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The Human 343delT HSPB5 Chaperone Associated with Early-onset Skeletal Myopathy Causes Defects in Protein Solubility.

ABSTRACT: Mutations of HSPB5 (also known as CRYAB or αB-crystallin), a bona fide heat shock protein and molecular chaperone encoded by the HSPB5 (crystallin, alpha B) gene, are linked to multisystem disorders featuring variable combinations of cataracts, cardiomyopathy, and skeletal myopathy. This study aimed to investigate the pathological mechanisms involved in an early-onset myofibrillar myopathy manifesting in a child harboring a homozygous recessive mutation in HSPB5, 343delT. To study HSPB5 343delT protein dynamics, we utilize model cell culture systems including induced pluripotent stem cells derived from the 343delT patient (343delT/343delT) along with isogenic, heterozygous, gene-corrected control cells (WT KI/343delT) and BHK21 cells, a cell line lacking endogenous HSPB5 expression. 343delT/343delT and WT KI/343delT-induced pluripotent stem cell-derived skeletal myotubes and cardiomyocytes did not express detectable levels of 343delT protein, contributable to the extreme insolubility of the mutant protein. Overexpression of HSPB5 343delT resulted in insoluble mutant protein aggregates and induction of a cellular stress response. Co-expression of 343delT with WT prevented visible aggregation of 343delT and improved its solubility. Additionally, in vitro refolding of 343delT in the presence of WT rescued its solubility. We demonstrate an interaction between WT and 343delT both in vitro and within cells. These data support a loss-of-function model for the myopathy observed in the patient because the insoluble mutant would be unavailable to perform normal functions of HSPB5, although additional gain-of-function effects of the mutant protein cannot be excluded. Additionally, our data highlight the solubilization of 343delT by WT, concordant with the recessive inheritance of the disease and absence of symptoms in carrier individuals.

SUBMITTER: Mitzelfelt KA

PROVIDER: S-EPMC4946913 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe protein kinase mechanistic target of rapamycin (mTOR) controls cellular growth and metabolism. Although balanced mTOR signalling is required for proper muscle homeostasis, partial mTOR inhibition by rapamycin has beneficial effects on various muscle disorders and age-related pathologies. Besides, more potent mTOR inhibitors targeting mTOR catalytic activity have been developed and are in clinical trials. However, the physiological impact of loss of mTOR catalytic activity in skeletal muscle is currently unknown.MethodsWe have generated the mTORmKOKI mouse model in which conditional loss of mTOR is concomitant with expression of kinase inactive mTOR in skeletal muscle. We performed a comparative phenotypic and biochemical analysis of mTORmKOKI mutant animals with muscle-specific mTOR knockout (mTORmKO) littermates.ResultsIn striking contrast with mTORmKO littermates, mTORmKOKI mice developed an early onset rapidly progressive myopathy causing juvenile lethality. More than 50% mTORmKOKI mice died before 8 weeks of age, and none survived more than 12 weeks, while mTORmKO mice died around 7 months of age. The growth rate of mTORmKOKI mice declined beyond 1 week of age, and the animals showed profound alterations in body composition at 4 weeks of age. At this age, their body weight was 64% that of mTORmKO mice (P < 0.001) due to significant reduction in lean and fat mass. The mass of isolated muscles from mTORmKOKI mice was remarkably decreased by 38-56% (P < 0.001) as compared with that from mTORmKO mice. Histopathological analysis further revealed exacerbated dystrophic features and metabolic alterations in both slow/oxidative and fast/glycolytic muscles from mTORmKOKI mice. We show that the severity of the mTORmKOKI as compared with the mild mTORmKO phenotype is due to more robust suppression of muscle mTORC1 signalling leading to stronger alterations in protein synthesis, oxidative metabolism, and autophagy. This was accompanied with stronger feedback activation of PKB/Akt and dramatic down-regulation of glycogen phosphorylase expression (0.16-fold in tibialis anterior muscle, P < 0.01), thus causing features of glycogen storage disease type V.ConclusionsOur study demonstrates a critical role for muscle mTOR catalytic activity in the regulation of whole-body growth and homeostasis. We suggest that skeletal muscle targeting with mTOR catalytic inhibitors may have detrimental effects. The mTORmKOKI mutant mouse provides an animal model for the pathophysiological understanding of muscle mTOR activity inhibition as well as for mechanistic investigation of the influence of skeletal muscle perturbations on whole-body homeostasis.

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The Human 343delT HSPB5 Chaperone Associated with Early-onset Skeletal Myopathy Causes Defects in Protein Solubility.

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