HSPB8 frameshift mutant aggregates weaken chaperone-assisted selective autophagy in neuromyopathies

Autophagy. 2023 Aug;19(8):2217-2239. doi: 10.1080/15548627.2023.2179780. Epub 2023 Feb 28.

Abstract

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.

Keywords: BAG3; CASA; HSPA; HSPB8; misfolding; myopathy; neuromuscular disorders; neuropathy; protein quality control.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • Apoptosis Regulatory Proteins / metabolism
  • Autophagy / genetics
  • Charcot-Marie-Tooth Disease* / genetics
  • Heat-Shock Proteins / metabolism
  • Humans
  • Molecular Chaperones / genetics
  • Molecular Chaperones / metabolism
  • Neuromuscular Diseases*
  • Sequestosome-1 Protein / genetics
  • Sequestosome-1 Protein / metabolism
  • Ubiquitin-Protein Ligases / metabolism

Substances

  • Sequestosome-1 Protein
  • Heat-Shock Proteins
  • STUB1 protein, human
  • Ubiquitin-Protein Ligases
  • BAG3 protein, human
  • Adaptor Proteins, Signal Transducing
  • Apoptosis Regulatory Proteins
  • HSPB8 protein, human
  • Molecular Chaperones

Grants and funding

This research was funded by the Fondazione Telethon - Italy (n. GGP19128 to AP), the Fondazione Cariplo - Italy (n. 2021-1544 to RC), the Fondazione Italiana di Ricerca per la Sclerosi Laterale Amiotrofica (AriSLA) - Italy (n. MLOpathy to AP; Target-RAN to AP), the Association Française contre les Myopathies - France (AFM Telethon n. 23236 to 1295 AP), the Kennedy’s Disease Association - USA (2018 grant to RC; 2020 grant to MG), the Università degli Studi di Milano - Progetti Interdipartimentali (#Gender-ALS to MG), the Fondazione Regionale per la Ricerca Biomedica (FRRB) - Italy (Regione Lombardia, TRANS_ALS, n. 2015-0023 to AP), the Ministero dell’Università e della Ricerca (MIUR) – Italy (PRIN—Progetti di ricerca di interesse nazionale (n. 2017F2A2C5 to AP and n. 2020PBS5MJ to VC); CN3: RNA – Codice Proposta: CN_00000041; Tematica Sviluppo di terapia genica e farmaci con tecnologia a RNA (Centro Nazionale di Ricerca – CN3 National Center for Gene Therapy and Drugs based on RNA Technology to AP); Progetto Dipartimenti di Eccellenza to DiSFeB), the Ministero della Salute, Agenzia Italiana del Farmaco (AIFA) – Italy (Co_ALS to AP).This research was also supported by grants from the University of Antwerp (TOP-BOF, n. 38694 and GOA n. 41667 to VT), the Flanders Fund for Scientific Research (FWO-Flanders, n. G040821N to VT), and Solve-RD (Horizon 2020 under grant agreement n. 779257 to VT as 1310 active partner). BT PhD fellowship was funded by FRRB (Regione Lombardia, TRANS_ALS, n. 2015-0023 to AP); LV and EA obtained, respectively, a PhD and junior postdoc fellowship (n. 1228021N to EA) of FWO-Flanders, Belgium. AP is a member of the Center of Excellence for Neurodegenerative Diseases (CEND) of the Università degli studi di Milano; VT is a member of the μNEURO Center of Excellence at the University of Antwerp. We thank VoLo Foundation for funding our translational studies in patient cell lines and animals.