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

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.

A novel c.515delCHSPB8-multisystem proteinopathy associated with inclusion body myopathy with cardiomyopathy

“…We report a family of Ashkenazi Jewish descent with hereditary rimmed vacuolar myopathy associated with a novel variant in the HSPB8 gene [1].…”

“…Chaperone-assisted selective autophagy (CASA) is a protein-mediated degradation pathway crucial for the maintenance of neuromuscular tissue [1]. HSPB8, or small heat shock protein family B member 8 complexes with co-chaperone BAG3, and together they associate with the complex formed by HSC70, a selected member of the heat shock protein family A (HSPA), E3 ubiquitin ligase STIP1 homology, and U-Box containing protein 1 (STUB1, also known as C terminus of HSC70-Interacting Protein, CHIP).…”

“…Failure in protein degradation result in BAG3-mediated shunting to aggresomes 1 [18]. HSPB8 mutations are associated with Charcot-Marie-Tooth type 2 L (CMT2L), distal 1 and myofibrillar pathology [19][20][21][22][23][24][25]. Frameshifts in the HSPB8 gene, resulting in elongated protein sequence, have been demonstrated to form insoluble protein aggregates, disrupting the CASA complex formation and function [1].…”

“…HSPB8 mutations are associated with Charcot-Marie-Tooth type 2 L (CMT2L), distal 1 and myofibrillar pathology [19][20][21][22][23][24][25]. Frameshifts in the HSPB8 gene, resulting in elongated protein sequence, have been demonstrated to form insoluble protein aggregates, disrupting the CASA complex formation and function [1]. Here we present a family with an HSPB8 frameshift mutation c.515delC manifesting as rimmed vacuolar myopathy, and compared their findings with all previous reports of HSPB8 myofibrillar myopathy to expand the clinical phenotype of this rare disease [20][21][22][23][24][25][26][27][28].…”

High-intensity interval training alleviates exhaustive exercise-induced HSP70-assisted selective autophagy in skeletal muscle

“…In fact, basic selective autophagy is essential for the degradation of damaged organelles and proteins in myocyte fibers to retain proteostasis and promote the repair process [ 30 ]. During normal muscle contraction, mechanical damage of proteins around the Z-disk is specifically recognized by the chaperone complex that composes the molecular chaperone HSP70 and cochaperone BAG3 [ 31 , 32 ]. Then, the complex undergoes ubiquitination with the assistance of CHIP (which acts as an E3–ubiquitin ligase) and is recognized by p62 to degrade through selective autophagy.…”