 chaperone machinery. In addition to the types I, II and III Hsp40 proteins all exhibiting the signature J-domain, the P. falciparum genome also encodes a number of specialized Hsp40 proteins with a J-like domain, which we have categorized as type IV Hsp40 proteins. Analysis of the potential P. falciparum Hsp40 protein interaction network revealed connections predominantly with cytoskeletal and membrane proteins, transcriptional machinery, DNA repair and replication machinery, translational machinery, the proteasome and proteolytic enzymes, and enzymes involved in cellular physiology. Comparison of the Hsp40 proteins of P. falciparum to those of other apicomplexa reveals that most of the proteins (especially the PEXEL/HT-containing proteins) are unique to P. falciparum. Furthermore, very few of the P. falciparum Hsp40 proteins have human homologs, except for those proteins implicated in fundamental biological processes. Our analysis suggests that P. falciparum has evolved an expanded and specialized Hsp40 protein machinery to enable it successfully to invade and remodel the human erythrocyte, and we propose a model in which these proteins are involved in chaperone
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Abdul-Wahab, Mohd Firdaus; Homma, Takayuki; Wright, Michael; Olerenshaw, Dee; Dafforn, Timothy R.; Nagata, Kazuhiro; Miller, Andrew D.
2013-01-01
Heat shock protein 47 (HSP47) is a single-substrate molecular chaperone crucial for collagen biosynthesis. Although its function is well established, the molecular mechanisms that govern binding to procollagen peptides and triple helices in the endoplasmic reticulum (followed by controlled release in the Golgi) are unclear. HSP47 binds procollagen at a neutral pH but releases at a pH similar to the pKa of the imidazole side chain of histidine residues. It thus seems likely that these residues are involved in this pH-dependent mechanism. Murine HSP47 has 14 histidine residues grouped into three clusters, known as the breach, gate, and shutter. Here, we report the use of histidine mutagenesis to demonstrate the relative contribution of these three clusters to HSP47 structure and the “pH switch.” Many of the tested mutants are silent; however, breach mutants H197A and H198A show binding but no apparent pH switch and are unable to control release. Another breach mutant, H191A, shows perturbed collagen release characteristics, consistent with observed perturbations in pH-driven trans-conformational changes. Thus, His-198, His-197 and His-191 are important (if not central) to HSP47 mechanism of binding/release to collagen. This is consistent with the breach cluster residues being well conserved across the HSP47 family. PMID:23212911
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Bird, Jonathan E.; Takagi, Yasuharu; Billington, Neil; Strub, Marie-Paule; Sellers, James R.; Friedman, Thomas B.
2014-01-01
Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin–specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end–directed motor that moves actin filaments in a gliding assay (∼430 nm·s−1 at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (kcat ∼6 s−1) was similar to the actin-detachment rate (kdet = 6.2 s−1) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells. PMID:25114250
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Structural Basis of Interdomain Communication in the Hsc70 Chaperone
Jiang, Jianwen; Prasad, Kondury; Lafer, Eileen M.; Sousa, Rui
2015-01-01
Summary Hsp70 family proteins are highly conserved chaperones involved in protein folding, degradation, targeting and translocation, and protein complex remodeling. They are comprised of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD). ATP binding to the NBD alters SBD conformation and substrate binding kinetics, but an understanding of the mechanism of interdomain communication has been hampered by the lack of a crystal structure of an intact chaperone. Were-port here the 2.6 Å structure of a functionally intact bovine Hsc70 (bHsc70) and a mutational analysis of the observed interdomain interface and the immediately adjacent interdomain linker. This analysis identifies interdomain interactions critical for chaperone function and supports an allosteric mechanism in which the interdomain linker invades and disrupts the interdomain interface when ATP binds. PMID:16307916
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Duran, Ivan; Martin, Jorge H.; Weis, Mary Ann; Krejci, Pavel; Konik, Peter; Li, Bing; Alanay, Yasemin; Lietman, Caressa; Lee, Brendan; Eyre, David; Cohn, Daniel H.; Krakow, Deborah
2017-01-01
Lysine hydroxylation of type I collagen telopeptides varies from tissue to tissue and these distinct hydroxylation patterns modulate collagen crosslinking to generate a unique extracellular matrix. Abnormalities in these patterns contribute to pathologies that include osteogenesis imperfecta (OI), fibrosis and cancer. Telopeptide procollagen modifications are carried out by lysyl hydroxylase 2 (LH2), however, little is known regarding how this enzyme regulates hydroxylation patterns. We identified an ER complex of resident chaperones that includes HSP47, FKBP65 and BiP regulating the activity of LH2. Our findings show that FKBP65 and HSP47 modulate the activity of LH2 to either favor or repress its activity. BiP was also identified as a member of the complex, playing a role in enhancing the formation of the complex. This newly identified ER chaperone complex contributes to our understanding of how LH2 regulates lysyl hydroxylation of type I collagen C-telopeptides to affect the quality of connective tissues. PMID:28177155
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NASA Astrophysics Data System (ADS)
Kurotobi, K.; Suzuki, Y.; Nakajima, H.; Suzuki, H.; Iwaki, M.
2003-05-01
He + ion implanted collagen-coated tubes with a fluence of 1 × 10 14 ions/cm 2 were exhibited antithrombogenicity. To investigate the mechanisms of antithrombogenicity of these samples, plasma protein adsorption assay and platelet adhesion experiments were performed. The adsorption of fibrinogen (Fg) and von Willebrand factor (vWf) was minimum on the He + ion implanted collagen with a fluence of 1 × 10 14 ions/cm 2. Platelet adhesion (using platelet rich plasma) was inhibited on the He + ion implanted collagen with a fluence of 1 × 10 14 ions/cm 2 and was accelerated on the untreated collagen and ion implanted collagen with fluences of 1 × 10 13, 1 × 10 15 and 1 × 10 16 ions/cm 2. Platelet activation with washed platelets was observed on untreated collagen and He + ion implanted collagen with a fluence of 1 × 10 14 ions/cm 2 and was inhibited with fluences of 1 × 10 13, 1 × 10 15 and 1 × 10 16 ions/cm 2. Generally, platelets can react with a specific ligand inside the collagen (GFOGER sequence). The results of platelets adhesion experiments using washed platelets indicated that there were no ligands such as GFOGER on the He + ion implanted collagen over a fluence of 1 × 10 13 ions/cm 2. On the 1 × 10 14 ions/cm 2 implanted collagen, no platelet activation was observed due to the influence of plasma proteins. From the above, it is concluded that the decrease of adsorbed Fg and vWf caused the antithrombogenicity of He + ion implanted collagen with a fluence of 1 × 10 14 ions/cm 2 and that plasma protein adsorption took an important role repairing the graft surface.
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Nucleic Acid Chaperone Activity of the ORF1 Protein from the Mouse LINE-1 Retrotransposon
Martin, Sandra L.; Bushman, Frederic D.
2001-01-01
Non-LTR retrotransposons such as L1 elements are major components of the mammalian genome, but their mechanism of replication is incompletely understood. Like retroviruses and LTR-containing retrotransposons, non-LTR retrotransposons replicate by reverse transcription of an RNA intermediate. The details of cDNA priming and integration, however, differ between these two classes. In retroviruses, the nucleocapsid (NC) protein has been shown to assist reverse transcription by acting as a “nucleic acid chaperone,” promoting the formation of the most stable duplexes between nucleic acid molecules. A protein-coding region with an NC-like sequence is present in most non-LTR retrotransposons, but no such sequence is evident in mammalian L1 elements or other members of its class. Here we investigated the ORF1 protein from mouse L1 and found that it does in fact display nucleic acid chaperone activities in vitro. L1 ORF1p (i) promoted annealing of complementary DNA strands, (ii) facilitated strand exchange to form the most stable hybrids in competitive displacement assays, and (iii) facilitated melting of an imperfect duplex but stabilized perfect duplexes. These findings suggest a role for L1 ORF1p in mediating nucleic acid strand transfer steps during L1 reverse transcription. PMID:11134335
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Toyama, Takuya; Nakamura, Yuki; Tamada, Kentaro; Shimizu, Hitomi; Ninagawa, Satoshi; Okada, Tetsuya; Ishikawa-Fujiwara, Tomoko; Aoyama, Eriko; Takigawa, Masaharu
2017-01-01
The unfolded protein response (UPR) handles unfolded/misfolded proteins accumulated in the endoplasmic reticulum (ER). However, it is unclear how vertebrates correctly use the total of ten UPR transducers. We have found that ER stress occurs physiologically during early embryonic development in medaka fish and that the smooth alignment of notochord cells requires ATF6 as a UPR transducer, which induces ER chaperones for folding of type VIII (short-chain) collagen. After secretion of hedgehog for tissue patterning, notochord cells differentiate into sheath cells, which synthesize type II collagen. In this study, we show that this vacuolization step requires both ATF6 and BBF2H7 as UPR transducers and that BBF2H7 regulates a complete set of genes (Sec23/24/13/31, Tango1, Sedlin, and KLHL12) essential for the enlargement of COPII vesicles to accommodate long-chain collagen for export, leading to the formation of the perinotochordal basement membrane. Thus, the most appropriate UPR transducer is activated to cope with the differing physiological ER stresses of different content types depending on developmental stage. PMID:28500182
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Ishikawa, Tokiro; Okada, Tetsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Kamei, Yasuhiro; Shigenobu, Shuji; Tanaka, Minoru; Saito, Taro L.; Yoshimura, Jun; Morishita, Shinichi; Toyoda, Atsushi; Sakaki, Yoshiyuki; Taniguchi, Yoshihito; Takeda, Shunichi; Mori, Kazutoshi
2013-01-01
ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra. PMID:23447699
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Ishikawa, Tokiro; Okada, Tetsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Kamei, Yasuhiro; Shigenobu, Shuji; Tanaka, Minoru; Saito, Taro L; Yoshimura, Jun; Morishita, Shinichi; Toyoda, Atsushi; Sakaki, Yoshiyuki; Taniguchi, Yoshihito; Takeda, Shunichi; Mori, Kazutoshi
2013-05-01
ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra.
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Meta-analysis of heat- and chemically upregulated chaperone genes in plant and human cells
Finka, Andrija; Mattoo, Rayees U. H.
2010-01-01
Molecular chaperones are central to cellular protein homeostasis. In mammals, protein misfolding diseases and aging cause inflammation and progressive tissue loss, in correlation with the accumulation of toxic protein aggregates and the defective expression of chaperone genes. Bacteria and non-diseased, non-aged eukaryotic cells effectively respond to heat shock by inducing the accumulation of heat-shock proteins (HSPs), many of which molecular chaperones involved in protein homeostasis, in reducing stress damages and promoting cellular recovery and thermotolerance. We performed a meta-analysis of published microarray data and compared expression profiles of HSP genes from mammalian and plant cells in response to heat or isothermal treatments with drugs. The differences and overlaps between HSP and chaperone genes were analyzed, and expression patterns were clustered and organized in a network. HSPs and chaperones only partly overlapped. Heat-shock induced a subset of chaperones primarily targeted to the cytoplasm and organelles but not to the endoplasmic reticulum, which organized into a network with a central core of Hsp90s, Hsp70s, and sHSPs. Heat was best mimicked by isothermal treatments with Hsp90 inhibitors, whereas less toxic drugs, some of which non-steroidal anti-inflammatory drugs, weakly expressed different subsets of Hsp chaperones. This type of analysis may uncover new HSP-inducing drugs to improve protein homeostasis in misfolding and aging diseases. Electronic supplementary material The online version of this article (doi:10.1007/s12192-010-0216-8) contains supplementary material, which is available to authorized users. PMID:20694844
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An RpoS-dependent sRNA regulates the expression of a chaperone involved in protein folding
Silva, Inês Jesus; Ortega, Álvaro Darío; Viegas, Sandra Cristina; García-del Portillo, Francisco; Arraiano, Cecília Maria
2013-01-01
Small noncoding RNAs (sRNAs) are usually expressed in the cell to face a variety of stresses. In this report we disclose the first target for SraL (also known as RyjA), a sRNA present in many bacteria, which is highly induced in stationary phase. We also demonstrate that this sRNA is directly transcribed by the major stress σ factor σS (RpoS) in Salmonella enterica serovar Typhimurium. We show that SraL sRNA down-regulates the expression of the chaperone Trigger Factor (TF), encoded by the tig gene. TF is one of the three major chaperones that cooperate in the folding of the newly synthesized cytosolic proteins and is the only ribosome-associated chaperone known in bacteria. By use of bioinformatic tools and mutagenesis experiments, SraL was shown to directly interact with the 5′ UTR of the tig mRNA a few nucleotides upstream of the Shine-Dalgarno region. Namely, point mutations in the sRNA (SraL*) abolished the repression of tig mRNA and could only down-regulate a tig transcript target with the respective compensatory mutations. We have also validated in vitro that SraL forms a stable duplex with the tig mRNA. This work constitutes the first report of a small RNA affecting protein folding. Taking into account that both SraL and TF are very well conserved in enterobacteria, this work will have important repercussions in the field. PMID:23893734
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An RpoS-dependent sRNA regulates the expression of a chaperone involved in protein folding.
Silva, Inês Jesus; Ortega, Alvaro Darío; Viegas, Sandra Cristina; García-Del Portillo, Francisco; Arraiano, Cecília Maria
2013-09-01
Small noncoding RNAs (sRNAs) are usually expressed in the cell to face a variety of stresses. In this report we disclose the first target for SraL (also known as RyjA), a sRNA present in many bacteria, which is highly induced in stationary phase. We also demonstrate that this sRNA is directly transcribed by the major stress σ factor σ(S) (RpoS) in Salmonella enterica serovar Typhimurium. We show that SraL sRNA down-regulates the expression of the chaperone Trigger Factor (TF), encoded by the tig gene. TF is one of the three major chaperones that cooperate in the folding of the newly synthesized cytosolic proteins and is the only ribosome-associated chaperone known in bacteria. By use of bioinformatic tools and mutagenesis experiments, SraL was shown to directly interact with the 5' UTR of the tig mRNA a few nucleotides upstream of the Shine-Dalgarno region. Namely, point mutations in the sRNA (SraL*) abolished the repression of tig mRNA and could only down-regulate a tig transcript target with the respective compensatory mutations. We have also validated in vitro that SraL forms a stable duplex with the tig mRNA. This work constitutes the first report of a small RNA affecting protein folding. Taking into account that both SraL and TF are very well conserved in enterobacteria, this work will have important repercussions in the field.
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ATP-dependent molecular chaperones in plastids--More complex than expected.
Trösch, Raphael; Mühlhaus, Timo; Schroda, Michael; Willmund, Felix
2015-09-01
Plastids are a class of essential plant cell organelles comprising photosynthetic chloroplasts of green tissues, starch-storing amyloplasts of roots and tubers or the colorful pigment-storing chromoplasts of petals and fruits. They express a few genes encoded on their organellar genome, called plastome, but import most of their proteins from the cytosol. The import into plastids, the folding of freshly-translated or imported proteins, the degradation or renaturation of denatured and entangled proteins, and the quality-control of newly folded proteins all require the action of molecular chaperones. Members of all four major families of ATP-dependent molecular chaperones (chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families) have been identified in plastids from unicellular algae to higher plants. This review aims not only at giving an overview of the most current insights into the general and conserved functions of these plastid chaperones, but also into their specific plastid functions. Given that chloroplasts harbor an extreme environment that cycles between reduced and oxidized states, that has to deal with reactive oxygen species and is highly reactive to environmental and developmental signals, it can be presumed that plastid chaperones have evolved a plethora of specific functions some of which are just about to be discovered. Here, the most urgent questions that remain unsolved are discussed, and guidance for future research on plastid chaperones is given. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.
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Shimamoto, Seiko; Kubota, Yasuo; Yamaguchi, Fuminori; Tokumitsu, Hiroshi; Kobayashi, Ryoji
2013-01-01
The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca2+/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca2+/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca2+/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca2+/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca2+-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway. PMID:23344957
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Membrane-associated stress proteins: more than simply chaperones.
Horváth, Ibolya; Multhoff, Gabriele; Sonnleitner, Alois; Vígh, László
2008-01-01
The protein- and/or lipid-mediated association of chaperone proteins to membranes is a widespread phenomenon and implicated in a number of physiological and pathological events that were earlier partially or completely overlooked. A temporary association of certain HSPs with membranes can re-establish the fluidity and bilayer stability and thereby restore the membrane functionality during stress conditions. The fluidity and microdomain organization of membranes are decisive factors in the perception and transduction of stresses into signals that trigger the activation of specific HS genes. Conversely, the membrane association of HSPs may result in the inactivation of membrane-perturbing signals, thereby switch off the heat shock response. Interactions between certain HSPs and specific lipid microdomains ("rafts") might be a previously unrecognized means for the compartmentalization of HSPs to specific signaling platforms, where key signaling proteins are known to be concentrated. Any modulations of the membranes, especially the raft-lipid composition of the cells can alter the extracellular release and thus the immuno-stimulatory activity of certain HSPs. Reliable techniques, allowing mapping of the composition and dynamics of lipid microdomains and simultaneously the spatio-temporal localization of HSPs in and near the plasma membrane can provide suitable means with which to address fundamental questions, such as how HSPs are transported to and translocated through the plasma membrane. The possession of such information is critical if we are to target the membrane association principles of HSPs for successful drug development in most various diseases.
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Terajima, Masahiko; Taga, Yuki; Chen, Yulong; Cabral, Wayne A.; Hou-Fu, Guo; Srisawasdi, Sirivimol; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Kurie, Jonathan M.; Marini, Joan C.; Yamauchi, Mitsuo
2016-01-01
Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet. 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1–3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation. PMID:26934917
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Terajima, Masahiko; Taga, Yuki; Chen, Yulong; Cabral, Wayne A; Hou-Fu, Guo; Srisawasdi, Sirivimol; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Kurie, Jonathan M; Marini, Joan C; Yamauchi, Mitsuo
2016-04-29
Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1-3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Functional cell-surface display of a lipase-specific chaperone.
Wilhelm, Susanne; Rosenau, Frank; Becker, Stefan; Buest, Sebastian; Hausmann, Sascha; Kolmar, Harald; Jaeger, Karl-Erich
2007-01-02
Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.
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Bozzacco, Leonia; Yi, Zhigang; Andreo, Ursula; Conklin, Claire R; Li, Melody M H; Rice, Charles M; MacDonald, Margaret R
2016-01-06
DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is
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Bozzacco, Leonia; Yi, Zhigang; Andreo, Ursula; Conklin, Claire R.; Li, Melody M. H.; Rice, Charles M.
2016-01-01
ABSTRACT DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. IMPORTANCE Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single
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Galigniana, Mario D; Echeverría, Pablo C; Erlejman, Alejandra G; Piwien-Pilipuk, Graciela
2010-01-01
In the absence of hormone, corticosteroid receptors such as GR (glucocorticoid receptor) and (mineralocorticoid receptor) are primarily located in the cytoplasm. Upon steroid-binding, they rapidly accumulate in the nucleus. Regardless of their primary location, these receptors and many other nuclear factors undergo a constant and dynamic nucleocytoplasmic shuttling. All members of the steroid receptor family are known to form large oligomeric structures with the heat-shock proteins of 90-kDa (hsp90) and 70-kDa (hsp70), the small acidic protein p23, and a tetratricopeptide repeat (TPR) -domain protein such as FK506-binding proteins (FKBPs), cyclophilins (CyPs) or the serine/threonine protein phosphatase 5 (PP5). It has always been stated that the dissociation of the chaperone heterocomplex (a process normally referred to as receptor "transformation") is the first step that permits the nuclear import of steroid receptors. However the experimental evidence is consistent with a model where the chaperone machinery is required for the retrotransport of the receptor through the cytoplasm and also facilitates the passage through the nuclear pore. Recent evidence indicates that the hsp90-based chaperone system also interacts with structures of the nuclear pore such as importin β and the integral nuclear pore glycoprotein Nup62 facilitating the passage of the untransformed receptor through the nuclear pore.
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Ricard-Blum, Sylvie
2011-01-01
Collagens are the most abundant proteins in mammals. The collagen family comprises 28 members that contain at least one triple-helical domain. Collagens are deposited in the extracellular matrix where most of them form supramolecular assemblies. Four collagens are type II membrane proteins that also exist in a soluble form released from the cell surface by shedding. Collagens play structural roles and contribute to mechanical properties, organization, and shape of tissues. They interact with cells via several receptor families and regulate their proliferation, migration, and differentiation. Some collagens have a restricted tissue distribution and hence specific biological functions. PMID:21421911
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Effect of glycation on α-crystallin structure and chaperone-like function
Kumar, P. Anil; Kumar, M. Satish; Reddy, G. Bhanuprakash
2007-01-01
The chaperone-like activity of α-crystallin is considered to play an important role in the maintenance of the transparency of the eye lens. However, in the case of aging and in diabetes, the chaperone function of α-crystallin is compromized, resulting in cataract formation. Several post-translational modifications, including non-enzymatic glycation, have been shown to affect the chaperone function of α-crystallin in aging and in diabetes. A variety of agents have been identified as the predominant sources for the formation of AGEs (advanced glycation end-products) in various tissues, including the lens. Nevertheless, glycation of α-crystallin with various sugars has resulted in divergent results. In the present in vitro study, we have investigated the effect of glucose, fructose, G6P (glucose 6-phosphate) and MGO (methylglyoxal), which represent the major classes of glycating agents, on the structure and chaperone function of α-crystallin. Modification of α-crystallin with all four agents resulted in the formation of glycated protein, increased AGE fluorescence, protein cross-linking and HMM (high-molecular-mass) aggregation. Interestingly, these glycation-related profiles were found to vary with different glycating agents. For instance, CML [Nϵ-(carboxymethyl)lysine] was the predominant AGE formed upon glycation of α-crystallin with these agents. Although fructose and MGO caused significant conformational changes, there were no significant structural perturbations with glucose and G6P. With the exception of MGO modification, glycation with other sugars resulted in decreased chaperone activity in aggregation assays. However, modification with all four sugars led to the loss of chaperone activity as assessed using an enzyme inactivation assay. Glycation-induced loss of α-crystallin chaperone activity was associated with decreased hydrophobicity. Furthermore, α-crystallin isolated from glycated TSP (total lens soluble protein) had also increased AGE
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Garrison, McKinzie A; Crowhurst, Karin A
2014-01-01
HdeA is a periplasmic chaperone found in several gram-negative pathogenic bacteria that are linked to millions of cases of dysentery per year worldwide. After the protein becomes activated at low pH, it can bind to other periplasmic proteins, protecting them from aggregation when the bacteria travel through the stomach on their way to colonize the intestines. It has been argued that one of the major driving forces for HdeA activation is the protonation of aspartate and glutamate side chains. The goal for this study, therefore, was to investigate, at the atomic level, the structural impact of this charge neutralization on HdeA during the transition from near-neutral conditions to pH 3.0, in preparation for unfolding and activation of its chaperone capabilities. NMR spectroscopy was used to measure pKa values of Asp and Glu residues and monitor chemical shift changes. Measurements of R2/R1 ratios from relaxation experiments confirm that the protein maintains its dimer structure between pH 6.0 and 3.0. However, calculated correlation times and changes in amide protection from hydrogen/deuterium exchange experiments provide evidence for a loosening of the tertiary and quaternary structures of HdeA; in particular, the data indicate that the dimer structure becomes progressively weakened as the pH decreases. Taken together, these results provide insight into the process by which HdeA is primed to unfold and carry out its chaperone duties below pH 3.0, and it also demonstrates that neutralization of aspartate and glutamate residues is not likely to be the sole trigger for HdeA dissociation and unfolding. PMID:24375557
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Jeon, Eun Young; Choi, Bong-Hyuk; Jung, Dooyup; Hwang, Byeong Hee; Cha, Hyung Joon
2017-07-01
Skin scarring after deep dermal injuries is a major clinical problem due to the current therapies limited to established scars with poor understanding of healing mechanisms. From investigation of aberrations within the extracellular matrix involved in pathophysiologic scarring, it was revealed that one of the main factors responsible for impaired healing is abnormal collagen reorganization. Here, inspired by the fundamental roles of decorin, a collagen-targeting proteoglycan, in collagen remodeling, we created a scar-preventive collagen-targeting glue consisting of a newly designed collagen-binding mussel adhesive protein and a specific glycosaminoglycan. The collagen-targeting glue specifically bound to type I collagen in a dose-dependent manner and regulated the rate and the degree of fibrillogenesis. In a rat skin excisional model, the collagen-targeting glue successfully accelerated initial wound regeneration as defined by effective reepithelialization, neovascularization, and rapid collagen synthesis. Moreover, the improved dermal collagen architecture was demonstrated by uniform size of collagen fibrils, their regular packing, and a restoration of healthy tissue component. Collectively, our natural healing-inspired collagen-targeting glue may be a promising therapeutic option for improving the healing rate with high-quality and effective scar inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.
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The chaperone-histone partnership: for the greater good of histone traffic and chromatin plasticity.
Hondele, Maria; Ladurner, Andreas G
2011-12-01
Histones are highly positively charged proteins that wrap our genome. Their surface properties also make them prone to nonspecific interactions and aggregation. A class of proteins known as histone chaperones is dedicated to safeguard histones by aiding their proper incorporation into nucleosomes. Histone chaperones facilitate ordered nucleosome assembly and disassembly reactions through the formation of semi-stable histone-chaperone intermediates without requiring ATP, but merely providing a complementary protein surface for histones to dynamically interact with. Recurrent 'chaperoning' mechanisms involve the masking of the histone's positive charge and the direct blocking of crucial histone surface sites, including those required for H3-H4 tetramerization or the binding of nucleosomal DNA. This shielding prevents histones from engaging in premature or unwanted interactions with nucleic acids and other cellular components. In this review, we analyze recent structural studies on chaperone-histone interactions and discuss the implications of this vital partnership for nucleosome assembly and disassembly pathways. Copyright © 2011 Elsevier Ltd. All rights reserved.
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Miller, Benjamin F; Olesen, Jens L; Hansen, Mette; Døssing, Simon; Crameri, Regina M; Welling, Rasmus J; Langberg, Henning; Flyvbjerg, Allan; Kjaer, Michael; Babraj, John A; Smith, Kenneth; Rennie, Michael J
2005-01-01
We hypothesized that an acute bout of strenuous, non-damaging exercise would increase rates of protein synthesis of collagen in tendon and skeletal muscle but these would be less than those of muscle myofibrillar and sarcoplasmic proteins. Two groups (n = 8 and 6) of healthy young men were studied over 72 h after 1 h of one-legged kicking exercise at 67% of maximum workload (Wmax). To label tissue proteins in muscle and tendon primed, constant infusions of [1-13C]leucine or [1-13C]valine and flooding doses of [15N] or [13C]proline were given intravenously, with estimation of labelling in target proteins by gas chromatography–mass spectrometry. Patellar tendon and quadriceps biopsies were taken in exercised and rested legs at 6, 24, 42 or 48 and 72 h after exercise. The fractional synthetic rates of all proteins were elevated at 6 h and rose rapidly to peak at 24 h post exercise (tendon collagen (0.077% h−1), muscle collagen (0.054% h−1), myofibrillar protein (0.121% h−1), and sarcoplasmic protein (0.134% h−1)). The rates decreased toward basal values by 72 h although rates of tendon collagen and myofibrillar protein synthesis remained elevated. There was no tissue damage of muscle visible on histological evaluation. Neither tissue microdialysate nor serum concentrations of IGF-I and IGF binding proteins (IGFBP-3 and IGFBP-4) or procollagen type I N-terminal propeptide changed from resting values. Thus, there is a rapid increase in collagen synthesis after strenuous exercise in human tendon and muscle. The similar time course of changes of protein synthetic rates in different cell types supports the idea of coordinated musculotendinous adaptation. PMID:16002437
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Krajewska, Joanna; Arent, Zbigniew; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina
2018-04-18
Bacterial ClpB is an ATP-dependent Hsp100 chaperone that reactivates aggregated proteins in cooperation with the DnaK chaperone system and promotes survival of bacteria under stress conditions. A large number of publications also indicate that ClpB supports the virulence of bacteria, including a pathogenic spirochaete Leptospira interrogans responsible for leptospirosis in both animals and humans. However, the exact role of ClpB in bacterial pathogenicity remains poorly characterized. It can be assumed that ClpB, due to its role as the molecular chaperone, mediates refolding of essential bacterial proteins, including the known virulence factors, which may become prone to aggregation under infection-induced stresses. In this study, we identified putative substrates of ClpB from L. interrogans (ClpB Li ). For this purpose, we used a proteomic approach combining the ClpB-Trap affinity pull-down assays, Liquid chromatography-tandem mass spectrometry (LC-MS-MS/MS), and bioinformatics analyses. Most of the identified proteins were enzymes predominantly associated with major metabolic pathways like the tricarboxylic acid (TCA) cycle, glycolysis–gluconeogenesis and amino acid and fatty acid metabolism. Based on our proteomic study, we suggest that ClpB can support the virulence of L. interrogans by protecting the conformational integrity and catalytic activity of multiple metabolic enzymes, thus maintaining energy homeostasis in pathogen cells.
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Chaperones in maturation of molybdoenzymes: Why specific is better than general?
Lemaire, Olivier N; Bouillet, Sophie; Méjean, Vincent; Iobbi-Nivol, Chantal; Genest, Olivier
2017-03-04
Molybdoenzymes play essential functions in living organisms and, as a result, in various geochemical cycles. It is thus crucial to understand how these complex proteins become highly efficient enzymes able to perform a wide range of catalytic activities. It has been established that specific chaperones are involved during their maturation process. Here, we raise the question of the involvement of general chaperones acting in concert with dedicated chaperones or not.
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von Holstein, Isabella; von Tersch, Matthew; Coutu, Ashley N; Penkman, Kirsty E H; Makarewicz, Cheryl A; Collins, Matthew J
2018-01-23
The oxygen isotope composition of collagen proteins is a potential indicator of adult residential location, useful for provenancing in ecology, archaeology and forensics. In acidic solution, proteins can exchange O from carboxylic acid moieties with reagent O. This study investigated whether this exchange occurs during demineralisation and gelatinisation preparation of bone/ivory collagen. EDTA and HCl demineralisation or gelatinisation reagents were made up in waters with different δ 18 O values, and were used to extract collagen from four skeletal tissue samples. Aliquots of extracted collagen were exposed to two different atmospheric waters, at 120°C and ambient temperature, and subsequently dried in a vacuum oven at 40°C or by freeze drying. Sample δ 18 O values were measured by HT/EA pyrolysis-IRMS using a zero-blank autosampler. Collagen samples exchanged O with both reagent waters and atmospheric water, which altered sample δ 18 O values. Exchange with reagent waters occurred in all extraction methods, but was greater at lower pH. Damage to the collagen samples during extraction increased O exchange. The nature of exchange of O with atmospheric water depended on the temperature of exposure: kinetic fractionation of O was identified at 120°C but not at ambient temperature. Exchange was difficult to quantify due to high variability of δ 18 O value between experimental replicates. Studies of δ 18 O values in collagen proteins should avoid extraction methods using acid solutions. This article is protected by copyright. All rights reserved.
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Shanmuganathan, Meera; Britz-McKibbin, Philip
2012-10-02
Pharmacological chaperones (PCs) are small molecules that stabilize and promote protein folding. Enzyme inhibition is widely used for PC selection; however, it does not accurately reflect chaperone activity. We introduce a functional assay for characterization of PCs based on their capacity to restore enzyme activity that is abolished upon chemical denaturation. Dose-dependent activity curves were performed as a function of urea to assess the chaperone potency of various ligands to β-glucocerebrosidase as a model system. Restoration of enzyme activity upon denaturation allows direct screening of PCs for treatment of genetic disorders associated with protein deficiency, such as Gaucher disease.
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Collagen and related extracellular matrix proteins in atherosclerotic plaque development.
Shami, Annelie; Gonçalves, Isabel; Hultgårdh-Nilsson, Anna
2014-10-01
The structure, composition and turnover of the extracellular matrix (ECM) as well as cell-matrix interactions are crucial in the developing atherosclerotic plaque. There is a need for further insight into specific proteins in the ECM and their functions in the developing plaque, and during the last few years a number of publications have highlighted this very important field of research. These novel findings will be addressed in the present review. This review covers literature focused on collagen and ECM proteins interacting with collagen, and what their roles may be in plaque development. Acute myocardial infarction and stroke are common diseases that cause disability and mortality, and the underlying mechanism is often the rupture of a vulnerable atherosclerotic plaque. The vascular ECM and the tissue repair in the atherosclerotic lesion are important players in plaque progression. Understanding how specific proteins in the ECM interact with cells in the plaque and affect the fate of the plaque can lead to new treatments for cardiovascular disease.
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Hayashi, Teruo; Lewis, Abasha; Hayashi, Eri; Betenbaugh, Michael J.; Su, Tsung-Ping
2011-01-01
Molecular chaperones localized at the endoplasmic reticulum (ER) lumen constitutively or cellular stress-dependently associate with a variety of proteins to promote their proper folding or to inhibit protein misfolding. ER chaperones preferentially form large complexes with co-chaperones and/or misfolded proteins in a highly crowded cellular environment that often hampers their detection by immunocytochemistry (ICC). This study establishes an antigen retrieval (AR) protocol to improve the ICC detection of ER chaperones in cultured cells using widely available antibodies against synthetic peptides. Among ten different antigen retrieval/fixation conditions, only the AR with Tris-HCl (pH 9.5) containing 6 M urea (80 °C for 10 min) significantly improved the ICC detection of the novel ER chaperone sigma-1 receptor (Sig-1R) in Chinese hamster ovary cells. Extended fixation with 4% paraformaldehyde for 1 hr effectively preserved the morphology of the ER under the AR condition. This method greatly enhanced the signal-to-noise ratio in Sig-1R ICC, thus allowing for semi-quantitative detection of protein upregulation under ER stress. The AR similarly improved the ICC detection of a series of other major ER chaperones, including BiP/GRP78, GRP94, calnexin, calreticulin, ERp57, protein disulfide isomerase, and cyclophilin B. The improved ICC methodology using the urea AR at 80°C may improve ICC of ER molecules as well as visualization of ER structure and substructures. PMID:21573736
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Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins?
2016-01-01
RNA silencing is a eukaryote‐specific phenomenon in which microRNAs and small interfering RNAs degrade messenger RNAs containing a complementary sequence. To this end, these small RNAs need to be loaded onto an Argonaute protein (AGO protein) to form the effector complex referred to as RNA‐induced silencing complex (RISC). RISC assembly undergoes multiple and sequential steps with the aid of Hsc70/Hsp90 chaperone machinery. The molecular mechanisms for this assembly process remain unclear, despite their significance for the development of gene silencing techniques and RNA interference‐based therapeutics. This review dissects the currently available structures of AGO proteins and proposes models and hypotheses for RISC assembly, covering the conformation of unloaded AGO proteins, the chaperone‐assisted duplex loading, and the slicer‐dependent and slicer‐independent duplex separation. The differences in the properties of RISC between prokaryotes and eukaryotes will also be clarified. WIREs RNA 2016, 7:637–660. doi: 10.1002/wrna.1356 For further resources related to this article, please visit the WIREs website. PMID:27184117
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Nucleic acid chaperone activity of retroviral Gag proteins.
Rein, Alan
2010-01-01
Retrovirus particles in which the Gag protein has not yet been cleaved by the viral protease are termed immature particles. The viral RNA within these particles shows clear evidence of the action of a nucleic acid chaperone (NAC): the genomic RNA is dimeric, and a cellular tRNA molecule is annealed, by its 3' 18 nucleotides, to a complementary stretch in the viral RNA, in preparation for priming reverse transcription in the next round of infection. It seems very likely that the NAC that has catalyzed dimerization and tRNA annealing is the NC domain of the Gag protein itself. However, neither the dimeric linkage nor the tRNA:viral RNA complex has the same structure as those in mature virus particles: thus the conformational effects of Gag within the particles are not equivalent to those of the free NC protein present in mature particles. It is not known whether these dissimilarities reflect intrinsic differences in the NAC activities of Gag and NC, or limitations on Gag imposed by the structure of the immature particle. Analysis of the interactions of recombinant Gag proteins with nucleic acids is complicated by the fact that they result in assembly of virus-like particles. Nevertheless, the available data indicates that the affinity of Gag for nucleic acids can be considerably higher than that of free NC. This enhanced affinity may be due to contributions of the matrix domain, a positively charged region at the N-terminus of Gag; interactions of neighboring Gag molecules with each other may also increase the affinity due to cooperativity of the binding. Recombinant HIV-1 Gag protein clearly exhibits NAC activity. In two well-studied experimental systems, Gag was more efficient than NC, as its NAC effects could be detected at a significantly lower molar ratio of protein to nucleotide than with NC. In one system, binding of nucleic acid by the matrix domain of Gag retarded the Gag-induced annealing of two RNAs; this effect could be ameliorated by the competitive
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Getz, Jean; Lin, Dingbo; Medeiros, Denis M
2011-10-01
Copper is ferried in a cell complexed to chaperone proteins, and in the heart much copper is required for cytochrome c oxidase (Cox). It is not completely understood how copper status affects the levels of these proteins. Here we determined if dietary copper deficiency could up- or down-regulate select copper chaperone proteins and Cox subunits 1 and 4 in cardiac tissue of rats. Sixteen weanling male Long-Evans rats were randomized into treatment groups, one group receiving a copper-deficient diet (<1 mg Cu/kg diet) and one group receiving a diet containing adequate copper (6 mg Cu/kg diet) for 5 weeks. Hearts were removed, weighed, and non-myofibrillar proteins separated to analyze for levels of CCS, Sco1, Ctr1, Cox17, Cox1, and Cox4 by SDS-PAGE and Western blotting. No changes were observed in the concentrations of CTR1 and Cox17 between copper-adequate and copper-deficient rats. CCS and Sco1 were up-regulated and Cox1 and Cox4 were both down-regulated as a result of copper deficiency. These data suggest that select chaperone proteins and may be up-regulated, and Cox1 and 4 down-regulated, by a dietary copper deficiency, whereas others appear not to be affected by copper status.
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Yen, Edwin H. K.; Sodek, Jaro; Melcher, Antony H.
1979-01-01
Mature periodontal tissues from adult-mouse first mandibular molars were cultured in a continuous-flow organ-culture system which allowed the regulation of both ascorbic acid concentration and pO2 (oxygen partial pressure). Protein synthesis was measured by analysing the incorporation of [3H]proline into collagenous and non-collagenous proteins during the last 24h of a 2-day culture. At low pO2 [16.0kPa (approx. 120mmHg)] approx. 60% of protein-incorporated [3H]proline was found in collagenous proteins. However, it was evident that this collagen was considerably underhydroxylated. At high pO2 [56.0kPa (approx. 420mmHg)], both the amount of collagen deposited in the tissues and the degree of hydroxylation were increased considerably. In contrast, no significant effect on non-collagenous protein was observed. Tissues cultured at low pO2 for the first 48h were unable to respond to a subsequent increase in pO2 during the last 24h. Analysis of pepsin-solubilized collagen α-chains labelled with [14C]glycine demonstrated the synthesis of both type-I and type-III collagens by explants cultured for 48h at high pO2. Type-III collagen comprised 20–30% of the radioactivity in α-chains in both the periodontal ligament and the tissues of the alveolar process. The pattern of protein synthesis in the alveolar tissues at high pO2 was similar to that observed in these tissues in vivo. However, in the cultured periodontal ligament the proportions of non-collagenous proteins and type-III collagens were increased in comparison with the tissue in vivo. PMID:454369
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Egbert, M; Ruetze, M; Sattler, M; Wenck, H; Gallinat, S; Lucius, R; Weise, J M
2014-01-01
Periostin is a secreted 90kDa matricellular protein, which is predominantly expressed in collagen-rich tissues. Collagen is the most abundant protein in mammals and has great tensile strength. Recent investigations have shown that periostin influences collagen fibrillogenesis and biomechanical properties of murine connective tissues. We investigated the function of periostin concerning collagen homeostasis during intrinsic and extrinsic skin aging. For this purpose, human skin samples of young and old donors as well as samples of photoaged and sun-protected skin areas were analyzed for periostin expression. Using in vitro models, we determined the cell types responsible for periostin expression and performed functional analyses with periostin knockdown cells. TaqMan Real-Time PCR, UV irradiation, knockdown experiments, immunostaining, electron microscopy, collagen degradation assay, collagen crosslink analysis. Periostin expression is highest in the papillary dermis and downregulated during skin aging. Fibroblasts and non-follicular skin derived precursors were identified as main source for periostin expression in human skin. Periostin knockdown in fibroblasts has no effect on collagen expression, but results in an increased fibril diameter and aberrant collagen structure. This leads to an increased susceptibility of collagen toward proteases, whereas recombinant periostin protects collagen fibrils from degradation. Our data show that periostin plays an important role for proper collagen assembly and homeostasis. During skin aging periostin expression decreases and contributes to the phenotype of aged skin. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
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Ishikawa, Yoshihiro; Holden, Paul; Bächinger, Hans Peter
2017-10-20
Collagen is the most abundant protein in the extracellular matrix in humans and is critical to the integrity and function of many musculoskeletal tissues. A molecular ensemble comprising more than 20 molecules is involved in collagen biosynthesis in the rough endoplasmic reticulum. Two proteins, heat shock protein 47 (Hsp47/ SERPINH1 ) and 65-kDa FK506-binding protein (FKBP65/ FKBP10 ), have been shown to play important roles in this ensemble. In humans, autosomal recessive mutations in both genes cause similar osteogenesis imperfecta phenotypes. Whereas it has been proposed that Hsp47 and FKBP65 interact in the rough endoplasmic reticulum, there is neither clear evidence for this interaction nor any data regarding their binding affinities for each other. In this study using purified endogenous proteins, we examined the interaction between Hsp47, FKBP65, and collagen and also determined their binding affinities and functions in vitro Hsp47 and FKBP65 show a direct but weak interaction, and FKBP65 prefers to interact with Hsp47 rather than type I collagen. Our results suggest that a weak interaction between Hsp47 and FKBP65 confers mutual molecular stability and also allows for a synergistic effect during collagen folding. We also propose that Hsp47 likely acts as a hub molecule during collagen folding and secretion by directing other molecules to reach their target sites on collagens. Our findings may explain why osteogenesis imperfecta-causing mutations in both genes result in similar phenotypes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Requena, Jose M.; Montalvo, Ana M.; Fraga, Jorge
2015-01-01
Molecular chaperones are key components in the maintenance of cellular homeostasis and survival, not only during stress but also under optimal growth conditions. Folding of nascent polypeptides is supported by molecular chaperones, which avoid the formation of aggregates by preventing nonspecific interactions and aid, when necessary, the translocation of proteins to their correct intracellular localization. Furthermore, when proteins are damaged, molecular chaperones may also facilitate their refolding or, in the case of irreparable proteins, their removal by the protein degradation machinery of the cell. During their digenetic lifestyle, Leishmania parasites encounter and adapt to harsh environmental conditions, such as nutrient deficiency, hypoxia, oxidative stress, changing pH, and shifts in temperature; all these factors are potential triggers of cellular stress. We summarize here our current knowledge on the main types of molecular chaperones in Leishmania and their functions. Among them, heat shock proteins play important roles in adaptation and survival of this parasite against temperature changes associated with its passage from the poikilothermic insect vector to the warm-blooded vertebrate host. The study of structural features and the function of chaperones in Leishmania biology is providing opportunities (and challenges) for drug discovery and improving of current treatments against leishmaniasis. PMID:26167482
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Mitochondrial Chaperones in the Brain: Safeguarding Brain Health and Metabolism?
Castro, José Pedro; Wardelmann, Kristina; Grune, Tilman; Kleinridders, André
2018-01-01
The brain orchestrates organ function and regulates whole body metabolism by the concerted action of neurons and glia cells in the central nervous system. To do so, the brain has tremendously high energy consumption and relies mainly on glucose utilization and mitochondrial function in order to exert its function. As a consequence of high rate metabolism, mitochondria in the brain accumulate errors over time, such as mitochondrial DNA (mtDNA) mutations, reactive oxygen species, and misfolded and aggregated proteins. Thus, mitochondria need to employ specific mechanisms to avoid or ameliorate the rise of damaged proteins that contribute to aberrant mitochondrial function and oxidative stress. To maintain mitochondria homeostasis (mitostasis), cells evolved molecular chaperones that shuttle, refold, or in coordination with proteolytic systems, help to maintain a low steady-state level of misfolded/aggregated proteins. Their importance is exemplified by the occurrence of various brain diseases which exhibit reduced action of chaperones. Chaperone loss (expression and/or function) has been observed during aging, metabolic diseases such as type 2 diabetes and in neurodegenerative diseases such as Alzheimer's (AD), Parkinson's (PD) or even Huntington's (HD) diseases, where the accumulation of damage proteins is evidenced. Within this perspective, we propose that proper brain function is maintained by the joint action of mitochondrial chaperones to ensure and maintain mitostasis contributing to brain health, and that upon failure, alter brain function which can cause metabolic diseases.
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Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone
2010-01-01
Background Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. Results In this study, we present the 3.3 Å crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC1-151). Specifically, we observe a rotationally-symmetric "head-to- head" dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC1-151. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may transition between
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Adam, Philip R.; Patil, Mrinalini K.; Dickenson, Nicholas E.; Choudhari, Shyamal; Barta, Michael; Geisbrecht, Brian V.; Picking, Wendy L.; Picking, William D.
2012-01-01
Shigella flexneri uses its type III secretion system (T3SS) to promote invasion of human intestinal epithelial cells as the first step in causing shigellosis, a life threatening form of dysentery. The Shigella type III secretion apparatus (T3SA) consists of a basal body that spans the bacterial envelope and an exposed needle that injects effector proteins into target cells. The nascent Shigella T3SA needle is topped with a pentamer of the needle tip protein invasion plasmid antigen D (IpaD). Bile salts trigger recruitment of the first hydrophobic translocator protein, IpaB, to the tip complex where it senses contact with a host membrane. In the bacterial cytoplasm, IpaB exists in a complex with its chaperone IpgC. Several structures of IpgC have been solved and we recently reported the 2.1-Å crystal structure of the N-terminal domain (IpaB74.224) of IpaB. Like IpgC, the IpaB N-terminal domain exists as a homodimer in solution. We now report that when the two are mixed, these homodimers dissociate and form heterodimers having a nanomolar dissociation constant. This is consistent with the equivalent complexes co-purified after being co-expressed in E. coli. Fluorescence data presented here also indicate that the N-terminal domain of IpaB possesses two regions that appear to contribute additively to chaperone binding. It is also likely that the IpaB N terminus adopts an alternative conformation as a result of chaperone binding. The importance of these findings within the functional context of these proteins is discussed. PMID:22497344
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Aouacheria, Abdel; Geourjon, Christophe; Aghajari, Nushin; Navratil, Vincent; Deléage, Gilbert; Lethias, Claire; Exposito, Jean-Yves
2006-12-01
Collagens are thought to represent one of the most important molecular innovations in the metazoan line. Basement membrane type IV collagen is present in all Eumetazoa and was found in Homoscleromorpha, a sponge group with a well-organized epithelium, which may represent the first stage of tissue differentiation during animal evolution. In contrast, spongin seems to be a demosponge-specific collagenous protein, which can totally substitute an inorganic skeleton, such as in the well-known bath sponge. In the freshwater sponge Ephydatia mülleri, we previously characterized a family of short-chain collagens that are likely to be main components of spongins. Using a combination of sequence- and structure-based methods, we present evidence of remote homology between the carboxyl-terminal noncollagenous NC1 domain of spongin short-chain collagens and type IV collagen. Unexpectedly, spongin short-chain collagen-related proteins were retrieved in nonsponge animals, suggesting that a family related to spongin constitutes an evolutionary sister to the type IV collagen family. Formation of the ancestral NC1 domain and divergence of the spongin short-chain collagen-related and type IV collagen families may have occurred before the parazoan-eumetazoan split, the earliest divergence among extant animal phyla. Molecular phylogenetics based on NC1 domain sequences suggest distinct evolutionary histories for spongin short-chain collagen-related and type IV collagen families that include spongin short-chain collagen-related gene loss in the ancestors of Ecdyzosoa and of vertebrates. The fact that a majority of invertebrates encodes spongin short-chain collagen-related proteins raises the important question to the possible function of its members. Considering the importance of collagens for animal structure and substratum attachment, both families may have played crucial roles in animal diversification.
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Stetz, Gabrielle; Verkhivker, Gennady M
2015-01-01
Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations
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Stetz, Gabrielle; Verkhivker, Gennady M.
2015-01-01
Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations
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Patki, Jyoti M; Shah, Priyanka
2017-10-01
Microbial heat shock proteins (Hsps) play an important role in pathogenesis and development of resistance to existing drugs. New compounds that target microbial molecular chaperones have the potential of combating the challenge of anti-microbial resistance. The present study was aimed at assessing the employment of in vitro enzyme refolding assay to detect anti-chaperone activity of Neem ( Azadirachta indica ) extracts. Protein extracts of thermotolerant Escherichia coli cells were used as a source of Hsps or chaperones. Thermotolerance was found to be induced by pre-treating E. coli cells at 47 °C before subjecting them to a lethal temperature of 55 °C. This thermotolerance correlated with over-expression of specific proteins and reduced aggregation as evident from the SDS-PAGE profiles. Refolding assays of denatured enzymes exhibited 45% activity regain in presence of cell protein extracts containing chaperones compared to less than 5% regain in BSA negative controls. The chaperone activity was found to be ATP dependent. Addition of Neem extracts to refolding reaction mixtures distinctly reduced the activity regain (20%) in a dose dependent manner (500 and 1000 ppm). The negative influence of plant extract on refolding of the enzyme in the presence of chaperones gives evidence to its anti-chaperone activity. We propose that the employment of in vitro enzyme refolding assays will help not only to analyze the activity of known and putative chaperones but also to screen natural compounds for anti-microbial-Hsp activity.
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Exploring a new jellyfish collagen in the production of microparticles for protein delivery.
Calejo, M Teresa; Almeida, António J; Fernandes, Ana I
2012-01-01
A microparticulate protein delivery system was developed using collagen, from the medusa Catostylus tagi, as a polymeric matrix. Collagen microparticles (CMPs) were produced by an emulsification-gelation-solvent extraction method and a high loading efficiency was found for the entrapment of lysozyme and α-lactalbumin. CMPs were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The uncross-linked CMPs were spherical, rough-surfaced, presenting an estimated median size of 28 µm by laser diffraction. Upon cross-linking, particle size (9.5 µm) and size distribution were reduced. CMPs showed a moderate hydrophobic behaviour and a positive surface charge. Cross-linking also resulted in greater stability in water, allowing a slow release, as shown by in vitro experiments. The assessment of lysozyme's biological activity showed that the protein remained active throughout the encapsulation and cross-linking processes. In summary, the work herein described shows the potential use of a marine collagen in the production of microparticles for the controlled release of therapeutic proteins.
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Pradhan, Nibedita; Shekhar, Shashi; Jana, Nihar R; Jana, Nikhil R
2017-03-29
Sugar-based osmolyte molecules are known to stabilize proteins under stress, but usually they have poor chaperone performance in inhibiting protein aggregation. Here, we show that the nanoparticle form of sugars molecule can enhance their chaperone performance typically by 10 2 -10 5 times, compared to molecular sugar. Sugar-based plate-like nanoparticles of 20-40 nm hydrodynamic size have been synthesized by simple heating of acidic aqueous solution of glucose/sucrose/maltose/trehalose. These nanoparticles have excitation-dependent green/yellow/orange emission and surface chemistry identical to the respective sugar molecule. Fibrillation of lysozyme/insulin/amyloid beta in extracellular space, aggregation of mutant huntingtin protein inside model neuronal cell, and cytotoxic effect of fibrils are investigated in the presence of these sugar nanoparticles. We found that sugar nanoparticles are 10 2 -10 5 times efficient than respective sugar molecules in inhibiting protein fibrillation and preventing cytotoxicity arising of fibrils. We propose that better performance of the nanoparticle form is linked to its stronger binding with fibril structure and enhanced cell uptake. This result suggests that nanoparticle form of osmolyte can be an attractive option in prevention and curing of protein aggregation-derived diseases.
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Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex.
Campanello, Gregory C; Lofgren, Michael; Yokom, Adam L; Southworth, Daniel R; Banerjee, Ruma
2017-10-27
G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5'-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM-MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Raju, Murugesan; Santhoshkumar, Puttur; Sharma, K. Krishna
2012-01-01
Background A substitution mutation in human αA-crystallin (αAG98R) is associated with autosomal dominant cataract. The recombinant mutant αAG98R protein exhibits altered structure, substrate-dependent chaperone activity, impaired oligomer stability and aggregation on prolonged incubation at 37°C. Our previous studies have shown that αA-crystallin–derived mini-chaperone (DFVIFLDVKHFSPEDLTVK) functions like a molecular chaperone by suppressing the aggregation of denaturing proteins. The present study was undertaken to determine the effect of αA-crystallin–derived mini-chaperone on the stability and chaperone activity of αAG98R-crystallin. Methodology/Principal Findings Recombinant αAG98R was incubated in presence and absence of mini-chaperone and analyzed by chromatographic and spectrometric methods. Transmission electron microscope was used to examine the effect of mini-chaperone on the aggregation propensity of mutant protein. Mini-chaperone containing photoactive benzoylphenylalanine was used to confirm the interaction of mini-chaperone with αAG98R. The rescuing of chaperone activity in mutantα-crystallin (αAG98R) by mini-chaperone was confirmed by chaperone assays. We found that the addition of the mini-chaperone during incubation of αAG98R protected the mutant crystallin from forming larger aggregates that precipitate with time. The mini-chaperone-stabilized αAG98R displayed chaperone activity comparable to that of wild-type αA-crystallin. The complexes formed between mini-αA–αAG98R complex and ADH were more stable than the complexes formed between αAG98R and ADH. Western-blotting and mass spectrometry confirmed the binding of mini-chaperone to mutant crystallin. Conclusion/Significance These results demonstrate that mini-chaperone stabilizes the mutant αA-crystallin and modulates the chaperone activity of αAG98R. These findings aid in our understanding of how to design peptide chaperones that can be used to stabilize mutant
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Fries, Gabriel R; Gassen, Nils C; Rein, Theo
2017-12-05
Among the chaperones and co-chaperones regulating the glucocorticoid receptor (GR), FK506 binding protein (FKBP) 51 is the most intensely investigated across different disciplines. This review provides an update on the role of the different co-chaperones of Hsp70 and Hsp90 in the regulation of GR function. The development leading to the focus on FKBP51 is outlined. Further, a survey of the vast literature on the mechanism and function of FKBP51 is provided. This includes its structure and biochemical function, its regulation on different levels-transcription, post-transcription, and post-translation-and its function in signaling pathways. The evidence portraying FKBP51 as a scaffolding protein organizing protein complexes rather than a chaperone contributing to the folding of individual proteins is collated. Finally, FKBP51's involvement in physiology and disease is outlined, and the promising efforts in developing drugs targeting FKBP51 are discussed.
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Hurtado, Pilar Perez; O'Connor, Peter B
2012-03-20
Collagen is the major component of skin, tendons, ligaments, teeth, and bones, it provides the framework that holds most multicellular animals together, and collagen type I constitutes the major fibrillar collagen of bone. Because of the complexity of collagen's structure, the study of post-translational modifications such as deamidation for this protein is challenging. Although there is no evidence of this protein being used for age assessment, it has been shown that deamidation of collagen is remarkably increased in old bones from mammals. Nonspectrometric methodologies have been used for the determination of the extent of deamidation as a measure of the amount of amide nitrogen released in ammonia as well as constant rates for deamidation of asparagine in collagen. In general, these methodologies required more sample and separation processes. To understand if collagen plays a significant role in the aging process of fossil materials, a simpler and more accurate method is needed to determine the extent of deamidation at the whole protein level. The present work shows a method to determine the extent of deamidation in collagen using Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) along with collisionally activated dissociation (CAD) and electron capture dissociation (ECD). The measured deamidation half-life for three different tryptic peptides from collagen (I) ranged from 2000 to 6000 s under high temperature conditions (∼62 °C) and pH 7.5.
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Pharmacological chaperoning: a primer on mechanism and pharmacology.
Leidenheimer, Nancy J; Ryder, Katelyn G
2014-05-01
Approximately forty percent of diseases are attributable to protein misfolding, including those for which genetic mutation produces misfolding mutants. Intriguingly, many of these mutants are not terminally misfolded since native-like folding, and subsequent trafficking to functional locations, can be induced by target-specific, small molecules variably termed pharmacological chaperones, pharmacoperones, or pharmacochaperones (PCs). PC targets include enzymes, receptors, transporters, and ion channels, revealing the breadth of proteins that can be engaged by ligand-assisted folding. The purpose of this review is to provide an integrated primer of the diverse mechanisms and pharmacology of PCs. In this regard, we examine the structural mechanisms that underlie PC rescue of misfolding mutants, including the ability of PCs to act as surrogates for defective intramolecular interactions and, at the intermolecular level, overcome oligomerization deficiencies and dominant negative effects, as well as influence the subunit stoichiometry of heteropentameric receptors. Not surprisingly, PC-mediated structural correction of misfolding mutants normalizes interactions with molecular chaperones that participate in protein quality control and forward-trafficking. A variety of small molecules have proven to be efficacious PCs and the advantages and disadvantages of employing orthostatic antagonists, active-site inhibitors, orthostatic agonists, and allosteric modulator PCs are considered. Also examined is the possibility that several therapeutic agents may have unrecognized activity as PCs, and this chaperoning activity may mediate/contribute to therapeutic action and/or account for adverse effects. Lastly, we explore evidence that pharmacological chaperoning exploits intrinsic ligand-assisted folding mechanisms. Given the widespread applicability of PC rescue of mutants associated with protein folding disorders, both in vitro and in vivo, the therapeutic potential of PCs is vast
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Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate
2012-06-29
The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.
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Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R.; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate
2012-01-01
The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone. PMID:22573329
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A gatekeeper chaperone complex directs translocator secretion during Type Three Secretion
Archuleta, Tara L.; Spiller, Benjamin W.; Kubori, Tomoko
2014-11-06
Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ~20 individual protein components thatmore » form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Thus, structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.« less
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Histone chaperones: assisting histone traffic and nucleosome dynamics.
Gurard-Levin, Zachary A; Quivy, Jean-Pierre; Almouzni, Geneviève
2014-01-01
The functional organization of eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. Histone chaperones, which are proteins that escort histones throughout their cellular life, are key actors in all facets of histone metabolism; they regulate the supply and dynamics of histones at chromatin for its assembly and disassembly. Histone chaperones can also participate in the distribution of histone variants, thereby defining distinct chromatin landscapes of importance for genome function, stability, and cell identity. Here, we discuss our current knowledge of the known histone chaperones and their histone partners, focusing on histone H3 and its variants. We then place them into an escort network that distributes these histones in various deposition pathways. Through their distinct interfaces, we show how they affect dynamics during DNA replication, DNA damage, and transcription, and how they maintain genome integrity. Finally, we discuss the importance of histone chaperones during development and describe how misregulation of the histone flow can link to disease.
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Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.
Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan
2016-05-01
Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. © FASEB.
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The conformational dynamics of the mitochondrial Hsp70 chaperone.
Mapa, Koyeli; Sikor, Martin; Kudryavtsev, Volodymyr; Waegemann, Karin; Kalinin, Stanislav; Seidel, Claus A M; Neupert, Walter; Lamb, Don C; Mokranjac, Dejana
2010-04-09
Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments. 2010 Elsevier Inc. All rights reserved.
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van Lith, Marcel; Karala, Anna-Riikka; Bown, Dave; Gatehouse, John A.; Ruddock, Lloyd W.; Saunders, Philippa T.K.
2007-01-01
Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide Δ-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility. PMID:17507649
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Conversion of scFv peptide-binding specificity for crystal chaperone development
Pai, Jennifer C.; Culver, Jeffrey A.; Drury, Jason E.; Motani, Rakesh S.; Lieberman, Raquel L.; Maynard, Jennifer A.
2011-01-01
In spite of advances in protein expression and purification over the last decade, many proteins remain recalcitrant to structure determination by X-ray crystallography. One emerging tactic to obtain high-quality protein crystals for structure determination, particularly in the case of membrane proteins, involves co-crystallization with a protein-specific antibody fragment. Here, we report the development of new recombinant single-chain antibody fragments (scFv) capable of binding a specific epitope that can be introduced into internal loops of client proteins. The previously crystallized hexa-histidine-specific 3D5 scFv antibody was modified in the complementary determining region and by random mutagenesis, in conjunction with phage display, to yield scFvs with new biochemical characteristics and binding specificity. Selected variants include those specific for the hexa-histidine peptide with increased expression, solubility (up to 16.6 mg/ml) and sub-micromolar affinity, and those with new specificity for the EE hexa-peptide (EYMPME) and nanomolar affinity. Complexes of one such chaperone with model proteins harboring either an internal or a terminal EE tag were isolated by gel filtration. The 3.1 Å resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a ∼52 Å channel in the crystal lattice. Notably, in spite of 85% sequence identity, and nearly identical crystallization conditions, the engineered scFv crystallizes in a different space group than the parent 3D5 scFv, and utilizes two new crystal contacts. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries. PMID:21217145
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Characterization of the recombinant copper chaperone (CCS) from the plant Glycine (G.) max.
Sagasti, Sara; Yruela, Inmaculada; Bernal, Maria; Lujan, Maria A; Frago, Susana; Medina, Milagros; Picorel, Rafael
2011-02-01
The goal of the present work was to characterize the recombinant copper chaperone (CCS) from soybean. Very little is known about plant copper chaperones, which makes this study of current interest, and allows for a comparison with the better known homologues from yeast and humans. To obtain sizeable amounts of pure protein suitable for spectroscopic characterization, we cloned and overexpressed the G. max CCS chaperone in E. coli in the presence of 0.5 mM CuSO(4) and 0.5 mM ZnSO(4) in the broth. A pure protein preparation was obtained by using two IMAC steps and pH gradient chromatography. Most of the proteins were obtained as apo-form, devoid of copper atoms. The chaperone showed a high content (i.e., over 40%) of loops, turns and random coil as determined both by circular dichroism and homology modelling. The homology 3-D structural model suggests the protein might fold in three structural protein domains. The 3-D model along with the primary structure and spectroscopic data may suggest that copper atoms occupy the two metal binding sites, MKCEGC and CTC, within the N-terminal domain I and C-terminal domain III, respectively. But only one Zn-binding site was obtained spectroscopically.
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Molecular transformers in the cell: lessons learned from the DegP protease-chaperone.
Sawa, Justyna; Heuck, Alexander; Ehrmann, Michael; Clausen, Tim
2010-04-01
Structure-function analysis of DegP revealed a novel mechanism for protease and chaperone regulation. Binding of unfolded proteins induces the oligomer reassembly from the resting hexamer (DegP6) into the functional protease-chaperone DegP12/24. The newly formed cage exhibits the characteristics of a proteolytic folding chamber, shredding those proteins that are severely misfolded while stabilizing and protecting proteins present in their native state. Isolation of native DegP complexes with folded outer membrane proteins (OMPs) highlights the importance of DegP in OMP biogenesis. The encapsulated OMP beta-barrel is significantly stabilized in the hydrophobic chamber of DegP12/24 and thus DegP seems to employ a reciprocal mechanism to those chaperones assisting the folding of water soluble proteins via polar interactions. In addition, we discuss in this review similarities to other complex proteolytic machines that, like DegP, are under control of a substrate-induced or stress-induced oligomer conversion.
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Harris, Julia M; Nguyen, Phil P; Patel, Milan J; Sporn, Zachary A; Hines, Justin K
2014-07-01
Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or 'strains'. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have
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Patel, Milan J.; Sporn, Zachary A.; Hines, Justin K.
2014-01-01
Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or ‘strains’. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI +] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI +] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ +] prion propagation. In contrast, weak [PSI +] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ +] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI +]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI +]/[RNQ +] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI +] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion
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Chemical chaperone ameliorates pathological protein aggregation in plectin-deficient muscle
Winter, Lilli; Staszewska, Ilona; Mihailovska, Eva; Fischer, Irmgard; Goldmann, Wolfgang H.; Schröder, Rolf; Wiche, Gerhard
2014-01-01
The ubiquitously expressed multifunctional cytolinker protein plectin is essential for muscle fiber integrity and myofiber cytoarchitecture. Patients suffering from plectinopathy-associated epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) and mice lacking plectin in skeletal muscle display pathological desmin-positive protein aggregation and misalignment of Z-disks, which are hallmarks of myofibrillar myopathies (MFMs). Here, we developed immortalized murine myoblast cell lines to examine the pathogenesis of plectinopathies at the molecular and single cell level. Plectin-deficient myotubes, derived from myoblasts, were fully functional and mirrored the pathological features of EBS-MD myofibers, including the presence of desmin-positive protein aggregates and a concurrent disarrangement of the myofibrillar apparatus. Using this cell model, we demonstrated that plectin deficiency leads to increased intermediate filament network and sarcomere dynamics, marked upregulation of HSPs, and reduced myotube resilience following mechanical stretch. Currently, no specific therapy or treatment is available to improve plectin-related or other forms of MFMs; therefore, we assessed the therapeutic potential of chemical chaperones to relieve plectinopathies. Treatment with 4-phenylbutyrate resulted in remarkable amelioration of the pathological phenotypes in plectin-deficient myotubes as well as in plectin-deficient mice. Together, these data demonstrate the biological relevance of the MFM cell model and suggest that this model has potential use for the development of therapeutic approaches for EBS-MD. PMID:24487589
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C–C Bond formation catalyzed by natural gelatin and collagen proteins
Kühbeck, Dennis; Bijayi Dhar, Basab; Schön, Eva-Maria; Cativiela, Carlos; Gotor-Fernández, Vicente
2013-01-01
Summary The activity of gelatin and collagen proteins towards C–C bond formation via Henry (nitroaldol) reaction between aldehydes and nitroalkanes is demonstrated for the first time. Among other variables, protein source, physical state and chemical modification influence product yield and kinetics, affording the nitroaldol products in both aqueous and organic media under mild conditions. Significantly, the scale-up of the process between 4-nitrobenzaldehyde and nitromethane is successfully achieved at 1 g scale and in good yield. A comparative kinetic study with other biocatalysts shows an increase of the first-order rate constant in the order chitosan < gelatin < bovine serum albumin (BSA) < collagen. The results of this study indicate that simple edible gelatin can promote C–C bond forming reactions under physiological conditions, which may have important implications from a metabolic perspective. PMID:23843902
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Thapliyal, Charu; Jain, Neha; Rashid, Naira; Chaudhuri Chattopadhyay, Pratima
2018-01-01
The maintenance of thermal stability is a major issue in protein engineering as many proteins tend to form inactive aggregates at higher temperatures. Zebrafish DHFR, an essential protein for the survival of cells, shows irreversible thermal unfolding transition. The protein exhibits complete unfolding and loss of activity at 50 °C as monitored by UV-Visible, fluorescence and far UV-CD spectroscopy. The heat induced inactivation of zDHFR follows first-order kinetics and Arrhenius law. The variation in the value of inactivation rate constant, k with increasing temperatures depicts faster inactivation at elevated temperatures. We have attempted to study the chaperoning ability of a shorter variant of GroEL (minichaperone) and compared it with that of conventional GroEL-GroES chaperone system. Both the chaperone system prevented the aggregation and assisted in refolding of zDHFR. The rate of thermal inactivation was significantly retarded in the presence of chaperones which indicate that it enhances the thermal stability of the enzyme. As minichaperone is less complex, and does not require high energy co-factors like ATP, for its function as compared to conventional GroEL-GroES system, it can act as a very good in vitro as well as in vivo chaperone model for monitoring assisted protein folding phenomenon. Copyright © 2017 Elsevier Inc. All rights reserved.
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Collagen Quantification in Tissue Specimens.
Coentro, João Quintas; Capella-Monsonís, Héctor; Graceffa, Valeria; Wu, Zhuning; Mullen, Anne Maria; Raghunath, Michael; Zeugolis, Dimitrios I
2017-01-01
Collagen is the major extracellular protein in mammals. Accurate quantification of collagen is essential in the biomaterials (e.g., reproducible collagen scaffold fabrication), drug discovery (e.g., assessment of collagen in pathophysiologies, such as fibrosis), and tissue engineering (e.g., quantification of cell-synthesized collagen) fields. Although measuring hydroxyproline content is the most widely used method to quantify collagen in biological specimens, the process is very laborious. To this end, the Sircol™ Collagen Assay is widely used due to its inherent simplicity and convenience. However, this method leads to overestimation of collagen content due to the interaction of Sirius red with basic amino acids of non-collagenous proteins. Herein, we describe the addition of an ultrafiltration purification step in the process to accurately determine collagen content in tissues.
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Stoichevska, Violet; Peng, Yong Y; Vashi, Aditya V; Werkmeister, Jerome A; Dumsday, Geoff J; Ramshaw, John A M
2017-03-01
Recombinant bacterial collagens provide a new opportunity for safe biomedical materials. They are readily expressed in Escherichia coli in good yield and can be readily purified by simple approaches. However, recombinant proteins are limited in that direct secondary modification during expression is generally not easily achieved. Thus, inclusion of unusual amino acids, cyclic peptides, sugars, lipids, and other complex functions generally needs to be achieved chemically after synthesis and extraction. In the present study, we have illustrated that bacterial collagens that have had their sequences modified to include cysteine residue(s), which are not normally present in bacterial collagen-like sequences, enable a range of specific chemical modification reactions to be produced. Various model reactions were shown to be effective for modifying the collagens. The ability to include alkyne (or azide) functions allows the extensive range of substitutions that are available via "click" chemistry to be accessed. When bifunctional reagents were used, some crosslinking occurred to give higher molecular weight polymeric proteins, but gels were not formed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 806-813, 2017. © 2016 Wiley Periodicals, Inc.
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Wu, Xiaomeng; Liu, Yaowei; Liu, Anjun; Wang, Wenhang
2017-05-01
The inferior thermal- stability of collagen hinders its extensive application in food industry, including edible packaging. To improve the thermal- stability and mechanical properties of collagen, we attempted to crosslink collagen with some proteins possessing excellent thermal stability (i. e., casein, keratin and soy protein isolate (SPI)). Observed from the SDS- PAGE and particle size distribution, some complexes with higher molecule weight and relative bigger size particle occurred in the protein mixture, especially after TGase crosslinking. Importantly, the crosslinking greatly improved the thermal- stable property of protein complex, especially that of the collagen- casein complex judged from differential scanning calorimetric (DSC). Moreover, the crosslinking enhanced the mechanical properties of the combined films in terms of tensile strength (TS) and elongation at break (EAB). Also, some obvious differences in morphology of proteins before and after TGase crosslinking were observed by scanning electron microscopy (SEM). These impacts of TGase crosslinking with heat- resistant proteins on collagen features were associated with the conformational changes of the protein complex analyzed by Fourier transform infrared spectroscopy (FTIR). In conclusion, TGase crosslinking with higher thermally stable proteins could be an effective method to contribute to collagen' application in food packaging field. Copyright © 2017 Elsevier B.V. All rights reserved.
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Collagens--structure, function, and biosynthesis.
Gelse, K; Pöschl, E; Aigner, T
2003-11-28
The extracellular matrix represents a complex alloy of variable members of diverse protein families defining structural integrity and various physiological functions. The most abundant family is the collagens with more than 20 different collagen types identified so far. Collagens are centrally involved in the formation of fibrillar and microfibrillar networks of the extracellular matrix, basement membranes as well as other structures of the extracellular matrix. This review focuses on the distribution and function of various collagen types in different tissues. It introduces their basic structural subunits and points out major steps in the biosynthesis and supramolecular processing of fibrillar collagens as prototypical members of this protein family. A final outlook indicates the importance of different collagen types not only for the understanding of collagen-related diseases, but also as a basis for the therapeutical use of members of this protein family discussed in other chapters of this issue.
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Dabbaghizadeh, Afrooz; Finet, Stéphanie; Morrow, Genevieve; Moutaoufik, Mohamed Taha; Tanguay, Robert M
2017-07-01
The structure and chaperone function of DmHsp22WT, a small Hsp of Drosophila melanogaster localized within mitochondria were examined. Mutations of conserved arginine mutants within the alpha-crystallin domain (ACD) domain (R105G, R109G, and R110G) were introduced, and their effects on oligomerization and chaperone function were assessed. Arginine to glycine mutations do not induce significant changes in tryptophan fluorescence, and the mutated proteins form oligomers that are of equal or smaller size than the wild-type protein. They all form oligomer with one single peak as determined by size exclusion chromatography. While all mutants demonstrate the same efficiency as the DmHsp22WT in a DTT-induced insulin aggregation assay, all are more efficient chaperones to prevent aggregation of malate dehydrogenase. Arginine mutants of DmHsp22 are efficient chaperones to retard aggregation of CS and Luc. In summary, this study shows that mutations of arginine to glycine in DmHsp22 ACD induce a number of structural changes, some of which differ from those described in mammalian sHsps. Interestingly, only the R110G-DmHsp22 mutant, and not the expected R109G equivalent to human R140-HspB1, R116-HspB4, and R120-HspB5, showed different structural properties compared with the DmHsp22WT.
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Thiol-based copper handling by the copper chaperone Atox1.
Hatori, Yuta; Inouye, Sachiye; Akagi, Reiko
2017-04-01
Human antioxidant protein 1 (Atox1) plays a crucial role in cellular copper homeostasis. Atox1 captures cytosolic copper for subsequent transfer to copper pumps in trans Golgi network, thereby facilitating copper supply to various copper-dependent oxidereductases matured within the secretory vesicles. Atox1 and other copper chaperones handle cytosolic copper using Cys thiols which are ideal ligands for coordinating Cu(I). Recent studies demonstrated reversible oxidation of these Cys residues in copper chaperones, linking cellular redox state to copper homeostasis. Highlighted in this review are unique redox properties of Atox1 and other copper chaperones. Also, summarized are the redox nodes in the cytosol which potentially play dominant roles in the redox regulation of copper chaperones. © 2016 IUBMB Life, 69(4):246-254, 2017. © 2017 International Union of Biochemistry and Molecular Biology.
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Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain.
Sriram, M; Osipiuk, J; Freeman, B; Morimoto, R; Joachimiak, A
1997-03-15
The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes
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Structure-function studies on hsp47: pH-dependent inhibition of collagen fibril formation in vitro.
Thomson, C A; Ananthanarayanan, V S
2000-01-01
Hsp47, a 47 kDa heat shock protein whose expression level parallels that of collagen, has been regarded as a collagen-specific molecular chaperone. Studies from other laboratories have established the association of Hsp47 with the nascent as well as the triple-helical procollagen molecule in the endoplasmic reticulum and its dissociation from procollagen in the Golgi. One of several roles suggested for Hsp47 in collagen biosynthesis is the prevention of aggregation of procollagen in the endoplasmic reticulum. However, no experimental evidence has been available to verify this suggestion. In the present study we have followed the aggregation of mature triple-helical collagen molecules into fibrils by using turbidimetric measurements in the absence and presence of Hsp47. In the pH range 6-7, fibril formation of type I collagen, as monitored by turbidimetry, proceeds with a lag of approx. 10 min and levels off by approx. 60 min. The addition of Hsp47 at pH 7 effectively inhibits fibril formation at and above a 1:1 molar ratio of Hsp47 to triple-helical collagen. This inhibition is markedly pH-dependent, being significantly diminished at pH 6. CD and fluorescence spectral data of Hsp47 in the pH range 4.2-7.4 reveal a significant alteration in its structure at pH values below 6.2, with a decrease in alpha-helix and an increase in beta-structure. This conformational change is likely to be the basis of the decreased binding of Hsp47 to collagen in vitro at pH 6.3 as well as its inability to inhibit collagen fibril formation at this pH. Our results also provide a functional assay for Hsp47 that can be used in studies on collagen and Hsp47 interactions. PMID:10903151
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TRP and rhodopsin transport depends on dual XPORT ER chaperones encoded by an operon
Chen, Zijing; Chen, Hsiang-Chin; Montell, Craig
2015-01-01
Summary TRP channels and G protein-coupled receptors (GPCR) play critical roles in sensory reception. However, the identities of the chaperones that assist GPCRs in translocating from the endoplasmic reticulum (ER) are limited, and TRP ER chaperones are virtually unknown. The one exception for TRPs is Drosophila XPORT. Here, we show that the xport locus is bicistronic, and encodes unrelated transmembrane proteins, which enable the signaling proteins that initiate and culminate phototransduction, rhodopsin 1 (Rh1) and TRP, to traffic to the plasma membrane. XPORT-A and XPORT-B are ER proteins, and loss of either has a profound impact on TRP and Rh1 targeting to the light-sensing compartment of photoreceptor cells. XPORT-B complexed in vivo with the Drosophila homolog of the mammalian HSP70 protein, GRP78/BiP, which in turn associated with Rh1. Our work highlights a coordinated network of chaperones required for the biosynthesis of the TRP channel and rhodopsin in Drosophila photoreceptor cells. PMID:26456832
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Nandi, Sandip Kumar; Panda, Alok Kumar; Chakraborty, Ayon; Ray, Sougata Sinha; Biswas, Ashis
2015-01-01
Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31–43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25–43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min-1. Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18. PMID:26098662
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Phosphomimics destabilize Hsp27 oligomeric assemblies and enhance chaperone activity.
Jovcevski, Blagojce; Kelly, Megan A; Rote, Anthea P; Berg, Tracey; Gastall, Heidi Y; Benesch, Justin L P; Aquilina, J Andrew; Ecroyd, Heath
2015-02-19
Serine phosphorylation of the mammalian small heat-shock protein Hsp27 at residues 15, 78, and 82 is thought to regulate its structure and chaperone function; however, the site-specific impact has not been established. We used mass spectrometry to assess the combinatorial effect of mutations that mimic phosphorylation upon the oligomeric state of Hsp27. Comprehensive dimerization yielded a relatively uncrowded spectrum, composed solely of even-sized oligomers. Modification at one or two serines decreased the average oligomeric size, while the triple mutant was predominantly a dimer. These changes were reflected in a greater propensity for oligomers to dissociate upon increased modification. The ability of Hsp27 to prevent amorphous or fibrillar aggregation of target proteins was enhanced and correlated with the amount of dissociated species present. We propose that, in vivo, phosphorylation promotes oligomer dissociation, thereby enhancing chaperone activity. Our data support a model in which dimers are the chaperone-active component of Hsp27. Copyright © 2015 Elsevier Ltd. All rights reserved.
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CUCURBITACIN D IS A DISRUPTOR OF THE HSP90 CHAPERONE MACHINERY
Hall, Jessica A.; Seedarala, Sahithi; Rice, Nichole; Kopel, Lucas; Halaweish, Fathi; Blagg, Brian S. J.
2018-01-01
Heat shock protein 90 (Hsp90) facilitates the maturation of many newly synthesized and unfolded proteins (clients) via the Hsp90 chaperone cycle, in which Hsp90 forms a heteroprotein complex and relies upon co-chaperones, immunophilins, etc. for assistance in client folding. Hsp90 inhibition has emerged as a strategy for anticancer therapies due to the involvement of clients in many oncogenic pathways. Inhibition of chaperone function results in client ubiquitinylation and degradation via the proteasome, ultimately leading to tumor digression. Small molecule inhibitors perturb ATPase activity at the N-terminus and include derivatives of the natural product geldanamycin. However, N-terminal inhibition also leads to induction of the pro-survival heat shock response (HSR), in which displacement of the Hsp90-bound transcription factor, Heat Shock Factor-1 translocates to the nucleus and induces transcription of heat shock proteins, including Hsp90. An alternative strategy for Hsp90 inhibition is disruption of the Hsp90 heteroprotein complex. Disruption of the Hsp90 heteroprotein complex is an effective strategy to prevent client maturation without induction of the HSR. Cucurbitacin D, isolated from Cucurbita texana, and 3-epi-isocucurbitacin D prevented client maturation without induction of the HSR. Cucurbitacin D also disrupted interactions between Hsp90 and two co-chaperones, Cdc37 and p23. PMID:25756299
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Addi, Cyril; Murschel, Frederic; De Crescenzo, Gregory
2017-04-01
Collagen-based biomaterials are widely used in the field of tissue engineering; they can be loaded with biomolecules such as growth factors (GFs) to modulate the biological response of the host and thus improve its potential for regeneration. Recombinant chimeric GFs fused to a collagen-binding domain (CBD) have been reported to improve their bioavailability and the host response, especially when combined with an appropriate collagen-based biomaterial. This review first provides an extensive description of the various CBDs that have been fused to proteins, with a focus on the need for accurate characterization of their interaction with collagen. The second part of the review highlights the benefits of various CBD/GF fusion proteins that have been designed for wound healing and bone regeneration.
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Study of Receptor-Chaperone Interactions Using the Optical Technique of Spectroscopic Ellipsometry
Kriechbaumer, Verena; Tsargorodskaya, Anna; Mustafa, Mohd K.; Vinogradova, Tatiana; Lacey, Joanne; Smith, David P.; Abell, Benjamin M.; Nabok, Alexei
2011-01-01
This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms. PMID:21767504
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Thermosensitivity of growth is determined by chaperone-mediated proteome reallocation
Chen, Ke; Gao, Ye; Mih, Nathan; O’Brien, Edward J.; Yang, Laurence; Palsson, Bernhard O.
2017-01-01
Maintenance of a properly folded proteome is critical for bacterial survival at notably different growth temperatures. Understanding the molecular basis of thermoadaptation has progressed in two main directions, the sequence and structural basis of protein thermostability and the mechanistic principles of protein quality control assisted by chaperones. Yet we do not fully understand how structural integrity of the entire proteome is maintained under stress and how it affects cellular fitness. To address this challenge, we reconstruct a genome-scale protein-folding network for Escherichia coli and formulate a computational model, FoldME, that provides statistical descriptions of multiscale cellular response consistent with many datasets. FoldME simulations show (i) that the chaperones act as a system when they respond to unfolding stress rather than achieving efficient folding of any single component of the proteome, (ii) how the proteome is globally balanced between chaperones for folding and the complex machinery synthesizing the proteins in response to perturbation, (iii) how this balancing determines growth rate dependence on temperature and is achieved through nonspecific regulation, and (iv) how thermal instability of the individual protein affects the overall functional state of the proteome. Overall, these results expand our view of cellular regulation, from targeted specific control mechanisms to global regulation through a web of nonspecific competing interactions that modulate the optimal reallocation of cellular resources. The methodology developed in this study enables genome-scale integration of environment-dependent protein properties and a proteome-wide study of cellular stress responses. PMID:29073085
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The prenyl-binding protein PrBP/δ: a chaperone participating in intracellular trafficking
Zhang, Houbin; Constantine, Ryan; Frederick, Jeanne M.; Baehr, Wolfgang
2012-01-01
Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d−/− mouse. PMID:22960045
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Protein chaperones: a composition of matter review (2008 – 2013)
Taldone, Tony; Patel, Hardik J; Bolaender, Alexander; Patel, Maulik R; Chiosis, Gabriela
2014-01-01
Introduction Heat shock proteins (Hsps) are proteins with important functions in regulating disease phenotypes. Historically, Hsp90 has first received recognition as a target in cancer, with consequent efforts extending its potential role to other diseases. Hsp70 has also attracted interest as a therapeutic target for its role as a co-chaperone to Hsp90 as well as its own anti-apoptotic roles. Areas covered Herein, patents from 2008 to 2013 are reviewed to identify those that disclose composition of matter claimed to inhibit Hsp90 or Hsp70. Expert opinion For Hsp90, there has been considerable creativity in the discovery of novel pharmacophores that fall outside the three initially discovered scaffolds (i.e., ansamycins, resorcinols and purines). Nonetheless, much of the patent literature appears to build on previously reported structure activity relationship through slight modifications of Hsp90 inhibitor space by finding weaknesses in existing patents. The major goal of future development of Hsp90 inhibitors is not necessarily identifying better molecules but rather understanding how to rationally use these agents in the clinic. The development of Hsp70 inhibitors has lagged behind. It will require a more concerted effort from the drug discovery community in order to begin to realize the potential of this target. PMID:24742089
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Wachnowsky, Christine; Liu, Yushi; Yoon, Taejin; Cowan, J A
2018-01-01
Iron-sulfur cluster biogenesis is a complex, but highly regulated process that involves de novo cluster formation from iron and sulfide ions on a scaffold protein, and subsequent delivery to final targets via a series of Fe-S cluster-binding carrier proteins. The process of cluster release from the scaffold/carrier for transfer to the target proteins may be mediated by a dedicated Fe-S cluster chaperone system. In human cells, the chaperones include heat shock protein HSPA9 and the J-type chaperone Hsc20. While the role of chaperones has been somewhat clarified in yeast and bacterial systems, many questions remain over their functional roles in cluster delivery and interactions with a variety of human Fe-S cluster proteins. One such protein, Nfu, has recently been recognized as a potential interaction partner of the chaperone complex. Herein, we examined the ability of human Nfu to function as a carrier by interacting with the human chaperone complex. Human Nfu is shown to bind to both chaperone proteins with binding affinities similar to those observed for IscU binding to the homologous HSPA9 and Hsc20, while Nfu can also stimulate the ATPase activity of HSPA9. Additionally, the chaperone complex was able to promote Nfu function by enhancing the second-order rate constants for Fe-S cluster transfer to target proteins and providing directionality in cluster transfer from Nfu by eliminating promiscuous transfer reactions. Together, these data support a hypothesis in which Nfu can serve as an alternative carrier protein for chaperone-mediated cluster release and delivery in Fe-S cluster biogenesis and trafficking. © 2017 Federation of European Biochemical Societies.
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Structural variants of yeast prions show conformer-specific requirements for chaperone activity
Stein, Kevin C.; True, Heather L.
2016-01-01
Summary Molecular chaperones monitor protein homeostasis and defend against the misfolding and aggregation of proteins that is associated with protein conformational disorders. In these diseases, a variety of different aggregate structures can form. These are called prion strains, or variants, in prion diseases, and cause variation in disease pathogenesis. Here, we use variants of the yeast prions [RNQ+] and [PSI+] to explore the interactions of chaperones with distinct aggregate structures. We found that prion variants show striking variation in their relationship with Hsp40s. Specifically, the yeast Hsp40 Sis1, and its human ortholog Hdj1, had differential capacities to process prion variants, suggesting that Hsp40 selectivity has likely changed through evolution. We further show that such selectivity involves different domains of Sis1, with some prion conformers having a greater dependence on particular Hsp40 domains. Moreover, [PSI+] variants were more sensitive to certain alterations in Hsp70 activity as compared to [RNQ+] variants. Collectively, our data indicate that distinct chaperone machinery is required, or has differential capacity, to process different aggregate structures. Elucidating the intricacies of chaperone-client interactions, and how these are altered by particular client structures, will be crucial to understanding how this system can go awry in disease and contribute to pathological variation. PMID:25060529
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Mechanistic basis for the recognition of a misfolded protein by the molecular chaperone Hsp90.
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.
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Kumar, C Sudheer; Swamy, Musti J
2016-07-05
HSP-1/2, a major protein of horse seminal plasma binds to choline phospholipids present on the sperm plasma membrane and perturbs its structure by intercalating into the hydrophobic core, which results in an efflux of choline phospholipids and cholesterol, an important event in sperm capacitation. HSP-1/2 also exhibits chaperone-like activity (CLA) in vitro and protects target proteins against various kinds of stress. In the present study we show that HSP-1/2 exhibits destabilizing activity toward model supported and cell membranes. The membranolytic activity of HSP-1/2 is found to be pH dependent, with lytic activity being high at mildly acidic pH (6.0-6.5) and low at mildly basic pH (8.0-8.5). Interestingly, the CLA is also found to be pH dependent, with high activity at mildly basic pH and low activity at mildly acidic pH. Taken together the present studies demonstrate that the membranolytic and chaperone-like activities of HSP-1/2 have an inverse relationship and are regulated via a pH switch, which is reversible. The higher CLA observed at mildly basic pH could be correlated to an increase in surface hydrophobicity of the protein. To the best of our knowledge, this is the first study reporting regulation of two different activities of a chaperone protein by a pH switch.
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Moparthi, Satish Babu; Fristedt, Rikard; Mishra, Rajesh; Almstedt, Karin; Karlsson, Martin; Hammarström, Per; Carlsson, Uno
2010-02-16
The single-domain cyclophilin 18 (Cyp18) has long been known to function as a peptidyl-prolyl cis/trans isomerase (PPI) and was proposed by us to also function as a chaperone [Freskgard, P.-O., Bergenhem, N., Jonsson, B.-H., Svensson, M., and Carlsson, U. (1992) Science 258, 466-468]. Later several multidomain PPIs were demonstrated to work as both a peptidyl-prolyl cis/trans isomerase and a chaperone. However, the chaperone ability of Cyp18 has been debated. In this work, we add additional results that show that Cyp18 can both accelerate the rate of refolding and increase the yield of native protein during the folding reaction, i.e., function as both a folding catalyst and a chaperone. Refolding experiments were performed using severely destabilized mutants of human carbonic anhydrase II under conditions where the unfolding reaction is significant and a larger fraction of a more destabilized variant populates molten globule-like intermediates during refolding. A correlation of native state protein stability of the substrate protein versus Cyp18 chaperone activity was demonstrated. The induced correction of misfolded conformations by Cyp18 likely functions through rescue from misfolding of transient molten globule intermediates. ANS binding data suggest that the interaction by Cyp18 leads to an early stage condensation of accessible hydrophobic portions of the misfolding-prone protein substrate during folding. The opposite effect was observed for GroEL known as an unfoldase at early stages of refolding. The chaperone effect of Cyp18 was also demonstrated for citrate synthase, suggesting a general chaperone effect of this PPI.
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The coming of age of chaperone-mediated autophagy.
Kaushik, Susmita; Cuervo, Ana Maria
2018-06-01
Chaperone-mediated autophagy (CMA) was the first studied process that indicated that degradation of intracellular components by the lysosome can be selective - a concept that is now well accepted for other forms of autophagy. Lysosomes can degrade cellular cytosol in a nonspecific manner but can also discriminate what to target for degradation with the involvement of a degradation tag, a chaperone and a sophisticated mechanism to make the selected proteins cross the lysosomal membrane through a dedicated translocation complex. Recent studies modulating CMA activity in vivo using transgenic mouse models have demonstrated that selectivity confers on CMA the ability to participate in the regulation of multiple cellular functions. Timely degradation of specific cellular proteins by CMA modulates, for example, glucose and lipid metabolism, DNA repair, cellular reprograming and the cellular response to stress. These findings expand the physiological relevance of CMA beyond its originally identified role in protein quality control and reveal that CMA failure with age may aggravate diseases, such as ageing-associated neurodegeneration and cancer.
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Aoki, Koh; Kragler, Friedrich; Xoconostle-Cázares, Beatriz; Lucas, William J.
2002-01-01
Plasmodesmata establish a pathway for the trafficking of non-cell-autonomously acting proteins and ribonucleoprotein complexes. Plasmodesmal enriched cell fractions and the contents of enucleate sieve elements, in the form of phloem sap, were used to isolate and characterize heat shock cognate 70 (Hsc70) chaperones associated with this cell-to-cell transport pathway. Three Cucurbita maxima Hsc70 chaperones were cloned and functional and sequence analysis led to the identification of a previously uncharacterized subclass of non-cell-autonomous chaperones. The highly conserved nature of the heat shock protein 70 (Hsp70) family, in conjunction with mutant analysis, permitted the characterization of a motif that allows these Hsc70 chaperones to engage the plasmodesmal non-cell-autonomous translocation machinery. Proof of concept that this motif is necessary for Hsp70 gain-of-movement function was obtained through the engineering of a human Hsp70 that acquired the capacity to traffic through plasmodesmata. These results are discussed in terms of the roles likely played by this subclass of Hsc70 chaperones in the trafficking of non-cell-autonomous proteins. PMID:12456884
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NASA Astrophysics Data System (ADS)
Ernenwein, Dawn M.
2011-12-01
Bottom-up self-assembly of peptides has driven the research progress for the following two projects: protein delivery vehicles of collagen microflorettes and the assembly of gold nanoparticles with coiled-coil peptides. Collagen is the most abundant protein in the mammals yet due to immunogenic responses, batch-to-batch variability and lack of sequence modifications, synthetic collagen has been designed to self-assemble into native collagen-like structures. In particular with this research, metal binding ligands were incorporated on the termini of collagen-like peptides to generate micron-sized particles, microflorettes. The over-arching goal of the first research project is to engineer MRI-active microflorettes, loaded with His-tagged growth factors with differential release rates while bound to stem cells that can be implemented toward regenerative cell-based therapies. His-tagged proteins, such as green fluorescent protein, have successfully been incorporated on the surface and throughout the microflorettes. Protein release was monitored under physiological conditions and was related to particle degradation. In human plasma full release was obtained within six days. Stability of the microflorettes under physiological conditions was also examined for the development of a therapeutically relevant delivery agent. Additionally, MRI active microflorettes have been generated through the incorporation of a gadolinium binding ligand, DOTA within the collagen-based peptide sequence. To probe peptide-promoted self-assemblies of gold nanoparticles (GNPs) by non-covalent, charge complementary interactions, a highly anionic coiled-coil peptide was designed and synthesized. Upon formation of peptide-GNP interactions, the hydrophobic domain of the coiled-coil were shown to promote the self-assembly of peptide-GNPs clustering. Hydrophobic forces were found to play an important role in the assembly process, as a peptide with an equally overall negative charge, but lacking an
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Xu, Wen-Xiao; Liu, Sheng-Zhi; Wu, Di; Qiao, Guo-Fen; Yan, Jinglong
2015-01-01
Promyelocytic leukemia (PML) protein is a tumor suppressor that fuses with retinoic acid receptor-α (PML-RARα) to contribute to the initiation of acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) upregulates expression of TGF-β1, promoting collagen synthesis in osteoblasts, and ATO binds directly to PML to induce oligomerization, sumoylation, and ubiquitination. However, how ATO upregulates TGF-β1 expression is uncertain. Thus, we suggested that PML sumoylation is responsible for regulation of TGF-β1 protein expression. Kunming mice were treated with ATO, and osteoblasts were counted under scanning electron microscopy. Masson's staining was used to quantify collagen content. hFOB1.19 cells were transfected with siRNA against UBC9 or RNF4, and then treated with ATO or FBS. TGF-β1, PML expression, and sumoylation were quantified with Western blot, and collagen quantified via immunocytochemistry. ATO enhanced osteoblast accumulation, collagen synthesis, and PML-NB formation in vivo. Knocking down UBC9 in hFOB1.19 cells inhibited ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. Conversely, knocking down RNF4 enhanced ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. These data suggest that PML sumoylation is required for ATO-induced collagen synthesis in osteoblasts. © 2015 S. Karger AG, Basel.
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Dyer, Jamie O; Dutta, Arnob; Gogol, Madelaine; Weake, Vikki M; Dialynas, George; Wu, Xilan; Seidel, Christopher; Zhang, Ying; Florens, Laurence; Washburn, Michael P; Abmayr, Susan M; Workman, Jerry L
2017-06-30
Mutations that affect myelodysplasia/myeloid leukemia factor (MLF) proteins are associated with leukemia and several other cancers. However, with no strong homology to other proteins of known function, the role of MLF proteins in the cell has remained elusive. Here, we describe a proteomics approach that identifies MLF as a member of a nuclear chaperone complex containing a DnaJ protein, BCL2-associated anthanogene 2, and Hsc70. This complex associates with chromatin and regulates the expression of target genes. The MLF complex is bound to sites of nucleosome depletion and sites containing active chromatin marks (e.g., H3K4me3 and H3K4me1). Hence, MLF binding is enriched at promoters and enhancers. Additionally, the MLF-chaperone complex functions to regulate transcription factor stability, including the RUNX transcription factor involved in hematopoiesis. Although Hsc70 and other co-chaperones have been shown to play a role in nuclear translocation of a variety of proteins including transcription factors, our findings suggest that MLF and the associated co-chaperones play a direct role in modulating gene transcription. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Cabral, Wayne A.; Ishikawa, Masaki; Garten, Matthias; Makareeva, Elena N.; Sargent, Brandi M.; Weis, MaryAnn; Barnes, Aileen M.; Webb, Emma A.; Shaw, Nicholas J.; Ala-Kokko, Leena; Lacbawan, Felicitas L.; Högler, Wolfgang; Leikin, Sergey; Blank, Paul S.; Zimmerberg, Joshua; Eyre, David R.; Yamada, Yoshihiko; Marini, Joan C.
2016-01-01
Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50–70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes. PMID:27441836
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Bennion, Brian J; Daggett, Valerie
2004-04-27
Proteins are very sensitive to their solvent environments. Urea is a common chemical denaturant of proteins, yet some animals contain high concentrations of urea. These animals have evolved an interesting mechanism to counteract the effects of urea by using trimethylamine N-oxide (TMAO). The molecular basis for the ability of TMAO to act as a chemical chaperone remains unknown. Here, we describe molecular dynamics simulations of a small globular protein, chymotrypsin inhibitor 2, in 8 M urea and 4 M TMAO/8 M urea solutions, in addition to other control simulations, to investigate this effect at the atomic level. In 8 M urea, the protein unfolds, and urea acts in both a direct and indirect manner to achieve this effect. In contrast, introduction of 4 M TMAO counteracts the effect of urea and the protein remains well structured. TMAO makes few direct interactions with the protein. Instead, it prevents unfolding of the protein by structuring the solvent. In particular, TMAO orders the solvent and discourages it from competing with intraprotein H bonds and breaking up the hydrophobic core of the protein.
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Study of receptor-chaperone interactions using the optical technique of spectroscopic ellipsometry.
Kriechbaumer, Verena; Tsargorodskaya, Anna; Mustafa, Mohd K; Vinogradova, Tatiana; Lacey, Joanne; Smith, David P; Abell, Benjamin M; Nabok, Alexei
2011-07-20
This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.
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Karpel-Massler, Georg; Ishida, Chiaki Tsuge; Bianchetti, Elena; Shu, Chang; Perez-Lorenzo, Rolando; Horst, Basil; Banu, Matei; Roth, Kevin A; Bruce, Jeffrey N; Canoll, Peter; Altieri, Dario C; Siegelin, Markus D
2017-07-01
Rational therapeutic approaches based on synthetic lethality may improve cancer management. On the basis of a high-throughput drug screen, we provide preclinical proof of concept that targeting the mitochondrial Hsp90 chaperone network (mtHsp90) and inhibition of Bcl-2, Bcl-xL, and Mcl-1 is sufficient to elicit synthetic lethality in tumors recalcitrant to therapy. Our analyses focused on BH3 mimetics that are broad acting (ABT263 and obatoclax) or selective (ABT199, WEHI-539, and A1210477), along with the established mitochondrial matrix chaperone inhibitor gamitrinib-TPP. Drug combinations were tested in various therapy-resistant tumors in vitro and in vivo in murine model systems of melanoma, triple-negative breast cancer, and patient-derived orthotopic xenografts (PDX) of human glioblastoma. We found that combining BH3 mimetics and gamitrinib-TPP blunted cellular proliferation in a synergistic manner by massive activation of intrinsic apoptosis. In like manner, suppressing either Bcl-2, Bcl-xL, or Mcl-1 recapitulated the effects of BH3 mimetics and enhanced the effects of gamitrinib-TPP. Mechanistic investigations revealed that gamitrinib-TPP activated a PERK-dependent integrated stress response, which activated the proapoptotic BH3 protein Noxa and its downstream targets Usp9X and Mcl-1. Notably, in the PDX glioblastoma and BRAFi-resistant melanoma models, this drug combination safely and significantly extended host survival. Our results show how combining mitochondrial chaperone and Bcl-2 family inhibitors can synergize to safely degrade the growth of tumors recalcitrant to other treatments. Cancer Res; 77(13); 3513-26. ©2017 AACR . ©2017 American Association for Cancer Research.
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Demand, Jens; Lüders, Jens; Höhfeld, Jörg
1998-01-01
The modulation of the chaperone activity of the heat shock cognate Hsc70 protein in mammalian cells involves cooperation with chaperone cofactors, such as Hsp40; BAG-1; the Hsc70-interacting protein, Hip; and the Hsc70-Hsp90-organizing protein, Hop. By employing the yeast two-hybrid system and in vitro interaction assays, we have provided insight into the structural basis that underlies Hsc70’s cooperation with different cofactors. The carboxy-terminal domain of Hsc70, previously shown to form a lid over the peptide binding pocket of the chaperone protein, mediates the interaction of Hsc70 with Hsp40 and Hop. Remarkably, the two cofactors bind to the carboxy terminus of Hsc70 in a noncompetitive manner, revealing the existence of distinct binding sites for Hsp40 and Hop within this domain. In contrast, Hip interacts exclusively with the amino-terminal ATPase domain of Hsc70. Hence, Hsc70 possesses separate nonoverlapping binding sites for Hsp40, Hip, and Hop. This appears to enable the chaperone protein to cooperate simultaneously with multiple cofactors. On the other hand, BAG-1 and Hip have recently been shown to compete in binding to the ATPase domain. Our data thus establish the existence of a network of cooperating and competing cofactors regulating the chaperone activity of Hsc70 in the mammalian cell. PMID:9528774
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The prenyl-binding protein PrBP/δ: a chaperone participating in intracellular trafficking.
Zhang, Houbin; Constantine, Ryan; Frederick, Jeanne M; Baehr, Wolfgang
2012-12-15
Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d(-/-) mouse. Copyright © 2012 Elsevier Ltd. All rights reserved.
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In vitro reconstitution of chaperone-mediated human RISC assembly.
Naruse, Ken; Matsuura-Suzuki, Eriko; Watanabe, Mariko; Iwasaki, Shintaro; Tomari, Yukihide
2018-01-01
To silence target mRNAs, small RNAs and Argonaute (Ago) proteins need to be assembled into RNA-induced silencing complexes (RISCs). Although the assembly of Drosophila melanogaster RISC was recently reconstituted by Ago2, the Dicer-2/R2D2 heterodimer, and five chaperone proteins, the absence of a reconstitution system for mammalian RISC assembly has posed analytical challenges. Here we describe reconstitution of human RISC assembly using Ago2 and five recombinant chaperone proteins: Hsp90β, Hsc70, Hop, Dnaja2, and p23. Our data show that ATP hydrolysis by both Hsp90β and Hsc70 is required for RISC assembly of small RNA duplexes but not for that of single-stranded RNAs. The reconstitution system lays the groundwork for further studies of small RNA-mediated gene silencing in mammals. © 2018 Naruse et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
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Dixit, Anshuman; Verkhivker, Gennady M.
2012-01-01
Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based “conformational selection” of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected residue clusters
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Genetic and orthopedic aspects of collagen disorders.
Carter, Erin M; Raggio, Cathleen L
2009-02-01
'Collagens' are a family of structurally related proteins that play a wide variety of roles in the extracellular matrix. To date, there are at least 29 known types of collagen. Accordingly, abnormality in the various collagens produces a large category of diseases with heterogeneous symptoms. This review presents genetic and orthopedic aspects of type II, IX, and XI collagen disorders. Although a diverse group of conditions, mutation of collagens affecting the articular cartilage typically produces an epiphyseal skeletal dysplasia phenotype. Often, the ocular or auditory systems or both are also involved. Treatment of these collagenopathies is symptomatic and individualized. Study of tissue from animal models allows examination of mutation effects on the abnormal protein structure and function. The collagen superfamily comprises an important structural protein in mammalian connective tissue. Mutation of collagens produces a wide variety of genetic disorders, and those mutations affecting types II, IX, and XI collagens produce an overlapping spectrum of skeletal dysplasias. Findings range from lethal to mild, depending on the mutation of the collagen gene and its subsequent effect on the structure and/or metabolism of the resultant procollagen and/or collagen protein and its function in the body.
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Gioia, Roberta; Tonelli, Francesca; Ceppi, Ilaria; Biggiogera, Marco; Leikin, Sergey; Fisher, Shannon; Tenedini, Elena; Yorgan, Timur A; Schinke, Thorsten; Tian, Kun; Schwartz, Jean-Marc; Forte, Fabiana; Wagener, Raimund; Villani, Simona; Rossi, Antonio; Forlino, Antonella
2017-08-01
Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment. © The Author 2017. Published by Oxford University Press.
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Gioia, Roberta; Tonelli, Francesca; Ceppi, Ilaria; Biggiogera, Marco; Leikin, Sergey; Fisher, Shannon; Tenedini, Elena; Yorgan, Timur A.; Schinke, Thorsten; Tian, Kun; Schwartz, Jean-Marc; Forte, Fabiana; Wagener, Raimund; Villani, Simona; Rossi, Antonio; Forlino, Antonella
2017-01-01
Abstract Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment. PMID:28475764
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GP96 is a GARP chaperone and controls regulatory T cell functions.
Zhang, Yongliang; Wu, Bill X; Metelli, Alessandra; Thaxton, Jessica E; Hong, Feng; Rachidi, Saleh; Ansa-Addo, Ephraim; Sun, Shaoli; Vasu, Chenthamarakshan; Yang, Yi; Liu, Bei; Li, Zihai
2015-02-01
Molecular chaperones control a multitude of cellular functions via folding chaperone-specific client proteins. CD4+FOXP3+ Tregs play key roles in maintaining peripheral tolerance, which is subject to regulation by multiple molecular switches, including mTOR and hypoxia-inducible factor. It is not clear whether GP96 (also known as GRP94), which is a master TLR and integrin chaperone, controls Treg function. Using murine genetic models, we demonstrated that GP96 is required for Treg maintenance and function, as loss of GP96 resulted in instability of the Treg lineage and impairment of suppressive functions in vivo. In the absence of GP96, Tregs were unable to maintain FOXP3 expression levels, resulting in systemic accumulation of pathogenic IFN-γ-producing and IL-17-producing T cells. We determined that GP96 serves as an essential chaperone for the cell-surface protein glycoprotein A repetitions predominant (GARP), which is a docking receptor for latent membrane-associated TGF-β (mLTGF-β). The loss of both GARP and integrins on GP96-deficient Tregs prevented expression of mLTGF-β and resulted in inefficient production of active TGF-β. Our work demonstrates that GP96 regulates multiple facets of Treg biology, thereby placing Treg stability and immunosuppressive functions strategically under the control of a major stress chaperone.
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Targeting Hsp90 and its co-chaperones to treat Alzheimer’s disease
Blair, Laura J.; Sabbagh, Jonathan J.; Dickey, Chad A.
2015-01-01
Introduction Alzheimer’s disease (AD), characterized by the accumulation of hyperphosphorylated tau and beta amyloid (Aβ), currently lacks effective treatment. Chaperone proteins, such as the heat shock protein (Hsp) 90, form macromolecular complexes with co-chaperones, which can regulate tau metabolism and Aβ processing. While small molecule inhibitors of Hsp90 have been successful at ameliorating tau and Aβ burden, their development into drugs to treat disease has been slow due to the off- and on-target effects of this approach as well as challenges with the pharmacology of current scaffolds. Thus, other approaches are being developed to improve these compounds and to target co-chaperones of Hsp90 in an effort to limit these liabilities. Areas Covered This article discusses the most current developments in Hsp90 inhibitors including advances in blood-brain barrier permeability, decreased toxicity, and homolog-specific small molecule inhibitors. In addition, we discuss current strategies targeting Hsp90 co-chaperones rather than Hsp90 itself to reduce off-target effects. Expert Opinion While Hsp90 inhibitors have proven their efficacy at reducing tau pathology, they have yet to meet with success in the clinic. The development of Hsp90/tau complex specific inhibitors and further development of Hsp90 co-chaperone specific drugs should yield more potent, less toxic therapeutics. PMID:25069659
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The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro.
Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc
2008-06-01
The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44-61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.
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The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro
Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc
2008-01-01
The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44–61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties. PMID:18442994
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A passive physical model for DnaK chaperoning
NASA Astrophysics Data System (ADS)
Uhl, Lionel; Dumont, Audrey; Dukan, Sam
2018-03-01
Almost all living organisms use protein chaperones with a view to preventing proteins from misfolding or aggregation either spontaneously or during cellular stress. This work uses a reaction-diffusion stochastic model to describe the dynamic localization of the Hsp70 chaperone DnaK in Escherichia coli cells during transient proteotoxic collapse characterized by the accumulation of insoluble proteins. In the model, misfolded (‘abnormal’) proteins are produced during alcoholic stress and have the propensity to aggregate with a polymerization-like kinetics. When aggregates diffuse more slowly they grow larger. According to Michaelis-Menten-type kinetics, DnaK has the propensity to bind with misfolded proteins or aggregates in order to catalyse refolding. To match experimental fluorescence microscopy data showing clusters of DnaK-GFP localized in multiple foci, the model includes spatial zones with local reduced diffusion rates to generate spontaneous assemblies of DnaK called ‘foci’. Numerical simulations of our model succeed in reproducing the kinetics of DnaK localization experimentally observed. DnaK starts from foci, moves to large aggregates during acute stress, resolves those aggregates during recovery and finally returns to its initial punctate localization pattern. Finally, we compare real biological events with hypothetical repartitions of the protein aggregates or DnaK. We then notice that DnaK action is more efficient on protein aggregates than on protein homogeneously distributed.
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Chaperone substrate provides missing link for cancer drug discovery.
Byrd, Katherine M; Blagg, Brian S J
2018-02-16
Both Hsp70 and Hsp90 chaperones are overexpressed in cancer, making them relevant targets for the development of cancer chemotherapeutics, but a lack of biomolecular readouts for Hsp70 inhibition has limited the pursuit of specific inhibitors for this enzyme. A new study from Cesa et al. identifies two inhibitors of apoptosis proteins (IAPs) as specific client substrates of Hsp70. These results establish biomarkers that can be utilized to monitor Hsp70 inhibition and provide a framework for future efforts to deconvolute chaperone networks. © 2018 Byrd and Blagg.
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McElroy, Kerensa; Mouton, Laurence; Du Pasquier, Louis; Qi, Weihong; Ebert, Dieter
2011-09-01
Collagen-like proteins containing glycine-X-Y repeats have been identified in several pathogenic bacteria potentially involved in virulence. Recently, a collagen-like surface protein, Pcl1a, was identified in Pasteuria ramosa, a spore-forming parasite of Daphnia. Here we characterise 37 novel putative P. ramosa collagen-like protein genes (PCLs). PCR amplification and sequencing across 10 P. ramosa strains showed they were polymorphic, distinguishing genotypes matching known differences in Daphnia/P. ramosa interaction specificity. Thirty PCLs could be divided into four groups based on sequence similarity, conserved N- and C-terminal regions and G-X-Y repeat structure. Group 1, Group 2 and Group 3 PCLs formed triplets within the genome, with one member from each group represented in each triplet. Maximum-likelihood trees suggested that these groups arose through multiple instances of triplet duplication. For Group 1, 2, 3 and 4 PCLs, X was typically proline and Y typically threonine, consistent with other bacterial collagen-like proteins. The amino acid composition of Pcl2 closely resembled Pcl1a, with X typically being glutamic acid or aspartic acid and Y typically being lysine or glutamine. Pcl2 also showed sequence similarity to Pcl1a and contained a predicted signal peptide, cleavage site and transmembrane domain, suggesting that it is a surface protein. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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Thomson, Nicholas M.; Saika, Azusa; Ushimaru, Kazunori; Sangiambut, Smith; Tsuge, Takeharu; Summers, David K.
2013-01-01
The type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter−1), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter−1) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer. PMID:23335776
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Stability of silk and collagen protein materials in space.
Hu, Xiao; Raja, Waseem K; An, Bo; Tokareva, Olena; Cebe, Peggy; Kaplan, David L
2013-12-05
Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered.
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Kim, Young Kyung; Gu, Li-sha; Bryan, Thomas E.; Kim, Jong Ryul; Chen, Liang; Liu, Yan; Yoon, James C.; Breschi, Lorenzo; Pashley, David H.; Tay, Franklin R.
2010-01-01
The complex morphologies of mineralised collagen fibrils are regulated through interactions between the collagen matrix and non-collagenous extracellular proteins. In the present study, polyvinylphosphonic acid, a biomimetic analogue of matrix phosphoproteins, was synthesised and confirmed with FTIR and NMR. Biomimetic mineralisation of reconstituted collagen fibrils devoid of natural non-collagenous proteins was demonstrated with TEM using a Portland cement-containing resin composite and a phosphate-containing fluid in the presence of polyacrylic acid as sequestration, and polyvinylphosphonic acid as templating matrix protein analogues. In the presence of these dual biomimetic analogues in the mineralisation medium, intrafibrillar and extrafibrillar mineralisation via bottom-up nanoparticle assembly based on the nonclassical crystallisation pathway could be identified. Conversely, only large mineral spheres with no preferred association with collagen fibrils were observed in the absence of biomimetic analogues in the medium. Mineral phases were evident within the collagen fibrils as early as 4 hours after the initially-formed amorphous calcium phosphate nanoprecursors were transformed into apatite nanocrystals. Selected area electron diffraction patterns of highly mineralised collagen fibrils were nearly identical to those of natural bone, with apatite crystallites preferentially aligned along the collagen fibril axes. PMID:20621767
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Lee, Seung Sik; Jung, Hyun Suk; Park, Soo-Kwon; Lee, Eun Mi; Singh, Sudhir; Lee, Yuno; Lee, Kyun Oh; Lee, Sang Yeol; Chung, Byung Yeoup
2015-11-13
AtTDX, a thioredoxin-like plant-specific protein present in Arabidopsis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW) complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins.
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Metal chaperones prevent zinc-mediated cognitive decline.
Adlard, Paul A; Parncutt, Jacqui; Lal, Varsha; James, Simon; Hare, Dominic; Doble, Philip; Finkelstein, David I; Bush, Ashley I
2015-09-01
Zinc transporter-3 (ZnT3) protein is responsible for loading zinc into presynaptic vesicles and consequently controls the availability of zinc at the glutamatergic synapse. ZnT3 has been shown to decline with age and in Alzheimer's disease (AD) and is crucially involved in learning and memory. In this study, we utilised whole animal behavioural analyses in the ZnT3 KO mouse line, together with electrophysiological analysis of long-term potentiation in brain slices from ZnT3 KO mice, to show that metal chaperones (clioquinol, 30 mg/kg/day for 6weeks) can prevent the age-dependent cognitive phenotype that characterises these animals. This likely occurs as a result of a homeostatic restoration of synaptic protein expression, as clioquinol significantly restored levels of various pre- and postsynaptic proteins that are critical for normal cognition, including PSD-95; AMPAR and NMDAR2b. We hypothesised that this clioquinol-mediated restoration of synaptic health resulted from a selective increase in synaptic zinc content within the hippocampus. While we demonstrated a small regional increase in hippocampal zinc content using synchrotron x-ray fluorescence microscopy, further sub-region analyses are required to determine whether this effect is seen in other regions of the hippocampal formation that are more closely linked to the synaptic plasticity effects observed in this study. These data support our recent report on the use of a different metal chaperone (PBT2) to prevent normal age-related cognitive decline and demonstrate that metal chaperones are efficacious in preventing the zinc-mediated cognitive decline that characterises ageing and disease. Copyright © 2014 Elsevier Inc. All rights reserved.
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Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD.
Johnson, Steven; Roversi, Pietro; Espina, Marianela; Olive, Andrew; Deane, Janet E; Birket, Susan; Field, Terry; Picking, William D; Blocker, Ariel J; Galyov, Edouard E; Picking, Wendy L; Lea, Susan M
2007-02-09
Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.
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Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone
DOE Office of Scientific and Technical Information (OSTI.GOV)
Qualley, Dominic F., E-mail: dqualley@berry.edu; Sokolove, Victoria L.; Ross, James L.
2015-03-13
Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two othermore » retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable. - Highlights: • BLV NC binds strongly to DNA and RNA. • BLV NC promotes mini-TAR annealing as well as HIV-1 NC. • Annealing kinetics suggest a low degree of similarity between BLV NC and HTLV-1 NC.« less
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Counteracting chemical chaperone effects on the single-molecule α-synuclein structural landscape.
Ferreon, Allan Chris M; Moosa, Mahdi Muhammad; Gambin, Yann; Deniz, Ashok A
2012-10-30
Protein structure and function depend on a close interplay between intrinsic folding energy landscapes and the chemistry of the protein environment. Osmolytes are small-molecule compounds that can act as chemical chaperones by altering the environment in a cellular context. Despite their importance, detailed studies on the role of these chemical chaperones in modulating structure and dimensions of intrinsically disordered proteins have been limited. Here, we used single-molecule Förster resonance energy transfer to test the counteraction hypothesis of counterbalancing effects between the protecting osmolyte trimethylamine-N-oxide (TMAO) and denaturing osmolyte urea for the case of α-synuclein, a Parkinson's disease-linked protein whose monomer exhibits significant disorder. The single-molecule experiments, which avoid complications from protein aggregation, do not exhibit clear solvent-induced cooperative protein transitions for these osmolytes, unlike results from previous studies on globular proteins. Our data demonstrate the ability of TMAO and urea to shift α-synuclein structures towards either more compact or expanded average dimensions. Strikingly, the experiments directly reveal that a 21 [urea][TMAO] ratio has a net neutral effect on the protein's dimensions, a result that holds regardless of the absolute osmolyte concentrations. Our findings shed light on a surprisingly simple aspect of the interplay between urea and TMAO on α-synuclein in the context of intrinsically disordered proteins, with potential implications for the biological roles of such chemical chaperones. The results also highlight the strengths of single-molecule experiments in directly probing the chemical physics of protein structure and disorder in more chemically complex environments.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Orgel, Joseph P.R.O.; Eid, Aya; Antipova, Olga
Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule,more » which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e{sub 1} bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.« less
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Orgel, Joseph P R O; Eid, Aya; Antipova, Olga; Bella, Jordi; Scott, John E
2009-09-15
Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e(1) bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e(1) bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.
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Enigmatic insight into collagen
Deshmukh, Shrutal Narendra; Dive, Alka M; Moharil, Rohit; Munde, Prashant
2016-01-01
Collagen is a unique, triple helical molecule which forms the major part of extracellular matrix. It is the most abundant protein in the human body, representing 30% of its dry weight. It is the fibrous structural protein that makes up the white fibers (collagen fibers) of skin, tendons, bones, cartilage and all other connective tissues. Collagens are not only essential for the mechanical resistance and resilience of multicellular organisms, but are also signaling molecules defining cellular shape and behavior. The human body has at least 16 types of collagen, but the most prominent types are I, II and III. Collagens are produced by several cell types and are distinguishable by their molecular compositions, morphologic characteristics, distribution, functions and pathogenesis. This is the major fibrous glycoprotein present in the extracellular matrix and in connective tissue and helps in maintaining the structural integrity of these tissues. It has a triple helical structure. Various studies have proved that mutations that modify folding of the triple helix result in identifiable genetic disorders. Collagen diseases share certain similarities with autoimmune diseases, because autoantibodies specific to each collagen disease are produced. Therefore, this review highlights the role of collagen in normal health and also the disorders associated with structural and functional defects in collagen. PMID:27601823
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A thermodynamic assay to test pharmacological chaperones for Fabry disease.
Andreotti, Giuseppina; Citro, Valentina; Correra, Antonella; Cubellis, Maria Vittoria
2014-03-01
The majority of the disease-causing mutations affect protein stability, but not functional sites and are amenable, in principle, to be treated with pharmacological chaperones. These drugs enhance the thermodynamic stability of their targets. Fabry disease, a disorder caused by mutations in the gene encoding lysosomal alpha-galactosidase, represents an excellent model system to develop experimental protocols to test the efficiency of such drugs. The stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced unfolding followed by limited proteolysis and Western blotting. We measured the concentration of urea needed to obtain half-maximal unfolding because this parameter represents an objective indicator of protein stability. Urea-induced unfolding is a versatile technique that can be adapted to cell extracts containing tiny amounts of wild-type or mutant proteins. It allows testing of protein stability as a function of pH, in the presence or in the absence of drugs. Results are not influenced by the method used to express the protein in transfected cells. Scarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases. This is particularly true for pharmacological chaperones that must be tested on each mutation associated with a given disease. Diverse in vitro tests are needed. We used a method based on chemically induced unfolding as a tool to assess whether a particular Fabry mutation is responsive to pharmacological chaperones, but, by no means is our protocol limited to this disease. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.
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Counteracting chemical chaperone effects on the single-molecule α-synuclein structural landscape
Ferreon, Allan Chris M.; Moosa, Mahdi Muhammad; Deniz, Ashok A.
2012-01-01
Protein structure and function depend on a close interplay between intrinsic folding energy landscapes and the chemistry of the protein environment. Osmolytes are small-molecule compounds that can act as chemical chaperones by altering the environment in a cellular context. Despite their importance, detailed studies on the role of these chemical chaperones in modulating structure and dimensions of intrinsically disordered proteins have been limited. Here, we used single-molecule Förster resonance energy transfer to test the counteraction hypothesis of counterbalancing effects between the protecting osmolyte trimethylamine-N-oxide (TMAO) and denaturing osmolyte urea for the case of α-synuclein, a Parkinson’s disease-linked protein whose monomer exhibits significant disorder. The single-molecule experiments, which avoid complications from protein aggregation, do not exhibit clear solvent-induced cooperative protein transitions for these osmolytes, unlike results from previous studies on globular proteins. Our data demonstrate the ability of TMAO and urea to shift α-synuclein structures towards either more compact or expanded average dimensions. Strikingly, the experiments directly reveal that a 2∶1 [urea]∶[TMAO] ratio has a net neutral effect on the protein’s dimensions, a result that holds regardless of the absolute osmolyte concentrations. Our findings shed light on a surprisingly simple aspect of the interplay between urea and TMAO on α-synuclein in the context of intrinsically disordered proteins, with potential implications for the biological roles of such chemical chaperones. The results also highlight the strengths of single-molecule experiments in directly probing the chemical physics of protein structure and disorder in more chemically complex environments. PMID:22826265
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Stability of Silk and Collagen Protein Materials in Space
Hu, Xiao; Raja, Waseem K.; An, Bo; Tokareva, Olena; Cebe, Peggy; Kaplan, David L.
2013-01-01
Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered. PMID:24305951
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Role and mechanism of the Hsp70 molecular chaperone machines in bacterial pathogens.
Ghazaei, Ciamak
2017-03-01
Heat shock proteins are highly conserved, stress-inducible, ubiquitous proteins that maintain homeostasis in both eukaryotes and prokaryotes. Hsp70 proteins belong to the heat shock protein family and enhance bacterial survival in hostile environments. Hsp70, known as DnaK in prokaryotes, supports numerous processes such as the assembly and disassembly of protein complexes, the refolding of misfolded and clustered proteins, membrane translocation and the regulation of regulatory proteins. The chaperone-based activity of Hsp70 depends on dynamic interactions between its two domains, known as the ATPase domain and the substrate-binding domain. It also depends on interactions between these domains and other co-chaperone molecules such as the Hsp40 protein family member DnaJ and nucleotide exchange factors. DnaJ is the primary chaperone that interacts with nascent polypeptide chains and functions to prevent their premature release from the ribosome and misfolding before it is targeted by DnaK. Adhesion of bacteria to host cells is mediated by both host and bacterial Hsp70. Following infection of the host, bacterial Hsp70 (DnaK) is in a position to initiate bacterial survival processes and trigger an immune response by the host. Any mutations in the dnaK gene have been shown to decrease the viability of bacteria inside the host. This review will give insights into the structure and mechanism of Hsp70 and its role in regulating the protein activity that contributes to pathogenesis.
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Wang, Dongmao; Mohammad, Mardhiah; Wang, Yanyan; Tan, Rachel; Murray, Lydia S; Ricardo, Sharon; Dagher, Hayat; van Agtmael, Tom; Savige, Judy
2017-07-01
X-linked Alport syndrome (OMIM 301050) is caused by COL4A5 missense variants in 40% of families. This study examined the effects of chemical chaperone treatment (sodium 4-phenylbutyrate) on fibroblast cell lines derived from men with missense mutations. Dermal fibroblast cultures were established from 2 affected men and 3 normals. Proliferation rates were examined, the collagen IV α5 chain localized with immunostaining, and levels of the intra- and extracellular chains quantitated with an in-house enzyme-linked immunosorbent assay. COL4A5 mRNA was measured using quantitative reverse transcriptase polymerase chain reaction. Endoplasmic reticulum (ER) size was measured on electron micrographs and after HSP47 immunostaining. Markers of ER stress (ATF6, HSPA5, DDIT3), autophagy (ATG5, BECN1, ATG7), and apoptosis (CASP3, BAD, BCL 2 ) were also quantitated by quantitative reverse transcriptase polymerase chain reaction. Measurements were repeated after 48 hours of incubation with 10 mM sodium 4-phenylbutyrate acid. Both COL4A5 missense variants were associated with reduced proliferation rates on day 6 ( P = 0.01 and P = 0.03), ER enlargement, and increased mRNA for ER stress and autophagy (all P values < 0.05) when compared with normal. Sodium 4-phenylbutyrate treatment increased COL4A5 transcript levels ( P < 0.01), and reduced ER size ( P < 0.01 by EM and P < 0.001 by immunostaining), ER stress (p HSPA5 and DDIT3, all P values < 0.01) and autophagy (ATG7, P < 0.01). Extracellular collagen IV α5 chain was increased in the M1 line only ( P = 0.06). Sodium 4-phenylbutyrate increases collagen IV α5 mRNA levels, reduces ER stress and autophagy, and possibly facilitates collagen IV α5 extracellular transport. Whether these actions delay end-stage renal failure in men with X-linked Alport syndrome and missense mutations will only be determined with clinical trials.
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A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress
Judge, Luke M.; Perez-Bermejo, Juan A.; Truong, Annie; Ribeiro, Alexandre J.S.; Yoo, Jennie C.; Jensen, Christina L.; Mandegar, Mohammad A.; Huebsch, Nathaniel; Kaake, Robyn M.; So, Po-Lin; Srivastava, Deepak; Krogan, Nevan J.
2017-01-01
Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity. PMID:28724793
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A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress.
Judge, Luke M; Perez-Bermejo, Juan A; Truong, Annie; Ribeiro, Alexandre Js; Yoo, Jennie C; Jensen, Christina L; Mandegar, Mohammad A; Huebsch, Nathaniel; Kaake, Robyn M; So, Po-Lin; Srivastava, Deepak; Pruitt, Beth L; Krogan, Nevan J; Conklin, Bruce R
2017-07-20
Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.
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Fidler, Aaron L; Boudko, Sergei P; Rokas, Antonis; Hudson, Billy G
2018-04-09
The cellular microenvironment, characterized by an extracellular matrix (ECM), played an essential role in the transition from unicellularity to multicellularity in animals (metazoans), and in the subsequent evolution of diverse animal tissues and organs. A major ECM component are members of the collagen superfamily -comprising 28 types in vertebrates - that exist in diverse supramolecular assemblies ranging from networks to fibrils. Each assembly is characterized by a hallmark feature, a protein structure called a triple helix. A current gap in knowledge is understanding the mechanisms of how the triple helix encodes and utilizes information in building scaffolds on the outside of cells. Type IV collagen, recently revealed as the evolutionarily most ancient member of the collagen superfamily, serves as an archetype for a fresh view of fundamental structural features of a triple helix that underlie the diversity of biological activities of collagens. In this Opinion, we argue that the triple helix is a protein structure of fundamental importance in building the extracellular matrix, which enabled animal multicellularity and tissue evolution. © 2018. Published by The Company of Biologists Ltd.
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A Structure of a Collagen VI VWA Domain Displays N and C Termini at Opposite Sides of the Protein
Becker, Ann-Kathrin A.; Mikolajek, Halina; Paulsson, Mats; Wagener, Raimund; Werner, Jörn M.
2014-01-01
Summary Von Willebrand factor A (VWA) domains are versatile protein interaction domains with N and C termini in close proximity placing spatial constraints on overall protein structure. The 1.2 Å crystal structures of a collagen VI VWA domain and a disease-causing point mutant show C-terminal extensions that place the N and C termini at opposite ends. This allows a “beads-on-a-string” arrangement of multiple VWA domains as observed for ten N-terminal domains of the collagen VI α3 chain. The extension is linked to the core domain by a salt bridge and two hydrophobic patches. Comparison of the wild-type and a muscular dystrophy-associated mutant structure identifies a potential perturbation of a protein interaction interface and indeed, the secretion of mutant collagen VI tetramers is affected. Homology modeling is used to locate a number of disease-associated mutations and analyze their structural impact, which will allow mechanistic analysis of collagen-VI-associated muscular dystrophy phenotypes. PMID:24332716
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Chaperones in Polyglutamine Aggregation: Beyond the Q-Stretch
Kuiper, E. F. E.; de Mattos, Eduardo P.; Jardim, Laura B.; Kampinga, Harm H.; Bergink, Steven
2017-01-01
Expanded polyglutamine (polyQ) stretches in at least nine unrelated proteins lead to inherited neuronal dysfunction and degeneration. The expansion size in all diseases correlates with age at onset (AO) of disease and with polyQ protein aggregation, indicating that the expanded polyQ stretch is the main driving force for the disease onset. Interestingly, there is marked interpatient variability in expansion thresholds for a given disease. Between different polyQ diseases the repeat length vs. AO also indicates the existence of modulatory effects on aggregation of the upstream and downstream amino acid sequences flanking the Q expansion. This can be either due to intrinsic modulation of aggregation by the flanking regions, or due to differential interaction with other proteins, such as the components of the cellular protein quality control network. Indeed, several lines of evidence suggest that molecular chaperones have impact on the handling of different polyQ proteins. Here, we review factors differentially influencing polyQ aggregation: the Q-stretch itself, modulatory flanking sequences, interaction partners, cleavage of polyQ-containing proteins, and post-translational modifications, with a special focus on the role of molecular chaperones. By discussing typical examples of how these factors influence aggregation, we provide more insight on the variability of AO between different diseases as well as within the same polyQ disorder, on the molecular level. PMID:28386214
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Self-Chaperoning of the Type III Secretion System needle tip proteins IpaD and BipD
Johnson, Steven; Roversi, Pietro; Espina, Marianela; Olive, Andrew; Deane, Janet E.; Birket, Susan; Field, Terry; Picking, William D.; Blocker, Ariel; Galyov, Edouard E.; Picking, Wendy L.; Lea, Susan M.
2007-01-01
Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence; IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intra-molecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure. PMID:17077085
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Morton, Elizabeth A.; Cornwell, Adam B.; Crick, Beresford; Lamitina, Todd; Douglas, Peter M.
2017-01-01
An extensive proteostatic network comprised of molecular chaperones and protein clearance mechanisms functions collectively to preserve the integrity and resiliency of the proteome. The efficacy of this network deteriorates during aging, coinciding with many clinical manifestations, including protein aggregation diseases of the nervous system. A decline in proteostasis can be delayed through the activation of cytoprotective transcriptional responses, which are sensitive to environmental stress and internal metabolic and physiological cues. The homeodomain-interacting protein kinase (hipk) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole Caenorhabditis elegans Hipk homolog, hpk-1, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of hpk-1 is deleterious to these phenotypes. We show that HPK-1 preserves proteostasis and extends longevity through distinct but complementary genetic pathways defined by the heat shock transcription factor (HSF-1), and the target of rapamycin complex 1 (TORC1). We demonstrate that HPK-1 antagonizes sumoylation of HSF-1, a post-translational modification associated with reduced transcriptional activity in mammals. We show that inhibition of sumoylation by RNAi enhances HSF-1-dependent transcriptional induction of chaperones in response to heat shock. We find that hpk-1 is required for HSF-1 to induce molecular chaperones after thermal stress and enhances hormetic extension of longevity. We also show that HPK-1 is required in conjunction with HSF-1 for maintenance of proteostasis in the absence of thermal stress, protecting against the formation of polyglutamine (Q35::YFP) protein aggregates and associated locomotory toxicity. These functions of HPK-1/HSF-1 undergo rapid down-regulation once animals reach reproductive maturity. We show
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Fu, Xinmiao; Chang, Zengyi
2004-04-02
Small heat shock proteins (sHsps) usually exist as oligomers that undergo dynamic oligomeric dissociation/re-association, with the dissociated oligomers as active forms to bind substrate proteins under heat shock conditions. In this study, however, we found that Hsp16.3, one sHsp from Mycobacterium tuberculosis, is able to sensitively modulate its chaperone-like activity in a range of physiological temperatures (from 25 to 37.5 degrees C) while its native oligomeric size is still maintained. Further analysis demonstrated that Hsp16.3 exposes higher hydrophobic surfaces upon temperatures increasing and that a large soluble complex between Hsp16.3 and substrate is formed only in the condition of heating temperature up to 35 and 37.5 degrees C. Structural analysis by fluorescence anisotropy showed that Hsp16.3 nonameric structure becomes more dynamic and variable at elevated temperatures. Moreover, subunit exchange between Hsp16.3 oligomers was found to occur faster upon temperatures increasing as revealed by fluorescence energy resonance transfer. These observations indicate that Hsp16.3 is able to modulate its chaperone activity by adjusting the dynamics of oligomeric dissociation/re-association process while maintaining its static oligomeric size unchangeable. A kinetic model is therefore proposed to explain the mechanism of sHsps-binding substrate proteins through oligomeric dissociation. The present study also implied that Hsp16.3 is at least capable of binding non-native proteins in vivo while expressing in the host organism that survives at 37 degrees C.
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Rowe, D J; Ko, S; Tom, X M; Silverstein, S J; Richards, D W
1999-09-01
Vitamin C or ascorbate is important in wound healing due to its essential role in collagen synthesis. To study wound healing in the periodontium, cells adherent to expanded polytetrafluoroethylene (ePTFE) augmentation membranes, recovered from edentulous ridge augmentation procedures, have been established in culture in our laboratories. The objective of this study was to determine whether treatment of these cells with a calcium ascorbate, which contains vitamin C metabolites (metabolite-supplemented ascorbate), would increase the production of collagenous protein and mineralized tissue in vitro, as compared to unsupplemented calcium ascorbate (ascorbate). Cells derived from ePTFE membranes were cultured with beta-glycerophosphate and the test agents for 2 to 5 weeks, and the surface areas of the cell cultures occupied by mineralized nodules were measured using computerized image analysis. One experiment tested the effects of calcium threonate, one of the vitamin C metabolites in metabolite-supplemented ascorbate. Incorporation of radioactive proline and glycine was used as a measure of total protein (radioactivity precipitated by trichloracetic acid) and collagenase-digestible protein (radioactivity released by collagenase digestion.) Co-localization of collagen and fibronectin was examined by immunofluorescence. In vitro treatment of these cells with metabolite-supplemented ascorbate increased the area of the cell cultures occupied by mineralized nodules after 5 weeks. Cell cultures treated with metabolite-supplemented ascorbate also exhibited significant increases in total protein. The increase in collagenous proteins in these cultures accounted for 85% of the increase in total protein. The greatest difference between treatment groups was observed in the cell-associated fraction containing the extracellular matrix. The additional collagen exhibited normal co-distribution with fibronectin. In cultures treated with ascorbate spiked with calcium threonate, the area
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Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation
Zheng, Xu; Krakowiak, Joanna; Patel, Nikit; Beyzavi, Ali; Ezike, Jideofor; Khalil, Ahmad S; Pincus, David
2016-01-01
Heat shock factor (Hsf1) regulates the expression of molecular chaperones to maintain protein homeostasis. Despite its central role in stress resistance, disease and aging, the mechanisms that control Hsf1 activity remain unresolved. Here we show that in budding yeast, Hsf1 basally associates with the chaperone Hsp70 and this association is transiently disrupted by heat shock, providing the first evidence that a chaperone repressor directly regulates Hsf1 activity. We develop and experimentally validate a mathematical model of Hsf1 activation by heat shock in which unfolded proteins compete with Hsf1 for binding to Hsp70. Surprisingly, we find that Hsf1 phosphorylation, previously thought to be required for activation, in fact only positively tunes Hsf1 and does so without affecting Hsp70 binding. Our work reveals two uncoupled forms of regulation - an ON/OFF chaperone switch and a tunable phosphorylation gain - that allow Hsf1 to flexibly integrate signals from the proteostasis network and cell signaling pathways. DOI: http://dx.doi.org/10.7554/eLife.18638.001 PMID:27831465
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Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes.
Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko; Sakaguchi, Yuriko; Katsuma, Susumu; Suzuki, Tsutomu; Tomari, Yukihide
2010-07-30
Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway. Copyright 2010 Elsevier Inc. All rights reserved.
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Voloshin, Olga; Gocheva, Yana; Gutnick, Marina; Movshovich, Natalia; Bakhrat, Anya; Baranes-Bachar, Keren; Bar-Zvi, Dudy; Parvari, Ruti; Gheber, Larisa; Raveh, Dina
2010-06-01
Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR (leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds alpha-tubulin and promotes alpha/beta dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds alpha-tubulin and MTs, and functions in suppression of benomyl sensitivity of pac2Delta mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics. pac2Delta mutants are sensitive to misfolded protein stress. This is suppressed by ectopic PAC2 with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.
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Chaperone-mediated autophagy degrades mutant p53
Vakifahmetoglu-Norberg, Helin; Kim, Minsu; Xia, Hong-guang; Iwanicki, Marcin P.; Ofengeim, Dimitry; Coloff, Jonathan L.; Pan, Lifeng; Ince, Tan A.; Kroemer, Guido; Brugge, Joan S.; Yuan, Junying
2013-01-01
Missense mutations in the gene TP53, which encodes p53, one of the most important tumor suppressors, are common in human cancers. Accumulated mutant p53 proteins are known to actively contribute to tumor development and metastasis. Thus, promoting the removal of mutant p53 proteins in cancer cells may have therapeutic significance. Here we investigated the mechanisms that govern the turnover of mutant p53 in nonproliferating tumor cells using a combination of pharmacological and genetic approaches. We show that suppression of macroautophagy by multiple means promotes the degradation of mutant p53 through chaperone-mediated autophagy in a lysosome-dependent fashion. In addition, depletion of mutant p53 expression due to macroautophagy inhibition sensitizes the death of dormant cancer cells under nonproliferating conditions. Taken together, our results delineate a novel strategy for killing tumor cells that depend on mutant p53 expression by the activation of chaperone-mediated autophagy and potential pharmacological means to reduce the levels of accumulated mutant p53 without the restriction of mutant p53 conformation in quiescent tumor cells. PMID:23913924
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The chaperone action of bovine milk αS1- and αS2-caseins and their associated form αS-casein.
Treweek, Teresa M; Thorn, David C; Price, William E; Carver, John A
2011-06-01
α(S)-Casein, the major milk protein, comprises α(S1)- and α(S2)-casein and acts as a molecular chaperone, stabilizing an array of stressed target proteins against precipitation. Here, we report that α(S)-casein acts in a similar manner to the unrelated small heat-shock proteins (sHsps) and clusterin in that it does not preserve the activity of stressed target enzymes. However, in contrast to sHsps and clusterin, α-casein does not bind target proteins in a state that facilitates refolding by Hsp70. α(S)-Casein was also separated into α- and α-casein, and the chaperone abilities of each of these proteins were assessed with amorphously aggregating and fibril-forming target proteins. Under reduction stress, all α-casein species exhibited similar chaperone ability, whereas under heat stress, α-casein was a poorer chaperone. Conversely, α(S2)-casein was less effective at preventing fibril formation by modified κ-casein, whereas α- and α(S1)-casein were comparably potent inhibitors. In the presence of added salt and heat stress, α(S1)-, α- and α(S)-casein were all significantly less effective. We conclude that α(S1)- and α-casein stabilise each other to facilitate optimal chaperone activity of α(S)-casein. This work highlights the interdependency of casein proteins for their structural stability. Copyright © 2011 Elsevier Inc. All rights reserved.
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Fan, Ming-Hui; Zhu, Qiang; Li, Hui-Hua; Ra, Hyun-Jeong; Majumdar, Sonali; Gulick, Dexter L.; Jerome, Jacob A.; Madsen, Daniel H.; Christofidou-Solomidou, Melpo; Speicher, David W.; Bachovchin, William W.; Feghali-Bostwick, Carol; Puré, Ellen
2016-01-01
Idiopathic pulmonary fibrosis is a disease characterized by progressive, unrelenting lung scarring, with death from respiratory failure within 2–4 years unless lung transplantation is performed. New effective therapies are clearly needed. Fibroblast activation protein (FAP) is a cell surface-associated serine protease up-regulated in the lungs of patients with idiopathic pulmonary fibrosis as well as in wound healing and cancer. We postulate that FAP is not only a marker of disease but influences the development of pulmonary fibrosis after lung injury. In two different models of pulmonary fibrosis, intratracheal bleomycin instillation and thoracic irradiation, we find increased mortality and increased lung fibrosis in FAP-deficient mice compared with wild-type mice. Lung extracellular matrix analysis reveals accumulation of intermediate-sized collagen fragments in FAP-deficient mouse lungs, consistent with in vitro studies showing that FAP mediates ordered proteolytic processing of matrix metalloproteinase (MMP)-derived collagen cleavage products. FAP-mediated collagen processing leads to increased collagen internalization without altering expression of the endocytic collagen receptor, Endo180. Pharmacologic FAP inhibition decreases collagen internalization as expected. Conversely, restoration of FAP expression in the lungs of FAP-deficient mice decreases lung hydroxyproline content after intratracheal bleomycin to levels comparable with that of wild-type controls. Our findings indicate that FAP participates directly, in concert with MMPs, in collagen catabolism and clearance and is an important factor in resolving scar after injury and restoring lung homeostasis. Our study identifies FAP as a novel endogenous regulator of fibrosis and is the first to show FAP's protective effects in the lung. PMID:26663085
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Relationship between the structure of SET/TAF-Ibeta/INHAT and its histone chaperone activity.
Muto, Shinsuke; Senda, Miki; Akai, Yusuke; Sato, Lui; Suzuki, Toru; Nagai, Ryozo; Senda, Toshiya; Horikoshi, Masami
2007-03-13
Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-Ibeta/INHAT at a resolution of 2.3 A. We found that SET/TAF-Ibeta/INHAT formed a dimer that assumed a "headphone"-like structure. Each subunit of the SET/TAF-Ibeta/INHAT dimer consisted of an N terminus, a backbone helix, and an "earmuff" domain. It resembles the structure of the related protein NAP-1. Comparison of the crystal structures of SET/TAF-Ibeta/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by approximately 40 degrees . Our biochemical analyses of mutants revealed that the region of SET/TAF-Ibeta/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-Ibeta/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nucleosome structures.
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Bocian-Ostrzycka, Katarzyna M.; Łasica, Anna M.; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J.; Drabik, Karolina; Dobosz, Aneta M.; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K.
2015-01-01
Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation – periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity. PMID:26500620
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Bocian-Ostrzycka, Katarzyna M; Łasica, Anna M; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J; Drabik, Karolina; Dobosz, Aneta M; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K
2015-01-01
Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation - periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.
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Adsorption and separation of proteins by collagen fiber adsorbent.
Li, Juan; Liao, Xue-pin; Zhang, Qi-xian; Shi, Bi
2013-06-01
The separation of proteins is a key step in biomedical and pharmaceutical industries. In the present investigation, the collagen fiber adsorbent (CFA) was exploited as column packing material to separate proteins. Bovine serum albumin (BSA), bovine hemoglobin (Hb) and lysozyme (LYS) that have different isoelectric points (pIs) were selected as model proteins to investigate the separation ability of CFA to proteins. In batch adsorption, the adsorption behaviors of these proteins on CFA under different pHs and ionic strengths indicated that the electrostatic interaction plays a predominant role in the adsorption of proteins on CFA. CFA exhibited high adsorption capacity to Hb and LYS. In column separation, the proteins were completely separated by adjusting pH and ionic strength of the eluent. The increase of flow rate could reduce the separation time with no influence on the recovery of protein in the experimental range. The protein recovery was higher than 90% even when the CFA column was re-used for 4 times in separation of BSA and LYS, and the retention time of BSA or LYS was almost constant during the repeated applications. In addition, as a practical application, LYS was successfully separated from chicken egg white powder by CFA column. Copyright © 2013 Elsevier B.V. All rights reserved.
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Brachvogel, Bent; Zaucke, Frank; Dave, Keyur; Norris, Emma L; Stermann, Jacek; Dayakli, Münire; Koch, Manuel; Gorman, Jeffrey J; Bateman, John F; Wilson, Richard
2013-05-10
Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. Matrilin-4, collagen XII, thrombospondin-4, fibronectin, βig-h3, and epiphycan are components of the in vivo collagen IX interactome. We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level
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DOE Office of Scientific and Technical Information (OSTI.GOV)
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 ERmore » 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.« less
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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.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Sweeney, Shawn M.; Orgel, Joseph P.; Fertala, Andrzej
Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The 'cell interaction domain' is proposed to regulatemore » dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The 'matrix interaction domain' may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.« less
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Brachvogel, Bent; Zaucke, Frank; Dave, Keyur; Norris, Emma L.; Stermann, Jacek; Dayakli, Münire; Koch, Manuel; Gorman, Jeffrey J.; Bateman, John F.; Wilson, Richard
2013-01-01
The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGFβ-induced protein βig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the
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Gabus, C; Derrington, E; Leblanc, P; Chnaiderman, J; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W K; Marc, D; Nandi, P; Darlix, J L
2001-06-01
Transmissible spongiform encephalopathies are fatal neurodegenerative diseases associated with the accumulation of a protease-resistant form of the prion protein (PrP). Although PrP is conserved in vertebrates, its function remains to be identified. In vitro PrP binds large nucleic acids causing the formation of nucleoprotein complexes resembling human immunodeficiency virus type 1 (HIV-1) nucleocapsid-RNA complexes and in vivo MuLV replication accelerates the scrapie infectious process, suggesting possible interactions between retroviruses and PrP. Retroviruses, including HIV-1 encode a major nucleic acid binding protein (NC protein) found within the virus where 2000 NC protein molecules coat the dimeric genome. NC is required in virus assembly and infection to chaperone RNA dimerization and packaging and in proviral DNA synthesis by reverse transcriptase (RT). In HIV-1, 5'-leader RNA/NC interactions appear to control these viral processes. This prompted us to compare and contrast the interactions of human and ovine PrP and HIV-1 NCp7 with HIV-1 5'-leader RNA. Results show that PrP has properties characteristic of NCp7 with respect to viral RNA dimerization and proviral DNA synthesis by RT. The NC-like properties of huPrP map to the N-terminal region of huPrP. Interestingly, PrP localizes in the membrane and cytoplasm of PrP-expressing cells. These findings suggest that PrP is a multifunctional protein possibly participating in nucleic acid metabolism.
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Prion-Associated Toxicity is Rescued by Elimination of Cotranslational Chaperones
Keefer, Kathryn M.; True, Heather L.
2016-01-01
The nascent polypeptide-associated complex (NAC) is a highly conserved but poorly characterized triad of proteins that bind near the ribosome exit tunnel. The NAC is the first cotranslational factor to bind to polypeptides and assist with their proper folding. Surprisingly, we found that deletion of NAC subunits in Saccharomyces cerevisiae rescues toxicity associated with the strong [PSI+] prion. This counterintuitive finding can be explained by changes in chaperone balance and distribution whereby the folding of the prion protein is improved and the prion is rendered nontoxic. In particular, the ribosome-associated Hsp70 Ssb is redistributed away from Sup35 prion aggregates to the nascent chains, leading to an array of aggregation phenotypes that can mimic both overexpression and deletion of Ssb. This toxicity rescue demonstrates that chaperone modification can block key steps of the prion life cycle and has exciting implications for potential treatment of many human protein conformational disorders. PMID:27828954
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Jarnuczak, Andrew F.; Eyers, Claire E.; Schwartz, Jean‐Marc; Grant, Christopher M.
2015-01-01
Molecular chaperones play an important role in protein homeostasis and the cellular response to stress. In particular, the HSP70 chaperones in yeast mediate a large volume of protein folding through transient associations with their substrates. This chaperone interaction network can be disturbed by various perturbations, such as environmental stress or a gene deletion. Here, we consider deletions of two major chaperone proteins, SSA1 and SSB1, from the chaperone network in Sacchromyces cerevisiae. We employ a SILAC‐based approach to examine changes in global and local protein abundance and rationalise our results via network analysis and graph theoretical approaches. Although the deletions result in an overall increase in intracellular protein content, correlated with an increase in cell size, this is not matched by substantial changes in individual protein concentrations. Despite the phenotypic robustness to deletion of these major hub proteins, it cannot be simply explained by the presence of paralogues. Instead, network analysis and a theoretical consideration of folding workload suggest that the robustness to perturbation is a product of the overall network structure. This highlights how quantitative proteomics and systems modelling can be used to rationalise emergent network properties, and how the HSP70 system can accommodate the loss of major hubs. PMID:25689132
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A Novel Functional Role of Collagen Glycosylation
Jürgensen, Henrik J.; Madsen, Daniel H.; Ingvarsen, Signe; Melander, Maria C.; Gårdsvoll, Henrik; Patthy, Laszlo; Engelholm, Lars H.; Behrendt, Niels
2011-01-01
Collagens make up the most abundant component of interstitial extracellular matrices and basement membranes. Collagen remodeling is a crucial process in many normal physiological events and in several pathological conditions. Some collagen subtypes contain specific carbohydrate side chains, the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens. The molecular basis for this interaction is known to involve the fibronectin type II domain but nothing is known about the function of the lectin domains in this respect. In this study, we have investigated a possible role of the single active lectin domain of uPARAP/Endo180 in the interaction with collagens. By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate the endocytic efficiency of the receptor toward highly glycosylated collagens such as basement membrane collagen IV. Surprisingly, this property was not shared by the mannose receptor, which internalized glycosylated collagens independently of its lectin function. This role of modulating its uptake efficiency by a specific receptor is a previously unrecognized function of collagen glycosylation. PMID:21768090
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SUNAMOTO, MASAAKI; KUZE, KOGO; IEHARA, NORIYUKI; TAKEOKA, HIROYA; NAGATA, KAZUHIRO; KITA, TORU; DOI, TOSHIO
1998-01-01
Glomerulosclerosis is characterized by accumulation of the mesangial extracellular matrix, including type I and IV collagen. The processing for the collagens in the glomeruli may play a critical role for development of glomerulosclerosis. We examined the expression of heat shock protein 47 (HSP47), a collagen-binding molecular chaperone in the progresive glomerulosclerosis model. Subtotally nephrectomized rats, unlike sham-operated rats, developed focal and segmental glomerulosclerosis. Immunological staining demonstrated an increased expression of HSP47 which paralleled the expression of type I and IV collagen in the glomeruli of the nephrectomized rats as the glomerulosclerosis developed. The mRNA levels encoding type I and type IV collagen and HSP47 were increased 3.4 fold, 3.6 fold and 2.8 fold, respectively, at week 7 after nephrectomy. By in situ hybridization, the expression of HSP47 mRNA was determined to be localized to the glomeruli with segmental sclerosis. These results suggest that HSP47 may play a central role in the process of extracellular matrix accumulation during the development of glomerulosclerosis. PMID:9741355
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Kreikemeyer, Bernd; Nakata, Masanobu; Oehmcke, Sonja; Gschwendtner, Caroline; Normann, Jana; Podbielski, Andreas
2005-09-30
The Streptococcus pyogenes collagen type I-binding protein Cpa (collagen-binding protein of group A streptococci) expressed by 28 serotypes of group A streptococci has been extensively characterized at the gene and protein levels. Evidence for three distinct families of cpa genes was found, all of which shared a common sequence encoding a 60-amino acid domain that accounted for selective binding to type I collagen. Surface plasmon resonance-based affinity measurements and functional studies indicated that the expression of Cpa was consistent with an attachment role for bacteria to tissue containing collagen type I. A cpa mutant displayed a significantly decreased internalization rate when incubated with HEp-2 cells but had no effect on the host cell viability. By utilizing serum from patients with a positive titer for streptolysin/DNase antibody, an increased anti-Cpa antibody titer was noted for patients with a clinical history of arthritis or osteomyelitis. Taken together, these results suggest Cpa may be a relevant matrix adhesin contributing to the pathogenesis of S. pyogenes infection of bones and joints.
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Fan, Ming-Hui; Zhu, Qiang; Li, Hui-Hua; Ra, Hyun-Jeong; Majumdar, Sonali; Gulick, Dexter L; Jerome, Jacob A; Madsen, Daniel H; Christofidou-Solomidou, Melpo; Speicher, David W; Bachovchin, William W; Feghali-Bostwick, Carol; Puré, Ellen
2016-04-08
Idiopathic pulmonary fibrosis is a disease characterized by progressive, unrelenting lung scarring, with death from respiratory failure within 2-4 years unless lung transplantation is performed. New effective therapies are clearly needed. Fibroblast activation protein (FAP) is a cell surface-associated serine protease up-regulated in the lungs of patients with idiopathic pulmonary fibrosis as well as in wound healing and cancer. We postulate that FAP is not only a marker of disease but influences the development of pulmonary fibrosis after lung injury. In two different models of pulmonary fibrosis, intratracheal bleomycin instillation and thoracic irradiation, we find increased mortality and increased lung fibrosis in FAP-deficient mice compared with wild-type mice. Lung extracellular matrix analysis reveals accumulation of intermediate-sized collagen fragments in FAP-deficient mouse lungs, consistent within vitrostudies showing that FAP mediates ordered proteolytic processing of matrix metalloproteinase (MMP)-derived collagen cleavage products. FAP-mediated collagen processing leads to increased collagen internalization without altering expression of the endocytic collagen receptor, Endo180. Pharmacologic FAP inhibition decreases collagen internalization as expected. Conversely, restoration of FAP expression in the lungs of FAP-deficient mice decreases lung hydroxyproline content after intratracheal bleomycin to levels comparable with that of wild-type controls. Our findings indicate that FAP participates directly, in concert with MMPs, in collagen catabolism and clearance and is an important factor in resolving scar after injury and restoring lung homeostasis. Our study identifies FAP as a novel endogenous regulator of fibrosis and is the first to show FAP's protective effects in the lung. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Chaperone-Mediated Autophagy in the Kidney: The Road More Traveled
Franch, Harold A.
2014-01-01
Summary Chaperone-mediated autophagy (CMA) is a lysosomal proteolytic pathway in which cytosolic substrate proteins contain specific chaperone recognition sequences required for degradation and are translocated directly across the lysosomal membrane for destruction. CMA proteolytic activity has a reciprocal relationship with macroautophagy: CMA is most active in cells in which macroautophagy is least active. Normal renal proximal tubular cells have low levels of macroautophagy, but high basal levels of CMA activity. CMA activity is regulated by starvation, growth factors, oxidative stress, lipids, aging, and retinoic acid signaling. The physiological consequences of changes in CMA activity depend on the substrate proteins present in a given cell type. In the proximal tubule, increased CMA results from protein or calorie starvation and from oxidative stress. Overactivity of CMA can be associated with tubular lysosomal pathology and certain cancers. Reduced CMA activity contributes to protein accumulation in renal tubular hypertrophy, but may contribute to oxidative tissue damage in diabetes and aging. Although there are more questions than answers about the role of high basal CMA activity, this remarkable feature of tubular protein metabolism appears to influence a variety of chronic diseases. PMID:24485032
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Geller, Ron; Pechmann, Sebastian; Acevedo, Ashley; Andino, Raul; Frydman, Judith
2018-05-03
Acquisition of mutations is central to evolution; however, the detrimental effects of most mutations on protein folding and stability limit protein evolvability. Molecular chaperones, which suppress aggregation and facilitate polypeptide folding, may alleviate the effects of destabilizing mutations thus promoting sequence diversification. To illuminate how chaperones can influence protein evolution, we examined the effect of reduced activity of the chaperone Hsp90 on poliovirus evolution. We find that Hsp90 offsets evolutionary trade-offs between protein stability and aggregation. Lower chaperone levels favor variants of reduced hydrophobicity and protein aggregation propensity but at a cost to protein stability. Notably, reducing Hsp90 activity also promotes clusters of codon-deoptimized synonymous mutations at inter-domain boundaries, likely to facilitate cotranslational domain folding. Our results reveal how a chaperone can shape the sequence landscape at both the protein and RNA levels to harmonize competing constraints posed by protein stability, aggregation propensity, and translation rate on successful protein biogenesis.
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Collagen-Gold Nanoparticle Conjugates for Versatile Biosensing
Unser, Sarah; Holcomb, Samuel; Cary, ReJeana; Sagle, Laura
2017-01-01
Integration of noble metal nanoparticles with proteins offers promising potential to create a wide variety of biosensors that possess both improved selectivity and versatility. The multitude of functionalities that proteins offer coupled with the unique optical properties of noble metal nanoparticles can allow for the realization of simple, colorimetric sensors for a significantly larger range of targets. Herein, we integrate the structural protein collagen with 10 nm gold nanoparticles to develop a protein-nanoparticle conjugate which possess the functionality of the protein with the desired colorimetric properties of the nanoparticles. Applying the many interactions that collagen undergoes in the extracellular matrix, we are able to selectively detect both glucose and heparin with the same collagen-nanoparticle conjugate. Glucose is directly detected through the cross-linking of the collagen fibrils, which brings the attached nanoparticles into closer proximity, leading to a red-shift in the LSPR frequency. Conversely, heparin is detected through a competition assay in which heparin-gold nanoparticles are added to solution and compete with heparin in the solution for the binding sites on the collagen fibrils. The collagen-nanoparticle conjugates are shown to detect both glucose and heparin in the physiological range. Lastly, glucose is selectively detected in 50% mouse serum with the collagen-nanoparticle devices possessing a linear range of 3–25 mM, which is also within the physiologically relevant range. PMID:28212282
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Hernández-Ramírez, Laura C.; Morgan, Rhodri M.L.; Barry, Sayka; D’Acquisto, Fulvio; Prodromou, Chrisostomos; Korbonits, Márta
2018-01-01
Despite the well-recognized role of loss-of-function mutations of the aryl hydrocarbon receptor interacting protein gene (AIP) predisposing to pituitary adenomas, the pituitary-specific function of this tumor suppressor remains an enigma. To determine the repertoire of interacting partners for the AIP protein in somatotroph cells, wild-type and variant AIP proteins were used for pull-down/quantitative mass spectrometry experiments against lysates of rat somatotropinoma-derived cells; relevant findings were validated by co-immunoprecipitation and co-localization. Global gene expression was studied in AIP mutation positive and negative pituitary adenomas via RNA microarrays. Direct interaction with AIP was confirmed for three known and six novel partner proteins. Novel interactions with HSPA5 and HSPA9, together with known interactions with HSP90AA1, HSP90AB1 and HSPA8, indicate that the function/stability of multiple chaperone client proteins could be perturbed by a deficient AIP co-chaperone function. Interactions with TUBB, TUBB2A, NME1 and SOD1 were also identified. The AIP variants p.R304* and p.R304Q showed impaired interactions with HSPA8, HSP90AB1, NME1 and SOD1; p.R304* also displayed reduced binding to TUBB and TUBB2A, and AIP-mutated tumors showed reduced TUBB2A expression. Our findings suggest that cytoskeletal organization, cell motility/adhesion, as well as oxidative stress responses, are functions that are likely to be involved in the tumor suppressor activity of AIP. PMID:29507682
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Parmar, Paresh A.; St-Pierre, Jean-Philippe; Chow, Lesley W.; Puetzer, Jennifer L.; Stoichevska, Violet; Peng, Yong Y.; Werkmeister, Jerome A.; Ramshaw, John A. M.; Stevens, Molly M.
2017-01-01
Collagen I foams are used in the clinic as scaffolds to promote articular cartilage repair as they provide a bioactive environment for cells with chondrogenic potential. However, collagen I as a base material does not allow for precise control over bioactivity. Alternatively, recombinant bacterial collagens can be used as “blank slate” collagen molecules to offer a versatile platform for incorporation of selected bioactive sequences and fabrication into 3D scaffolds. Here, we show the potential of Streptococcal collagen-like 2 (Scl2) protein foams modified with peptides designed to specifically and noncovalently bind hyaluronic acid and chondroitin sulfate to improve chondrogenesis of human mesenchymal stem cells (hMSCs) compared to collagen I foams. Specific compositions of functionalized Scl2 foams lead to improved chondrogenesis compared to both nonfunctionalized Scl2 and collagen I foams, as indicated by gene expression, extracellular matrix accumulation, and compression moduli. hMSCs cultured in functionalized Scl2 foams exhibit decreased collagens I and X gene and protein expression, suggesting an advantage over collagen I foams in promoting a chondrocytic phenotype. These highly modular foams can be further modified to improve specific aspects chondrogenesis. As such, these scaffolds also have the potential to be tailored for other regenerative medicine applications. PMID:27219220
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Yadav, Ashok Kumar; Tyagi, Ashish; Kaushik, Jai Kumar; Saklani, Asha Chandola; Grover, Sunita; Batish, Virender Kumar
2013-12-14
Human feacal isolates were ascertain as genus Lactobacillus using specific primer LbLMA1/R16-1 and further identified as Lactobacillus plantarum with species specific primers Lpl-3/Lpl-2. 25 L. plantarum strains were further assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to immobilized human collagen type-1. Surface proteins were isolated from selected L. plantarum 91(Lp91) strain. The purified collagen binding protein (Cbp) protein was assessed for its anti-adhesion activity against enteric Escherichia coli 0157:H7 pathogen on immobilized collagen. Four L. plantarum strains displayed high degree of hydrophobicity and significant adhesion to collagen. A 72 kDa protein was purified which reduced 59.71% adhesion of E. coli 0157:H7 on immobilized collagen as compared to control well during adhesion assay. Cbp protein is the major influencing factor in inhibition of E. coli 0157:H7 adhesion with extracellular matrix (ECM) components. Hydrophobicity and adhesion potential are closely linked attributes precipitating in better colonization potential of the lactobacillus strains. Cbp is substantiated as a crucial surface protein contributing in adhesion of lactobacillus strains. The study can very well be the platform for commercialization of indigenous probiotic strain once their functional attributes are clinically explored. Copyright © 2013 Elsevier GmbH. All rights reserved.
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Chiang, T M; Wang, Y B; Kang, E S
2000-12-01
Nitric oxide plays an important role in platelet function and platelets possess the endothelial isoform of nitric oxide synthase. Several reports have indicated that nitric oxide is released upon exposure of platelets to collagen. We have reported that a non-integrin platelet protein of 65 kDa is a receptor for type I collagen. By direct measurement of NO release from washed human platelets suspended in Tyrode buffer with a ISO-NO Mark II, World Precision Instruments, Sarasota, FL, USA, p30 sensor, type I collagen, but not ADP and epinephrine, induces the release of NO in a time-dependent manner. The production of NO is inhibited either by preincubation of type I collagen with the platelet type I collagen receptor recombinant protein or by preincubation of platelets with the antibody to the receptor protein, the anti-65 antibody. However, preincubation of platelets with anti-P-selectin and anti-glycoprotein IIb/IIIa did not affect the release of NO by platelets. These results suggest that the 65 kDa platelet receptor for type I collagen is specifically linked to the generation of NO, and that the 65 kDa platelet receptor for type I collagen plays an important new role in platelet function.
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Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli
Veisi, Kamal; Farajnia, Safar; Zarghami, Nosratollah; Khoram Khorshid, Hamid Reza; Samadi, Nasser; Ahdi Khosroshahi, Shiva; Zarei Jaliani, Hossein
2015-01-01
Purpose: Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. Methods: To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. Results: SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. Conclusion: The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies. PMID:26793607
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Hoff, Kevin G.; Silberg, Jonathan J.; Vickery, Larry E.
2000-01-01
The iscU gene in bacteria is located in a gene cluster encoding proteins implicated in iron–sulfur cluster assembly and an hsc70-type (heat shock cognate) molecular chaperone system, iscSUA-hscBA. To investigate possible interactions between these systems, we have overproduced and purified the IscU protein from Escherichia coli and have studied its interactions with the hscA and hscB gene products Hsc66 and Hsc20. IscU and its iron–sulfur complex (IscU–Fe/S) stimulated the basal steady-state ATPase activity of Hsc66 weakly in the absence of Hsc20 but, in the presence of Hsc20, increased the ATPase activity up to 480-fold. Hsc20 also decreased the apparent Km for IscU stimulation of Hsc66 ATPase activity, and surface plasmon resonance studies revealed that Hsc20 enhances binding of IscU to Hsc66. Surface plasmon resonance and isothermal titration calorimetry further showed that IscU and Hsc20 form a complex, and Hsc20 may thereby aid in the targeting of IscU to Hsc66. These results establish a direct and specific role for the Hsc66/Hsc20 chaperone system in functioning with isc gene components for the assembly of iron–sulfur cluster proteins. PMID:10869428
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Bender, Tom; Lewrenz, Ilka; Franken, Sebastian; Baitzel, Catherina; Voos, Wolfgang
2011-01-01
Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria. PMID:21209324
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Brokopp, Chad E.; Schoenauer, Roman; Richards, Peter; Bauer, Stefan; Lohmann, Christine; Emmert, Maximilian Y.; Weber, Benedikt; Winnik, Stephan; Aikawa, Elena; Graves, Kirk; Genoni, Michele; Vogt, Peter; Lüscher, Thomas F.; Renner, Christoph; Hoerstrup, Simon P.; Matter, Christian M.
2011-01-01
Aims Collagen degradation in atherosclerotic plaques with thin fibrous caps renders them more prone to rupture. Fibroblast activation protein (FAP) plays a role in arthritis and tumour formation through its collagenase activity. However, the significance of FAP in thin-cap human fibroatheromata remains unknown. Methods and results We detected enhanced FAP expression in type IV–V human aortic atheromata (n = 12), compared with type II–III lesions (n = 9; P < 0.01) and healthy aortae (n = 8; P < 0.01) by immunostaining and western blot analyses. Fibroblast activation protein was also increased in thin-cap (<65 µm) vs. thick-cap (≥65 µm) human coronary fibroatheromata (n = 12; P < 0.01). Fibroblast activation protein was expressed by human aortic smooth muscle cells (HASMC) as shown by colocalization on immunofluorescent aortic plaque stainings (n = 10; P < 0.01) and by flow cytometry in cell culture. Although macrophages did not express FAP, macrophage burden in human aortic plaques correlated with FAP expression (n = 12; R2= 0.763; P < 0.05). Enzyme-linked immunosorbent assays showed a time- and dose-dependent up-regulation of FAP in response to human tumour necrosis factor α (TNFα) in HASMC (n = 6; P < 0.01). Moreover, supernatants from peripheral blood-derived macrophages induced FAP expression in cultured HASMC (n = 6; P < 0.01), an effect abolished by blocking TNFα (n = 6; P < 0.01). Fibroblast activation protein associated with collagen-poor regions in human coronary fibrous caps and digested type I collagen and gelatin in vitro (n = 6; P < 0.01). Zymography revealed that FAP-mediated collagenase activity was neutralized by an antibody directed against the FAP catalytic domain both in HASMC (n = 6; P < 0.01) and in fibrous caps of atherosclerotic plaques (n = 10; P < 0.01). Conclusion Fibroblast activation protein expression in HASMC is induced by macrophage-derived TNFα. Fibroblast activation protein associates with thin-cap human coronary
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Identification of a collagen type I adhesin of Bacteroides fragilis.
Galvão, Bruna P G V; Weber, Brandon W; Rafudeen, Mohamed S; Ferreira, Eliane O; Patrick, Sheila; Abratt, Valerie R
2014-01-01
Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼ 31 and ∼ 34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼ 31 kDa and the ∼ 34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼ 31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein.
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Identification of a Collagen Type I Adhesin of Bacteroides fragilis
Galvão, Bruna P. G. V.; Weber, Brandon W.; Rafudeen, Mohamed S.; Ferreira, Eliane O.; Patrick, Sheila; Abratt, Valerie R.
2014-01-01
Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼31 and ∼34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼31 kDa and the ∼34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein. PMID:24618940
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Haemorrhoids - a collagen disease?
Willis, S; Junge, K; Ebrahimi, R; Prescher, A; Schumpelick, V
2010-12-01
The cause of haemorrhoidal disease is unknown, epidemiological data and histopathological findings support the hypothesis that reduced connective tissue stability is associated with the incidence of haemorrhoids. Therefore the aim of this study was to analyse the quantity and quality of collagen formation in the corpus cavernosum recti in patients with III°/IV° haemorrhoids in comparison with persons without haemorrhoids. Haemorrhoidectomy specimens of 31 patients with III°/IV° haemorrhoids were examined. The specimens of 20 persons who died a natural death and who had no haemorrhoidal disease served as the controls. The amount of collagen was estimated photometrically by calculating the collagen/protein ratio. The collagen I/III ratio served as parameter for the quality of collagen formation and was calculated using cross polarization spectroscopy. Patients with haemorrhoids had a significantly reduced collagen/protein ratio (42.2 ± 16.2μg/mg vs 72.5±31.0μg/mg; P= 0.02) and a significantly reduced collagen I/III ratio (2.0±0.1 vs 4.6±0.3; P<0.001) compared with persons without haemorrhoidal disease. There was no correlation with patients' age or gender. There is a fundamental disorder of collagen metabolism in patients with haemorrhoidal disease. It remains unclear whether this is due to exogenous or endogenous influences. © 2010 The Authors. Colorectal Disease © 2010 The Association of Coloproctology of Great Britain and Ireland.
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Jarnuczak, Andrew F; Eyers, Claire E; Schwartz, Jean-Marc; Grant, Christopher M; Hubbard, Simon J
2015-09-01
Molecular chaperones play an important role in protein homeostasis and the cellular response to stress. In particular, the HSP70 chaperones in yeast mediate a large volume of protein folding through transient associations with their substrates. This chaperone interaction network can be disturbed by various perturbations, such as environmental stress or a gene deletion. Here, we consider deletions of two major chaperone proteins, SSA1 and SSB1, from the chaperone network in Sacchromyces cerevisiae. We employ a SILAC-based approach to examine changes in global and local protein abundance and rationalise our results via network analysis and graph theoretical approaches. Although the deletions result in an overall increase in intracellular protein content, correlated with an increase in cell size, this is not matched by substantial changes in individual protein concentrations. Despite the phenotypic robustness to deletion of these major hub proteins, it cannot be simply explained by the presence of paralogues. Instead, network analysis and a theoretical consideration of folding workload suggest that the robustness to perturbation is a product of the overall network structure. This highlights how quantitative proteomics and systems modelling can be used to rationalise emergent network properties, and how the HSP70 system can accommodate the loss of major hubs. © 2015 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Pea Chaperones under Centrifugation
NASA Astrophysics Data System (ADS)
Talalaiev, Oleksandr
2008-06-01
Etiolated Pisum sativum seedlings were subjected to altered g-forces by centrifugation (3-14g). By using semiquantitative RT-PCR, we studied transcripts of pea genes coding for chaperones that are representatives of small heat shock proteins (sHsps) family. Four members from the different classes of sHsps: cytosolic Hsp17.7 and Hsp18.1 (class I and class II accordingly), chloroplast Hsp21 (class III) and endoplasmic reticulum Hsp22.7 (class IV) were investigated. We conclude that exposure to 3, 7, 10 and 14g for 1h did not affect the level of sHsp transcripts.
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A new class of animal collagen masquerading as an insect silk
Sutherland, Tara D.; Peng, Yong Y.; Trueman, Holly E.; Weisman, Sarah; Okada, Shoko; Walker, Andrew A.; Sriskantha, Alagacone; White, Jacinta F.; Huson, Mickey G.; Werkmeister, Jerome A.; Glattauer, Veronica; Stoichevska, Violet; Mudie, Stephen T.; Haritos, Victoria S.; Ramshaw, John A. M.
2013-01-01
Collagen is ubiquitous throughout the animal kingdom, where it comprises some 28 diverse molecules that form the extracellular matrix within organisms. In the 1960s, an extracorporeal animal collagen that forms the cocoon of a small group of hymenopteran insects was postulated. Here we categorically demonstrate that the larvae of a sawfly species produce silk from three small collagen proteins. The native proteins do not contain hydroxyproline, a post translational modification normally considered characteristic of animal collagens. The function of the proteins as silks explains their unusual collagen features. Recombinant proteins could be produced in standard bacterial expression systems and assembled into stable collagen molecules, opening the door to manufacture a new class of artificial collagen materials. PMID:24091725
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The histone shuffle: histone chaperones in an energetic dance
Das, Chandrima; Tyler, Jessica K.; Churchill, Mair E.A.
2014-01-01
Our genetic information is tightly packaged into a rather ingenious nucleoprotein complex called chromatin in a manner that enables it to be rapidly accessed during genomic processes. Formation of the nucleosome, which is the fundamental unit of chromatin, occurs via a stepwise process that is reversed to enable the disassembly of nucleosomes. Histone chaperone proteins have prominent roles in facilitating these processes as well as in replacing old histones with new canonical histones or histone variants during the process of histone exchange. Recent structural, biophysical and biochemical studies have begun to shed light on the molecular mechanisms whereby histone chaperones promote chromatin assembly, disassembly and histone exchange to facilitate DNA replication, repair and transcription. PMID:20444609
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Supercharging Chaperones: A Meeting Toolkit for Maximizing Learning for Youth and Chaperones
ERIC Educational Resources Information Center
Brandt, Brian
2016-01-01
Trip and conference chaperones are a wonderful resource in youth development programs. These well-intended volunteers, many parents of youth participating in the event, want the best experience for the youth but are not necessarily trained in positive youth development. A consequence of this circumstance is that not all chaperones provide the best…
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Sweeney, Shawn M.; Orgel, Joseph P.; Fertala, Andrzej; McAuliffe, Jon D.; Turner, Kevin R.; Di Lullo, Gloria A.; Chen, Steven; Antipova, Olga; Perumal, Shiamalee; Ala-Kokko, Leena; Forlino, Antonella; Cabral, Wayne A.; Barnes, Aileen M.; Marini, Joan C.; Antonio, James D. San
2008-01-01
Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The “cell interaction domain” is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The “matrix interaction domain” may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging. PMID:18487200
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Rousseau, Julie; Gioia, Roberta; Layrolle, Pierre; Lieubeau, Blandine; Heymann, Dominique; Rossi, Antonio; Marini, Joan C; Trichet, Valerie; Forlino, Antonella
2014-01-01
Gene silencing approaches have the potential to become a powerful curative tool for a variety of monogenic diseases caused by gain-of-function mutations. Classical osteogenesis imperfecta (OI), a dominantly inherited bone dysplasia, is characterized in its more severe forms by synthesis of structurally abnormal type I collagen, which exerts a negative effect on extracellular matrix. Specific suppression of the mutant (Mut) allele would convert severe OI forms to the mild type caused by a quantitative defect in normal collagen. Here, we describe the in vitro and ex vivo investigation of a small interfering RNA (siRNA) approach to allele-specific gene silencing using Mut Col1a1 from the Brtl mouse, a well-characterized model for classical human OI. A human embryonic kidney cell line, which expresses the firefly luciferase gene, combined with either wild-type or Mut Brtl Col1a1 exon 23 sequences, was used for the first screening. The siRNAs selected based on their specificity and the corresponding short hairpin RNAs (shRNAs) subcloned in a lentiviral vector were evaluated ex vivo in Brtl fibroblasts for their effect on collagen transcripts and protein. A preferential reduction of the Mut allele of up to 52% was associated with about 40% decrease of the Mut protein, with no alteration of cell proliferation. Interestingly, a downregulation of HSP47, a specific collagen chaperone known to be upregulated in some OI cases, was detected. Our data support further testing of shRNAs and their delivery by lentivirus as a strategy to specifically suppress the Mut allele in mesenchymal stem cells of OI patients for autologous transplantation. PMID:24022296
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Structure and function of Hip, an attenuator of the Hsp70 chaperone cycle.
Li, Zhuo; Hartl, F Ulrich; Bracher, Andreas
2013-08-01
The Hsp70-interacting protein, Hip, cooperates with the chaperone Hsp70 in protein folding and prevention of aggregation. Hsp70 interacts with non-native protein substrates in an ATP-dependent reaction cycle regulated by J-domain proteins and nucleotide exchange factors (NEFs). Hip is thought to delay substrate release by slowing ADP dissociation from Hsp70. Here we present crystal structures of the dimerization domain and the tetratricopeptide repeat (TPR) domain of rat Hip. As shown in a cocrystal structure, the TPR core of Hip interacts with the Hsp70 ATPase domain through an extensive interface, to form a bracket that locks ADP in the binding cleft. Hip and NEF binding to Hsp70 are mutually exclusive, and thus Hip attenuates active cycling of Hsp70-substrate complexes. This mechanism explains how Hip enhances aggregation prevention by Hsp70 and facilitates transfer of specific proteins to downstream chaperones or the proteasome.
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2015-01-01
Biocomposite matrices with high mechanical strength, high stability, and the ability to direct matrix-specific stem cell differentiation are essential for the reconstruction of lesioned tissues in tissue engineering and cell therapeutics. Toward this end, we used the electrospinning technique to fabricate well-aligned composite fibers from collagen and spider dragline silk protein, obtained from the milk of transgenic goats, mimicking the native extracellular matrix (ECM) on a similar scale. Collagen and the dragline silk proteins were found to mix homogeneously at all ratios in the electrospun (E-spun) fibers. As a result, the ultimate tensile strength and elasticity of the fibers increased monotonically with silk percentage, whereas the stretchability was slightly reduced. Strikingly, we found that the incorporation of silk proteins to collagen dramatically increased the matrix stability against excessive fiber swelling and shape deformation in cell culture medium. When human decidua parietalis placental stem cells (hdpPSCs) were seeded on the collagen–silk matrices, the matrices were found to support cell proliferation at a similar rate as that of the pure collagen matrix, but they provided cell adhesion with reduced strengths and induced cell polarization at varied levels. Matrices containing 15 and 30 wt % silk in collagen (CS15, CS30) were found to induce a level of neural differentiation comparable to that of pure collagen. In particular, CS15 matrix induced the highest extent of cell polarization and promoted the development of extended 1D neural filaments strictly in-line with the aligned fibers. Taking the increased mechanical strength and fiber stability into consideration, CS15 and CS30 E-spun fibers offer better alternatives to pure collagen fibers as scaffolds that can be potentially utilized in neural tissue repair and the development of future nanobiodevices. PMID:25405355
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Banci, Lucia; Bertini, Ivano; Cefaro, Chiara; Cenacchi, Lucia; Ciofi-Baffoni, Simone; Felli, Isabella Caterina; Gallo, Angelo; Gonnelli, Leonardo; Luchinat, Enrico; Sideris, Dionisia; Tokatlidis, Kostas
2010-01-01
Several proteins of the mitochondrial intermembrane space are targeted by internal targeting signals. A class of such proteins with α-helical hairpin structure bridged by two intramolecular disulfides is trapped by a Mia40-dependent oxidative process. Here, we describe the oxidative folding mechanism underpinning this process by an exhaustive structural characterization of the protein in all stages and as a complex with Mia40. Two consecutive induced folding steps are at the basis of the protein-trapping process. In the first one, Mia40 functions as a molecular chaperone assisting α-helical folding of the internal targeting signal of the substrate. Subsequently, in a Mia40-independent manner, folding of the second substrate helix is induced by the folded targeting signal functioning as a folding scaffold. The Mia40-induced folding pathway provides a proof of principle for the general concept that internal targeting signals may operate as a folding nucleus upon compartment-specific activation. PMID:21059946
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The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum
DOE Office of Scientific and Technical Information (OSTI.GOV)
Bakkouri, Majida El; Pow, Andre; Mulichak, Anne
The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, weremore » also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.« less
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Liu, Xiaojun; Zeng, Shimei; Dong, Shaojian; Jin, Can; Li, Jiale
2015-01-01
In this study, we clone and characterize a novel matrix protein, hic31, from the mantle of Hyriopsis cumingii. The amino acid composition of hic31 consists of a high proportion of Glycine residues (26.67%). Tissue expression detection by RT-PCR indicates that hic31 is expressed specifically at the mantle edge. In situ hybridization results reveals strong signals from the dorsal epithelial cells of the outer fold at the mantle edge, and weak signals from inner epithelial cells of the same fold, indicating that hic31 is a prismatic-layer matrix protein. Although BLASTP results identify no shared homology with other shell-matrix proteins or any other known proteins, the hic31 tertiary structure is similar to that of collagen I, alpha 1 and alpha 2. It has been well proved that collagen forms the basic organic frameworks in way of collagen fibrils and minerals present within or outside of these fibrils. Therefore, hic31 might be a framework-matrix protein involved in the prismatic-layer biomineralization. Besides, the gene expression of hic31 increase in the early stages of pearl sac development, indicating that hic31 may play important roles in biomineralization of the pearl prismatic layer.
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Regulation of the copper chaperone CCS by XIAP-mediated ubiquitination.
Brady, Graham F; Galbán, Stefanie; Liu, Xuwen; Basrur, Venkatesha; Gitlin, Jonathan D; Elenitoba-Johnson, Kojo S J; Wilson, Thomas E; Duckett, Colin S
2010-04-01
In order to balance the cellular requirements for copper with its toxic properties, an elegant set of mechanisms has evolved to regulate and buffer intracellular copper. The X-linked inhibitor of apoptosis (XIAP) protein was recently identified as a copper-binding protein and regulator of copper homeostasis, although the mechanism by which XIAP binds copper in the cytosol is unclear. Here we describe the identification of the copper chaperone for superoxide dismutase (CCS) as a mediator of copper delivery to XIAP in cells. We also find that CCS is a target of the E3 ubiquitin ligase activity of XIAP, although interestingly, ubiquitination of CCS by XIAP was found to lead to enhancement of its chaperone activity toward its physiologic target, superoxide dismutase 1, rather than proteasomal degradation. Collectively, our results reveal novel links among apoptosis, copper metabolism, and redox regulation through the XIAP-CCS complex.
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Saveliev, A S; Kovaleva, I E; Novikova, L A; Isaeva, L V; Luzikov, V N
1999-03-15
When studying the fate of mammalian apocytochrome P450scc (apo-P450scc) imported in small amounts into isolated yeast mitochondria, we found that it undergoes degradation, this process being retarded if recipient mitochondria are preloaded in vivo (to about 0.2% of total organelle protein) with a fusion protein composed of mammalian adrenodoxin reductase and adrenodoxin (AdR-Ad); in parallel we observed aggregation of apo-P450scc. These effects suggest some overload of Pim1p protease and/or mtHsp70 system by AdR-Ad, as both of them are involved in the degradation of apo-P450scc (see Savel'ev et al. J. Biol. Chem. 273, 20596-20602, 1998). However, under the same conditions AdR-Ad was not able to impede the import of proteins into mitochondria and the development of the mitochondrial respiratory machinery in yeast, the processes requiring the mtHsp70 system and Pim1p, respectively. These data imply that chaperones and Pim1p protease prefer their natural targets in mitochondria to imported foreign proteins. Copyright 1999 Academic Press.
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NASA Astrophysics Data System (ADS)
Sharma, Sunny; Sarkar, Suparna; Paul, Simanta Sarani; Roy, Syamal; Chattopadhyay, Krishnananda
2013-12-01
Protein aggregation is believed to occur through the formation of misfolded conformations. It is expected that, in order to minimize aggregation, an effective small molecule chaperone would destabilize these intermediates. To study the mechanism of a chemical chaperone, we have designed a series of mutant proteins in which a tryptophan residue experiences different local environments and solvent exposures. We show that these mutants correspond to a series of conformationally altered proteins with varying degree of misfolding stress and aggregation propensities. Using arginine as a model small molecule, we show that a combination of unfolded state contraction and denaturant like properties results in selective targeting and destabilization of the partially folded proteins. In comparison, the effect of arginine towards the folded like control mutant, which is not aggregation prone, is significantly less. Other small molecules, lacking either of the above two properties, do not offer any specificity towards the misfolded proteins.
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Zhang, Zong-Kang; Li, Jie; Yan, De-Xin; Leung, Wing-Nang; Zhang, Bao-Ting
2016-01-01
Most acute coronary syndromes result from rupture of vulnerable atherosclerotic plaques. The collagen content of plaques may critically affect plaque stability. This study tested whether Icaritin (ICT), an intestinal metabolite of Epimedium-derived flavonoids, could alter the collagen synthesis/degradation balance in atherosclerotic lesions. Rabbits were fed with an atherogenic diet for four months. Oral administration of ICT (10 mg·kg−1·day−1) was started after two months of an atherogenic diet and lasted for two months. The collagen degradation-related parameters, including macrophages accumulation, content and activity of interstitial collagenase-1 (MMP-1), and the collagen synthesis-related parameters, including amount and distribution of smooth muscle cells (SMC) and collagen mRNA/protein levels, were evaluated in the aorta. ICT reduced plasma lipid levels, inhibited macrophage accumulation, lowered MMP-1 mRNA and protein expression, and suppressed proteolytic activity of pro-MMP-1 and MMP-1 in the aorta. ICT changed the distribution of the SMCs towards the fibrous cap of lesions without increasing the amount of SMCs. Higher collagen protein content in lesions and aorta homogenates was observed with ICT treatment compared with the atherogenic diet only, without altered collagen mRNA level. These results suggest that ICT could inhibit the collagen degradation-related factors and facilitate collagen accumulation in atherosclerotic lesions, indicating a new potential of ICT in atherosclerotic plaques. PMID:26828485
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Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis
Blaby-Haas, Crysten E.; Padilla-Benavides, Teresita; Stübe, Roland; ...
2014-12-02
Metallochaperones traffic copper (Cu +) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. In this paper, we present a previously unidentified Cu + chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu + chaperone delivers Cu + with specificity for PAA1, whichmore » is flipped in the envelope relative to prototypical bacterial ATPases, compatible with a role in Cu + import into the stroma and consistent with the canonical catalytic mechanism of these enzymes. The ubiquity of the chaperone suggests conservation of this Cu +-delivery mechanism and provides a unique snapshot into the evolution of a Cu + distribution pathway. Finally, we also provide evidence for an interaction between PAA2, the Cu +-ATPase in thylakoids, and the Cu +-chaperone for Cu/Zn superoxide dismutase (CCS), uncovering a Cu + network that has evolved to fine-tune Cu + distribution.« less
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O'Brien, Daniel; Jones, Laura M; Good, Sarah; Miles, Jo; Vijayabaskar, M S; Aston, Rebecca; Smith, Catrin E; Westhead, David R; van Oosten-Hawle, Patricija
2018-06-26
In metazoans, tissues experiencing proteotoxic stress induce "transcellular chaperone signaling" (TCS) that activates molecular chaperones, such as hsp-90, in distal tissues. How this form of inter-tissue communication is mediated to upregulate systemic chaperone expression and whether it can be utilized to protect against protein misfolding diseases remain open questions. Using C. elegans, we identified key components of a systemic stress signaling pathway that links the innate immune response with proteostasis maintenance. We show that mild perturbation of proteostasis in the neurons or the intestine activates TCS via the GATA zinc-finger transcription factor PQM-1. PQM-1 coordinates neuron-activated TCS via the innate immunity-associated transmembrane protein CLEC-41, whereas intestine-activated TCS depends on the aspartic protease ASP-12. Both TCS pathways can induce hsp-90 in muscle cells and facilitate amelioration of Aβ 3-42 -associated toxicity. This may have powerful implications for the treatment of diseases related to proteostasis dysfunction. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
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Sporn, Zachary A; Hines, Justin K
2015-01-01
Yeast prions are heritable protein-based elements, most of which are formed of amyloid aggregates that rely on the action of molecular chaperones for transmission to progeny. Prions can form distinct amyloid structures, known as 'strains' in mammalian systems, that dictate both pathological progression and cross-species infection barriers. In yeast these same amyloid structural polymorphisms, called 'variants', dictate the intensity of prion-associated phenotypes and stability in mitosis. We recently reported that [PSI(+)] prion variants differ in the fundamental domain requirements for one chaperone, the Hsp40/J-protein Sis1, which are mutually exclusive between 2 different yeast prions, demonstrating a functional plurality for Sis1. Here we extend that analysis to incorporate additional data that collectively support the hypothesis that Sis1 has multiple functional roles that can be accomplished by distinct sets of domains. These functions are differentially required by distinct prions and prion variants. We also present new data regarding Hsp104-mediated prion elimination and show that some Sis1 functions, but not all, are conserved in the human homolog Hdj1/DNAJB1. Importantly, of the 10 amyloid-based prions indentified to date in Saccharomyces cerevisiae, the chaperone requirements of only 4 are known, leaving a great diversity of amyloid structures, and likely modes of amyloid-chaperone interaction, largely unexplored.
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Yoo, Hee Young; Huang, Jun; Li, Lin; Foo, Mathias; Zeng, Hongbo; Hwang, Dong Soo
2016-03-14
Recent works on mussel adhesion have identified a load bearing matrix protein (PTMP1) containing von Willebrand factor (vWF) with collagen binding capability that contributes to the mussel holdfast by manipulating mussel collagens. Using a surface forces apparatus, we investigate for the first time, the nanomechanical properties of vWF-collagen interaction using homologous proteins of mussel byssus, PTMP1 and preCollagens (preCols), as collagen. Mimicking conditions similar to mussel byssus secretion (pH < 5.0) and seawater condition (pH 8.0), PTMP1 and preCol interact weakly in the "positioning" phase based on vWF-collagen binding and strengthen in "locked" phase due to the combined effects of electrostatic attraction, metal binding, and mechanical shearing. The progressive enhancement of binding between PTMP1 with porcine collagen under the aforementioned conditions is also observed. The binding mechanisms of PTMP1-preCols provide insights into the molecular interaction of the mammalian collagen system and the development of an artificial extracellular matrix based on collagens.
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Kelley, Patrick B; Abraham, Edathara C
2003-10-01
alphaB-crystallin, a member of the small heat-shock protein (hsp) family of proteins, is able to function as a molecular chaperone by protecting other proteins from stress-induced aggregation by recognizing and binding to partially unfolded species of damaged proteins. The present work has investigated the role of phenylalanine-28 (F28) of the 22RLFDQFF28 region of alphaB-crystallin in maintaining chaperone function and oligomeric structure under physiological condition and under thermal stress. Bovine alphaB-crystallin was cloned for the first time and the cDNA sequence revealed greater than 90% homology to that of human, rat and mouse alphaB-crystallins. F28 was mutated to a serine followed by expression of the mutant F28S and the wild-type alphaB (alphaB-wt) in E. coli and subsequent purification of the protein by size-exclusion chromatography. Secondary and tertiary structure analyses showed some structural changes in the mutant. Chaperone activity and oligomeric size of the mutant was unchanged at 37 degrees C whereas at 58 degrees C the chaperone activity was significantly decreased and the oligomeric size ranged from low molecular weight to high molecular weight showing disintegration of the oligomeric structure. The data support the idea that the participation of large oligomeric structure rather than smaller units is required to have optimal chaperone activity and the hydrophobic F28 residue is needed for maintaining the native oligomeric structure under thermal stress.
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The Escherichia coli P and Type 1 Pilus Assembly Chaperones PapD and FimC Are Monomeric in Solution
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sarowar, Samema; Hu, Olivia J.; Werneburg, Glenn T.
ABSTRACT The chaperone/usher pathway is used by Gram-negative bacteria to assemble adhesive surface structures known as pili or fimbriae. Uropathogenic strains ofEscherichia coliuse this pathway to assemble P and type 1 pili, which facilitate colonization of the kidney and bladder, respectively. Pilus assembly requires a periplasmic chaperone and outer membrane protein termed the usher. The chaperone allows folding of pilus subunits and escorts the subunits to the usher for polymerization into pili and secretion to the cell surface. Based on previous structures of mutant versions of the P pilus chaperone PapD, it was suggested that the chaperone dimerizes in themore » periplasm as a self-capping mechanism. Such dimerization is counterintuitive because the chaperone G1 strand, important for chaperone-subunit interaction, is buried at the dimer interface. Here, we show that the wild-type PapD chaperone also forms a dimer in the crystal lattice; however, the dimer interface is different from the previously solved structures. In contrast to the crystal structures, we found that both PapD and the type 1 pilus chaperone, FimC, are monomeric in solution. Our findings indicate that pilus chaperones do not sequester their G1 β-strand by forming a dimer. Instead, the chaperones may expose their G1 strand for facile interaction with pilus subunits. We also found that the type 1 pilus adhesin, FimH, is flexible in solution while in complex with its chaperone, whereas the P pilus adhesin, PapGII, is rigid. Our study clarifies a crucial step in pilus biogenesis and reveals pilus-specific differences that may relate to biological function. IMPORTANCEPili are critical virulence factors for many bacterial pathogens. UropathogenicE. colirelies on P and type 1 pili assembled by the chaperone/usher pathway to adhere to the urinary tract and establish infection. Studying pilus assembly is important for understanding mechanisms of protein secretion, as well as for identifying points
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Rodríguez, F; Morán, L; González, G; Troncoso, E; Zúñiga, R N
2017-04-01
Mussel byssus is a by-product of mussel production and is a potential source of collagen. The goal of this study was to extract collagen from the byssus of Chilean mussel using an enzymatic method and characterize it. A pepsin-aided extraction method was employed where first an enzymatic hydrolysis at two pepsin/substrate ratios (1:50 or 4:50) and times (4 or 24 h) was done. Extraction was conducted at 80 °C for 24 h, in a 0.5 N acetic acid solution. All samples were analyzed for collagen content, amino acid profile, turbidity, viscosity, solubility, denaturation temperature and surface tension. Hydrolysis time had significant effect on collagen content, hydroxyproline content and extraction yield. Hydrolysis with a pepsin/byssus ratio of 4:50 for 24 h gave the better extraction performance with values of 69 mg/g protein, 1.8 mg/g protein and 30%, for collagen content, hydroxyproline content and extraction yield, respectively. No differences were found for the viscosity and surface tension of collagen dispersions, suggesting that the enzymatic hydrolysis did not affect the integrity of the collagen molecule. Denaturation temperature of freeze-dried byssus collagen presented a high value (83-91 °C), making this kind of collagen a very interesting material for encapsulation of bioactive molecules and for biomedical applications.
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Perrody, Elsa; Cirinesi, Anne-Marie; Desplats, Carine; Keppel, France; Schwager, Françoise; Tranier, Samuel; Georgopoulos, Costa; Genevaux, Pierre
2012-01-01
The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ32, which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ32 facilitates RB43 bacteriophage proliferation is discussed. PMID:23133404
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Murphy, Brooke A; Bunda, Severa; Mitts, Thomas; Hinek, Aleksander
2010-12-17
The results of our in vitro experiments indicate that exposing cultured human aortic smooth muscle cells and dermal fibroblasts to 39 to 41 °C induces a significant up-regulation in the net deposition of elastic fibers, but not of collagen I or fibronectin, and also decreases the deposition of chondroitin sulfate-containing moieties. We further demonstrate that mild hyperthermia also rectifies the insufficient elastogenesis notable in cultures of fibroblasts derived from the stretch-marked skin of adult patients and in cultures of dermal fibroblasts from children with Costello syndrome, which is characterized by the accumulation of chondroitin 6-sulfate glycosaminoglycans that induce shedding and inactivation of the 67-kDa elastin-binding protein. We have previously established that this protein serves as a reusable chaperone for tropoelastin and that its recycling is essential for the normal deposition of elastic fibers. We now report that hyperthermia not only inhibits deposition of chondroitin 6-sulfate moieties and the consequent preservation of elastin-binding protein molecules but also induces their faster recycling. This, in turn, triggers a more efficient preservation of tropoelastin, enhancement of its secretion and extracellular assembly into elastic fibers. The presented results encourage using mild hyperthermia to restore elastic fiber production in damaged adult skin and to enhance elastogenesis in children with genetic elastinopathies.
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Structure and function of archaeal prefoldin, a co-chaperone of group II chaperonin.
Ohtaki, Akashi; Noguchi, Keiichi; Yohda, Masafumi
2010-01-01
Molecular chaperones are key cellular components involved in the maintenance of protein homeostasis and other unrelated functions. Prefoldin is a chaperone that acts as a co-factor of group II chaperonins in eukaryotes and archaea. It assists proper folding of protein by capturing nonnative proteins and delivering it to the group II chaperonin. Eukaryotic prefoldin is a multiple subunit complex composed of six different polypeptide chains. Archaeal prefoldin, on the other hand, is a heterohexameric complex composed of two alpha and four beta subunits, and forms a double beta barrel assembly with six long coiled coils protruding from it like a jellyfish with six tentacles. Based on the structural information of the archaeal prefoldin, substrate recognition and prefoldin-chaperonin binding mechanisms have been investigated. In this paper, we review a series of studies on the molecular mechanisms of archaeal PFD function. Particular emphasis will be placed on the molecular structures revealed by X-ray crystallography and molecular dynamics induced by binding to nonnative protein substrates.
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Tripathi, Manish Kumar; Rajput, Charul; Mishra, Saumya; Rasheed, Mohd Sami Ur; Singh, Mahendra Pratap
2018-06-11
Homeostatic regulation of class II programmed cell death/autophagy for the degradation and elimination of substandard organelles and defective proteins is decisive for the survival of dopaminergic neurons. Chaperone-mediated autophagy (CMA), one of the most highly dedicated self-sacrificing events, is accountable for the partial elimination of redundant soluble cytoplasmic proteins in Parkinson's disease (PD). CMA is characterized by the selective delivery of superfluous protein containing lysine-phenylalanine-glutamate-arginine-glutamine (KFERQ)/KFERQ-like motif to the lysosome through molecular chaperones, such as heat shock cognate-70 (Hsc-70). KFERQ/KFERQ-like motif present in the poor quality cytoplasmic substrate protein and Hsc-70 complex is recognized by a janitor protein, which is referred to as the lysosome-associated membrane protein-2A (LAMP-2A). This protein is known to facilitate an entry of substrate-chaperone complex in the lumen for hydrolytic cleavage of substrate and elimination of end-products. Impaired CMA is repeatedly blamed for an accumulation of surplus soluble proteins. However, it is still an enigma if CMA is a bonus or curse for PD. Case-control studies and cellular and animal models have deciphered the contribution of impaired CMA in PD. Current article updates the role of CMA in toxicant models and recapitulates the evidences that have highlighted a link between impaired CMA and PD. Although PD is an irreversible happening and CMA is a dual edging phenomenon, it is anticipated that fine-tuning of the latter encounters the former to a certain extent. Besides, the truth, embellishment, and propaganda regarding the issue are also emphasized in the final segment of the article.
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Collagen mimetic peptide engineered M13 bacteriophage for collagen targeting and imaging in cancer.
Jin, Hyo-Eon; Farr, Rebecca; Lee, Seung-Wuk
2014-11-01
Collagens are over-expressed in various human cancers and subsequently degraded and denatured by proteolytic enzymes, thus making them a target for diagnostics and therapeutics. Genetically engineered bacteriophage (phage) is a promising candidate for the development of imaging or therapeutic materials for cancer collagen targeting due to its promising structural features. We genetically engineered M13 phages with two functional peptides, collagen mimetic peptide and streptavidin binding peptide, on their minor and major coat proteins, respectively. The resulting engineered phage functions as a therapeutic or imaging material to target degraded and denatured collagens in cancerous tissues. We demonstrated that the engineered phages are able to target and label abnormal collagens expressed on A549 human lung adenocarcinoma cells after the conjugation with streptavidin-linked fluorescent agents. Our engineered collagen binding phage could be a useful platform for abnormal collagen imaging and drug delivery in various collagen-related diseases. Published by Elsevier Ltd.
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Chemical chaperones reduce endoplasmic reticulum stress and prevent mutant HFE aggregate formation.
de Almeida, Sérgio F; Picarote, Gonçalo; Fleming, John V; Carmo-Fonseca, Maria; Azevedo, Jorge E; de Sousa, Maria
2007-09-21
HFE C282Y, the mutant protein associated with hereditary hemochromatosis (HH), fails to acquire the correct conformation in the endoplasmic reticulum (ER) and is targeted for degradation. We have recently shown that an active unfolded protein response (UPR) is present in the cells of patients with HH. Now, by using HEK 293T cells, we demonstrate that the stability of HFE C282Y is influenced by the UPR signaling pathway that promotes its degradation. Treatment of HFE C282Y-expressing cells with tauroursodeoxycholic acid (TUDCA), a bile acid derivative with chaperone properties, or with the chemical chaperone sodium 4-phenylbutyrate (4PBA) impeded the UPR activation. However, although TUDCA led to an increased stability of the mutant protein, 4PBA contributed to a more efficient disposal of HFE C282Y to the degradation route. Fluorescence microscopy and biochemical analysis of the subcellular localization of HFE revealed that a major portion of the C282Y mutant protein forms intracellular aggregates. Although neither TUDCA nor 4PBA restored the correct folding and intracellular trafficking of HFE C282Y, 4PBA prevented its aggregation. These data suggest that TUDCA hampers the UPR activation by acting directly on its signal transduction pathway, whereas 4PBA suppresses ER stress by chemically enhancing the ER capacity to cope with the expression of misfolded HFE, facilitating its degradation. Together, these data shed light on the molecular mechanisms involved in HFE C282Y-related HH and open new perspectives on the use of orally active chemical chaperones as a therapeutic approach for HH.
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Gyurko, David M; Soti, Csaba; Stetak, Attila; Csermely, Peter
2014-05-01
During the last decade, network approaches became a powerful tool to describe protein structure and dynamics. Here, we describe first the protein structure networks of molecular chaperones, then characterize chaperone containing sub-networks of interactomes called as chaperone-networks or chaperomes. We review the role of molecular chaperones in short-term adaptation of cellular networks in response to stress, and in long-term adaptation discussing their putative functions in the regulation of evolvability. We provide a general overview of possible network mechanisms of adaptation, learning and memory formation. We propose that changes of network rigidity play a key role in learning and memory formation processes. Flexible network topology provides ' learning-competent' state. Here, networks may have much less modular boundaries than locally rigid, highly modular networks, where the learnt information has already been consolidated in a memory formation process. Since modular boundaries are efficient filters of information, in the 'learning-competent' state information filtering may be much smaller, than after memory formation. This mechanism restricts high information transfer to the 'learning competent' state. After memory formation, modular boundary-induced segregation and information filtering protect the stored information. The flexible networks of young organisms are generally in a 'learning competent' state. On the contrary, locally rigid networks of old organisms have lost their 'learning competent' state, but store and protect their learnt information efficiently. We anticipate that the above mechanism may operate at the level of both protein-protein interaction and neuronal networks.
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Li, Ran; Yuan, Fengjie; Fu, Wan; Zhang, Luyao; Zhang, Nan; Wang, Yanan; Ma, Ke; Li, Xue; Wang, Lina; Zhu, Wei-Guo; Zhao, Ying
2017-02-17
The serine/threonine kinase Unc-51-like kinase-1 (Ulk1) is thought to be essential for induction of autophagy, an intracellular bulk degradation process that is activated by various stresses. Although several proteins have been suggested as Ulk1 substrates during autophagic process, it still remains largely unknown about Ulk1's physiological substrates. Here, by performing in vitro and in vivo phosphorylation assay, we report that the co-chaperone cell division cycle protein 37 (Cdc37) is a Ulk1 substrate. Ulk1-mediated phosphorylation of Ser-339 in Cdc37 compromised the recruitment of client kinases to a complex comprising Cdc37 and heat shock protein 90 (Hsp90) but only modestly affected Cdc37 binding to Hsp90. Because the recruitment of protein kinase clients to the Hsp90 complex is essential for their stability and functions, Ser-339 phosphorylation of Cdc37 disrupts its ability as a co-chaperone to coordinate Hsp90. Hsp90 inhibitors are cancer chemotherapeutic agents by inducing depletion of clients, many of which are oncogenes. Upon treatment with an Hsp90 inhibitor in cancer cells, Ulk1 promoted the degradation of Hsp90-Cdc37 client kinases, resulting in increased cellular sensitivity to Hsp90 inhibitors. Thus, our study provides evidence for an anti-proliferative role of Ulk1 in response to Hsp90 inhibition in cancer cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Suzuki, Toru; Muto, Shinsuke; Miyamoto, Saku; Aizawa, Kenichi; Horikoshi, Masami; Nagai, Ryozo
2003-08-01
Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.
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Fibrinogen inhibits fibroblast-mediated contraction of collagen
Nien, Yih-Dar; Han, Yuan-Ping; Tawil, Bill; Chan, Linda S.; Tuan, Tai-Lan; Garner, Warren L.
2008-01-01
Extracellular matrix changes in composition and organization as it transitions from the provisional matrix of the fibrin/platelet plug to collagen scar in healed wounds. The manner in which individual matrix proteins affect these activities is not well established. In this article we describe the interactions of two important extracellular matrix components, fibrin and collagen, using an in vitro model of wound contraction, the fibroblast-populated collagen lattice. We utilized different fibrinogen sources and measured tissue reorganization in floating and tensioned collagen lattices. Our results showed that both fibrin and fibrinogen decreased the contraction of fibroblast populated collagen lattices in a dose-dependent manner. Polymerization of fibrinogen to fibrin using thrombin had no effect on this inhibition. Further, there was no effect due to changes in protein concentration, alternate components of the fibrin sealant, or the enzymatic action of thrombin. These results suggest that the initial stability of the fibrin provisional matrix is due to the fibrin, because this protein appears to inhibit contraction of the matrix. This may be important in the early phases of wound healing when clot stability is vital for hemostasis. Later, as fibrin is replaced by collagen, wound contraction can occur. PMID:12950643
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The groESL Chaperone Operon of Lactobacillus johnsonii†
Walker, D. Carey; Girgis, Hany S.; Klaenhammer, Todd R.
1999-01-01
The Lactobacillus johnsonii VPI 11088 groESL operon was localized on the chromosome near the insertion element IS1223. The operon was initially cloned as a series of three overlapping PCR fragments, which were sequenced and used to design primers to amplify the entire operon. The amplified fragment was used as a probe to recover the chromosomal copy of the groESL operon from a partial library of L. johnsonii VPI 11088 (NCK88) DNA, cloned in the shuttle vector pTRKH2. The 2,253-bp groESL fragment contained three putative open reading frames, two of which encoded the ubiquitous GroES and GroEL chaperone proteins. Analysis of the groESL promoter region revealed three transcription initiation sites, as well as three sets of inverted repeats (IR) positioned between the transcription and translation start sites. Two of the three IR sets bore significant homology to the CIRCE elements, implicated in negative regulation of the heat shock response in many bacteria. Northern analysis and primer extension revealed that multiple temperature-sensitive promoters preceded the groESL chaperone operon, suggesting that stress protein production in L. johnsonii is strongly regulated. Maximum groESL transcription activity was observed following a shift to 55°C, and a 15 to 30-min exposure of log-phase cells to this temperature increased the recovery of freeze-thawed L. johnsonii VPI 11088. These results suggest that a brief, preconditioning heat shock can be used to trigger increased chaperone production and provide significant cross-protection from the stresses imposed during the production of frozen culture concentrates. PMID:10388700
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Tao, Jiahui; Petrova, Kseniya; Ron, David
2010-05-25
P58(IPK) might function as an endoplasmic reticulum molecular chaperone to maintain protein folding homeostasis during unfolded protein responses. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the endoplasmic reticulum, we have determined the crystal structure of P58(IPK) TPR fragment to 2.5 {angstrom} resolution by the SAD method. The crystal structure of P58(IPK) revealed three domains (I-III) with similar folds and each domain contains three TPR motifs. An ELISA assay indicated that P58(IPK) acts as a molecular chaperone by interacting withmore » misfolded proteins such as luciferase and rhodanese. The P58(IPK) structure reveals a conserved hydrophobic patch located in domain I that might be involved in binding the misfolded polypeptides. Structure-based mutagenesis for the conserved hydrophobic residues located in domain I significantly reduced the molecular chaperone activity of P58(IPK).« less
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Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy
Fang, Xi; Wu, Tongbin; Liu, Canzhao; Veevers, Jennifer; Stroud, Matthew J.; Zhang, Zhiyuan; Ma, Xiaolong; Mu, Yongxin; Lao, Dieu-Hung; Dalton, Nancy D.; Gu, Yusu; Wang, Celine; Wang, Michael; Liang, Yan; Ouyang, Kunfu; Peterson, Kirk L.; Evans, Sylvia M.
2017-01-01
Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2–associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure. PMID:28737513
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Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy.
Fang, Xi; Bogomolovas, Julius; Wu, Tongbin; Zhang, Wei; Liu, Canzhao; Veevers, Jennifer; Stroud, Matthew J; Zhang, Zhiyuan; Ma, Xiaolong; Mu, Yongxin; Lao, Dieu-Hung; Dalton, Nancy D; Gu, Yusu; Wang, Celine; Wang, Michael; Liang, Yan; Lange, Stephan; Ouyang, Kunfu; Peterson, Kirk L; Evans, Sylvia M; Chen, Ju
2017-08-01
Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2-associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure.
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The Endoplasmic Reticulum Chaperone GRP170: From Immunobiology to Cancer Therapeutics
Wang, Hongxia; Pezeshki, Abdul Mohammad; Yu, Xiaofei; Guo, Chunqing; Subjeck, John R.; Wang, Xiang-Yang
2014-01-01
Glucose-regulated protein 170 (GRP170) is the largest member of glucose-regulated protein family that resides in the endoplasmic reticulum (ER). As a component of the ER chaperone network, GRP170 assists in protein folding, assembly, and transportation of secretory or transmembrane proteins. The well documented cytoprotective activity of intracellular GRP170 due to its intrinsic chaperoning property has been shown to provide a survival benefit in cancer cells during tumor progression or metastasis. Accumulating evidence shows that extracellular GRP170 displays a superior capacity in delivering tumor antigens to specialized antigen-presenting cells for cross-presentation, resulting in generation of an anti-tumor immune response dependent on cytotoxic CD8+ T cells. This unique feature of GRP170 provides a molecular basis for using GRP170 as an immunostimulatory adjuvant to develop a recombinant vaccine for therapeutic immunization against cancers. This review summarizes the latest findings in understanding the biological effects of GRP170 on cell functions and tumor progression. The immunomodulating activities of GRP170 during interactions with the innate and adaptive arms of the immune system as well as its therapeutic applications in cancer immunotherapy will be discussed. PMID:25629003
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USDA-ARS?s Scientific Manuscript database
Periplasmic chaperones HdeA and HdeB are known to be important for cell survival at low pH (pH<3) in E. coli and Shigella spp. Here we investigated the roles of these two acid chaperones in survival of various enterohemorrhagic E. coli (EHEC) following exposure to pH 2.0. Similar to K-12 strains, th...
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Effects of ingestion of collagen peptide on collagen fibrils and glycosaminoglycans in the dermis.
Matsuda, Naoya; Koyama, Yoh-ichi; Hosaka, Yoshinao; Ueda, Hiromi; Watanabe, Takafumi; Araya, Takayuki; Irie, Shinkichi; Takehana, Kazushige
2006-06-01
In order to investigate the effects of collagen peptide ingestion on fibroblasts and the extracellular matrix in the dermis, collagen peptide was administered orally to pigs at 0.2 g/kg body weight/d for 62 d, and its effects were compared with those of lactalbumin and water controls. Fibroblast density, and diameter and density of collagen fibrils were significantly larger in the collagen peptide group than in the lactalbumin and water control groups. The two major components of dermal glycosaminoglycans, hyaluronic acid and dermatan sulfate, which are present in the inter-fibrillar space, did not differ significantly among the three groups. However, the ratio of dermatan sulfate, which is derived from fibril-bound decorin, was largest in the collagen peptide group. These results suggest that ingestion of collagen peptide induces increased fibroblast density and enhances formation of collagen fibrils in the dermis in a protein-specific manner.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Grover, Abhinav; Shandilya, Ashutosh; Agrawal, Vibhuti
2011-01-07
Research highlights: {yields} Withaferin A and 17-DMAG synergistically inhibit the Hsp90-Cdc37 chaperone pair. {yields} Binding of WA to Cdc37 cleft suppresses its kinase binding activity. {yields} 17-DMAG binding to the association complex results in H-bonds with 60% clustering. {yields} The ligands' bound complex was found structurally and thermodynamically stable. -- Abstract: The chaperone Hsp90 is involved in regulating the stability and activation state of more than 200 'client' proteins and takes part in the cancer diseased states. The major clientele-protein kinases depend on Hsp90 for their proper folding and functioning. Cdc37, a kinase targeting co-chaperone of Hsp90, mediates the interactionsmore » between Hsp90 and protein kinases. Targeting of Cdc37 has the prospect of delivering predominantly kinase-selective molecular responses as compared to the current pharmacologic Hsp90 inhibitors. The present work reports a bio-computational study carried out with the aim of exploring the dual inhibition of Hsp90/Cdc37 chaperone/co-chaperone association complex by the naturally occurring drug candidates withaferin A and 17-DMAG along with their possible modes of action. Our molecular docking studies reveal that withaferin A in combination with 17-DMAG can act as potent chaperone system inhibitors. The structural and thermodynamic stability of the ligands' bound complex was also observed from molecular dynamics simulations in water. Our results suggest a novel tumor suppressive action mechanism of herbal ligands which can be looked forward for further clinical investigations for possible anticancer drug formulations.« less
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Allosteric mechanism controls traffic in the chaperone/usher pathway.
Di Yu, Xiao; Dubnovitsky, Anatoly; Pudney, Alex F; Macintyre, Sheila; Knight, Stefan D; Zavialov, Anton V
2012-11-07
Many virulence organelles of Gram-negative bacterial pathogens are assembled via the chaperone/usher pathway. The chaperone transports organelle subunits across the periplasm to the outer membrane usher, where they are released and incorporated into growing fibers. Here, we elucidate the mechanism of the usher-targeting step in assembly of the Yersinia pestis F1 capsule at the atomic level. The usher interacts almost exclusively with the chaperone in the chaperone:subunit complex. In free chaperone, a pair of conserved proline residues at the beginning of the subunit-binding loop form a "proline lock" that occludes the usher-binding surface and blocks usher binding. Binding of the subunit to the chaperone rotates the proline lock away from the usher-binding surface, allowing the chaperone-subunit complex to bind to the usher. We show that the proline lock exists in other chaperone/usher systems and represents a general allosteric mechanism for selective targeting of chaperone:subunit complexes to the usher and for release and recycling of the free chaperone. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Cheng, Mingyu; Wang, Hao; Yoshida, Ryu
2010-01-01
Collagen–platelet (PL)-rich plasma composites have shown in vivo potential to stimulate anterior cruciate ligament (ACL) healing at early time points in large animal models. However, little is known about the cellular mechanisms by which the plasma component of these composites may stimulate healing. We hypothesized that the components of PL-rich plasma (PRP), namely the PLs and PL-poor plasma (PPP), would independently significantly influence ACL cell viability and metabolic activity, including collagen gene expression. To test this hypothesis, ACL cells were cultured in a collagen type I hydrogel with PLs, PPP, or the combination of the two (PRP) for 14 days. The inclusion of PLs, PPP, and PRP all significantly reduced the rate of cell apoptosis and enhanced the metabolic activity of fibroblasts in the collagen hydrogel. PLs promoted fibroblast-mediated collagen scaffold contraction, whereas PPP inhibited this contraction. PPP and PRP both promoted cell elongation and the formation of wavy fibrous structure in the scaffolds. The addition of only PLs or only plasma proteins did not significantly enhance gene expression of collagen types I and III but the combination, as PRP, did. Our findings suggest that the addition of both PLs and plasma proteins to collagen hydrogel may be useful in stimulating ACL healing by enhancing ACL cell viability, metabolic activity, and collagen synthesis. PMID:19958169
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Type I Collagen and Collagen Mimetics as Angiogenesis Promoting Superpolymers
DOE Office of Scientific and Technical Information (OSTI.GOV)
Twardowski, T.; Fertala, A.; Orgel, J.P.R.O.
Angiogenesis, the development of blood vessels from the pre-existing vasculature, is a key component of embryogenesis and tissue regeneration. Angiogenesis also drives pathologies such as tumor growth and metastasis, and hemangioma development in newborns. On the other hand, promotion of angiogenesis is needed in tissues with vascular insufficiencies, and in bioengineering, to endow tissue substitutes with appropriate microvasculatures. Therefore, much research has focused on defining mechanisms of angiogenesis, and identifying pro- and anti-angiogenic molecules. Type I collagen, the most abundant protein in humans, potently stimulates angiogenesis in vitro and in vivo. Crucial to its angiogenic activity appears to be ligationmore » and possibly clustering of endothelial cell (EC) surface {alpha}1{beta}1/{alpha}2{beta}1 integrin receptors by the GFPGER502-507 sequence of the collagen fibril. However, additional aspects of collagen structure and function that may modulate its angiogenic properties are discussed. Moreover, type I collagen and fibrin, another angiogenic polymer, share several structural features. These observations suggest strategies for creating 'angiogenic superpolymers', including: modifying type I collagen to influence its biological half-life, immunogenicity, and integrin binding capacity; genetically engineering fibrillar collagens to include additional integrin binding sites or angiogenic determinants, and remove unnecessary or deleterious sequences without compromising fibril integrity; and exploring the suitability of poly(ortho ester), PEG-lysine copolymer, tubulin, and cholesteric cuticle as collagen mimetics, and suggesting means of modifying them to display ideal angiogenic properties. The collagenous and collagen mimetic angiogenic superpolymers described here may someday prove useful for many applications in tissue engineering and human medicine.« less
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Sekhar, Ashok; Nagesh, Jayashree; Rosenzweig, Rina; Kay, Lewis E
2017-11-01
The Hsp70 chaperone system plays a critical role in cellular homeostasis by binding to client protein molecules. We have recently shown by methyl-TROSY NMR methods that the Escherichia coli Hsp70, DnaK, can form multiple bound complexes with a small client protein, hTRF1. In an effort to characterize the interactions further we report here the results of an NMR-based titration study of hTRF1 and DnaK, where both molecular components are monitored simultaneously, leading to a binding model. A central finding is the formation of a previously undetected 3:1 hTRF1-DnaK complex, suggesting that under heat shock conditions, DnaK might be able to protect cytosolic proteins whose net concentrations would exceed that of the chaperone. Moreover, these results provide new insight into the heterogeneous ensemble of complexes formed by DnaK chaperones and further emphasize the unique role of NMR spectroscopy in obtaining information about individual events in a complex binding scheme by exploiting a large number of probes that report uniquely on distinct binding processes. © 2017 The Protein Society.
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Hill, Ryan C.; Wither, Matthew J.; Nemkov, Travis; Barrett, Alexander; D'Alessandro, Angelo; Dzieciatkowska, Monika; Hansen, Kirk C.
2015-01-01
Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in well-preserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. The data are available via ProteomeXchange with identifier PXD001827. PMID:25948757
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Cooper, Colin A; Zhang, Kun; Andres, Sara N; Fang, Yuan; Kaniuk, Natalia A; Hannemann, Mandy; Brumell, John H; Foster, Leonard J; Junop, Murray S; Coombes, Brian K
2010-02-05
Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 A revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.
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Armas, Pablo; Nasif, Sofía; Calcaterra, Nora B
2008-02-15
Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression. Copyright 2007 Wiley-Liss, Inc.
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Biomimetic soluble collagen purified from bones.
Ferreira, Ana Marina; Gentile, Piergiorgio; Sartori, Susanna; Pagliano, Cristina; Cabrele, Chiara; Chiono, Valeria; Ciardelli, Gianluca
2012-11-01
Type I collagen has been extensively exploited as a biomaterial for biomedical applications and drug delivery; however, small molecular alterations occurring during the isolation procedure and its interaction with residual bone extracellular matrix molecules or proteins might affect the overall material biocompatibility and performance. The aim of the current work is to study the potential alterations in collagen properties and organization associated with the absence of proteoglycans, which mimic pathological conditions associated with age-related diseases. A new approach for evaluating the effect of proteoglycans on the properties of isolated type I collagen from the bone matrix is described. Additional treatment with guanidine hydrochloride was introduced to remove residual proteoglycans from the collagen matrix. The properties of the isolated collagen with/without guanidine hydrochloride treatment were investigated and compared with a commercial rabbit collagen as control. We demonstrate that the absence of proteoglycans in the isolated type I collagen affects its thermal properties, the extraction into its native structure, and its ability to hydrate and self-assemble into fibers. The fine control and tuning of all these features, linked to the absence of non-collagenous proteins as proteoglycans, offer the possibility of designing new strategies and biomaterials with advanced biomimetic properties aimed at regenerating bone tissue in the case of fragility and/or defects. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu
2016-02-17
Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn't showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody-drug conjugates (ADC) or immunotoxins.
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A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix
Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu
2016-01-01
Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn’t showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody–drug conjugates (ADC) or immunotoxins. PMID:26883295
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Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke
2014-01-01
Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68. PMID
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Sewing, Judith; Klinger, Matthias; Notbohm, Holger
2017-03-01
Cartilage is a tissue with a very low capability of self-repair and the search for suitable materials supporting the chondrogenic phenotype and thus avoiding fibrotic dedifferentiation for matrix-associated chondrocyte transplantation (MACI) is ongoing. Jellyfish collagen was thought to be a suitable material mainly because of its good availability and easy handling. Collagen was extracted from jellyfish Rhopilema esculentum and the spreading of porcine chondrocytes on two (2D) and three dimensional (3D) collagen matrices examined in comparison with vertebrate collagens, placenta collagen and a commercially available matrix from porcine collagen type I (Optimaix®). In 2D, most chondrocytes kept their round shape on jellyfish collagen and vertebrate collagen type II compared with vertebrate collagen type I. This was also confirmed in 3D experiments, where chondrocytes preserved their phenotype on jellyfish collagen, as indicated by high collagen II/(II + I) ratios (≥54 % and ~92 % collagen type II in mRNA and protein, respectively) and no proliferation during 28 days of cultivation. These observations were discussed with a view to potential structural differences of jellyfish collagen, which might influence the integrin-mediated adhesion mechanisms of vertebrate cells on jellyfish collagen. This probably results from a lack of integrin-binding sites and the existence of an alternative binding mechanism such that cells kept their round shape on jellyfish collagen, preventing chondrocytes from dedifferentiation. Thus, collagen from R. esculentum is a very suitable and promising material for cartilage tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.
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Tiku, Moti L; Narla, Haritha; Jain, Mohit; Yalamanchili, Praveen
2007-01-01
Osteoarthritis (OA) affects a large segment of the aging population and is a major cause of pain and disability. At present, there is no specific treatment available to prevent or retard the cartilage destruction that occurs in OA. Recently, glucosamine sulfate has received attention as a putative agent that may retard cartilage degradation in OA. The precise mechanism of action of glucosamine is not known. We investigated the effect of glucosamine in an in vitro model of cartilage collagen degradation in which collagen degradation induced by activated chondrocytes is mediated by lipid peroxidation reaction. Lipid peroxidation in chondrocytes was measured by conjugated diene formation. Protein oxidation and aldehydic adduct formation were studied by immunoblot assays. Antioxidant effect of glucosamine was also tested on malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) formation on purified lipoprotein oxidation for comparison. Glucosamine sulfate and glucosamine hydrochloride in millimolar (0.1 to 50) concentrations specifically and significantly inhibited collagen degradation induced by calcium ionophore-activated chondrocytes. Glucosamine hydrochloride did not inhibit lipid peroxidation reaction in either activated chondrocytes or in copper-induced oxidation of purified lipoproteins as measured by conjugated diene formation. Glucosamine hydrochloride, in a dose-dependent manner, inhibited malondialdehyde (TBARS) formation by oxidized lipoproteins. Moreover, we show that glucosamine hydrochloride prevents lipoprotein protein oxidation and inhibits malondialdehyde adduct formation in chondrocyte cell matrix, suggesting that it inhibits advanced lipoxidation reactions. Together, the data suggest that the mechanism of decreasing collagen degradation in this in vitro model system by glucosamine may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix
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Olive, Joshua A; Cowan, J A
2018-07-01
Iron‑sulfur clusters are essential cofactors found across all domains of life. Their assembly and transfer are accomplished by highly conserved protein complexes and partners. In eukaryotes a [2Fe-2S] cluster is first assembled in the mitochondria on the iron‑sulfur cluster scaffold protein ISCU in tandem with iron, sulfide, and electron donors. Current models suggest that a chaperone pair interacts with a cluster-bound ISCU to facilitate cluster transfer to a monothiol glutaredoxin. In humans this protein is glutaredoxin 5 (GLRX5) and the cluster can then be exchanged with a variety of target apo proteins. By use of circular dichroism spectroscopy, the kinetics of cluster exchange reactivity has been evaluated for human GLRX5 with a variety of cluster donor and acceptor partners, and the role of chaperones determined for several of these. In contrast to the prokaryotic model, where heat-shock type chaperone proteins HscA and HscB are required for successful and efficient transfer of a [2Fe-2S] cluster from the ISCU scaffold to a monothiol glutaredoxin. However, in the human system the chaperone homologs, HSPA9 and HSC20, are not necessary for human ISCU to promote cluster transfer to GLRX5, and appear to promote the reverse transfer. Cluster exchange with the human iron‑sulfur cluster carrier protein NFU1 and ferredoxins (FDX's), and the role of chaperones, has also been evaluated, demonstrating in certain cases control over the directionality of cluster transfer. In contrast to other prokaryotic and eukaryotic organisms, NFU1 is identified as a more likely physiological donor of [2Fe-2S] cluster to human GLRX5 than ISCU. Copyright © 2018 Elsevier Inc. All rights reserved.
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Batyuk, Alexander; Wu, Yufan; Honegger, Annemarie; Heberling, Matthew M; Plückthun, Andreas
2016-04-24
DARPin libraries, based on a Designed Ankyrin Repeat Protein consensus framework, are a rich source of binding partners for a wide variety of proteins. Their modular structure, stability, ease of in vitro selection and high production yields make DARPins an ideal starting point for further engineering. The X-ray structures of around 30 different DARPin complexes demonstrate their ability to facilitate crystallization of their target proteins by restricting flexibility and preventing undesired interactions of the target molecule. However, their small size (18 kDa), very hydrophilic surface and repetitive structure can limit the DARPins' ability to provide essential crystal contacts and their usefulness as a search model for addressing the crystallographic phase problem in molecular replacement. To optimize DARPins for their application as crystallization chaperones, rigid domain-domain fusions of the DARPins to larger proteins, proven to yield high-resolution crystal structures, were generated. These fusions were designed in such a way that they affect only one of the terminal capping repeats of the DARPin and do not interfere with residues involved in target binding, allowing to exchange at will the binding specificities of the DARPin in the fusion construct. As a proof of principle, we designed rigid fusions of a stabilized version of Escherichia coli TEM-1 β-lactamase to the C-terminal capping repeat of various DARPins in six different relative domain orientations. Five crystal structures representing four different fusion constructs, alone or in complex with the cognate target, show the predicted relative domain orientations and prove the validity of the concept. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Collagen-Based Biomaterials for Wound Healing
Chattopadhyay, Sayani; Raines, Ronald T.
2014-01-01
With its wide distribution in soft and hard connective tissues, collagen is the most abundant of animal proteins. In vitro, natural collagen can be formed into highly organized, three-dimensional scaffolds that are intrinsically biocompatible, biodegradable, non-toxic upon exogenous application, and endowed with high tensile strength. These attributes make collagen the material of choice for wound healing and tissue engineering applications. In this article, we review the structure and molecular interactions of collagen in vivo; the recent use of natural collagen in sponges, injectables, films and membranes, dressings, and skin grafts; and the on-going development of synthetic collagen mimetic peptides as pylons to anchor cytoactive agents in wound beds. PMID:24633807
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Lu, Yongbo; Kamel-El Sayed, Suzan A; Wang, Kun; Tiede-Lewis, LeAnn M; Grillo, Michael A; Veno, Patricia A; Dusevich, Vladimir; Phillips, Charlotte L; Bonewald, Lynda F; Dallas, Sarah L
2018-06-01
Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel
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Mazur, Anna; Holthoff, Emily; Vadali, Shanthi; Kelly, Thomas; Post, Steven R.
2016-01-01
Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM). These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP), a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204). Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages. PMID:26934296
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Renal myofibroblasts contract collagen I matrix lattices in vitro.
Kelynack, K J; Hewitson, T D; Pedagogos, E; Nicholls, K M; Becker, G J
1999-01-01
Myofibroblasts, cells with both fibroblastic and smooth muscle cell features, have been implicated in renal scarring. In addition to synthetic properties, contractile features and integrin expression may allow myofibroblasts to rearrange and contract interstitial collagenous proteins. Myofibroblasts from normal rat kidneys were grown in cell-populated collagen lattices to measure cell generated contraction. Following detachment of cell populated collagen lattices, myofibroblasts progressively contracted collagen lattices, reducing lattice diameter by 42% at 24 h. Alignment of myofibroblasts, rearrangement of fibrils and beta(1) integrin expression were observed within lattices. We postulate that interstitial myofibroblasts contribute to renal scarring through manipulation of collagenous proteins. Copyright 1999 S. Karger AG, Basel
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FK506-binding protein 10 (FKBP10) regulates lung fibroblast migration via collagen VI synthesis.
Knüppel, Larissa; Heinzelmann, Katharina; Lindner, Michael; Hatz, Rudolf; Behr, Jürgen; Eickelberg, Oliver; Staab-Weijnitz, Claudia A
2018-04-19
In idiopathic pulmonary fibrosis (IPF), fibroblasts gain a more migratory phenotype and excessively secrete extracellular matrix (ECM), ultimately leading to alveolar scarring and progressive dyspnea. Here, we analyzed the effects of deficiency of FK506-binding protein 10 (FKBP10), a potential IPF drug target, on primary human lung fibroblast (phLF) adhesion and migration. Using siRNA, FKBP10 expression was inhibited in phLF in absence or presence of 2ng/ml transforming growth factor-β1 (TGF-β1) and 0.1mM 2-phosphoascorbate. Effects on cell adhesion and migration were monitored by an immunofluorescence (IF)-based attachment assay, a conventional scratch assay, and single cell tracking by time-lapse microscopy. Effects on expression of key players in adhesion dynamics and migration were analyzed by qPCR and Western Blot. Colocalization was evaluated by IF microscopy and by proximity ligation assays. FKBP10 knockdown significantly attenuated adhesion and migration of phLF. Expression of collagen VI was decreased, while expression of key components of the focal adhesion complex was mostly upregulated. The effects on migration were 2-phosphoascorbate-dependent, suggesting collagen synthesis as the underlying mechanism. FKBP10 colocalized with collagen VI and coating culture dishes with collagen VI, and to a lesser extent with collagen I, abolished the effect of FKBP10 deficiency on migration. These findings show, to our knowledge for the first time, that FKBP10 interacts with collagen VI and that deficiency of FKBP10 reduces phLF migration mainly by downregulation of collagen VI synthesis. The results strengthen FKBP10 as an important intracellular regulator of ECM remodeling and support the concept of FKBP10 as drug target in IPF.
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Integrity of N- and C-termini is important for E. coli Hsp31 chaperone activity
Sastry, M S R; Zhou, Weibin; Baneyx, François
2009-01-01
Hsp31 is a stress-inducible molecular chaperone involved in the management of protein misfolding at high temperatures and in the development of acid resistance in starved E. coli. Each subunit of the Hsp31 homodimer consists of two structural domains connected by a flexible linker that sits atop a continuous tract of nonpolar residues adjacent to a hydrophobic bowl defined by the dimerization interface. Previously, we proposed that while the bowl serves as a binding site for partially folded species at physiological temperatures, chaperone function under heat shock conditions requires that folding intermediates further anneal to high-affinity binding sites that become uncovered upon thermally induced motion of the linker. In support of a mechanism requiring that client proteins first bind to the bowl, we show here that fusion of a 20-residue-long hexahistidine tag to the N-termini of Hsp31 abolishes chaperone activity at all temperatures by inducing reversible structural changes that interfere with substrate binding. We further demonstrate that extending the C-termini of Hsp31 with short His tags selectively suppresses chaperone function at high temperatures by interfering with linker movement. The structural and functional sensitivity of Hsp31 to lengthening is consistent with the high degree of conservation of class I Hsp31 orthologs and will serve as a cautionary tale on the implications of affinity tagging. PMID:19517531
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Jung, Eunsun; Cho, Jae Youl; Park, Deokhoon; Kim, Min Hee; Park, Beomseok; Lee, Sang Yeol; Lee, Jongsung
2015-02-01
Skin aging appears to be principally attributed to a decrease in type I collagen level and the regeneration ability of dermal fibroblasts. We hypothesized that vegetable peptones promote cell proliferation and production of type I collagen in human dermal fibroblasts. Therefore, we investigated the effects of vegetable peptones on cell proliferation and type I collagen production and their possible mechanisms in human dermal fibroblasts. Vegetable peptones significantly promoted cell proliferation in a concentration-dependent manner. In addition, the human luciferase type I collagen α2 promoter and type I procollagen synthesis assays showed that the vegetable peptones induced type I procollagen production by activating the type I collagen α2 promoter. Moreover, the vegetable peptones activated p90 ribosomal s6 kinase, which was mediated by activating the Raf-p44/42 mitogen-activated protein kinase signaling pathway. Furthermore, the vegetable peptone-induced increase in cell proliferation and type I collagen production decreased upon treatment with the ERK inhibitor PD98059. Taken together, these findings suggest that increased proliferation of human dermal fibroblasts and enhanced production of type I collagen by vegetable peptones occur primarily by inducing the p90 ribosomal s6 kinase-CCAAT/enhancer binding protein β phosphorylation pathway, which is mediated by activating Raf-ERK signaling. Copyright © 2015 Elsevier Inc. All rights reserved.
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Hierarchical and non-hierarchical mineralisation of collagen
Liu, Yan; Kim, Young-Kyung; Dai, Lin; Li, Nan; Khan, Sara; Pashley, David H.; Tay, Franklin R.
2010-01-01
Biomineralisation of collagen involves functional motifs incorporated in extracellular matrix protein molecules to accomplish the objectives of stabilising amorphous calcium phosphate into nanoprecursors and directing the nucleation and growth of apatite within collagen fibrils. Here we report the use of small inorganic polyphosphate molecules to template hierarchical intrafibrillar apatite assembly in reconstituted collagen in the presence of polyacrylic acid to sequester calcium and phosphate into transient amorphous nanophases. The use of polyphosphate without a sequestration analogue resulted only in randomly-oriented extrafibrillar precipitations along the fibrillar surface. Conversely, the use of polyacrylic acid without a templating analogue resulted only in non-hierarchical intrafibrillar mineralisation with continuous apatite strands instead of discrete crystallites. The ability of using simple non-protein molecules to recapitulate different levels of structural hierarchy in mineralised collagen signifies the ultimate simplicity in Nature’s biomineralisation design principles and challenges the need for using more complex recombinant matrix proteins in bioengineering applications. PMID:21040969
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Characterization of the Grp94/OS-9 chaperone-lectin complex
Seidler, Paul M.; Shinsky, Stephen A.; Hong, Feng; Li, Zihai; Cosgrove, Michael S.; Gewirth, Daniel T.
2014-01-01
Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER associated degradation (ERAD) quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins (TMPs). To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal MRH lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains ‘mammalian-specific insets’ that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate, and additionally hints at a plausible biological role for the Grp94/OS-9 complex. PMID:25193139
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The fibrillar collagen family.
Exposito, Jean-Yves; Valcourt, Ulrich; Cluzel, Caroline; Lethias, Claire
2010-01-28
Collagens, or more precisely collagen-based extracellular matrices, are often considered as a metazoan hallmark. Among the collagens, fibrillar collagens are present from sponges to humans, and are involved in the formation of the well-known striated fibrils. In this review we discuss the different steps in the evolution of this protein family, from the formation of an ancestral fibrillar collagen gene to the formation of different clades. Genomic data from the choanoflagellate (sister group of Metazoa) Monosiga brevicollis, and from diploblast animals, have suggested that the formation of an ancestral alpha chain occurred before the metazoan radiation. Phylogenetic studies have suggested an early emergence of the three clades that were first described in mammals. Hence the duplication events leading to the formation of the A, B and C clades occurred before the eumetazoan radiation. Another important event has been the two rounds of "whole genome duplication" leading to the amplification of fibrillar collagen gene numbers, and the importance of this diversification in developmental processes. We will also discuss some other aspects of fibrillar collagen evolution such as the development of the molecular mechanisms involved in the formation of procollagen molecules and of striated fibrils.
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Gaude, H; Aznar, N; Delay, A; Bres, A; Buchet-Poyau, K; Caillat, C; Vigouroux, A; Rogon, C; Woods, A; Vanacker, J-M; Höhfeld, J; Perret, C; Meyer, P; Billaud, M; Forcet, C
2012-03-22
LKB1 is a tumor suppressor that is constitutionally mutated in a cancer-prone condition, called Peutz-Jeghers syndrome, as well as somatically inactivated in a sizeable fraction of lung and cervical neoplasms. The LKB1 gene encodes a serine/threonine kinase that associates with the pseudokinase STRAD (STE-20-related pseudokinase) and the scaffolding protein MO25, the formation of this heterotrimeric complex promotes allosteric activation of LKB1. We have previously reported that the molecular chaperone heat shock protein 90 (Hsp90) binds to and stabilizes LKB1. Combining pharmacological studies and RNA interference approaches, we now provide evidence that the co-chaperone Cdc37 participates to the regulation of LKB1 stability. It is known that the Hsp90-Cdc37 complex recognizes a surface within the N-terminal catalytic lobe of client protein kinases. In agreement with this finding, we found that the chaperones Hsp90 and Cdc37 interact with an LKB1 isoform that differs in the C-terminal region, but not with a novel LKB1 variant that lacks a portion of the kinase N-terminal lobe domain. Reconstitution of the two complexes LKB1-STRAD and LKB1-Hsp90-Cdc37 with recombinant proteins revealed that the former is catalytically active whereas the latter is inactive. Furthermore, consistent with a documented repressor function of Hsp90, LKB1 kinase activity was transiently stimulated upon dissociation of Hsp90. Finally, disruption of the LKB1-Hsp90 complex favors the recruitment of both Hsp/Hsc70 and the U-box dependent E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein) that triggers LKB1 degradation. Taken together, our results establish that the Hsp90-Cdc37 complex controls both the stability and activity of the LKB1 kinase. This study further shows that two chaperone complexes with antagonizing activities, Hsp90-Cdc37 and Hsp/Hsc70-CHIP, finely control the cellular level of LKB1 protein.
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In Vivo Substrates of the Lens Molecular Chaperones αA-Crystallin and αB-Crystallin
Andley, Usha P.; Malone, James P.; Townsend, R. Reid
2014-01-01
αA-crystallin and αB-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in αA-R49C and αB-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old αA-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of αA-crystallin; increased association between αA-crystallin and filensin, actin, or creatine kinase B; increased acidification of βB1-crystallin; increased levels of grifin; and an association between βA3/A1-crystallin and αA-crystallin. Homozygous αA-R49C mutant lenses exhibited increased associations between αA-crystallin and βB3-, βA4-, βA2-crystallins, and grifin, whereas levels of βB1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, α-enolase, and cytochrome c increased more than 50-fold in homozygous αA-R49C mutant lenses. In αB-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several β- and γ-crystallins, and degradation of αA- and αB-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal α-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of αA- and αB-crystallin. PMID:24760011
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Trigger Factor can antagonize both SecB and DnaK/DnaJ chaperone functions in Escherichia coli
Ullers, Ronald S.; Ang, Debbie; Schwager, Françoise; Georgopoulos, Costa; Genevaux, Pierre
2007-01-01
Polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytoplasmic, integral membrane, or exported proteins. In Escherichia coli, the chaperones SecB, Trigger Factor (TF), and DnaK are key players in this process. Here, we report that, as with dnaK or dnaJ mutants, a secB null strain exhibits a strong cold-sensitive (Cs) phenotype. Through suppressor analyses, we found that inactivating mutations in the tig gene encoding TF fully relieve both the Cs phenotype and protein aggregation observed in the absence of SecB. This antagonistic effect of TF depends on its ribosome-binding and chaperone activities but unrelated to its peptidyl-prolyl cis/trans isomerase (PPIase) activity. Furthermore, in contrast to the previously known synergistic action of TF and DnaK/DnaJ above 30°C, a tig null mutation partially suppresses the Cs phenotype exhibited by a compromised DnaK/DnaJ chaperone machine. The antagonistic role of TF is further exemplified by the fact that the secB dnaJ double mutant is viable only in the absence of TF. Finally, we show that, in the absence of TF, more SecA and ribosomes are associated with the inner membrane, suggesting that the presence of TF directly or indirectly interferes with the process of cotranslational protein targeting to the Sec translocon. PMID:17360615
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Plant Leucine Aminopeptidases Moonlight as Molecular Chaperones to Alleviate Stress-induced Damage*
Scranton, Melissa A.; Yee, Ashley; Park, Sang-Youl; Walling, Linda L.
2012-01-01
Leucine aminopeptidases (LAPs) are present in animals, plants, and microbes. In plants, there are two classes of LAPs. The neutral LAPs (LAP-N and its orthologs) are constitutively expressed and detected in all plants, whereas the stress-induced acidic LAPs (LAP-A) are expressed only in a subset of the Solanaceae. LAPs have a role in insect defense and act as a regulator of the late branch of wound signaling in Solanum lycopersicum (tomato). Although the mechanism of LAP-A action is unknown, it has been presumed that LAP peptidase activity is essential for regulating wound signaling. Here we show that plant LAPs are bifunctional. Using three assays to monitor protein protection from heat-induced damage, it was shown that the tomato LAP-A and LAP-N and the Arabidopsis thaliana LAP1 and LAP2 are molecular chaperones. Assays using LAP-A catalytic site mutants demonstrated that LAP-A chaperone activity was independent of its peptidase activity. Furthermore, disruption of the LAP-A hexameric structure increased chaperone activity. Together, these data identify a new class of molecular chaperones and a new function for the plant LAPs as well as suggesting new mechanisms for LAP action in the defense of solanaceous plants against stress. PMID:22493451
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Whitehead, Timothy A; Boonyaratanakornkit, Boonchai B; Höllrigl, Volker; Clark, Douglas S
2007-04-01
Prefoldin is a molecular chaperone found in the domains eukarya and archaea that acts in conjunction with Group II chaperonin to correctly fold other nascent proteins. Previously, our group identified a putative single subunit of prefoldin, gamma PFD, that was up-regulated in response to heat stress in the hyperthermophilic archaeon Methanocaldococcus jannaschii. In order to characterize this protein, we subcloned and expressed it and the other two prefoldin subunits from M. jannaschii, alpha and beta PFD, into Eschericia coli and characterized the proteins. Whereas alpha and beta PFD readily assembled into the expected hexamer, gamma PFD would not assemble with either protein. Instead, gamma PFD forms long filaments of defined dimensions measuring 8.5 nm x 1.7-3.5 nm and lengths exceeding 1 microm. Filamentous gamma PFD acts as a molecular chaperone through in vitro assays, in a manner comparable to PFD. A possible molecular model for filament assembly is discussed.
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Whitehead, Timothy A.; Boonyaratanakornkit, Boonchai B.; Höllrigl, Volker; Clark, Douglas S.
2007-01-01
Prefoldin is a molecular chaperone found in the domains eukarya and archaea that acts in conjunction with Group II chaperonin to correctly fold other nascent proteins. Previously, our group identified a putative single subunit of prefoldin, γ PFD, that was up-regulated in response to heat stress in the hyperthermophilic archaeon Methanocaldococcus jannaschii. In order to characterize this protein, we subcloned and expressed it and the other two prefoldin subunits from M. jannaschii, α and β PFD, into Eschericia coli and characterized the proteins. Whereas α and β PFD readily assembled into the expected hexamer, γ PFD would not assemble with either protein. Instead, γ PFD forms long filaments of defined dimensions measuring 8.5 nm × 1.7–3.5 nm and lengths exceeding 1 μm. Filamentous γ PFD acts as a molecular chaperone through in vitro assays, in a manner comparable to PFD. A possible molecular model for filament assembly is discussed. PMID:17384227
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Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen*
Gilio, Karen; Harper, Matthew T.; Cosemans, Judith M. E. M.; Konopatskaya, Olga; Munnix, Imke C. A.; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D.; Heemskerk, Johan W. M.; Poole, Alastair W.
2010-01-01
Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen. PMID:20479008
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Functional divergence of platelet protein kinase C (PKC) isoforms in thrombus formation on collagen.
Gilio, Karen; Harper, Matthew T; Cosemans, Judith M E M; Konopatskaya, Olga; Munnix, Imke C A; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D; Heemskerk, Johan W M; Poole, Alastair W
2010-07-23
Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent alpha-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCalpha and PKCbeta, whereas the novel isoform, PKC, negatively regulates these events. PKCdelta also negatively regulates thrombus formation but not alpha-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCalpha or PKCbeta showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKC. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.
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Ribeiro, Raquel; Gilberto, Samuel; Gomes, Ricardo A.; Ferreira, António; Mateus, Élia; Barroso, Eduardo; Coelho, Ana V.; Freire, Ana Ponces; Cordeiro, Carlos
2015-01-01
Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis. PMID:26147092
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Silberg, Jonathan J; Tapley, Tim L; Hoff, Kevin G; Vickery, Larry E
2004-12-24
The ATPase activity of HscA, a specialized hsp70 molecular chaperone from Escherichia coli, is regulated by the iron-sulfur cluster assembly protein IscU and the J-type co-chaperone HscB. IscU behaves as a substrate for HscA, and HscB enhances the binding of IscU to HscA. To better understand the mechanism by which HscB and IscU regulate HscA, we examined binding of HscB to the different conformational states of HscA and the effects of HscB and IscU on the kinetics of the individual steps of the HscA ATPase reaction cycle. Affinity sensor studies revealed that whereas IscU binds both ADP (R-state) and ATP (T-state) HscA complexes, HscB interacts only with an ATP-bound state. Studies of ATPase activity under single-turnover and rapid mixing conditions showed that both IscU and HscB interact with the low peptide affinity T-state of HscA (HscA++.ATP) and that both modestly accelerate (3-10-fold) the rate-determining steps in the HscA reaction cycle, k(hyd) and k(T-->R). When present together, IscU and HscB synergistically stimulate both k(hyd) (approximately = 500-fold) and k(T-->R) (approximately = 60-fold), leading to enhanced formation of the HscA.ADP-IscU complex (substrate capture). Following ADP/ATP exchange, IscU also stimulates k(R-->T) (approximately = 50-fold) and thereby accelerates the rate at which the low peptide affinity HscA++.ATP T-state is regenerated. Because HscA nucleotide exchange is fast, the overall rate of the chaperone cycle in vivo will be determined by the availability of the IscU-HscB substrate-co-chaperone complex.
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Matsui, Chieko; Deng, Lin; Minami, Nanae; Abe, Takayuki; Koike, Kazuhiko; Shoji, Ikuo
2018-07-01
Hepatitis C virus (HCV) infection is closely associated with type 2 diabetes. We reported that HCV infection induces the lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) via interaction with HCV nonstructural protein 5A (NS5A) protein, thereby suppressing GLUT2 gene expression. The molecular mechanisms of selective degradation of HNF-1α caused by NS5A are largely unknown. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway. Here, we investigated whether CMA is involved in the selective degradation of HNF-1α in HCV-infected cells and observed that the pentapeptide spanning from amino acid (aa) 130 to aa 134 of HNF-1α matches the rule for the CMA-targeting motif, also known as KFERQ motif. A cytosolic chaperone protein, heat shock cognate protein of 70 kDa (HSC70), and a lysosomal membrane protein, lysosome-associated membrane protein type 2A (LAMP-2A), are key components of CMA. Immunoprecipitation analysis revealed that HNF-1α was coimmunoprecipitated with HSC70, whereas the Q130A mutation (mutation of Q to A at position 130) of HNF-1α disrupted the interaction with HSC70, indicating that the CMA-targeting motif of HNF-1α is important for the association with HSC70. Immunoprecipitation analysis revealed that increasing amounts of NS5A enhanced the association of HNF-1α with HSC70. To determine whether LAMP-2A plays a role in the degradation of HNF-1α protein, we knocked down LAMP-2A mRNA by RNA interference; this knockdown by small interfering RNA (siRNA) recovered the level of HNF-1α protein in HCV J6/JFH1-infected cells. This result suggests that LAMP-2A is required for the degradation of HNF-1α. Immunofluorescence study revealed colocalization of NS5A and HNF-1α in the lysosome. Based on our findings, we propose that HCV NS5A interacts with HSC70 and recruits HSC70 to HNF-1α, thereby promoting the lysosomal degradation of HNF-1α via CMA. IMPORTANCE Many viruses use a protein degradation system, such as
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Rutsdottir, Gudrun; Härmark, Johan; Weide, Yoran; Hebert, Hans; Rasmussen, Morten I; Wernersson, Sven; Respondek, Michal; Akke, Mikael; Højrup, Peter; Koeck, Philip J B; Söderberg, Christopher A G; Emanuelsson, Cecilia
2017-05-12
Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended I X V X I motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the I X V X I motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Electrochemically Preadsorbed Collagen Promotes Adult Human Mesenchymal Stem Cell Adhesion
Benavidez, Tomás E.; Wechsler, Marissa E.; Farrer, Madeleine M.; Bizios, Rena
2016-01-01
The present article reports on the effect of electric potential on the adsorption of collagen type I (the most abundant component of the organic phase of bone) onto optically transparent carbon electrodes (OTCE) and its mediation on subsequent adhesion of adult, human, mesenchymal stem cells (hMSCs). For this purpose, adsorption of collagen type I was investigated as a function of the protein concentration (0.01, 0.1, and 0.25 mg/mL) and applied potential (open circuit potential [OCP; control], +400, +800, and +1500 mV). The resulting substrate surfaces were characterized using spectroscopic ellipsometry, atomic force microscopy, and cyclic voltammetry. Adsorption of collagen type I onto OTCE was affected by the potential applied to the sorbent surface and the concentration of protein. The higher the applied potential and protein concentration, the higher the adsorbed amount (Γcollagen). It was also observed that the application of potential values higher than +800 mV resulted in the oxidation of the adsorbed protein. Subsequent adhesion of hMSCs on the OTCEs (precoated with the collagen type I films) under standard cell culture conditions for 2 h was affected by the extent of collagen preadsorbed onto the OTCE substrates. Specifically, enhanced hMSCs adhesion was observed when the Γcollagen was the highest. When the collagen type I was oxidized (under applied potential equal to +1500 mV), however, hMSCs adhesion was decreased. These results provide the first correlation between the effects of electric potential on protein adsorption and subsequent modulation of anchorage-dependent cell adhesion. PMID:26549607
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Takalloo, Zeinab; Sajedi, Reza H; Hosseinkhani, Saman; Asghari, S Mohsen
2016-11-15
Artemin is an abundant thermostable protein in Artemia encysted embryos and considered as a stress protein, as its highly regulated expression is associated with stress resistance. Artemin cDNA was previously isolated and cloned from Artemia urmiana and artemin was found as an efficient molecular chaperone in vitro. Here, co-transformation of E. coli was performed with two expression vectors containing artemin and firefly luciferase for in vivo studies. The time-course of luciferase inactivation at low and elevated temperatures showed that luciferase was rapidly inactivated in control cells, but it was found that luciferase was protected significantly in artemin expressing cells. More interestingly, luciferase activity was completely regained in heat treated artemin expressing cells at room temperature. In addition, in both stress conditions, similar to residual activity of luciferase, cell viability in induced cultures over-expressing artemin was significantly higher than non-expressed artemin cells. It can be suggested that artemin confers impressive resistance in stressful conditions when introduced into E. coli cells, which is due to that it protects proteins against aggregation. Such luciferase co-expression system can be used as a real-time reporter to investigate the activity of chaperone proteins in vivo and provide a rapid and simple test for molecular chaperones. Copyright © 2016 Elsevier Inc. All rights reserved.
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King, Amy M; Bartpho, Thanatchaporn; Sermswan, Rasana W; Bulach, Dieter M; Eshghi, Azad; Picardeau, Mathieu; Adler, Ben; Murray, Gerald L
2013-08-01
Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp., but knowledge of leptospiral pathogenesis remains limited. However, the development of mutagenesis systems has allowed the investigation of putative virulence factors and their involvement in leptospirosis. LipL41 is the third most abundant lipoprotein found in the outer membranes of pathogenic leptospires and has been considered a putative virulence factor. LipL41 is encoded on the large chromosome 28 bp upstream of a small open reading frame encoding a hypothetical protein of unknown function. This gene was named lep, for LipL41 expression partner. In this study, lipL41 was found to be cotranscribed with lep. Two transposon mutants were characterized: a lipL41 mutant and a lep mutant. In the lep mutant, LipL41 protein levels were reduced by approximately 90%. Lep was shown through cross-linking and coexpression experiments to bind to LipL41. Lep is proposed to be a molecular chaperone essential for the stable expression of LipL41. The roles of LipL41 and Lep in the pathogenesis of Leptospira interrogans were investigated; surprisingly, neither of these two unique proteins was essential for acute leptospirosis.
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Zhu, J K; Shi, J; Bressan, R A; Hasegawa, P M
1993-01-01
DnaJ is a 36-kD heat shock protein that functions together with Dnak (Hsp70) as a molecular chaperone in Escherichia coli. We have obtained a cDNA clone from the higher plant Atriplex nummularia that encodes a 46.6-kD polypeptide (ANJ1) with an overall 35.2% amino acid sequence identity with the E. coli DnaJ. ANJ1 has 43.4% overall sequence identity with the Saccharomyces cerevisiae cytoplasmic DnaJ homolog YDJ1/MAS5. Complementation of the yeast mas5 mutation indicated that ANJ1 is a functional homolog of YDJ1/MAS5. The presence of other DnaJ homologs in A. nummularia was demonstrated by the detection of proteins that are antigenically related to the yeast mitochondrial DnaJ homolog SCJ1 and the yeast DnaJ-related protein Sec63. Expression of the ANJ1 gene was compared with that of an A. nummularia Hsp70 gene. Expression of both ANJ1 and Hsp70 transcripts was coordinately induced by heat shock. However, noncoordinate accumulation of ANJ1 and Hsp70 mRNAs occurred during the cell growth cycle and in response to NaCl stress. PMID:8467224
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Zhu, J K; Shi, J; Bressan, R A; Hasegawa, P M
1993-03-01
DnaJ is a 36-kD heat shock protein that functions together with Dnak (Hsp70) as a molecular chaperone in Escherichia coli. We have obtained a cDNA clone from the higher plant Atriplex nummularia that encodes a 46.6-kD polypeptide (ANJ1) with an overall 35.2% amino acid sequence identity with the E. coli DnaJ. ANJ1 has 43.4% overall sequence identity with the Saccharomyces cerevisiae cytoplasmic DnaJ homolog YDJ1/MAS5. Complementation of the yeast mas5 mutation indicated that ANJ1 is a functional homolog of YDJ1/MAS5. The presence of other DnaJ homologs in A. nummularia was demonstrated by the detection of proteins that are antigenically related to the yeast mitochondrial DnaJ homolog SCJ1 and the yeast DnaJ-related protein Sec63. Expression of the ANJ1 gene was compared with that of an A. nummularia Hsp70 gene. Expression of both ANJ1 and Hsp70 transcripts was coordinately induced by heat shock. However, noncoordinate accumulation of ANJ1 and Hsp70 mRNAs occurred during the cell growth cycle and in response to NaCl stress.
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Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy
Pedrozo, Zully; Torrealba, Natalia; Fernández, Carolina; Gatica, Damian; Toro, Barbra; Quiroga, Clara; Rodriguez, Andrea E.; Sanchez, Gina; Gillette, Thomas G.; Hill, Joseph A.; Donoso, Paulina; Lavandero, Sergio
2013-01-01
Time for primary review: 15 days Aims Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation–contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. Methods and results To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. Conclusion These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels. PMID:23404999
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Hübscher, Volker; Mudholkar, Kaivalya; Rospert, Sabine
2017-02-01
Activation of the heterotrimeric kinase SNF1 via phosphorylation of a specific residue within the α subunit is essential for the release from glucose repression in the yeast Saccharomyces cerevisiae. When glucose is available, SNF1 is maintained in the dephosphorylated, inactive state by the phosphatase Glc7-Reg1. Recent findings suggest that Bmh and Ssb combine their unique client-binding properties to interact with the regulatory region of the SNF1 α subunit and by that stabilize a conformation of SNF1, which is accessible for Glc7-Reg1-dependent dephosphorylation. Together, the 14-3-3 protein Bmh and the Hsp70 homolog Ssb comprise a novel chaperone module, which is required to maintain proper glucose repression in the yeast S. cerevisiae.
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EML4-ALK fusions: propelling cancer but creating exploitable chaperone dependence.
Workman, Paul; van Montfort, Rob
2014-06-01
The crystal structure of a conserved tubulin-binding region of the EML1 protein reveals a highly atypical fold in one of its β-propeller domains. Disruption of the EML1 core region domain in many of the oncogenic EML4-ALK fusion protein variants that drive non-small cell lung cancer explains their dependence on the HSP90 molecular chaperone, provides a basis to allow more precise patient stratification for therapy, and suggests a more general model for other oncogenic fusion proteins. ©2014 American Association for Cancer Research.
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The role of fibrinogen glycation in ATTR: evidence for chaperone activity loss in disease.
Fonseca, Daniel; Gilberto, Samuel; Ribeiro-Silva, Cristina; Ribeiro, Raquel; Guinote, Inês Batista; Saraiva, Susana; Gomes, Ricardo A; Mateus, Élia; Viana, Ana; Barroso, Eduardo; Freire, Ana Ponces; Freire, Patrick; Cordeiro, Carlos; da Costa, Gonçalo
2016-07-15
Transthyretin amyloidosis (ATTR) belongs to a class of disorders caused by protein misfolding and aggregation. ATTR is a disabling disorder of autosomal dominant trait, where transthyretin (TTR) forms amyloid deposits in different organs, causing dysfunction of the peripheral nervous system. We previously discovered that amyloid fibrils from ATTR patients are glycated by methylglyoxal. Even though no consensus has been reached about the actual role of methylglyoxal-derived advanced glycation end-products in amyloid diseases, evidence collected so far points to a role for protein glycation in conformational abnormalities, being ubiquitously found in amyloid deposits in Alzheimer's disease, dialysis-related amyloidosis and Parkinson's diseases. Human fibrinogen, an extracellular chaperone, was reported to specifically interact with a wide spectrum of stressed proteins and suppress their aggregation, being an interacting protein with TTR. Fibrinogen is differentially glycated in ATTR, leading to its chaperone activity loss. Here we show the existence of a proteostasis imbalance in ATTR linked to fibrinogen glycation by methylglyoxal. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.
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Hyriopsis cumingii Hic52-A novel nacreous layer matrix protein with a collagen-like structure.
Liu, Xiaojun; Pu, Jingwen; Zeng, Shimei; Jin, Can; Dong, Shaojian; Li, Jiale
2017-09-01
Nacre is a product of a precisely regulated biomineralization process and a major contributor to the luster of pearls. Nacre is composed of calcium carbonate and an organic matrix of proteins that is secreted from mollusc mantle tissue and is exclusively associated with shell formation. In this study, hic52, a novel matrix protein gene from mantle of Hyriopsis cumingii, was cloned and functionally analyzed. The full-length cDNA of hic52 encoded 542 amino acids and contained a signal peptide of 18 amino acids. Excluding the signal peptide, the theoretical molecular mass of the polypeptide was 52.2kDa. The predicted isoelectric point was 10.37, indicating a basic shell protein. The amino acid sequence of hic52 featured high proportion of Gly (28.8%) and Gln (12.4%) residues. The predicted tertiary structure was characterized as having similarities to collagen I, alpha 1 and alpha 2 in the structure. The polypeptide sequence shared no homology with collagen. The hic52 expression pattern by quantitative real-time PCR and in situ hybridization exhibits at the dorsal epithelial cells of the mantle. Expression increased during the stages of pearl sac development. The data showed that hic52 is probably a framework shell protein that mediates and controls the nacreous biomineralization process. Copyright © 2017 Elsevier B.V. All rights reserved.
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Stetz, Gabrielle; Tse, Amanda
2017-01-01
The overarching goal of delineating molecular principles underlying differentiation of protein kinase clients and chaperone-based modulation of kinase activity is fundamental to understanding activity of many oncogenic kinases that require chaperoning of Hsp70 and Hsp90 systems to attain a functionally competent active form. Despite structural similarities and common activation mechanisms shared by cyclin-dependent kinase (CDK) proteins, members of this family can exhibit vastly different chaperone preferences. The molecular determinants underlying chaperone dependencies of protein kinases are not fully understood as structurally similar kinases may often elicit distinct regulatory responses to the chaperone. The regulatory divergences observed for members of CDK family are of particular interest as functional diversification among these kinases may be related to variations in chaperone dependencies and can be exploited in drug discovery of personalized therapeutic agents. In this work, we report the results of a computational investigation of several members of CDK family (CDK5, CDK6, CDK9) that represented a broad repertoire of chaperone dependencies—from nonclient CDK5, to weak client CDK6, and strong client CDK9. By using molecular simulations of multiple crystal structures we characterized conformational ensembles and collective dynamics of CDK proteins. We found that the elevated dynamics of CDK9 can trigger imbalances in cooperative collective motions and reduce stability of the active fold, thus creating a cascade of favorable conditions for chaperone intervention. The ensemble-based modeling of residue interaction networks and community analysis determined how differences in modularity of allosteric networks and topography of communication pathways can be linked with the client status of CDK proteins. This analysis unveiled depleted modularity of the allosteric network in CDK9 that alters distribution of communication pathways and leads to impaired
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Modern collagen wound dressings: function and purpose.
Fleck, Cynthia Ann; Simman, Richard
2010-09-01
Collagen, which is produced by fibroblasts, is the most abundant protein in the human body. A natural structural protein, collagen is involved in all 3 phases of the wound-healing cascade. It stimulates cellular migration and contributes to new tissue development. Because of their chemotactic properties on wound fibroblasts, collagen dressings encourage the deposition and organization of newly formed collagen, creating an environment that fosters healing. Collagen-based biomaterials stimulate and recruit specific cells, such as macrophages and fibroblasts, along the healing cascade to enhance and influence wound healing. These biomaterials can provide moisture or absorption, depending on the delivery system. Collagen dressings are easy to apply and remove and are conformable. Collagen dressings are usually formulated with bovine, avian, or porcine collagen. Oxidized regenerated cellulose, a plant-based material, has been combined with collagen to produce a dressing capable of binding to and protecting growth factors by binding and inactivating matrix metalloproteinases in the wound environment. The increased understanding of the biochemical processes involved in chronic wound healing allows the design of wound care products aimed at correcting imbalances in the wound microenvironment. Traditional advanced wound care products tend to address the wound's macroenvironment, including moist wound environment control, fluid management, and controlled transpiration of wound fluids. The newer class of biomaterials and wound-healing agents, such as collagen and growth factors, targets specific defects in the chronic wound environment. In vitro laboratory data point to the possibility that these agents benefit the wound healing process at a biochemical level. Considerable evidence has indicated that collagen-based dressings may be capable of stimulating healing by manipulating wound biochemistry.
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Caplan, Jeffrey L.; Zhu, Xiaohong; Mamillapalli, Padmavathi; Marathe, Rajendra; Anandalakshmi, Radhamani; Dinesh-Kumar, S. P.
2009-01-01
Summary The plant innate immune response requires a rapid, global reprogramming of cellular processes. Here we employed two complementary proteomic methods, two-dimensional differential in-gel electrophoresis (2D-DIGE) and iTRAQ, to identify differentially regulated proteins early during a defense response. Besides defense-related proteins, the constituents of the largest category of up-regulated proteins were cytoplasmic- and endoplasmic reticulum (ER)-residing molecular chaperones. Silencing of ER-resident protein disulfide isomerases, NbERp57 and NbP5, and the calreticulins, NbCRT2 and NbCRT3, lead to a partial loss of N immune receptor-mediated defense against Tobacco mosaic virus (TMV). Furthermore, NbCRT2 and NbCRT3 are required for the expression of a novel induced receptor-like kinase (IRK). IRK is a plasma membrane-localized protein required for the N-mediated hypersensitive response programmed cell death (HR-PCD) and resistance to TMV. These data support a model in which ER-resident chaperones are required for the accumulation of membrane bound or secreted proteins that are necessary for innate immunity. PMID:19917500
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Jeanne, Marion; Jorgensen, Jeff; Gould, Douglas B
2015-05-05
Collagen type IV alpha1 (COL4A1) and alpha2 (COL4A2) form heterotrimers critical for vascular basement membrane stability and function. Patients with COL4A1 or COL4A2 mutations suffer from diverse cerebrovascular diseases, including cerebral microbleeds, porencephaly, and fatal intracerebral hemorrhage (ICH). However, the pathogenic mechanisms remain unknown, and there is a lack of effective treatment. Using Col4a1 and Col4a2 mutant mouse models, we investigated the genetic complexity and cellular mechanisms underlying the disease. We found that Col4a1 mutations cause abnormal vascular development, which triggers small-vessel disease, recurrent hemorrhagic strokes, and age-related macroangiopathy. We showed that allelic heterogeneity, genetic context, and environmental factors such as intense exercise or anticoagulant medication modulated disease severity and contributed to phenotypic heterogeneity. We found that intracellular accumulation of mutant collagen in vascular endothelial cells and pericytes was a key triggering factor of ICH. Finally, we showed that treatment of mutant mice with a US Food and Drug Administration-approved chemical chaperone resulted in a decreased collagen intracellular accumulation and a significant reduction in ICH severity. Our data are the first to show therapeutic prevention in vivo of ICH resulting from Col4a1 mutation and imply that a mechanism-based therapy promoting protein folding might also prevent ICH in patients with COL4A1 and COL4A2 mutations. © 2015 American Heart Association, Inc.
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Lundin, Victor F.; Stirling, Peter C.; Gomez-Reino, Juan; Mwenifumbo, Jill C.; Obst, Jennifer M.; Valpuesta, José M.; Leroux, Michel R.
2004-01-01
Prefoldin (PFD) is a jellyfish-shaped molecular chaperone that has been proposed to play a general role in de novo protein folding in archaea and is known to assist the biogenesis of actins, tubulins, and potentially other proteins in eukaryotes. Using point mutants, chimeras, and intradomain swap variants, we show that the six coiledcoil tentacles of archaeal PFD act in concert to bind and stabilize nonnative proteins near the opening of the cavity they form. Importantly, the interaction between chaperone and substrate depends on the mostly buried interhelical hydrophobic residues of the coiled coils. We also show by electron microscopy that the tentacles can undergo an en bloc movement to accommodate an unfolded substrate. Our data reveal how archael PFD uses its unique architecture and intrinsic coiled-coil properties to interact with nonnative polypeptides. PMID:15070724
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Bencomo, Alberto; Lara-Martínez, Reyna; Rivera-Marrero, Suchitil; Domínguez, Guadalupe; Pérez-Perera, Rafaela; Jiménez-García, Luis Felipe; Altamirano-Bustamante, Nelly F.; Diaz-Delgado, Massiel; Vedrenne, Fernand; Rivillas-Acevedo, Lina; Pasten-Hidalgo, Karina; Segura-Valdez, María de Lourdes; Islas-Andrade, Sergio; Garrido-Magaña, Eulalia; Perera-Pintado, Alejandro; Prats-Capote, Anaís; Rodríguez-Tanty, Chryslaine; Altamirano-Bustamante, Myriam M.
2015-01-01
The increasing prevalence of conformational diseases, including Alzheimer's disease, type 2 Diabetes Mellitus and Cancer, poses a global challenge at many different levels. It has devastating effects on the sufferers as well as a tremendous economic impact on families and the health system. In this work, we apply a cross-functional approach that combines ideas, concepts and technologies from several disciplines in order to study, in silico and in vitro, the role of a novel chemical chaperones family (NCHCHF) in processes of protein aggregation in conformational diseases. Given that Serum Albumin (SA) is the most abundant protein in the blood of mammals, and Bovine Serum Albumin (BSA) is an off-the-shelf protein available in most labs around the world, we compared the ligandability of BSA:NCHCHF with the interaction sites in the Human Islet Amyloid Polypeptide (hIAPP):NCHCHF, and in the amyloid pharmacophore fragments (Aβ17–42 and Aβ16–21):NCHCHF. We posit that the merging of this interaction sites is a meta-structure of pharmacophore which allows the development of chaperones that can prevent protein aggregation at various states from: stabilizing the native state to destabilizing oligomeric state and protofilament. Furthermore to stabilize fibrillar structures, thus decreasing the amount of toxic oligomers in solution, as is the case with the NCHCHF. The paper demonstrates how a set of NCHCHF can be used for studying and potentially treating the various physiopathological stages of a conformational disease. For instance, when dealing with an acute phase of cytotoxicity, what is needed is the recruitment of cytotoxic oligomers, thus chaperone F, which accelerates fiber formation, would be very useful; whereas in a chronic stage it is better to have chaperones A, B, C, and D, which stabilize the native and fibril structures halting self-catalysis and the creation of cytotoxic oligomers as a consequence of fiber formation. Furthermore, all the chaperones are
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Moutaoufik, Mohamed Taha; Morrow, Geneviève; Maaroufi, Halim; Férard, Céline; Finet, Stéphanie; Tanguay, Robert M
2017-07-01
The small Hsp DmHsp27 from Drosophila melanogaster is one of the few small heat shock proteins (sHsps) found within the nucleus. We report that its dimerization is independent of disulfide bond formation and seems to rely on salt bridges. Unlike metazoan sHsps, DmHsp27 forms two populations of oligomers not in equilibrium. Mutations at highly conserved arginine residues in mammalian sHsps have been reported to be associated with protein conformational defects and intracellular aggregation. Independent mutation of three highly conserved arginines (R122, R131, and R135) to glycine in DmHsp27 results in only one population of higher molecular weight form. In vitro, the chaperone-like activity of wild-type DmHsp27 was comparable with that of its two isolated populations and to the single population of the R122G, R131G, and R135G using luciferase as substrate. However, using insulin, the chaperone-like activity of wild-type DmHsp27 was lower than that of R122G and R131G mutants. Altogether, the results characterize wild-type DmHsp27 and its alpha-crystallin domain (ACD) arginine mutants and may give insight into protection mechanism of sHsps.
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Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro.
Petzoldt, Svenja; Kahra, Dana; Kovermann, Michael; Dingeldein, Artur P G; Niemiec, Moritz S; Ådén, Jörgen; Wittung-Stafshede, Pernilla
2015-06-01
After Ctr1-mediated copper ion (Cu) entry into the human cytoplasm, chaperones Atox1 and CCS deliver Cu to P1B-type ATPases and to superoxide dismutase, respectively, via direct protein-protein interactions. Although the two Cu chaperones are presumed to work along independent pathways, we here assessed cross-reactivity between Atox1 and the first domain of CCS (CCS1) using biochemical and biophysical methods in vitro. By NMR we show that CCS1 is monomeric although it elutes differently from Atox1 in size exclusion chromatography (SEC). This property allows separation of Atox1 and CCS1 by SEC and, combined with the 254/280 nm ratio as an indicator of Cu loading, we demonstrate that Cu can be transferred from one protein to the other. Cu exchange also occurs with full-length CCS and, as expected, the interaction involves the metal binding sites since mutation of Cu-binding cysteine in Atox1 eliminates Cu transfer from CCS1. Cross-reactivity between CCS and Atox1 may aid in regulation of Cu distribution in the cytoplasm.
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Nahomi, Rooban B; DiMauro, Michael A; Wang, Benlian; Nagaraj, Ram H
2015-01-01
Previous studies have identified peptides in the 'crystallin-domain' of the small heat-shock protein (sHSP) α-crystallin with chaperone and anti-apoptotic activities. We found that peptides in heat-shock protein Hsp20 (G71HFSVLLDVKHFSPEEIAVK91) and Hsp27 (D93RWRVSLDVNHFAPDELTVK113) with sequence homology to α-crystallin also have robust chaperone and anti-apoptotic activities. Both peptides inhibited hyperthermic and chemically induced aggregation of client proteins. The scrambled peptides of Hsp20 and Hsp27 showed no such effects. The chaperone activities of the peptides were better than those from αA- and αB-crystallin. HeLa cells took up the FITC-conjugated Hsp20 peptide and, when the cells were thermally stressed, the peptide was translocated from the cytoplasm to the nucleus. The two peptides inhibited apoptosis in HeLa cells by blocking cytochrome c release from the mitochondria and caspase-3 activation. We found that scrambling the last four amino acids in the two peptides (KAIV in Hsp20 and KTLV in Hsp27) made them unable to enter cells and ineffective against stress-induced apoptosis. Intraperitoneal injection of the peptides prevented sodium-selenite-induced cataract formation in rats by inhibiting protein aggregation and oxidative stress. Our study has identified peptides from Hsp20 and Hsp27 that may have therapeutic benefit in diseases where protein aggregation and apoptosis are contributing factors.
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Yang, Chunzhang; Wang, Herui; Zhu, Dongwang; Hong, Christopher S.; Dmitriev, Pauline; Zhang, Chao; Li, Yan; Ikejiri, Barbara; Brady, Roscoe O.; Zhuang, Zhengping
2015-01-01
Gaucher disease is caused by mutations of the GBA1 gene, which encodes the lysosomal anchored gluococerebrosidase (GCase). GBA1 mutations commonly result in protein misfolding, abnormal chaperone recognition, and premature degradation, but are less likely to affect catalytic activity. In the present study, we demonstrate that the Hsp90/HOP/Cdc37 complex recruits Hsp27 after recognition of GCase mutants with subsequent targeting of GCase mutant peptides to degradation mechanisms such as VCP and the 26S proteasome. Inhibition of Hsp27 not only increased the quantity of enzyme but also enhanced GCase activity in fibroblasts derived from patients with Gaucher disease. These findings provide insight into a possible therapeutic strategy for protein misfolding diseases by correcting chaperone binding and altering subsequent downstream patterns of protein degradation. PMID:25583479
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Yang, Chunzhang; Wang, Herui; Zhu, Dongwang; Hong, Christopher S; Dmitriev, Pauline; Zhang, Chao; Li, Yan; Ikejiri, Barbara; Brady, Roscoe O; Zhuang, Zhengping
2015-01-27
Gaucher disease is caused by mutations of the GBA1 gene, which encodes the lysosomal anchored gluococerebrosidase (GCase). GBA1 mutations commonly result in protein misfolding, abnormal chaperone recognition, and premature degradation, but are less likely to affect catalytic activity. In the present study, we demonstrate that the Hsp90/HOP/Cdc37 complex recruits Hsp27 after recognition of GCase mutants with subsequent targeting of GCase mutant peptides to degradation mechanisms such as VCP and the 26S proteasome. Inhibition of Hsp27 not only increased the quantity of enzyme but also enhanced GCase activity in fibroblasts derived from patients with Gaucher disease. These findings provide insight into a possible therapeutic strategy for protein misfolding diseases by correcting chaperone binding and altering subsequent downstream patterns of protein degradation.
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Effect of Silicone on the Collagen Fibrillogenesis and Stability
Kadziński, Leszek; Prokopowicz, Magdalena; Jakóbkiewicz-Banecka, Joanna; Gabig-Cimińska, Magdalena; Łukasiak, Jerzy; Banecki, Bogdan
2015-01-01
Collagen, the most abundant protein in mammals, is able to form fibrils, which have central role in tissue repair, fibrosis, and tumor invasion. As a component of skin, tendons, and cartilages, this protein contacts with any implanted materials. An inherent problem associated with implanted prostheses is their propensity to be coated with host proteins shortly after implantation. Also, silicone implants undergoing relatively long periods of contact with blood can lead to formation of thrombi and emboli. In this paper, we demonstrate the existence of interactions between siloxanes and collagen. Low-molecular-weight cyclic siloxane (hexamethylcyclotrisiloxane—D3) and polydimethylsiloxanes (PDMS) forming linear chains, ranging in viscosity from 20 to 12,000 cSt, were analyzed. We show that D3 as well as short-chain PDMS interact with collagen, resulting in a decrease in fibrillogenesis. However, loss of collagen native structure does not occur because of these interactions. Rather, collagen seems to be sequestered in its native form in an interlayer formed by collagen–siloxane complexes. On the other hand, silicone molecules with longer chains (i.e., PDMS with viscosity of 1000 and 12,000 cSt, the highest viscosity analyzed here) demonstrate little interaction with this protein and do not seem to affect collagen activity. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1275–1281, 2015 PMID:25589402
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HdeB chaperone activity is coupled to its intrinsic dynamic properties
Ding, Jienv; Yang, Chengfeng; Niu, Xiaogang; Hu, Yunfei; Jin, Changwen
2015-01-01
Enteric bacteria encounter extreme acidity when passing through hosts’ stomach. Since the bacterial periplasmic space quickly equilibrates with outer environment, an efficient acid resistance mechanism is essential in preventing irreversible protein denaturation/aggregation and maintaining bacteria viability. HdeB, along with its homolog HdeA, was identified as a periplasmic acid-resistant chaperone. Both proteins exist as homodimers and share similar monomeric structures under neutral pH, while showing different dimeric packing interfaces. Previous investigations show that HdeA functions through an acid-induced dimer-to-monomer transition and partial unfolding at low pH (pH 2–3), resulting in exposure of hydrophobic surfaces that bind substrate proteins. In contrast, HdeB appears to have a much higher optimal activation pH (pH 4–5), under which condition the protein maintains a well-folded dimer and the mechanism for its chaperone activity remains elusive. Herein, we present an NMR study of HdeB to investigate its dynamic properties. Our results reveal that HdeB undergoes significant micro- to milli-second timescale conformational exchanges at neutral to near-neutral pH, under the later condition it exhibits optimal activity. The current study indicates that HdeB activation is coupled to its intrinsic dynamics instead of structural changes, and therefore its functional mechanism is apparently different from HdeA. PMID:26593705
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Targeting HSF1 disrupts HSP90 chaperone function in chronic lymphocytic leukemia.
Ganguly, Siddhartha; Home, Trisha; Yacoub, Abdulraheem; Kambhampati, Suman; Shi, Huidong; Dandawate, Prasad; Padhye, Subhash; Saluja, Ashok K; McGuirk, Joseph; Rao, Rekha
2015-10-13
CLL is a disease characterized by chromosomal deletions, acquired copy number changes and aneuploidy. Recent studies have shown that overexpression of Heat Shock Factor (HSF) 1 in aneuploid tumor cells can overcome deficiencies in heat shock protein (HSP) 90-mediated protein folding and restore protein homeostasis. Interestingly, several independent studies have demonstrated that HSF1 expression and activity also affects the chaperoning of HSP90 kinase clients, although the mechanism underlying this observation is unclear. Here, we determined how HSF1 regulates HSP90 function using CLL as a model system. We report that HSF1 is overexpressed in CLL and treatment with triptolide (a small molecule inhibitor of HSF1) induces apoptosis in cultured and primary CLL B-cells. We demonstrate that knockdown of HSF1 or its inhibition with triptolide results in the reduced association of HSP90 with its kinase co-chaperone cell division cycle 37 (CDC37), leading to the partial depletion of HSP90 client kinases, Bruton's Tyrosine Kinase (BTK), c-RAF and cyclin-dependent kinase 4 (CDK4). Treatment with triptolide or HSF1 knockdown disrupts the cytosolic complex between HSF1, p97, HSP90 and the HSP90 deacetylase- Histone deacetylase 6 (HDAC6). Consequently, HSF1 inhibition results in HSP90 acetylation and abrogation of its chaperone function. Finally, tail vein injection of Mec-1 cells into Rag2-/-IL2Rγc-/- mice followed by treatment with minnelide (a pro-drug of triptolide), reduced leukemia, increased survival and attenuated HSP90-dependent survival signaling in vivo. In conclusion, our study provides a strong rationale to target HSF1 and test the activity of minnelide against human CLL.
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Targeting Hsp90-Cdc37: A Promising Therapeutic Strategy by Inhibiting Hsp90 Chaperone Function.
Wang, Lei; Li, Li; Gu, Kai; Xu, Xiao-Li; Sun, Yuan; You, Qi-Dong
2017-01-01
The Hsp90 chaperone protein regulates the folding, maturation and stability of a wide variety of oncoproteins. In recent years, many Hsp90 inhibitors have entered into the clinical trials while all of them target ATPase showing similar binding capacity and kinds of side-effects so that none have reached to the market. During the regulation progress, numerous protein- protein interactions (PPI) such as Hsp90 and client proteins or cochaperones are involved. With the Hsp90-cochaperones PPI networks being more and more clear, many cancerous proteins have been reported to be tightly correlated to Hsp90-cochaperones PPI. Among them, Hsp90-Cdc37 PPI has been widely reported to associate with numerous protein kinases, making it a novel target for the treatment of cancers. In this paper, we briefly review the strategies and modulators targeting Hsp90-Cdc37 complex including direct and indirect regulation mechanism. Through these discussions we expect to present inspirations for new insights into an alternative way to inhibit Hsp90 chaperone function. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
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Dual function of the UNC-45b chaperone with myosin and GATA4 in cardiac development
Chen, Daisi; Li, Shumin; Singh, Ram; Spinette, Sarah; Sedlmeier, Reinhard; Epstein, Henry F.
2012-01-01
Summary Cardiac development requires interplay between the regulation of gene expression and the assembly of functional sarcomeric proteins. We report that UNC-45b recessive loss-of-function mutations in C3H and C57BL/6 inbred mouse strains cause arrest of cardiac morphogenesis at the formation of right heart structures and failure of contractile function. Wild-type C3H and C57BL/6 embryos at the same stage, E9.5, form actively contracting right and left atria and ventricles. The known interactions of UNC-45b as a molecular chaperone are consistent with diminished accumulation of the sarcomeric myosins, but not their mRNAs, and the resulting decreased contraction of homozygous mutant embryonic hearts. The novel finding that GATA4 accumulation is similarly decreased at the protein but not mRNA levels is also consistent with the function of UNC-45b as a chaperone. The mRNAs of known downstream targets of GATA4 during secondary cardiac field development, the cardiogenic factors Hand1, Hand2 and Nkx-2.5, are also decreased, consistent with the reduced GATA4 protein accumulation. Direct binding studies show that the UNC-45b chaperone forms physical complexes with both the alpha and beta cardiac myosins and the cardiogenic transcription factor GATA4. Co-expression of UNC-45b with GATA4 led to enhanced transcription from GATA promoters in naïve cells. These novel results suggest that the heart-specific UNC-45b isoform functions as a molecular chaperone mediating contractile function of the sarcomere and gene expression in cardiac development. PMID:22553207
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Gistelinck, C.; Gioia, R.; Gagliardi, A.; Tonelli, F.; Marchese, L.; Bianchi, L.; Landi, C.; Bini, L.; Huysseune, A.; Witten, P. E.; Staes, A.; Gevaert, K.; De Rocker, N.; Menten, B.; Malfait, F.; Leikin, S.; Carra, S.; Tenni, R.; Rossi, A.; De Paepe, A.; Coucke, P.; Willaert, A.; Forlino, A.
2016-01-01
Over the last years the zebrafish imposed itself as a powerful model to study skeletal diseases, but a limit to its use is the poor characterization of collagen type I, the most abundant protein in bone and skin. In tetrapods collagen type I is a trimer mainly composed of two α1 chains and one α2 chain, encoded by COL1A1 and COL1A2 genes, respectively. In contrast, in zebrafish three type I collagen genes exist, col1a1a, col1a1b and col1a2 coding for α1(I), α3(I) and α2(I) chains. During embryonic and larval development the three collagen type I genes showed a similar spatio-temporal expression pattern, indicating their co-regulation and interdependence at these stages. In both embryonic and adult tissues, the presence of the three α(I) chains was demonstrated, although in embryos α1(I) was present in two distinct glycosylated states, suggesting a developmental-specific collagen composition. Even though in adult bone, skin and scales equal amounts of α1(I), α3(I) and α2(I) chains are present, the presented data suggest a tissue-specific stoichiometry and/or post-translational modification status for collagen type I. In conclusion, this data will be useful to properly interpret results and insights gained from zebrafish models of skeletal diseases. PMID:26876635
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Liu, Shing-Hwa; Yang, Ching-Chin; Chan, Ding-Cheng; Wu, Cheng-Tien; Chen, Li-Ping; Huang, Jenq-Wen; Hung, Kuan-Yu; Chiang, Chih-Kang
2016-04-19
Renal tubulointerstitial fibrosis is the common and final pathologic change of kidney in end-stage renal disease. Interesting, endoplasmic reticulum (ER) stress is known to contribute to the pathophysiological mechanisms during the development of renal fibrosis. Here, we investigated the effects of chemical chaperon sodium 4-phenylbutyrate (4-PBA) on renal fibrosis in vivo and in vitro. In a rat unilateral ureteral obstruction (UUO) model, 4-PBA mimicked endogenous ER chaperon in the kidneys and significantly reduced glucose regulated protein 78 (GRP78), CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), activating transcription factor 4 (ATF4), and phosphorylated JNK protein expressions as well as restored spliced X-box-binding protein 1 (XBP1) expressions in the kidneys of UUO rats. 4-PBA also attenuated the increases of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) protein expressions, tubulointerstitial fibrosis, and apoptosis in the kidneys of UUO rats. Moreover, transforming growth factor (TGF)-β markedly increased ER stress-associated molecules, profibrotic factors, and apoptotic markers in the renal tubular cells (NRK-52E), all of which could be significantly counteracted by 4-PBA treatment. 4-PBA also diminished TGF-β-increased CTGF promoter activity and CTGF mRNA expression in NRK-52E cells. Taken together, our results indicated that 4-PBA acts as an ER chaperone to ameliorate ER stress-induced renal tubular cell apoptosis and renal fibrosis.
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Vakiloroayaei, Ana; Shah, Neha S.; Oeffinger, Marlene
2017-01-01
Abstract Non-coding RNAs have critical roles in biological processes, and RNA chaperones can promote their folding into the native shape required for their function. La proteins are a class of highly abundant RNA chaperones that contact pre-tRNAs and other RNA polymerase III transcripts via their common UUU-3′OH ends, as well as through less specific contacts associated with RNA chaperone activity. However, whether La proteins preferentially bind misfolded pre-tRNAs or instead engage all pre-tRNA substrates irrespective of their folding status is not known. La deletion in yeast is synthetically lethal when combined with the loss of tRNA modifications predicted to contribute to the native pre-tRNA fold, such as the N2, N2-dimethylation of G26 by the methyltransferase Trm1p. In this work, we identify G26 containing pre-tRNAs that misfold in the absence of Trm1p and/or La (Sla1p) in Schizosaccharomyces pombe cells, then test whether La preferentially associates with such tRNAs in vitro and in vivo. Our data suggest that La does not discriminate a native from misfolded RNA target, and highlights the potential challenges faced by RNA chaperones in preferentially binding defective substrates. PMID:28977649
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Zhang, Ziyu; Shen, Longyan; Law, Kelvin; Zhang, Zengdi; Liu, Xiaotong; Hua, Hu; Li, Sanen; Huang, Huijie; Yue, Shen; Hui, Chi-chung
2016-01-01
ABSTRACT Cellular responses to the graded Sonic Hedgehog (Shh) morphogenic signal are orchestrated by three Gli genes that give rise to both transcription activators and repressors. An essential downstream regulator of the pathway, encoded by the tumor suppressor gene Suppressor of fused (Sufu), plays critical roles in the production, trafficking, and function of Gli proteins, but the mechanism remains controversial. Here, we show that Sufu is upregulated in active Shh responding tissues and accompanies Gli activators translocating into and Gli repressors out of the nucleus. Trafficking of Sufu to the primary cilium, potentiated by Gli activators but not repressors, was found to be coupled to its nuclear import. We have identified a nuclear export signal (NES) motif of Sufu in juxtaposition to the protein kinase A (PKA) and glycogen synthase kinase 3 (GSK3) dual phosphorylation sites and show that Sufu binds the chromatin with both Gli1 and Gli3. Close comparison of neural tube development among individual Ptch1−/−, Sufu−/−, and Ptch1−/−; Sufu−/− double mutant embryos indicates that Sufu is critical for the maximal activation of Shh signaling essential to the specification of the most-ventral neurons. These data define Sufu as a novel class of molecular chaperone required for every aspect of Gli regulation and function. PMID:27849569
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Lu, Mei; Zhao, Xiaoyun; Xing, Haonan; Xun, Zhe; Yang, Tianzhi; Cai, Cuifang; Wang, Dongkai; Ding, Pingtian
2018-04-03
Cell-free (CF) protein synthesis has emerged as a powerful technique platform for efficient protein production in vitro. Liposomes have been widely studied as therapeutic carriers due to their biocompatibility, biodegradability, low toxicity, flexible surface manipulation, easy preparation, and higher cargo encapsulation capability. However, rapid immune clearance, insufficient targeting capacity, and poor cytoplasmic delivery efficiency substantially restrict their clinical application. The incorporation of functional membrane proteins (MPs) or peptides allows the transfer of biological properties to liposomes and imparts them with improved circulation, increased targeting, and efficient intracellular delivery. Liposome-chaperoned CF synthesis enables production of proteoliposomes in one-step reaction, which not only substantially simplifies the production procedure but also keeps protein functionality intact. Building off these observations, proteoliposomes with integrated MPs represent an excellent candidate for therapeutic delivery. In this review, we describe recent advances in CF synthesis with emphasis on detailing key factors for improving CF expression efficiency. Furthermore, we provide insights into strategies for rational design of proteoliposomal nanodelivery systems via CF synthesis. Liposome-chaperoned CF synthesis has emerged as a powerful approach for the design of recombinant proteoliposomes in one-step reaction. The incorporation of bioactive MPs or peptides into liposomes via CF synthesis can facilitate the development of proteoliposomal nanodelivery systems with improved circulation, increased targeting, and enhanced cellular delivery capacity. Moreover, by adapting lessons learned from natural delivery vehicles, novel bio-inspired proteoliposomes with enhanced delivery properties could be produced in CF systems. In this review, we first give an overview of CF synthesis with focus on enhancing protein expression in liposome-chaperoned CF
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Cucurbitacin D Is a Disruptor of the HSP90 Chaperone Machinery.
Hall, Jessica A; Seedarala, Sahithi; Rice, Nichole; Kopel, Lucas; Halaweish, Fathi; Blagg, Brian S J
2015-04-24
Heat shock protein 90 (Hsp90) facilitates the maturation of many newly synthesized and unfolded proteins (clients) via the Hsp90 chaperone cycle, in which Hsp90 forms a heteroprotein complex and relies upon cochaperones, immunophilins, etc., for assistance in client folding. Hsp90 inhibition has emerged as a strategy for anticancer therapies due to the involvement of clients in many oncogenic pathways. Inhibition of chaperone function results in client ubiquitinylation and degradation via the proteasome, ultimately leading to tumor digression. Small molecule inhibitors perturb ATPase activity at the N-terminus and include derivatives of the natural product geldanamycin. However, N-terminal inhibition also leads to induction of the pro-survival heat shock response (HSR), in which displacement of the Hsp90-bound transcription factor, heat shock factor-1, translocates to the nucleus and induces transcription of heat shock proteins, including Hsp90. An alternative strategy for Hsp90 inhibition is disruption of the Hsp90 heteroprotein complex. Disruption of the Hsp90 heteroprotein complex is an effective strategy to prevent client maturation without induction of the HSR. Cucurbitacin D, isolated from Cucurbita texana, and 3-epi-isocucurbitacin D prevented client maturation without induction of the HSR. Cucurbitacin D also disrupted interactions between Hsp90 and two cochaperones, Cdc37 and p23.
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Stetz, Gabrielle; Verkhivker, Gennady M
2016-08-22
Although molecular mechanisms of allosteric regulation in the Hsp70 chaperones have been extensively studied at both structural and functional levels, the current understanding of allosteric inhibition of chaperone activities by small molecules is still lacking. In the current study, using a battery of computational approaches, we probed allosteric inhibition mechanisms of E. coli Hsp70 (DnaK) and human Hsp70 proteins by small molecule inhibitors PET-16 and novolactone. Molecular dynamics simulations and binding free energy analysis were combined with network-based modeling of residue interactions and allosteric communications to systematically characterize and compare molecular signatures of the apo form, substrate-bound, and inhibitor-bound chaperone complexes. The results suggested a mechanism by which the allosteric inhibitors may leverage binding energy hotspots in the interaction networks to stabilize a specific conformational state and impair the interdomain allosteric control. Using the network-based centrality analysis and community detection, we demonstrated that substrate binding may strengthen the connectivity of local interaction communities, leading to a dense interaction network that can promote an efficient allosteric communication. In contrast, binding of PET-16 to DnaK may induce significant dynamic changes and lead to a fractured interaction network and impaired allosteric communications in the DnaK complex. By using a mechanistic-based analysis of distance fluctuation maps and allosteric propensities of protein residues, we determined that the allosteric network in the PET-16 complex may be small and localized due to the reduced communication and low cooperativity of the substrate binding loops, which may promote the higher rates of substrate dissociation and the decreased substrate affinity. In comparison with the significant effect of PET-16, binding of novolactone to HSPA1A may cause only moderate network changes and preserve allosteric
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Merino, Irene; Contreras, Angela; Jing, Zhong-Ping; Gallardo, Fernando; Cánovas, Francisco M; Gómez, Luis
2014-02-01
Climate-driven heat stress is a key factor affecting forest plantation yields. While its effects are expected to worsen during this century, breeding more tolerant genotypes has proven elusive. We report here a substantial and durable increase in the thermotolerance of hybrid poplar (Populus tremula×Populus alba) through overexpression of a major small heat shock protein (sHSP) with convenient features. Experimental evidence was obtained linking protective effects in the transgenic events with the unique chaperone activity of sHSPs. In addition, significant positive correlations were observed between phenotype strength and heterologous sHSP accumulation. The remarkable baseline levels of transgene product (up to 1.8% of total leaf protein) have not been reported in analogous studies with herbaceous species. As judged by protein analyses, such an accumulation is not matched either by endogenous sHSPs in both heat-stressed poplar plants and field-grown adult trees. Quantitative real time-polymerase chain reaction analyses supported these observations and allowed us to identify the poplar members most responsive to heat stress. Interestingly, sHSP overaccumulation was not associated with pleiotropic effects that might decrease yields. The poplar lines developed here also outperformed controls under in vitro and ex vitro culture conditions (callus biomass, shoot production, and ex vitro survival), even in the absence of thermal stress. These results reinforce the feasibility of improving valuable genotypes for plantation forestry, a field where in vitro recalcitrance, long breeding cycles, and other practical factors constrain conventional genetic approaches. They also provide new insights into the biological functions of the least understood family of heat shock protein chaperones.
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Raynes, J K; Day, L; Augustin, M A; Carver, J A
2015-04-01
Within each milk protein there are many individual protein variants and marked alterations to milk functionality can occur depending on the genetic variants of each protein present. Bovine A(1) and A(2) β-casein (β-CN) are 2 variants that contribute to differences in the gelation performance of milk. The A(1) and A(2) β-CN variants differ by a single AA, the substitution of histidine for proline at position 67. β-Casein not only participates in formation of the casein micelle but also forms an oligomeric micelle itself and functions as a molecular chaperone to prevent the aggregation of a wide range of proteins, including the other caseins. Micelle assembly of A(1) and A(2) β-CN was investigated using dynamic light scattering and small-angle X-ray scattering, whereas protein functionality was assessed using fluorescence techniques and molecular chaperone assays. The A(2) β-CN variant formed smaller micelles than A(1) β-CN, with the monomer-micelle equilibrium of A(2) β-CN being shifted toward the monomer. This shift most likely arose from structural differences between the 2 β-CN variants associated with the adoption of greater polyproline-II helix in A(2) β-CN and most likely led to enhanced chaperone activity of A(2) β-CN compared with A(1) β-CN. The difference in micelle assembly, and hence chaperone activity, may provide explain differences in the functionality of homozygous A(1) and A(2) milk. The results of this study highlight that substitution of even a single AA can significantly alter the properties of an intrinsically unstructured protein such as β-CN and, in this case, may have an effect on the functionality of milk. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
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Hsp31, a member of the DJ-1 superfamily, is a multitasking stress responder with chaperone activity
Aslam, Kiran; Hazbun, Tony R.
2016-01-01
ABSTRACT Among different types of protein aggregation, amyloids are a biochemically well characterized state of protein aggregation that are associated with a large number of neurodegenerative diseases including Parkinson's disease, Alzheimer and Creutzfeldt-Jakob disease. Yeast, Saccharomyces cerevisiae is an insightful model to understand the underlying mechanism of protein aggregation. Many yeast molecular chaperones can modulate aggregation and misfolding of proteins including α-Syn and the Sup35 prion. Hsp31 is a homodimeric protein structurally similar to human DJ-1, a Parkinson's disease-linked protein, and both are members of the DJ-1/ThiJ/PfpI superfamily. An emerging view is that Hsp31 and its associated superfamily members each have divergent multitasking functions that have the common theme of responding and managing various types of cellular stress. Hsp31 has several biochemical activities including chaperone and detoxifying enzyme activities that modulate at various points of a stress pathway such as toxicity associated with protein misfolding. However, we have shown the protective role of Hsp31's chaperone activity can operate independent of detoxifying enzyme activities in preventing the early stages of protein aggregate formation and associated cellular toxicities. We provide additional data that collectively supports the multiple functional roles that can be accomplished independent of each other. We present data indicating Hsp31 purified from yeast is more active compared to expression and purification from E. coli suggesting that posttranslational modifications could be important for Hsp31 to be fully active. We also compare the similarities and differences in activities among paralogs of Hsp31 supporting a model in which this protein family has overlapping but diverging roles in responding to various sources of cellular stresses. PMID:27097320
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Assimon, Victoria A; Southworth, Daniel R; Gestwicki, Jason E
2015-12-08
Heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) require the help of tetratricopeptide repeat (TPR) domain-containing cochaperones for many of their functions. Each monomer of Hsp70 or Hsp90 can interact with only a single TPR cochaperone at a time, and each member of the TPR cochaperone family brings distinct functions to the complex. Thus, competition for TPR binding sites on Hsp70 and Hsp90 appears to shape chaperone activity. Recent structural and biophysical efforts have improved our understanding of chaperone-TPR contacts, focusing on the C-terminal EEVD motif that is present in both chaperones. To better understand these important protein-protein interactions on a wider scale, we measured the affinity of five TPR cochaperones, CHIP, Hop, DnaJC7, FKBP51, and FKBP52, for the C-termini of four members of the chaperone family, Hsc70, Hsp72, Hsp90α, and Hsp90β, in vitro. These studies identified some surprising selectivity among the chaperone-TPR pairs, including the selective binding of FKBP51/52 to Hsp90α/β. These results also revealed that other TPR cochaperones are only able to weakly discriminate between the chaperones or between their paralogs. We also explored whether mimicking phosphorylation of serine and threonine residues near the EEVD motif might impact affinity and found that pseudophosphorylation had selective effects on binding to CHIP but not other cochaperones. Together, these findings suggest that both intrinsic affinity and post-translational modifications tune the interactions between the Hsp70 and Hsp90 proteins and the TPR cochaperones.
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Gotoh, Kazuhisa; Nonoguchi, Kohsuke; Higashitsuji, Hiroaki; Kaneko, Yoshiyuki; Sakurai, Toshiharu; Sumitomo, Yasuhiko; Itoh, Katsuhiko; Subjeck, John R; Fujita, Jun
2004-02-27
Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. We constructed subtracted cDNA libraries enriched with genes overexpressed in HCCs. Among the 17 genes identified were molecular chaperones, Hsp110, Hsp90B, and Hsp70-1. Expression of the Hsp110 family members was further analyzed, and increased transcript levels of Hsp110 and Apg-2, but not Apg-1, were found in 12 and 14, respectively, of 18 HCCs. Immunohistochemical analysis demonstrated the overexpression of the proteins in tumor cells. Apg-2 had chaperone ability similar to Hsp110 in a thermal denaturation assay using luciferase, and showed anti-apoptotic activity. These results suggest that the Hsp110 family members play important roles in hepatocarcinogenesis through their chaperoning activities.
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Molecular mechanics of mineralized collagen fibrils in bone
Nair, Arun K.; Gautieri, Alfonso; Chang, Shu-Wei; Buehler, Markus J.
2013-01-01
Bone is a natural composite of collagen protein and the mineral hydroxyapatite. The structure of bone is known to be important to its load-bearing characteristics, but relatively little is known about this structure or the mechanism that govern deformation at the molecular scale. Here we perform full-atomistic calculations of the three-dimensional molecular structure of a mineralized collagen protein matrix to try to better understand its mechanical characteristics under tensile loading at various mineral densities. We find that as the mineral density increases, the tensile modulus of the network increases monotonically and well beyond that of pure collagen fibrils. Our results suggest that the mineral crystals within this network bears up to four times the stress of the collagen fibrils, whereas the collagen is predominantly responsible for the material’s deformation response. These findings reveal the mechanism by which bone is able to achieve superior energy dissipation and fracture resistance characteristics beyond its individual constituents. PMID:23591891
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Kajitani, Kaori; Kato, Kohsuke; Nagata, Kyosuke
2017-04-01
Linker histone H1 is involved in the regulation of gene activity through the maintenance of higher-order chromatin structure. Previously, we have shown that template activating factor-I (TAF-I or protein SET) is involved in linker histone H1 dynamics as a histone H1 chaperone. In human and murine cells, two TAF-I subtypes exist, namely TAF-Iα and TAF-Iβ. TAF-I has a highly acidic amino acid cluster in its C-terminal region and forms homo- or heterodimers through its dimerization domain. Both dimer formation and the C-terminal region of TAF-I are essential for the histone chaperone activity. TAF-Iα exhibits less histone chaperone activity compared with TAF-Iβ even though TAF-Iα and β differ only in their N-terminal regions. However, it is unclear how subtype-specific TAF-I activities are regulated. Here, we have shown that the N-terminal region of TAF-Iα autoinhibits its histone chaperone activity via intramolecular interaction with its C-terminal region. When the interaction between the N- and C-terminal regions of TAF-Iα is disrupted, TAF-Iα shows a histone chaperone activity similar to that of TAF-Iβ. Taken together, these results provide mechanistic insights into the concept that fine tuning of TAF-I histone H1 chaperone activity relies on the subtype compositions of the TAF-I dimer. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.
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Matsuura, Takashi; Tokutomi, Kentaro; Sasaki, Michiko; Katafuchi, Michitsuna; Mizumachi, Emiri; Sato, Hironobu
2014-01-01
Bone undergoes constant remodeling throughout life. The cellular and biochemical mechanisms of bone remodeling vary in a region-specific manner. There are a number of notable differences between the mandible and long bones, including developmental origin, osteogenic potential of mesenchymal stem cells, and the rate of bone turnover. Collagen, the most abundant matrix protein in bone, is responsible for determining the relative strength of particular bones. Posttranslational modifications of collagen, such as intermolecular crosslinking and lysine hydroxylation, are the most essential determinants of bone strength, although the amount of collagen is also important. In comparison to long bones, the mandible has greater collagen content, a lower amount of mature crosslinks, and a lower extent of lysine hydroxylation. The great abundance of immature crosslinks in mandibular collagen suggests that there is a lower rate of cross-link maturation. This means that mandibular collagen is relatively immature and thus more readily undergoes degradation and turnover. The greater rate of remodeling in mandibular collagen likely renders more flexibility to the bone and leaves it more suited to constant exercise. As reviewed here, it is important in clinical dentistry to understand the distinctive features of the bones of the jaw.
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Tokutomi, Kentaro; Sasaki, Michiko; Katafuchi, Michitsuna; Mizumachi, Emiri; Sato, Hironobu
2014-01-01
Bone undergoes constant remodeling throughout life. The cellular and biochemical mechanisms of bone remodeling vary in a region-specific manner. There are a number of notable differences between the mandible and long bones, including developmental origin, osteogenic potential of mesenchymal stem cells, and the rate of bone turnover. Collagen, the most abundant matrix protein in bone, is responsible for determining the relative strength of particular bones. Posttranslational modifications of collagen, such as intermolecular crosslinking and lysine hydroxylation, are the most essential determinants of bone strength, although the amount of collagen is also important. In comparison to long bones, the mandible has greater collagen content, a lower amount of mature crosslinks, and a lower extent of lysine hydroxylation. The great abundance of immature crosslinks in mandibular collagen suggests that there is a lower rate of cross-link maturation. This means that mandibular collagen is relatively immature and thus more readily undergoes degradation and turnover. The greater rate of remodeling in mandibular collagen likely renders more flexibility to the bone and leaves it more suited to constant exercise. As reviewed here, it is important in clinical dentistry to understand the distinctive features of the bones of the jaw. PMID:24818151
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Chaperones of F[subscript 1]-ATPase
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ludlam, Anthony; Brunzelle, Joseph; Pribyl, Thomas
2009-09-25
Mitochondrial F{sub 1}-ATPase contains a hexamer of alternating {alpha} and {beta} subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to {beta} and {alpha}. In the absence of Atp11p and Atp12p, the hexamer is not formed, and {alpha} and {beta} precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F{sub 1} assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486--1493) hypothesized that the chaperones themselves look very much like the {alpha} and {beta} subunits, and proposed an exchange of Atp11pmore » for {alpha} and of Atp12p for {beta}; the driving force for the exchange was expected to be a higher affinity of {alpha} and {beta} for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to {beta} and Atp12p is bound to {alpha}, the two F{sub 1} subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to {alpha} and {beta} prevents further interactions between these F1 subunits. However, Atp11p and Atp12p do not resemble {alpha} or {beta}, and it is instead the F{sub 1} {gamma} subunit that initiates the release of the chaperones from {alpha} and {beta} and their further assembly into the mature complex.« less
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Schroeter, Elena R; DeHart, Caroline J; Cleland, Timothy P; Zheng, Wenxia; Thomas, Paul M; Kelleher, Neil L; Bern, Marshall; Schweitzer, Mary H
2017-02-03
Sequence data from biomolecules such as DNA and proteins, which provide critical information for evolutionary studies, have been assumed to be forever outside the reach of dinosaur paleontology. Proteins, which are predicted to have greater longevity than DNA, have been recovered from two nonavian dinosaurs, but these results remain controversial. For proteomic data derived from extinct Mesozoic organisms to reach their greatest potential for investigating questions of phylogeny and paleobiology, it must be shown that peptide sequences can be reliably and reproducibly obtained from fossils and that fragmentary sequences for ancient proteins can be increasingly expanded. To test the hypothesis that peptides can be repeatedly detected and validated from fossil tissues many millions of years old, we applied updated extraction methodology, high-resolution mass spectrometry, and bioinformatics analyses on a Brachylophosaurus canadensis specimen (MOR 2598) from which collagen I peptides were recovered in 2009. We recovered eight peptide sequences of collagen I: two identical to peptides recovered in 2009 and six new peptides. Phylogenetic analyses place the recovered sequences within basal archosauria. When only the new sequences are considered, B. canadensis is grouped more closely to crocodylians, but when all sequences (current and those reported in 2009) are analyzed, B. canadensis is placed more closely to basal birds. The data robustly support the hypothesis of an endogenous origin for these peptides, confirm the idea that peptides can survive in specimens tens of millions of years old, and bolster the validity of the 2009 study. Furthermore, the new data expand the coverage of B. canadensis collagen I (a 33.6% increase in collagen I alpha 1 and 116.7% in alpha 2). Finally, this study demonstrates the importance of reexamining previously studied specimens with updated methods and instrumentation, as we obtained roughly the same amount of sequence data as the
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USDA-ARS?s Scientific Manuscript database
An intronless cluster of three class I small heat shock protein (sHSP) chaperone genes, Sl17.6, Sl20.0 and Sl20.1, resident on the short arm of chromosome 6 in tomato, was previously characterized (Goyal et al., 2012). This shsp chaperone gene cluster was found decorated with cis sequences known to ...
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Collagen matrix as a tool in studying fibroblastic cell behavior.
Kanta, Jiří
2015-01-01
Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes.
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Collagen matrix as a tool in studying fibroblastic cell behavior
Kanta, Jiří
2015-01-01
Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes. PMID:25734486
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Multiscale modeling of a conditionally disordered pH-sensing chaperone.
Ahlstrom, Logan S; Law, Sean M; Dickson, Alex; Brooks, Charles L
2015-04-24
The pH-sensing chaperone HdeA promotes the survival of enteropathogenic bacteria during transit through the harshly acidic environment of the mammalian stomach. At low pH, HdeA transitions from an inactive, folded, dimer to chaperone-active, disordered, monomers to protect against the acid-induced aggregation of periplasmic proteins. Toward achieving a detailed mechanistic understanding of the pH response of HdeA, we develop a multiscale modeling approach to capture its pH-dependent thermodynamics. Our approach combines pK(a) (logarithmic acid dissociation constant) calculations from all-atom constant pH molecular dynamics simulations with coarse-grained modeling and yields new, atomic-level, insights into HdeA chaperone function that can be directly tested by experiment. "pH triggers" that significantly destabilize the dimer are each located near the N-terminus of a helix, suggesting that their neutralization at low pH destabilizes the helix macrodipole as a mechanism of monomer disordering. Moreover, we observe a non-monotonic change in the pH-dependent stability of HdeA, with maximal stability of the dimer near pH5. This affect is attributed to the protonation Glu37, which exhibits an anomalously high pK(a) value and is located within the hydrophobic dimer interface. Finally, the pH-dependent binding pathway of HdeA comprises a partially unfolded, dimeric intermediate that becomes increasingly stable relative to the native dimer at lower pH values and displays key structural features for chaperone-substrate interaction. We anticipate that the insights from our model will help inform ongoing NMR and biochemical investigations. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Electrophoretic mobility patterns of collagen following laser welding
NASA Astrophysics Data System (ADS)
Bass, Lawrence S.; Moazami, Nader; Pocsidio, Joanne O.; Oz, Mehmet C.; LoGerfo, Paul; Treat, Michael R.
1991-06-01
Clinical application of laser vascular anastomosis in inhibited by a lack of understanding of its mechanism. Whether tissue fusion results from covalent or non-covalent bonding of collagen and other structural proteins is unknown. We compared electrophoretic mobility of collagen in laser treated and untreated specimens of rat tail tendon (>90% type I collagen) and rabbit aorta. Welding was performed, using tissue shrinkage as the clinical endpoint, using the 808 nm diode laser (power density 14 watts/cm2) and topical indocyanine green dye (max absorption 805 nm). Collagen was extracted with 8 M urea (denaturing), 0.5 M acetic acid (non-denaturing) and acetic acid/pepsin (cleaves non- helical protein). Mobility patterns on gel electrophoresis (SDS-PAGE) after urea or acetic acid extraction were identical in the lasered and control tendon and vessel (confirmed by optical densitometry), revealing no evidence of formation of novel covalent bonds. Alpha and beta band intensity was diminished in pepsin incubated lasered specimens compared with controls (optical density ratio 0.00 +/- 9 tendon, 0.65 +/- 0.12 aorta), indicating the presence of denatured collagen. With the laser parameters used, collagen is denatured without formation of covalent bonds, suggesting that non-covalent interaction between denatured collagen molecules may be responsible for the weld. Based on this mechanism, welding parameters can be chosen which produce collagen denaturation without cell death.
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Effective Chaperone Selection and Training for Enhanced Youth Experiences
ERIC Educational Resources Information Center
Anderson, Emily J.; Roop, Kelsey; MacArthur, Stacey
2017-01-01
This article identifies key strategies for selecting and training chaperones for youth programs. Although substantial research on volunteer core competencies and training exists, very little has been written to specifically address volunteers who serve in a chaperone capacity. We surveyed chaperones who had participated in an international youth…
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Araujo, Thaís L S; Borges, Julio Cesar; Ramos, Carlos H; Meyer-Fernandes, José Roberto; Oliveira Júnior, Reinaldo S; Pascutti, Pedro G; Foguel, Debora; Palhano, Fernando L
2014-05-13
We investigated the folding of the 70 kDa human cytosolic inducible protein (Hsp70) in vitro using high hydrostatic pressure as a denaturing agent. We followed the structural changes in Hsp70 induced by high hydrostatic pressure using tryptophan fluorescence, molecular dynamics, circular dichroism, high-performance liquid chromatography gel filtration, dynamic light scattering, ATPase activity, and chaperone activity. Although monomeric, Hsp70 is very sensitive to hydrostatic pressure; after pressure had been removed, the protein did not return to its native sate but instead formed oligomeric species that lost chaperone activity but retained ATPase activity.
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Babizhayev, Mark A
2012-08-01
Cataract is a leading cause of blindness worldwide and is responsible for ∼40-80% of the estimated 45 million cases of blindness that occur across the globe. In addition to providing refractive properties to the lens for focusing the image, it is believed that the molecular chaperone function of α-crystallin is essential in preventing the light scattering due to aggregation of other proteins and thus in the maintenance of lens transparency and thereby prevention of cataract. By now, it is fairly acknowledged that chaperoning ability of α-crystallin is instrumental in the maintenance of crystalline lens transparency, and decreased chaperone-like activity of α-crystallin is associated with various types and stages of cataract. A better pharmacological targeting of safeguarding the α-crystallin chaperone activity may aid the development of therapeutic strategies that could evade the need for cataract surgery and revive lens transparency of the cataractous lenses. This article originally summarizes the significance of modulation and enhancing of α-crystallin chaperone activity with imidazole-containing dipeptides N-acetylcarnosine, carnosine and carcinine in consequence to prevent, delay or dissolve the human cataract. A growing evidence and discussion of recent patents are presented in this study that demonstrate the ability of N-acetylcarnosine (lubricant eye drops) or carcinine (lubricant eye drops) (universal antioxidant and deglycation agent) resistant to enzymatic hydrolysis with carnosinase to act as pharmacological chaperones, to decrease oxidative stress and ameliorate oxidative and excessive glycation stress-related eye disease phenotypes, suggesting that the field of chaperone therapy might hold novel treatments for age-related cataracts, age-related macular degeneration (AMD) and ocular complications of diabetes (OCD). The therapeutic strategies are highlighted in the study for identifying potential chaperone compounds and for experimentally
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Roles of the N- and C-terminal sequences in Hsp27 self-association and chaperone activity
Lelj-Garolla, Barbara; Mauk, A Grant
2012-01-01
The small heat shock protein 27 (Hsp27 or HSPB1) is an oligomeric molecular chaperone in vitro that is associated with several neuromuscular, neurological, and neoplastic diseases. Although aspects of Hsp27 biology are increasingly well known, understanding of the structural basis for these involvements or of the functional properties of the protein remains limited. As all 11 human small heat shock proteins (sHsps) possess an α-crystallin domain, their varied functional and physiological characteristics must arise from contributions of their nonconserved sequences. To evaluate the role of two such sequences in Hsp27, we have studied three Hsp27 truncation variants to assess the functional contributions of the nonconserved N- and C-terminal sequences. The N-terminal variants Δ1–14 and Δ1–24 exhibit little chaperone activity, somewhat slower but temperature-dependent subunit exchange kinetics, and temperature-independent self-association with formation of smaller oligomers than wild-type Hsp27. The C-terminal truncation variants exhibit chaperone activity at 40 °C but none at 20 °C, limited subunit exchange, and temperature-independent self-association with an oligomer distribution at 40 °C that is very similar to that of wild-type Hsp27. We conclude that more of the N-terminal sequence than simply the WPDF domain is essential in the formation of larger, native-like oligomers after binding of substrate and/or in binding of Hsp27 to unfolding peptides. On the other hand, the intrinsically flexible C-terminal region drives subunit exchange and thermally-induced unfolding, both of which are essential to chaperone activity at low temperature and are linked to the temperature dependence of Hsp27 self-association. PMID:22057845
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Porowińska, Dorota; Czarnecka, Joanna; Komoszyński, Michał
2014-07-01
NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5'-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.
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Nandi, Sandip K; Rehna, Elengikal A A; Panda, Alok K; Shiburaj, Sugathan; Dharmalingam, Kuppamuthu; Biswas, Ashis
2013-12-01
Mycobacterium leprae HSP18 is a small heat shock protein (sHSP). It is a major immunodominant antigen of M. leprae pathogen. Previously, we have reported the existence of two M. leprae HSP18 variants in various leprotic patients. One of the variants has serine at position 52, whereas the other one has proline at the same position. We have also reported that HSP18 having proline at position 52 (HSP18P(52)) is a nonameric protein and exhibits chaperone function. However, the structural and functional characterization of wild-type HSP18 having serine at position 52 (HSP18S(52)) is yet to be explored. Furthermore, the implications of the S52P mutation on the structure and chaperone function of HSP18 are not well understood. Therefore, we cloned and purified these two HSP18 variants. We found that HSP18S(52) is also a molecular chaperone and an oligomeric protein. Intrinsic tryptophan fluorescence and far-UV CD measurements revealed that the S52P mutation altered the tertiary and secondary structure of HSP18. This point mutation also reduced the oligomeric assembly and decreased the surface hydrophobicity of HSP18, as revealed by HPLC and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid binding studies, respectively. Mutant protein was less stable against thermal and chemical denaturation and was more susceptible towards tryptic cleavage than wild-type HSP18. HSP18P(52) had lower chaperone function and was less effective in protecting thermal killing of Escherichia coli than HSP18S(52). Taken together, our data suggest that serine 52 is important for the larger oligomerization and chaperone function of HSP18. Because both variants differ in stability and function, they may have different roles in the survival of M. leprae in infected hosts. © 2013 FEBS.
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Analysis of Protein Interactions at Native Chloroplast Membranes by Ellipsometry
Kriechbaumer, Verena; Nabok, Alexei; Mustafa, Mohd K.; Al-Ammar, Rukaiah; Tsargorodskaya, Anna; Smith, David P.; Abell, Ben M.
2012-01-01
Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE). We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins. PMID:22479632
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Chaperone turns gatekeeper: PCBP2 and DMT1 form an iron-transport pipeline.
Lane, Darius J R; Richardson, Des R
2014-08-15
How is cellular iron (Fe) uptake and efflux regulated in mammalian cells? In this issue of the Biochemical Journal, Yanatori et al. report for the first time that a member of the emerging PCBP [poly(rC)-binding protein] Fe-chaperone family, PCBP2, physically interacts with the major Fe importer DMT1 (divalent metal transporter 1) and the Fe exporter FPN1 (ferroportin 1). In both cases, the interaction of the Fe transporter with PCBP2 is Fe-dependent. Interestingly, another PCBP Fe-chaperone, PCBP1, does not appear to bind to DMT1. Strikingly, the PCBP2-DMT1 interaction is required for DMT1-dependent cellular Fe uptake, suggesting that, in addition to functioning as an intracellular Fe chaperone, PCBP2 may be a molecular 'gate- keeper' for transmembrane Fe transport. These new data hint at the possibility that PCBP2 may be a component of a yet-to-be-described Fe-transport metabolon that engages in Fe channelling to and from Fe transporters and intracellular sites.
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Lysyl oxidases regulate fibrillar collagen remodelling in idiopathic pulmonary fibrosis.
Tjin, Gavin; White, Eric S; Faiz, Alen; Sicard, Delphine; Tschumperlin, Daniel J; Mahar, Annabelle; Kable, Eleanor P W; Burgess, Janette K
2017-11-01
Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung with few effective therapeutic options. Structural remodelling of the extracellular matrix [i.e. collagen cross-linking mediated by the lysyl oxidase (LO) family of enzymes (LOX, LOXL1-4)] might contribute to disease pathogenesis and represent a therapeutic target. This study aimed to further our understanding of the mechanisms by which LO inhibitors might improve lung fibrosis. Lung tissues from IPF and non-IPF subjects were examined for collagen structure (second harmonic generation imaging) and LO gene (microarray analysis) and protein (immunohistochemistry and western blotting) levels. Functional effects (collagen structure and tissue stiffness using atomic force microscopy) of LO inhibitors on collagen remodelling were examined in two models, collagen hydrogels and decellularized human lung matrices. LOXL1 / LOXL2 gene expression and protein levels were increased in IPF versus non-IPF. Increased collagen fibril thickness in IPF versus non-IPF lung tissues correlated with increased LOXL1/LOXL2, and decreased LOX, protein expression. β-Aminoproprionitrile (β-APN; pan-LO inhibitor) but not Compound A (LOXL2-specific inhibitor) interfered with transforming growth factor-β-induced collagen remodelling in both models. The β-APN treatment group was tested further, and β-APN was found to interfere with stiffening in the decellularized matrix model. LOXL1 activity might drive collagen remodelling in IPF lungs. The interrelationship between collagen structural remodelling and LOs is disrupted in IPF lungs. Inhibition of LO activity alleviates fibrosis by limiting fibrillar collagen cross-linking, thereby potentially impeding the formation of a pathological microenvironment in IPF. © 2017. Published by The Company of Biologists Ltd.
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Liu, Xiaolei; Huang, Sihua; Wang, Xingqin; Tang, Beisha; Li, Wenming; Mao, Zixu
2015-08-01
Lysosomes degrade dysfunctional intracellular components via three pathways: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Unlike the other two, CMA degrades cytosolic proteins with a recognized KFERQ-like motif in lysosomes and is important for cellular homeostasis. CMA activity declines with age and is altered in neurodegenerative diseases. Its impairment leads to the accumulation of aggregated proteins, some of which may be directly tied to the pathogenic processes of neurodegenerative diseases. Its induction may accelerate the clearance of pathogenic proteins and promote cell survival, representing a potential therapeutic approach for the treatment of neurodegenerative diseases. In this review, we summarize the current findings on how CMA is involved in neurodegenerative diseases, especially in Parkinson's disease.
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Squeglia, Flavia; Bachert, Beth; De Simone, Alfonso; Lukomski, Slawomir; Berisio, Rita
2014-02-21
The arsenal of virulence factors deployed by streptococci includes streptococcal collagen-like (Scl) proteins. These proteins, which are characterized by a globular domain and a collagen-like domain, play key roles in host adhesion, host immune defense evasion, and biofilm formation. In this work, we demonstrate that the Scl2.3 protein is expressed on the surface of invasive M3-type strain MGAS315 of Streptococcus pyogenes. We report the crystal structure of Scl2.3 globular domain, the first of any Scl. This structure shows a novel fold among collagen trimerization domains of either bacterial or human origin. Despite there being low sequence identity, we observed that Scl2.3 globular domain structurally resembles the gp41 subunit of the envelope glycoprotein from human immunodeficiency virus type 1, an essential subunit for viral fusion to human T cells. We combined crystallographic data with modeling and molecular dynamics techniques to gather information on the entire lollipop-like Scl2.3 structure. Molecular dynamics data evidence a high flexibility of Scl2.3 with remarkable interdomain motions that are likely instrumental to the protein biological function in mediating adhesive or immune-modulatory functions in host-pathogen interactions. Altogether, our results provide molecular tools for the understanding of Scl-mediated streptococcal pathogenesis and important structural insights for the future design of small molecular inhibitors of streptococcal invasion.
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Carmichael, D J; Dodd, C M; Veis, A
1977-03-28
Bone and dentin collagen are less susceptible to solubilization by pepsin digestion then is skin collagen. Digestion at 4 degrees C for 72 h solubilized only 35.3% of bovine cortical bone and 5.6% of bovine dentin compared with nearly 100% dissolution of bovine skin. Sodium dodecyl sulfate-acrylamide gel electrophoresis and molecular sieve chromatography showed that, for bone and dentin, intact alpha chains and cross-linked aggregates of beta, gamma and higher weight remained intact after pepsin solubilization but lower molecular weight fragments also were prevalent indicating chain scission in helical regions. Electron microscopic examination of segment long spacing precipitates of the soluble collagens confirmed the presence of solubilized polymerized collagen. The principal reducible cross-link in both bone and dentin was the precursor of dihydroxylsinonorleucine and this cross-link was also present in the solubilized collagens. Small amounts of non-collagenous proteins and glycosaminoglycans of different compositions in dentin and bone resisted extraction before pepsin digestion. However, the differences in solubilization of the collagens have been related to differences in cross-linkage placement.
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Vakiloroayaei, Ana; Shah, Neha S; Oeffinger, Marlene; Bayfield, Mark A
2017-11-02
Non-coding RNAs have critical roles in biological processes, and RNA chaperones can promote their folding into the native shape required for their function. La proteins are a class of highly abundant RNA chaperones that contact pre-tRNAs and other RNA polymerase III transcripts via their common UUU-3'OH ends, as well as through less specific contacts associated with RNA chaperone activity. However, whether La proteins preferentially bind misfolded pre-tRNAs or instead engage all pre-tRNA substrates irrespective of their folding status is not known. La deletion in yeast is synthetically lethal when combined with the loss of tRNA modifications predicted to contribute to the native pre-tRNA fold, such as the N2, N2-dimethylation of G26 by the methyltransferase Trm1p. In this work, we identify G26 containing pre-tRNAs that misfold in the absence of Trm1p and/or La (Sla1p) in Schizosaccharomyces pombe cells, then test whether La preferentially associates with such tRNAs in vitro and in vivo. Our data suggest that La does not discriminate a native from misfolded RNA target, and highlights the potential challenges faced by RNA chaperones in preferentially binding defective substrates. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Azasugar inhibitors as pharmacological chaperones for Krabbe disease
Hill, Chris H.; Viuff, Agnete H.; Spratley, Samantha J.; ...
2015-03-23
Krabbe disease is a devastating neurodegenerative disorder characterized by rapid demyelination of nerve fibers. This disease is caused by defects in the lysosomal enzyme β-galactocerebrosidase (GALC), which hydrolyzes the terminal galactose from glycosphingolipids. These lipids are essential components of eukaryotic cell membranes: substrates of GALC include galactocerebroside, the primary lipid component of myelin, and psychosine, a cytotoxic metabolite. Mutations of GALC that cause misfolding of the protein may be responsive to pharmacological chaperone therapy (PCT), whereby small molecules are used to stabilize these mutant proteins, thus correcting trafficking defects and increasing residual catabolic activity in cells. Here we describe amore » new approach for the synthesis of galacto-configured azasugars and the characterization of their interaction with GALC using biophysical, biochemical and crystallographic methods. We identify that the global stabilization of GALC conferred by azasugar derivatives, measured by fluorescence-based thermal shift assays, is directly related to their binding affinity, measured by enzyme inhibition. X-ray crystal structures of these molecules bound in the GALC active site reveal which residues participate in stabilizing interactions, show how potency is achieved and illustrate the penalties of aza/iminosugar ring distortion. The structure–activity relationships described here identify the key physical properties required of pharmacological chaperones for Krabbe disease and highlight the potential of azasugars as stabilizing agents for future enzyme replacement therapies. This work lays the foundation for new drug-based treatments of Krabbe disease.« less
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Moscardini, Mila; Pistello, Mauro; Bendinelli, M; Ficheux, Damien; Miller, Jennifer T; Gabus, Caroline; Le Grice, Stuart F J; Surewicz, Witold K; Darlix, Jean-Luc
2002-04-19
All lentiviruses and oncoretroviruses examined so far encode a major nucleic-acid binding protein (nucleocapsid or NC* protein), approximately 2500 molecules of which coat the dimeric RNA genome. Studies on HIV-1 and MoMuLV using in vitro model systems and in vivo have shown that NC protein is required to chaperone viral RNA dimerization and packaging during virus assembly, and proviral DNA synthesis by reverse transcriptase (RT) during infection. The human cellular prion protein (PrP), thought to be the major component of the agent causing transmissible spongiform encephalopathies (TSE), was recently found to possess a strong affinity for nucleic acids and to exhibit chaperone properties very similar to HIV-1 NC protein in the HIV-1 context in vitro. Tight binding of PrP to nucleic acids is proposed to participate directly in the prion disease process. To extend our understanding of lentiviruses and of the unexpected nucleic acid chaperone properties of the human prion protein, we set up an in vitro system to investigate replication of the feline immunodeficiency virus (FIV), which is functionally and phylogenetically distant from HIV-1. The results show that in the FIV model system, NC protein chaperones viral RNA dimerization, primer tRNA(Lys,3) annealing to the genomic primer-binding site (PBS) and minus strand DNA synthesis by the homologous FIV RT. FIV NC protein is able to trigger specific viral DNA synthesis by inhibiting self-priming of reverse transcription. The human prion protein was found to mimic the properties of FIV NC with respect to primer tRNA annealing to the viral RNA and chaperoning minus strand DNA synthesis. Copyright 2002 Elsevier Science Ltd.
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The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones.
Maurizy, Chloé; Quinternet, Marc; Abel, Yoann; Verheggen, Céline; Santo, Paulo E; Bourguet, Maxime; C F Paiva, Ana; Bragantini, Benoît; Chagot, Marie-Eve; Robert, Marie-Cécile; Abeza, Claire; Fabre, Philippe; Fort, Philippe; Vandermoere, Franck; M F Sousa, Pedro; Rain, Jean-Christophe; Charpentier, Bruno; Cianférani, Sarah; Bandeiras, Tiago M; Pradet-Balade, Bérengère; Manival, Xavier; Bertrand, Edouard
2018-05-29
R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers. The human genome encodes two other proteins bearing RPAP3-C-terminal-like domains and three containing PIH-like domains. Systematic interaction analyses show that one RPAP3-like protein, SPAG1, binds PIH1D2 and RUVBL1/2 to form an R2TP-like complex termed R2SP. This co-chaperone is enriched in testis and among 68 of the potential clients identified, some are expressed in testis and others are ubiquitous. One substrate is liprin-α2, which organizes large signaling complexes. Remarkably, R2SP is required for liprin-α2 expression and for the assembly of liprin-α2 complexes, indicating that R2SP functions in quaternary protein folding. Effects are stronger at 32 °C, suggesting that R2SP could help compensating the lower temperate of testis.
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Stability of local secondary structure determines selectivity of viral RNA chaperones.
Bravo, Jack P K; Borodavka, Alexander; Barth, Anders; Calabrese, Antonio N; Mojzes, Peter; Cockburn, Joseph J B; Lamb, Don C; Tuma, Roman
2018-05-18
To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.
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Collagen based polyurethanes—A review of recent advances and perspective.
Zuber, Mohammad; Zia, Fatima; Zia, Khalid Mahmood; Tabasum, Shazia; Salman, Mahwish; Sultan, Neelam
2015-09-01
Collagen is mostly found in fibrous tissues such as tendons, ligaments and skin. Collagen makes up approximately 30% of the proteins within the body. These are tough and strong structures found all over the body: in bones, tendons and ligaments. Collagen being the most abundant protein provides tensile strength via cell matrix interactions to tissue architecture. Biomimetic materials of collagen origin gained wide spread acceptance in clinical applications. Vitamin C deficiency causes scurvy a serious and painful disease in which defective collagen prevents the formation of strong connective tissue, gums deteriorate and bleed, with loss of teeth; skin discolors, and wounds do not heal. Effective collagens prevent the manifestation of such disorders. Polyurethanes on the other hand are frequently used for various applications as they offered in wide-ranging of compositions, properties and complex structures. Collagen/PU bio-composites have potential array for biomedical applications. Considering versatile properties of the elongated fibrils and wide industrial and biomedical applications including biocompatibility of polyurethane, this review shed a light on collagen based polyurethane materials with their potential applications especially focusing the bio-medical field. Copyright © 2015 Elsevier B.V. All rights reserved.
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Thorpe, Chavaunne T.; Streeter, Ian; Pinchbeck, Gina L.; Goodship, Allen E.; Clegg, Peter D.; Birch, Helen L.
2010-01-01
Little is known about the rate at which protein turnover occurs in living tendon and whether the rate differs between tendons with different physiological roles. In this study, we have quantified the racemization of aspartic acid to calculate the age of the collagenous and non-collagenous components of the high strain injury-prone superficial digital flexor tendon (SDFT) and low strain rarely injured common digital extensor tendon (CDET) in a group of horses with a wide age range. In addition, the turnover of collagen was assessed indirectly by measuring the levels of collagen degradation markers (collagenase-generated neoepitope and cross-linked telopeptide of type I collagen). The fractional increase in d-Asp was similar (p = 0.7) in the SDFT (5.87 × 10−4/year) and CDET (5.82 × 10−4/year) tissue, and d/l-Asp ratios showed a good correlation with pentosidine levels. We calculated a mean (±S.E.) collagen half-life of 197.53 (±18.23) years for the SDFT, which increased significantly with horse age (p = 0.03) and was significantly (p < 0.001) higher than that for the CDET (34.03 (±3.39) years). Using similar calculations, the half-life of non-collagenous protein was 2.18 (±0.41) years in the SDFT and was significantly (p = 0.04) lower than the value of 3.51 (±0.51) years for the CDET. Collagen degradation markers were higher in the CDET and suggested an accumulation of partially degraded collagen within the matrix with aging in the SDFT. We propose that increased susceptibility to injury in older individuals results from an inability to remove partially degraded collagen from the matrix leading to reduced mechanical competence. PMID:20308077
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Miller, James H.; Avilés-Reyes, Alejandro; Scott-Anne, Kathy; Gregoire, Stacy; Watson, Gene E.; Sampson, Edith; Progulske-Fox, Ann; Koo, Hyun; Bowen, William H.; Lemos, José A.
2015-01-01
Streptococcus mutans is the etiological agent of dental caries and one of the many bacterial species implicated in infective endocarditis. The expression of the collagen-binding protein Cnm by S. mutans has been associated with extraoral infections, but its relevance for dental caries has only been theorized to date. Due to the collagenous composition of dentinal and root tissues, we hypothesized that Cnm may facilitate the colonization of these surfaces, thereby enhancing the pathogenic potential of S. mutans in advancing carious lesions. As shown for extraoral endothelial cell lines, Cnm mediates the invasion of oral keratinocytes and fibroblasts by S. mutans. In this study, we show that in the Cnm+ native strain, OMZ175, Cnm mediates stringent adhesion to dentinal and root tissues as well as collagen-coated surfaces and promotes both cariogenicity and carriage in vivo. In vitro, ex vivo, and in vivo experiments revealed that while Cnm is not universally required for S. mutans cariogenicity, it contributes to (i) the invasion of the oral epithelium, (ii) enhanced binding on collagenous surfaces, (iii) implantation of oral biofilms, and (IV) the severity of caries due to a native Cnm+ isolate. Taken together, our findings reveal that Cnm is a colonization factor that contributes to the pathogenicity of certain S. mutans strains in their native habitat, the oral cavity. PMID:25733523
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Yang, Seung Won; Jang, Yo Han; Kwon, Soon Bin; Lee, Yoon Jae; Chae, Wonil; Byun, Young Ho; Kim, Paul; Park, Chan; Lee, Young Jae; Kim, Choon Kang; Kim, Young Seok; Choi, Seong Il; Seong, Baik Lin
2018-05-01
A novel protein-folding function of RNA has been recognized, which can outperform previously known molecular chaperone proteins. The RNA as a molecular chaperone (chaperna) activity is intrinsic to some ribozymes and is operational during viral infections. Our purpose was to test whether influenza hemagglutinin (HA) can be assembled in a soluble, trimeric, and immunologically activating conformation by means of an RNA molecular chaperone (chaperna) activity. An RNA-interacting domain (RID) from the host being immunized was selected as a docking tag for RNA binding, which served as a transducer for the chaperna function for de novo folding and trimeric assembly of RID-HA1. Mutations that affect tRNA binding greatly increased the soluble aggregation defective in trimer assembly, suggesting that RNA interaction critically controls the kinetic network in the folding/assembly pathway. Immunization of mice resulted in strong hemagglutination inhibition and high titers of a neutralizing antibody, providing sterile protection against a lethal challenge and confirming the immunologically relevant HA conformation. The results may be translated into a rapid response to a new influenza pandemic. The harnessing of the novel chaperna described herein with immunologically tailored antigen-folding functions should serve as a robust prophylactic and diagnostic tool for viral infections.-Yang, S. W., Jang, Y. H., Kwon, S. B., Lee, Y. J., Chae, W., Byun, Y. H., Kim, P., Park, C., Lee, Y. J., Kim, C. K., Kim, Y. S., Choi, S. I., Seong, B. L. Harnessing an RNA-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation.
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Yamasaki, Takashi; Nakazaki, Yosuke; Yoshida, Masasuke; Watanabe, Yo-hei
2011-07-01
ClpB, a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), forms a ring-shaped hexamer and cooperates with the DnaK chaperone system to reactivate aggregated proteins in an ATP-dependent manner. The ClpB protomer consists of an N-terminal domain, an AAA+ module (AAA-1), a middle domain, and a second AAA+ module (AAA-2). Each AAA+ module contains highly conserved WalkerA and WalkerB motifs, and two arginines (AAA-1) or one arginine (AAA-2). Here, we investigated the roles of these arginines (Arg322, Arg323, and Arg747) of ClpB from Thermus thermophilus in the ATPase cycle and chaperone function by alanine substitution. These mutations did not affect nucleotide binding, but did inhibit the hydrolysis of the bound ATP and slow the threading of the denatured protein through the central pore of the T. thermophilus ClpB ring, which severely impaired the chaperone functions. Previously, it was demonstrated that ATP binding to the AAA-1 module induced motion of the middle domain and stabilized the ClpB hexamer. However, the arginine mutations of the AAA-1 module destabilized the ClpB hexamer, even though ATP-induced motion of the middle domain was not affected. These results indicated that the three arginines are crucial for ATP hydrolysis and chaperone activity, but not for ATP binding. In addition, the two arginines in AAA-1 and the ATP-induced motion of the middle domain independently contribute to the stabilization of the hexamer. © 2011 The Authors Journal compilation © 2011 FEBS.
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Conserved conformational selection mechanism of Hsp70 chaperone-substrate interactions
Velyvis, Algirdas; Zoltsman, Guy; Rosenzweig, Rina; Bouvignies, Guillaume
2018-01-01
Molecular recognition is integral to biological function and frequently involves preferred binding of a molecule to one of several exchanging ligand conformations in solution. In such a process the bound structure can be selected from the ensemble of interconverting ligands a priori (conformational selection, CS) or may form once the ligand is bound (induced fit, IF). Here we focus on the ubiquitous and conserved Hsp70 chaperone which oversees the integrity of the cellular proteome through its ATP-dependent interaction with client proteins. We directly quantify the flux along CS and IF pathways using solution NMR spectroscopy that exploits a methyl TROSY effect and selective isotope-labeling methodologies. Our measurements establish that both bacterial and human Hsp70 chaperones interact with clients by selecting the unfolded state from a pre-existing array of interconverting structures, suggesting a conserved mode of client recognition among Hsp70s and highlighting the importance of molecular dynamics in this recognition event. PMID:29460778
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Biodegradable packing materials from hydrolysates of collagen waste proteins.
Langmaier, F; Mokrejs, P; Kolomaznik, K; Mladek, M
2008-01-01
Enzymatic hydrolysates of waste collagen proteins (H), from current industrial manufacture (leather, edible meat product casings, etc.) of mean molecular mass 20-30 kDa by a reaction with dialdehyde starch (DAS), produces hydrogels applicable as biodegradable (or even edible) packaging materials for food, cosmetic and pharmaceutical products. Thermo-reversibility of prepared hydrogels is given by concentrations of H and DAS in a reaction mixture. At concentrations of H 25-30% (w/w) and that of DAS 15-20% (related to weight of hydrolysate), thermo-reversible hydrogels arise, which can be processed into packaging materials by a technique similar to that of soft gelatin capsules (SGC). Exceeding the limit of 20% DAS leads to hydrogels that are thermo-reversible only in part, a further increase in DAS concentration then leads to thermo-irreversible gels whose processing into biodegradable packaging materials necessitates employment of other procedures.
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Current trends in chaperone use by plastic and reconstructive surgeons.
Choudry, Umar; Barta, Ruth J; Kim, Nicholas
2013-06-01
There is a paucity of literature regarding the use of chaperones by surgeons when examining patients. Use of a chaperone not only makes the patient comfortable but also potentially protects the surgeon from perceived misconduct. This is especially true for plastic surgeons who examine sensitive areas commonly. The purpose of this study was to determine the current trends in chaperone use by plastic surgeons when examining patients. A 23-question online survey was sent to all members of the American Society of Plastic Surgeons. Data collected online were analyzed using Student t test and Pearson χ test. A P < 0.05 was considered significant. Of the 4990 surgeons polled, 830 (16.6%) responded to some or all of the questions (707 [85%] male; 117 [14%] female). The overall chaperone use by plastic surgeons during all examinations of patients was 30%. This rate increased up to 60% while examining sensitive areas. Male surgeons reported a higher frequency of chaperone use than female surgeons (P < 0.001). Cosmetic surgeons had a higher rate of chaperone use compared to reconstructive surgeons (P = 0.001). Similarly, surgeons who had been in practice for more than 20 years reported a higher rate of chaperone use compared to surgeons in practice for less than 20 years (P = 0.032). Sixty-one (7.6%; 56 male and 5 female) surgeons reported being accused of inappropriate behavior by patients, of whom 49 (80%) did not have a chaperone present. There was no significant difference among male and female surgeons in rates of being accused of inappropriate behavior (7.9% vs 4.2%, P = 0.19). There was a higher rate of chaperone use by male plastic surgeons, surgeons with more than 20 years experience, and cosmetic surgeons. Despite the difference in chaperone use between the sexes, both had similar rates of being accused of inappropriate behavior during examinations by patients, and although these incidents were quite low, most had no chaperone present during those examinations.
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Wang, Qi; Hu, Weina; Lei, Mingming; Wang, Yong; Yan, Bing; Liu, Jun; Zhang, Ren; Jin, Yuanzhe
2013-01-01
To investigate if microRNAs (miRNAs) play a role in regulating h-ERG trafficking in the setting of chronic oxidative stress as a common deleterious factor for many cardiac disorders. We treated neonatal rat ventricular myocytes and HEK293 cells with stable expression of h-ERG with H2O2 for 12 h and 48 h. Expression of miR-17-5p seed miRNAs was quantified by real-time RT-PCR. Protein levels of chaperones and h-ERG trafficking were measured by Western blot analysis. Luciferase reporter gene assay was used to study miRNA and target interactions. Whole-cell patch-clamp techniques were employed to record h-ERG K(+) current. H-ERG trafficking was impaired by H2O2 after 48 h treatment, accompanied by reciprocal changes of expression between miR-17-5p seed miRNAs and several chaperones (Hsp70, Hsc70, CANX, and Golga2), with the former upregulated and the latter downregulated. We established these chaperones as targets for miR-17-5p. Application miR-17-5p inhibitor rescued H2O2-induced impairment of h-ERG trafficking. Upregulation of endogenous by H2O2 or forced miR-17-5p expression either reduced h-ERG current. Sequestration of AP1 by its decoy molecule eliminated the upregulation of miR-17-5p, and ameliorated impairment of h-ERG trafficking. Collectively, deregulation of the miR-17-5p seed family miRNAs can cause severe impairment of h-ERG trafficking through targeting multiple ER stress-related chaperones, and activation of AP1 likely accounts for the deleterious upregulation of these miRNAs, in the setting of prolonged duration of oxidative stress. These findings revealed the role of miRNAs in h-ERG trafficking, which may contribute to the cardiac electrical disturbances associated with oxidative stress.
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NASA Astrophysics Data System (ADS)
Balasubramanian, Preethi; Prabhakaran, Molamma P.; Sireesha, Merum; Ramakrishna, Seeram
The extracellular matrix is a complex biological structure encoded with various proteins, among which the collagen family is the most significant and abundant of all, contributing 30-35% of the whole-body protein. "Collagen" is a generic term for proteins that forms a triple-helical structure with three polypeptide chains, and around 29 types of collagen have been identified up to now. Although most of the members of the collagen family form such supramolecular structures, extensive diversity exists between each type of collagen. The diversity is not only based on the molecular assembly and supramolecular structures of collagen types but is also observed within its tissue distribution, function, and pathology. Collagens possess complex hierarchical structures and are present in various forms such as collagen fibrils (1.5-3.5 nm wide), collagen fibers (50-70 nm wide), and collagen bundles (150-250 nm wide), with distinct properties characteristic of each tissue providing elasticity to skin, softness of the cartilage, stiffness of the bone and tendon, transparency of the cornea, opaqueness of the sclera, etc. There exists an exclusive relation between the structural features of collagen in human tissues (such as the collagen composition, collagen fibril length and diameter, collagen distribution, and collagen fiber orientation) and its tissue-specific mechanical properties. In bone, a transverse collagen fiber orientation prevails in regions of higher compressive stress whereas longitudinally oriented collagen fibers correlate to higher tensile stress. The immense versatility of collagen compels a thorough understanding of the collagen types and this review discusses the major types of collagen found in different human tissues, highlighting their tissue-specific uniqueness based on their structure and mechanical function. The changes in collagen during a specific tissue damage or injury are discussed further, focusing on the many tissue engineering applications for
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Sigma-1 receptor chaperones in neurodegenerative and psychiatric disorders
Pokrass, Michael J; Klauer, Neal R; De Credico, Nicole E
2017-01-01
Introduction Sigma-1 receptors (Sig-1Rs) are molecular chaperones that reside mainly in the endoplasmic reticulum (ER) but exist also in the proximity of the plasma membrane. Sig-1Rs are highly expressed in the CNS and are involved in many cellular processes including cell differentiation, neuritogenesis, microglia activation, protein quality control, calcium-mediated ER stress and ion channel modulation. Disturbance in any of the above cellular processes can accelerate the progression of many neurological disorders; therefore, the Sig-1R has been implicated in several neurological diseases. Areas covered This review broadly covers the functions of Sig-1Rs including several neurodegenerative disorders in humans and drug addiction-associated neurological disturbance in the case of HIV infection. We discuss how several Sig-1R ligands could be utilized in therapeutic approaches to treat those disorders. Expert opinion Emerging understanding of the cellular functions of this unique transmembrane chaperone may lead to the use of new agents or broaden the use of certain available ligands as therapeutic targets in those neurological disorders. PMID:25331742
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Borzova, Vera A.; Markossian, Kira A.; Kara, Dmitriy A.; Chebotareva, Natalia A.; Makeeva, Valentina F.; Poliansky, Nikolay B.; Muranov, Konstantin O.; Kurganov, Boris I.
2013-01-01
The methodology for quantification of the anti-aggregation activity of protein and chemical chaperones has been elaborated. The applicability of this methodology was demonstrated using a test-system based on dithiothreitol-induced aggregation of bovine serum albumin at 45°C as an example. Methods for calculating the initial rate of bovine serum albumin aggregation (v agg) have been discussed. The comparison of the dependences of v agg on concentrations of intact and cross-linked α-crystallin allowed us to make a conclusion that a non-linear character of the dependence of v agg on concentration of intact α-crystallin was due to the dynamic mobility of the quaternary structure of α-crystallin and polydispersity of the α-crystallin–target protein complexes. To characterize the anti-aggregation activity of the chemical chaperones (arginine, arginine ethyl ester, arginine amide and proline), the semi-saturation concentration [L]0.5 was used. Among the chemical chaperones studied, arginine ethyl ester and arginine amide reveal the highest anti-aggregation activity ([L]0.5 = 53 and 58 mM, respectively). PMID:24058554
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Odetti, P; Aragno, I; Rolandi, R; Garibaldi, S; Valentini, S; Cosso, L; Traverso, N; Cottalasso, D; Pronzato, M A; Marinari, U M
2000-01-01
The main functional property of collagen is to provide a supporting framework to almost all tissues: the effects of non-enzymatic glycation on this protein are deleterious and in diabetes mellitus contribute to the mechanism of late complications. The aim of this work is to provide evidence by scanning force microscopy of modifications in collagen structure caused by high glucose concentration, in vivo and in vitro, and to correlate the data with markers of non-enzymatic glycation. Tendon fibrils were obtained from the tails of 8-month-old rats (BB/WOR/MOL¿BB) which developed diabetes spontaneously at least 12 weeks before they were killed, and from diabetes-resistant rats of the same strain (BB/WOR/MOL¿WB). A scanning force microscope (SFM; Nanoscope III) equipped with a Contact Mode Head was used for imaging. Band interval, diameter and depth of D-band gap were measured in non-diabetic and diabetic tail tendon fibrils and in fibrils incubated with glucose (0.5 M for 2 weeks). Fructosamine was determined in the tendon fibrils by a colorimetric method and pentosidine was evaluated in acid-hydrolyzed samples by coupled reverse phase-ionic exchange column HPLC. Incubated fibrils revealed modifications in radius (228+/-5 nm) and gap depth (3.65+/-0.10 nm) that closely reproduce diabetes-induced damage (236+/-3 and 3.20+/-0.04 nm respectively) and were significantly different from the pattern seen in non-diabetic fibrils (151+/-1 and 2.06+/-0.03 nm; p<0.001). Both fructosamine and pentosidine were higher in diabetic (3.82+/-1.43 nmol/mg and 2.23+/-0.24 pmol/mg collagen respectively) and in glucose-incubated fibrils (9.27+/-0.55 nmol/mg and 5.15+/-0.12 pmol/mg collagen respectively) vs non-diabetic tendons (1.29+/-0.08 nmol/mg and 0.88+/-0.11 pmol/mg collagen respectively; p<0.01); during the time course of incubation, an early increase in fructosamine was seen, whereas pentosidine increased later. The D-band parameter was similar in all three groups, indicating that
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Leaden, Laura; Busi, Maria V; Gomez-Casati, Diego F
2014-11-01
Arabidopsis plants contain two genes coding for mitochondrial Hsp70-type chaperon-like proteins, AtHscA1 (At4g37910) and AtHscA2 (At5g09590). Both genes are homologs of the Ssq1 gene involved in Fe-S cluster assembly in yeast. Protein-protein interaction studies showed that AtHscA2 interacts with AtIsu1 and AtHscB, two Arabidopsis homologs of the Isu1 protein and the Jac1 yeast co-chaperone. Moreover, this interaction could modulate the activity of AtHscA2. In the presence of a 1:5:5 molar ratio of AtHscA2:AtIsu1:AtHscB we observed an increase in the V(max) and a decrease in the S(0.5) for ATP of AtHscA2. Furthermore, an increase of about 28-fold in the catalytic efficiency of AtHscA2 was also observed. Results suggest that AtHscA2 in cooperation with AtIsu1 and AtHscB play an important role in the regulation of the Fe-S assembly pathway in plant mitochondria. Copyright © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.