Dataset Information

HspB8 interacts with BAG3 in a "native-like" conformation forming a complex that displays chaperone-like activity.

ABSTRACT: The HspB8-BAG3 complex plays an important role in the protein quality control acting alone or within multi-components complexes. To clarify the mechanism underlying its activity, in this work we used biochemical and biophysical approaches to study the tendency of both proteins to auto-assemble and to form the complex. Solubility and Thioflavin T assays, Fourier transform infrared spectroscopy and atomic force microscopy analyses clearly showed the tendency of HspB8 to self-assemble at high concentration and to form oligomers in a "native-like" conformation; otherwise, BAG3 aggregates poorly. Noteworthy, also HspB8 and BAG3 associate in a "native-like" conformation, forming a stable complex. Furthermore, the high difference between dissociation constant values of HspB8-HspB8 interaction with respect to the binding to BAG3 obtained by surface plasmon resonance confirms that HspB8 is an obligated partner of BAG3 in vivo. Lastly, both proteins alone or in the complex are able to bind and affect the aggregation of the Josephin domain, the structured domain that triggers the ataxin-3 fibrillation. In particular, the complex displayed higher activity than HspB8 alone. All this considered, we can assert that the two proteins form a stable assembly with chaperone-like activity that could contribute to the physiological role of the complex in vivo.

SUBMITTER: Sciandrone B

PROVIDER: S-EPMC10273338 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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HspB8 interacts with BAG3 in a "native-like" conformation forming a complex that displays chaperone-like activity.

Sciandrone Barbara B Ami Diletta D D'Urzo Annalisa A Angeli Elena E Relini Annalisa A Vanoni Marco M Natalello Antonino A Regonesi Maria Elena ME

Protein science : a publication of the Protein Society 20230701 7

The HspB8-BAG3 complex plays an important role in the protein quality control acting alone or within multi-components complexes. To clarify the mechanism underlying its activity, in this work we used biochemical and biophysical approaches to study the tendency of both proteins to auto-assemble and to form the complex. Solubility and Thioflavin T assays, Fourier transform infrared spectroscopy and atomic force microscopy analyses clearly showed the tendency of HspB8 to self-assemble at high conce ...[more]

PMID: 37243950

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Project description:Chaperone-assisted selective autophagy (CASA) is a highly selective pathway for the disposal of misfolding and aggregating proteins. In muscle, CASA assures muscle integrity by favoring the turnover of structural components damaged by mechanical strain. In neurons, CASA promotes the removal of aggregating substrates. A crucial player of CASA is HSPB8 (heat shock protein family B (small) member 8), which acts in a complex with HSPA, their cochaperone BAG3, and the E3 ubiquitin ligase STUB1. Recently, four novel HSPB8 frameshift (fs) gene mutations have been linked to neuromyopathies, and encode carboxy-terminally mutated HSPB8, sharing a common C-terminal extension. Here, we analyzed the biochemical and functional alterations associated with the HSPB8_fs mutant proteins. We demonstrated that HSPB8_fs mutants are highly insoluble and tend to form proteinaceous aggregates in the cytoplasm. Notably, all HSPB8 frameshift mutants retain their ability to interact with CASA members but sequester them into the HSPB8-positive aggregates together with two autophagy receptors SQSTM1/p62 and TAX1BP1. This copartitioning process negatively affects the CASA capability to remove its clients and causes a general failure in proteostasis response. Further analyses revealed that the aggregation of the HSPB8_fs mutants occurs independently of the other CASA members or from the autophagy receptors interaction, but it is an intrinsic feature of the mutated amino acid sequence. HSPB8_fs mutants aggregation alters the differentiation capacity of muscle cells and impairs sarcomere organization. Collectively, these results shed light on a potential pathogenic mechanism shared by the HSPB8_fs mutants described in neuromuscular diseases.Abbreviations : ACD: α-crystallin domain; ACTN: actinin alpha; BAG3: BAG cochaperone 3; C: carboxy; CASA: chaperone-assisted selective autophagy; CE: carboxy-terminal extension; CLEM: correlative light and electron microscopy; CMT2L: Charcot-Marie-Tooth type 2L; CTR: carboxy-terminal region; dHMNII: distal hereditary motor neuropathy type II; EV: empty vector; FRA: filter retardation assay; fs: frameshift; HSPA/HSP70: heat shock protein family A (Hsp70); HSPB1/Hsp27: heat shock protein family B (small) member 1; HSPB8/Hsp22: heat shock protein family B (small) member 8; HTT: huntingtin; KO: knockout; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MD: molecular dynamics; MTOC: microtubule organizing center; MYH: myosin heavy chain; MYOG: myogenin; NBR1: NBR1 autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; NSC34: Neuroblastoma X Spinal Cord 34; OPTN: optineurin; polyQ: polyglutamine; SQSTM1/p62: sequestosome 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TAX1BP1: Tax1 binding protein 1; TUBA: tubulin alpha; WT: wild-type.

Dataset Information

HspB8 interacts with BAG3 in a "native-like" conformation forming a complex that displays chaperone-like activity.

Publications

HspB8 interacts with BAG3 in a "native-like" conformation forming a complex that displays chaperone-like activity.

Similar Datasets

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