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 ␣B-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 earlyonset 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.HSPB5 (also known as CRYAB or ␣B-crystallin) is a small molecular weight heat shock protein encoded by the HSPB5 (crystallin, alpha B) gene and functions as a molecular chaperone. Its promoter contains a heat shock element, a stress-responsive binding site of heat shock transcription factor 1 (HSF1), that functionally up-regulates the expression of HSPB5. Increased levels of HSPB5 can then go on to provide

Neuromuscular Diseases Due to Chaperone Mutations: A Review and Some New Results

“…Propensity to aggregation in vitro and in vivo is a nearly universal feature of the analyzed CRYAB mutations (Table 5), which has been demonstrated for p.R120G [222,262,263,271,272] as well as several other mutations [227,258,259,[272][273][274]. CRYAB p.R120G expressed in cells or transgenic tissues forms aggregates that coalesce into perinuclear aggresomes [262,271].…”

“…Functional studies on p.S115Pfs*14 revealed that the mutant protein is extremely insoluble, but its solubility is increased upon the coexpression of wild-type CRYAB [274]. Overexpression of the mutant protein in BHK21 cells produced aggregates, some of which contained desmin, and it also elicited a stress response [274].…”

“…Functional studies on p.S115Pfs*14 revealed that the mutant protein is extremely insoluble, but its solubility is increased upon the coexpression of wild-type CRYAB [274]. Overexpression of the mutant protein in BHK21 cells produced aggregates, some of which contained desmin, and it also elicited a stress response [274]. Based on these findings, the pathogenic effect of p.S115Pfs*14 was suggested to depend on loss of function due to the unavailability of the aggregating mutant protein, with an additional toxic gain-of-function possibly contributing to the severity [274].…”

“…Overexpression of the mutant protein in BHK21 cells produced aggregates, some of which contained desmin, and it also elicited a stress response [274]. Based on these findings, the pathogenic effect of p.S115Pfs*14 was suggested to depend on loss of function due to the unavailability of the aggregating mutant protein, with an additional toxic gain-of-function possibly contributing to the severity [274]. However, the similar phenotype of the c.3G > A (p.Met1?)…”

“…mutation now argues against a major contribution from a gain-of-function component [234]. Despite interacting with wild-type CRYAB, the p.S115Pfs*14 protein does not seem to interfere with its function in heterozygous carriers, although late-onset dominant effects are not excluded [274]. Table 5.…”