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Sequestosome-1 (p62) expression reveals chaperone-assisted selective autophagy in immune-mediated necrotizing myopathies.

ABSTRACT: Diffuse myofiber necrosis in the context of inflammatory myopathy is the hallmark of immune-mediated necrotizing myopathy (IMNM). We have previously shown that skeletal muscle fibers of IMNM patients may display nonrimmed vacuoles and sarcoplasmic irregularities. The dysfunctional chaperone activity has been linked to the defective assembly of skeletal muscle proteins and their degradation via lysosomes, autophagy and the proteasomal machinery. This study was undertaken to highlight a chaperone-assisted selective autophagy (CASA) pathway, functionally involved in protein homeostasis, cell stress and the immune response in skeletal muscle of IMNM patients. Skeletal muscle biopsies from 54 IMNM patients were analyzed by immunostaining, as well as by qPCR. Eight biopsies of sIBM patients served as pathological controls, and eight biopsies of nondisease control subjects were included. Alteration of autophagy was detectable in all IMNM biopsy samples highlighted via a diffuse sarcoplasmic staining pattern by p62 and LC3 independent of vacuoles. This pattern was at variance with the coarse focal staining pattern mostly confined to rimmed vacuoles in sIBM. Colocalization of p62 with the chaperone proteins HSP70 and αB-crystalline points to the specific targeting of misfolded proteins to the CASA machinery. Bcl2-associated athanogene 3 (BAG3) positivity of these fibers emphasizes the selectivity of autophagy processes and these fibers also express MHC class I sarcolemma. Expression of genes involved in autophagy and endoplasmic reticulum (ER) stress pathways studied here is significantly upregulated in IMNM. We highlight that vacuoles without sarcolemmal features may arise in IMNM muscle biopsies, and they must not be confounded with sIBM-specific vacuoles. Further, we show the activation of selective autophagy and emphasize the role of chaperones in this context. CASA occurs in IMNM muscle, and specific molecular pathways of autophagy differ from the ones in sIBM, with p62 as a unique identifier of this process.

SUBMITTER: Fischer N

PROVIDER: S-EPMC8018061 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:Proteotoxicity resulting from accumulation of damaged/unwanted proteins contributes prominently to cellular aging and neurodegeneration. Proteasomal removal of these proteins upon covalent polyubiquitination is highly regulated. Recent reports proposed a role for autophagy in clearance of diffuse ubiquitinated proteins delivered by p62/SQSTM1. Here, we compared the turnover dynamics of endogenous ubiquitinated proteins by proteasomes and autophagy by assessing the effect of their inhibitors. Autophagy inhibitors bafilomycin A1, ammonium chloride, and 3-methyladenine failed to increase ubiquitinated protein levels. The proteasome inhibitor epoxomicin raised ubiquitinated protein levels at least 3-fold higher than the lysosomotropic agent chloroquine. These trends were observed in SK-N-SH cells under serum or serum-free conditions and in WT or Atg5(-/-) mouse embryonic fibroblasts (MEFs). Notably, chloroquine considerably inhibited proteasomes in SK-N-SH cells and MEFs. In these cells, elevation of p62/SQSTM1 was greater upon proteasome inhibition than with all autophagy inhibitors tested and was reduced in Atg5(-/-) MEFs. With epoxomicin, soluble p62/SQSTM1 associated with proteasomes and p62/SQSTM1 aggregates contained inactive proteasomes, ubiquitinated proteins, and autophagosomes. Prolonged autophagy inhibition (96 h) failed to elevate ubiquitinated proteins in rat cortical neurons, although epoxomicin did. Moreover, prolonged autophagy inhibition in cortical neurons markedly increased p62/SQSTM1, supporting its degradation mainly by autophagy and not by proteasomes. In conclusion, we clearly demonstrate that pharmacologic or genetic inhibition of autophagy fails to elevate ubiquitinated proteins unless the proteasome is affected. We also provide strong evidence that p62/SQSTM1 associates with proteasomes and that autophagy degrades p62/SQSTM1. Overall, the function of p62/SQSTM1 in the proteasomal pathway and autophagy requires further elucidation.

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Sequestosome-1 (p62) expression reveals chaperone-assisted selective autophagy in immune-mediated necrotizing myopathies.

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