Myopathy mutations in DNAJB6 slow conformer specific substrate processing that is rescued by NEF modulation

Molecular chaperones, or heat shock proteins (HSPs), protect against the toxic misfolding and aggregation of proteins. As such, mutations or deficiencies within the chaperone network can lead to disease. In fact, dominant mutations in DNAJB6 (Hsp40/Sis1), an Hsp70 co-chaperone, leads to a protein aggregate myopathy termed Limb-Girdle Muscular Dystrophy Type D1 (LGMDD1). DNAJB6 client proteins and co-chaperone interactions in skeletal muscle are not known. Here, we used the yeast prion model client in conjunction with in vitro chaperone activity assays to gain mechanistic insights, and found that LGMDD1 mutants affect Hsp40 functions. Strikingly, the mutants changed the structure of client protein aggregates, as determined by altered distribution of prion strains. They also impair the Hsp70 ATPase cycle, dimerization, and substrate processing and consequently poison the function of wild-type protein. These results define the mechanisms by which LGMDD1 mutations alter chaperone activity and provide avenues for therapeutic intervention.

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Myopathy mutations in DNAJB6 slow conformer specific substrate processing that is corrected by NEF modulation

10.21203/rs.3.rs-1041146/v1 ◽

2022 ◽

Author(s):

Ankan Bhadra ◽

Michael Rau ◽

Jil Daw ◽

James Fitzpatrick ◽

Conrad C. Weihl ◽

...

Keyword(s):

Chaperone Activity ◽

Specific Substrate ◽

Yeast Prion ◽

Protein Aggregate ◽

Client Proteins ◽

Chaperone Interactions ◽

Chaperone Network ◽

Shock Proteins ◽

Substrate Processing

Abstract Molecular chaperones, or heat shock proteins (HSPs), protect against the toxic misfolding and aggregation of proteins. As such, mutations or deficiencies within the chaperone network can lead to disease. In fact, dominant mutations in DNAJB6 (Hsp40/Sis1), an Hsp70 co-chaperone, leads to a protein aggregate myopathy termed Limb-Girdle Muscular Dystrophy Type D1 (LGMDD1). DNAJB6 client proteins and co-chaperone interactions in skeletal muscle are not known. Here, we used the yeast prion model client in conjunction with in vitro chaperone activity assays to gain mechanistic insights, and found that LGMDD1 mutants affect Hsp40 functions. Strikingly, the mutants changed the structure of client protein aggregates, as determined by altered distribution of prion strains. They also impair the Hsp70 ATPase cycle, dimerization, and substrate processing and consequently poison the function of wild-type protein. These results define the mechanisms by which LGMDD1 mutations alter chaperone activity and provide avenues for therapeutic intervention.

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The Mitochondrial Small Heat Shock Protein HSP22 from Pea is a Thermosoluble Chaperone Prone to Co-Precipitate with Unfolding Client Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms21010097 ◽

2019 ◽

Vol 21 (1) ◽

pp. 97

Author(s):

Marie-Hélène Avelange-Macherel ◽

Aurélia Rolland ◽

Marie-Pierre Hinault ◽

Dimitri Tolleter ◽

David Macherel

Keyword(s):

Heat Shock ◽

Recombinant Protein ◽

Quaternary Structure ◽

Small Heat Shock Protein ◽

Effect Of Temperature ◽

Molar Ratio ◽

Client Proteins ◽

Shock Proteins ◽

Cellular Compartments

The small heat shock proteins (sHSPs) are molecular chaperones that share an alpha-crystallin domain but display a high diversity of sequence, expression, and localization. They are especially prominent in plants, populating most cellular compartments. In pea, mitochondrial HSP22 is induced by heat or oxidative stress in leaves but also strongly accumulates during seed development. The molecular function of HSP22 was addressed by studying the effect of temperature on its structural properties and chaperone effects using a recombinant or native protein. Overexpression of HSP22 significantly increased bacterial thermotolerance. The secondary structure of the recombinant protein was not affected by temperature in contrast with its quaternary structure. The purified protein formed large polydisperse oligomers that dissociated upon heating (42 °C) into smaller species (mainly monomers). The recombinant protein appeared thermosoluble but precipitated with thermosensitive proteins upon heat stress in assays either with single protein clients or within complex extracts. As shown by in vitro protection assays, HSP22 at high molar ratio could partly prevent the heat aggregation of rhodanese but not of malate dehydrogenase. HSP22 appears as a holdase that could possibly prevent the aggregation of some proteins while co-precipitating with others to facilitate their subsequent refolding by disaggregases or clearance by proteases.

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Mitochondrial peroxiredoxin functions as crucial chaperone reservoir in Leishmania infantum

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1419682112 ◽

2015 ◽

Vol 112 (7) ◽

pp. E616-E624 ◽

Cited By ~ 42

Author(s):

Filipa Teixeira ◽

Helena Castro ◽

Tânia Cruz ◽

Eric Tse ◽

Philipp Koldewey ◽

...

Keyword(s):

Leishmania Infantum ◽

Protein Unfolding ◽

Protozoan Parasite ◽

Chaperone Activity ◽

Dual Function ◽

Active Site Cysteine ◽

Chaperone Function ◽

Client Proteins

Cytosolic eukaryotic 2-Cys-peroxiredoxins have been widely reported to act as dual-function proteins, either detoxifying reactive oxygen species or acting as chaperones to prevent protein aggregation. Several stimuli, including peroxide-mediated sulfinic acid formation at the active site cysteine, have been proposed to trigger the chaperone activity. However, the mechanism underlying this activation and the extent to which the chaperone function is crucial under physiological conditions in vivo remained unknown. Here we demonstrate that in the vector-borne protozoan parasite Leishmania infantum, mitochondrial peroxiredoxin (Prx) exerts intrinsic ATP-independent chaperone activity, protecting a wide variety of different proteins against heat stress-mediated unfolding in vitro and in vivo. Activation of the chaperone function appears to be induced by temperature-mediated restructuring of the reduced decamers, promoting binding of unfolding client proteins in the center of Prx’s ringlike structure. Client proteins are maintained in a folding-competent conformation until restoration of nonstress conditions, upon which they are released and transferred to ATP-dependent chaperones for refolding. Interference with client binding impairs parasite infectivity, providing compelling evidence for the in vivo importance of Prx’s chaperone function. Our results suggest that reduced Prx provides a mitochondrial chaperone reservoir, which allows L. infantum to deal successfully with protein unfolding conditions during the transition from insect to the mammalian hosts and to generate viable parasites capable of perpetuating infection.

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The Activity and Specificity of the Outer Membrane Protein Chaperone SurA Are Modulated by a Proline Isomerase Domain

mBio ◽

10.1128/mbio.00540-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 19

Author(s):

Dante P. Ricci ◽

Jaclyn Schwalm ◽

Michelle Gonzales-Cope ◽

Thomas J. Silhavy

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Chaperone Activity ◽

Content Type ◽

Bam Complex ◽

Ppiase Domain ◽

Chaperone Network ◽

Novel Therapeutic Strategies

ABSTRACTSurA is a component of the periplasmic chaperone network that plays a central role in biogenesis of integral outer membrane β-barrel proteins (OMPs) inEscherichia coli. Although SurA contains two well-conserved proline isomerase (PPIase) domains, the contribution of these domains to SurA function is unclear. In the present work, we show that defects in OMP assembly caused by mutation of the β-barrel assembly factors BamA or BamB can be corrected by gain-of-function mutations in SurA that map to the first PPIase domain. These mutations apparently bypass the requirement for a stable interaction between SurA and the Bam complex and enhance SurA chaperone activityin vivodespite destabilization of the proteinin vitro. Our findings suggest an autoinhibitory mechanism for regulation of SurA chaperone activity through interdomain interactions involving a PPIase domain. We propose a model in which SurA activity is modulated by an interaction between SurA and the Bam complex that alters the substrate specificity of the chaperone.IMPORTANCEThe dominantsurAmutations described here alter amino acid residues that are highly conserved in eukaryotic homologs of SurA, including Pin1, the human proline isomerase (PPIase) implicated in Alzheimer’s disease and certain cancers. Consequently, a mechanistic description of SurA function may enhance our understanding of clinically important PPIases and their role(s) in disease. In addition, the virulence of Gram-negative bacterial pathogens, such asSalmonella,Shigella, andEscherichia coliO157:H7, is largely dependent on SurA, making this PPIase/chaperone an attractive antibiotic target. Investigating the function of SurA in outer membrane (OM) biogenesis will be useful in the development of novel therapeutic strategies for the disruption of the OM or the processes that are essential for its assembly.

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Transgenic Zebrafish Models Reveal Distinct Molecular Mechanisms for Cataract-linked αA-Crystallin Mutants

10.1101/364125 ◽

2018 ◽

Author(s):

Shu-Yu Wu ◽

Ping Zou ◽

Sanjay Mishra ◽

Hassane S Mchaourab

Keyword(s):

Mouse Models ◽

Protein Interactions ◽

Molecular Mechanisms ◽

Cellular Proliferation ◽

Transgenic Zebrafish ◽

Αb Crystallin ◽

Client Proteins ◽

Shock Proteins ◽

And Function

AbstractMutations in the small heat shock proteins α-crystallins have been linked to autosomal dominant cataracts in humans. Extensive studies in vitro have revealed a spectrum of alterations to the structure and function of these proteins including shifts in the size of the oligomer, modulation of subunit exchange and modification of their affinity to client proteins. Although mouse models of these mutants were instrumental in identifying changes in cellular proliferation and lens development, a direct comparative analysis of their effects on lens proteostasis has not been performed. Here, we have transgenically expressed cataract-linked mutants of αA- and αB-crystallin in the zebrafish lens to dissect the underlying molecular changes that contribute to the loss of lens optical properties. Zebrafish lines expressing these mutants displayed a range of morphological lens defects. Phenotype penetrance and severity were dependent on the mutation even in fish lines lacking endogenous α-crystallin. The mechanistic origins of these differences were investigated by the transgenic co-expression of a destabilized human γD-crystallin mutant. We found that the R49C but not the R116C mutant of αA-crystallin promoted aggregation of γD-crystallin, although both mutants have similar affinity to client proteins in vitro. Our working model attributes these differences to the propensity of R49C, located in the buried N-terminal domain of αA-crystallin, to disulfide crosslinking as previously demonstrated in vitro. Our findings complement and extend previous work in mouse models and emphasize the need of investigating chaperone/client protein interactions in appropriate cellular context.

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The Caenorhabditis elegans 12‐kDa small heat shock proteins with little in vitro chaperone activity play crucial roles for its dauer formation, longevity and reproduction

Protein Science ◽

10.1002/pro.4160 ◽

2021 ◽

Author(s):

Xinmiao Fu ◽

Anastasia N Ezemaduka ◽

Xinping Lu ◽

Zengyi Chang

Keyword(s):

Caenorhabditis Elegans ◽

Heat Shock ◽

Heat Shock Proteins ◽

Small Heat Shock Proteins ◽

Chaperone Activity ◽

Dauer Formation ◽

Shock Proteins

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Molecular and pharmacological chaperones for SOD1

Biochemical Society Transactions ◽

10.1042/bst20200318 ◽

2020 ◽

Vol 48 (4) ◽

pp. 1795-1806

Author(s):

Gareth S.A. Wright

Keyword(s):

Neuronal Loss ◽

Therapeutic Benefit ◽

Copper Chaperone ◽

Pharmacological Chaperones ◽

Sporadic Als ◽

Chaperone Interactions ◽

Chaperone Network ◽

Shock Proteins ◽

Lateral Sclerosis ◽

Selection Of

The efficacy of superoxide dismutase-1 (SOD1) folding impacts neuronal loss in motor system neurodegenerative diseases. Mutations can prevent SOD1 post-translational processing leading to misfolding and cytoplasmic aggregation in familial amyotrophic lateral sclerosis (ALS). Evidence of immature, wild-type SOD1 misfolding has also been observed in sporadic ALS, non-SOD1 familial ALS and Parkinson's disease. The copper chaperone for SOD1 (hCCS) is a dedicated and specific chaperone that assists SOD1 folding and maturation to produce the active enzyme. Misfolded or misfolding prone SOD1 also interacts with heat shock proteins and macrophage migration inhibitory factor to aid folding, refolding or degradation. Recognition of specific SOD1 structures by the molecular chaperone network and timely dissociation of SOD1-chaperone complexes are, therefore, important steps in SOD1 processing. Harnessing these interactions for therapeutic benefit is actively pursued as is the modulation of SOD1 behaviour with pharmacological and peptide chaperones. This review highlights the structural and mechanistic aspects of a selection of SOD1-chaperone interactions together with their impact on disease models.

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In Vitro Holdase Activity of E. coli Small Heat-Shock Proteins IbpA, IbpB and IbpAB: A Biophysical Study with Some Unconventional Techniques

Protein and Peptide Letters ◽

10.2174/0929866521666131224094408 ◽

2014 ◽

Vol 21 (6) ◽

pp. 564-571 ◽

Cited By ~ 4

Author(s):

Sourav Roy ◽

Monobesh Patra ◽

Suman Nandy ◽

Milon Banik ◽

Rakhi Dasgupta ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Proteins ◽

Small Heat Shock Proteins ◽

E Coli ◽

Biophysical Study ◽

Shock Proteins

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Translation of Some Maize Small Heat Shock Proteins Is Initiated From Internal In-Frame AUGs

Genetics ◽

10.1093/genetics/148.1.471 ◽

1998 ◽

Vol 148 (1) ◽

pp. 471-477

Author(s):

J Roger H Frappier ◽

David B Walden ◽

Burr G Atkinson

Keyword(s):

Heat Shock ◽

Heat Shock Proteins ◽

Single Gene ◽

Small Heat Shock Proteins ◽

Shock Proteins

Abstract Etiolated maize radicles (inbred Oh43) subjected to a brief heat shock synthesize a family of small heat shock proteins (≃18 kD) that is composed of at least 12 members. We previously described the cDNA-derived sequence of three maize shsp mRNAs (cMHSP18-1, cMHSP18-3, and cMHSP18-9). In this report, we demonstrate that the mRNA transcribed in vitro from one of these cDNAs (cMHSP 18-9) is responsible for the synthesis of three members of the shsp family, and we suggest that cMHSP18-3 may be responsible for the synthesis of three additional members and cMHSP18-1 for the synthesis of two other members of this family. The fact that these genes do not contain introns, coupled with the observations reported herein, suggest that maize may have established another method of using a single gene to produce a number of different proteins.

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