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The permanently chaperone-active small heat shock protein Hsp17 from C. elegans exhibits topological separation of its N-terminal regions

ABSTRACT: Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind non-native proteins in an ATP-independent manner. C. elegans encodes 16 different sHsps, among them Hsp17, which is evolutionarily distinct from other sHsps in the nematode. The structure and mechanism of Hsp17 and how these may differ from other sHsps remain unclear. Here, we find that Hsp17 has a distinct expression pattern, structural organization, and chaperone function. Consistent with its presence under non-stress conditions, and in contrast to many other sHsps, we determined that Hsp17 is a mono-disperse, permanently active chaperone in vitro, which interacts with hundreds of different C. elegans proteins under physiological conditions. Additionally, our cryo-EM structure of Hsp17 reveals that in the 24-mer complex, 12 N-terminal regions are involved in its chaperone function. These flexible regions are located on the outside of the spherical oligomer, whereas the other 12 N-terminal regions are engaged in stabilizing interactions in its interior. This allows the same region in Hsp17 to perform different functions depending on the topological context. Taken together, our results reveal structural and functional features that further define the structural basis of permanently active sHsps.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Caenorhabditis Elegans

SUBMITTER: Moritz Mühlhofer

LAB HEAD: Johannes Buchner

PROVIDER: PXD028080 | Pride | 2022-12-08

REPOSITORIES: Pride

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The permanently chaperone-active small heat shock protein Hsp17 from C. elegans exhibits topological separation of its N-terminal regions

Project description:Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind non-native proteins in an ATP-independent manner. C. elegans encodes 16 different sHsps, among them Hsp17, which is evolutionarily distinct from other sHsps in the nematode. The structure and mechanism of Hsp17 and how these may differ from other sHsps remain unclear. Here, we find that Hsp17 has a distinct expression pattern, structural organization, and chaperone function. Consistent with its presence under non-stress conditions, and in contrast to many other sHsps, we determined that Hsp17 is a mono-disperse, permanently active chaperone in vitro, which interacts with hundreds of different C. elegans proteins under physiological conditions. Additionally, our cryo-EM structure of Hsp17 reveals that in the 24-mer complex, 12 N-terminal regions are involved in its chaperone function. These flexible regions are located on the outside of the spherical oligomer, whereas the other 12 N-terminal regions are engaged in stabilizing interactions in its interior. This allows the same region in Hsp17 to perform different functions depending on the topological context. Taken together, our results reveal structural and functional features that further define the structural basis of permanently active sHsps.

2022-12-08 | PXD038019 | Pride

The permanently chaperone-active small heat shock protein Hsp17 from C. elegans exhibits topological separation of its N-terminal regions

2022-12-08 | PXD030504 | Pride

Disordered region encodes α-crystallin chaperone activity toward lens client γD-crystallin

Project description:Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR (“Critical sequence”) contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.

2023-01-31 | PXD035668 | Pride

Thylakoid membranes from Arabidopsis LC-MSMS

Project description:The small heat shock protein (sHsp) chaperones are crucial for cell survival and can prevent aggregation of client proteins that partially unfold under destabilizing conditions. Most investigations on the chaperone activity sHsps are based on a limited set of thermosensitive model substrate client proteins since the endogenous targets are often not known. There is a high diversity among sHsps, with a single conserved -sandwich fold domain defining the family, the α-crystallin domain, whereas the N-terminal and C-terminal regions are highly variable in length and sequence among various sHsps and conserved only within orthologues. The endogenous targets are probably also varying among various sHsps, cellular compartments, cell type and organism. Here we have investigated Hsp21, a non-metazoan sHsp expressed in the chloroplasts in green plants, which experience huge environmental fluctuations not least in temperature. We describe how Hsp21 can also interact with the chloroplast thylakoid membranes, both when isolated thylakoid membranes are incubated with Hsp21 protein and when plants are heat-stressed. The amount of Hsp21 associated with the thylakoid membranes was precisely determined by quantitative mass spectrometry after metabolic 15N-isotope labelling, of either recombinantly expressed and purified Hsp21 protein or intact Arabidopsis thaliana plants. We found that Hsp21 is among few proteins that become associated with the thylakoid membranes in heat-stressed plants, and that approximately two thirds of the pool of chloroplast Hsp21 is affected. We conclude that for a complete picture of the role of sHsps in plant stress resistance also their association with the membranes should be considered

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SWI/SNF and the histone chaperone Rtt106 drive expression of the Pleiotropic Drug Resistance network genes

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2022-02-21 | E-MTAB-9787 | biostudies-arrayexpress

Epigenetically induced paucity of histone H2A.Z stabilizes fission yeast ectopic centromeres

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Site-specific N-glycosylation Characterization of Micro Disease-related Proteins Based on EThcD-sceHCD-MS/MS

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2022-09-13 | PXD035757 | Pride

Phosphorylation activates the yeast small heat shock protein Hsp26 by weakening domain contacts in the oligomer ensemble

Project description:Hsp26 is a small heat shock protein (sHsp) from S. cerevisiae. It is known to be activated by dissociation of the oligomer at heat shock temperatures. We wondered whether phosphorylation regulates its activity at physiological temperatures. In Hsp26, 9 phosphorylation sites which are located in different structural elements are known. Our analysis of phospho-mimetic mutations showed that phosphorylation activates Hsp26 at permissive temperature. The cryo-EM structure of the Hsp26 40mer revealed contacts between the conserved core domain of Hsp26 and the so-called thermosensor domain in the N-terminal part of the protein, which are targeted by phosphorylation. Furthermore, the C-terminal extension, which also links subunits within the oligomer, contains several phosphorylation sites. The introduction of negative charges at these strategic positions makes regions in the N-terminal domain accessible for the binding of substrate proteins. Relieving the intrinsic inhibition via different phosphorylation sites allows the fine-tuning of chaperone activity in response to proteotoxic stresses independent of heat activation. The described weakening of domain interactions within and between subunits could be a general regulation principle of sHsps.

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