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Small heat shock protein activity is regulated by variable oligomeric substructure.

ABSTRACT: The alpha-crystallins are members of the small heat shock protein family of molecular chaperones that have evolved to minimize intracellular protein aggregation; however, they are also implicated in a number of protein deposition diseases. In this study, we employed novel mass spectrometry techniques to investigate the changes in quaternary structure associated with this switch from chaperone to adjuvant of aggregation. We replicated the oligomeric rearrangements observed for post-translationally modified alpha-crystallins, without altering the protein sequence, by refolding the alpha-crystallins in vitro. This refolding resulted in a loss of dimeric substructure concomitant with an augmentation of substrate affinity. We show that packaging of small heat shock proteins into dimeric units is used to control the level of chaperone function by regulating the exposure of hydrophobic surfaces. We propose that a bias toward monomeric substructure is responsible for the aberrant chaperone behavior associated with the alpha-crystallins in protein deposition diseases.

SUBMITTER: Benesch JL

PROVIDER: S-EPMC2661405 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Small heat shock protein activity is regulated by variable oligomeric substructure.

Benesch Justin L P JL Ayoub Marina M Robinson Carol V CV Aquilina J Andrew JA

The Journal of biological chemistry 20080819 42

The alpha-crystallins are members of the small heat shock protein family of molecular chaperones that have evolved to minimize intracellular protein aggregation; however, they are also implicated in a number of protein deposition diseases. In this study, we employed novel mass spectrometry techniques to investigate the changes in quaternary structure associated with this switch from chaperone to adjuvant of aggregation. We replicated the oligomeric rearrangements observed for post-translationall ...[more]

PMID: 18713743

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Project description: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 fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.

Project description:Two isoforms of the 90-kDa heat shock protein, HSP90alpha and HSP90beta, are present in the cytosol of mammalian cells. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions (native PAGE) revealed that HSP90alpha predominantly exists as a homodimer and that HSP90beta is present mainly as a monomer [Minami, Kawasaki, Miyata, Suzuki and Yahara (1991) J. Biol. Chem. 266, 10099-10103]. However, only the dimeric form has been observed under other analytical conditions such as gradient centrifugation. In this study, therefore, we investigated native forms of HSP90 by use of immunochemical techniques with isoform-specific monoclonal antibodies recently developed in our laboratory. Glycerol gradient centrifugation at the physiological salt concentration as well as native PAGE analysis of rat liver cytosol revealed oligomeric forms of HSP90alpha sedimenting at 8-10S as predominant ones. On the other hand, the glycerol gradient centrifugation revealed multiple forms of HSP90beta oligomers sedimenting at 6-12S. All of the HSP90beta oligomers, however, migrated at 100-kDa monomer and 190-kDa dimer positions on native PAGE. A novel two-dimensional double native PAGE revealed that the entity was converted from the HSP90beta dimer to monomers during the electrophoresis. The same PAGE further revealed that the HSP90alpha oligomer also dissociated into dimers during the electrophoresis. Full-length form of bacterially-expressed human HSP90alpha migrated as dimers, but a considerable amount did not penetrate into the gel under native PAGE conditions, indicating the existence of oligomeric forms. Electrophoretic studies of deletion mutants of HSP90 demonstrated that the C-terminal 200 amino acids were capable of forming oligomers. Taken together, we conclude that both of the HSP90 isoforms predominantly exist as oligomeric forms in the cytosol even under unstressed conditions but that they artificially dissociate into smaller forms when subjected to native PAGE.

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Small heat shock protein activity is regulated by variable oligomeric substructure.

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Small heat shock protein activity is regulated by variable oligomeric substructure.

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