Mechanism of HSPB8 Myopathy Pathology
Mutations in HSPB8 gene have been previously linked to a rare type of Charcot Marie Tooth disease (CMT type 2L) and distal hereditary autosomal dominant neuronopathy type 2 (OMIM). HSPB8 Myopathy is caused by mutations in HSPB8 gene, typically frameshift mutations in exon 3 (Figure 1). This mutation leads to a stop codon readthrough and results in a longer protein product, with extended C terminal region, that is predicted to be highly disordered and more prone to aggregation than the WT protein (Tedesco et al, 2023a).
List of mutations known to cause HSPB8 Myopathy (personal communication with dr Lan Weiss, 2024):
c.515dupC (p.P173Sfs*43)
c.151insC (p.P173SfsX43)
c.508_509delCA (p.GIn170Glyfs*45)
c.525-529delAACAT (p.T176Wfs*38)
c.421A>G (p.K141E)
c.515delC (p.Pro172Leufs*75)
c.577-580dupGTCA (p.T194Sfs*23)
Figure From Inoue-Shibui et al, 2021- HSPB8 variants and predicted proteins. A Scheme of HSPB8, HSPB8, and sHSPs. HSPB8 consists of three exons. The previously reported muscular phenotype related to c.515dup (p.Pro173Serfs*43), c.508_509del (p.Gln170Glyfs*45), and c.577_580dup (p.Thr194Serfs*23) are located in the last exon, as a result of frameshift mutations. Hot spot mutations of Charcot-Marie-Tooth type 2L and distal hereditary motor neuropathy IIa are located at the 421 and 423 bases in exon 2. HSPB8 contains an α-crystallin domain, in grey. The dark blue parts in the α-crystallin domain are β4and β8 chains, respectively, to which the IXI/V domain binds. sHSPs, other than HSPB8, have an α-crystallin domain and an IXI/V domain (yellow part) in the Cterminal. B Predicted amino acid sequences. The reference sequence (CCDS9189.1) consists of 196 amino acids. The amino acids of previously reported variants are in red. The variant identified in this study, c.525_529del (p.Thr176Trpfs*38) is located in the latter part of the last exon causing a frameshift mutation, and is predicted to result in the elongation of 17 amino acids in the C-terminal. The previously reported p.Gln170Glyfs*45, p.Pro173Serfs*43, and p. Thr194Serfs*23 are also predicted to have an elongation effect on amino acids in the C-terminus. The IXI/V motifs are boxed yellow.
Mechanisms of HSPB8 Myopathy pathology are under investigation. In 2017, Echaniz-Laguna suggested that myopathy is caused by haploinsufficiency of HSPB8 protein, as they observed decreased levels of HSPB8 protein in patient muscle tissue compared to controls, and were not able to detected a shorter or a longer form using an antibody targeting the N-terminal of HSPB8 (Echaniz-Laguna et al, 2017). However a recent study by Tedesco and colleagues points to a dominant negative and toxic gain of function mechanism (Tedesco et al, 2023a).
This study investigated the biochemical and functional alterations associated with the HSPB8_fs mutant proteins. It shows that HSPB8_fs mutants are highly insoluble and tend to form proteinaceous aggregates in the cytoplasm. Although all HSPB8 frameshift mutants retain their ability to interact with CASA members, they sequester these members into HSPB8-positive aggregates along with two autophagy receptors, SQSTM1/p62 and TAX1BP1. This process adversely affects CASA’s ability to remove its clients, leading to a general failure in proteostasis response. The aggregation of these mutants is an intrinsic feature of the mutated amino acid sequence and occurs independently of interactions with other CASA members or autophagy receptors (Tedesco et al, 2023a).
Figure 4 From Tedesco et al., 2023a - Frameshift mutants of HSPB8 form high molecular weight insoluble species and cytoplasmic aggregates. (A) Schematic representation of HSPB8_WT and its HSPB8_fs mutant structures. The α-crystallin domain (ACD) is reported in Orange, the N-terminal region (NTR) in blue, the C-terminal region (CTR) in green. The striped green region represents the mutated CTR of p.P173Sfs*43 (fs1), p.Q170Gfs*45 (fs3) and p.T176Wfs*38 mutants. The red region represents the common C-terminal extension (CE) shared by all fs mutants. The box at the bottom reports the nomenclature of the mutated C-termini of the HSPB8 mutants: mCTR (mutated CTR, comprises both the mutated CTR and the CE) and the CE. (B) Immunofluorescence analysis of HeLa cells transiently transfected with V5-tagged HSPB8 constructs. HSPB8 is in green, nuclei were stained with DAPI, scale bar: 20 μm. (C) Western blot and filter retardation assay (FRA) analyses of NP-40 soluble/insoluble protein fractions of HeLa cells transiently transfected with V5-tagged HSPB8 constructs or an empty vector (EV). Bar graphs report mean values (± SD) of densitometry of HSPB8-V5 on soluble TUBA/tubulin alpha for western blot. All graphs are normalized to the HSPB8_WT-V5. One-way ANOVA with Tukey’s test was performed: * p < 0.05, ** p < 0.01, *** p < 0.001; n = 3.
