Selective degradation of aggregate-prone CryAB mutants by HSPB1 is mediated by ubiquitin-proteasome pathways

Disease-causing mutations of genes encoding small MW heat shock proteins (sHSPs) constitute a growing family of inherited myofibrillar disorders In the present work we found that three structurally-distinct CryAB mutants R120G 450delA and 464delCT are mostly present in the detergent insoluble fractions when overexpressed in H9c2 rat heart cells We found that either over expression or knockdown of HSPB1 a related sHSP affects the solubility stability and degradation of aggregation-prone CryAB mutants HSPB1 overexpression has negligible effects on the solubility and protein aggregates of either R120G and/or 450delA but increased the solubility and prevented formation of 464delCT aggregates HSPB1 knockdown decreased solubility and increased protein aggregates of all CryAB mutants indicating a key role for HSPB1 in clearance of CryAB mutants under basal conditions We provide four lines of evidence that such selective clearance of R120G 450delA and 464delCT mutants by HSPB1 is mediated by the ubiquitin-proteasome system (UPS) First we found that treatment with the proteasome inhibitors increased the levels of all CryAB mutants Second R120G and 450delA overexpression corresponded to the accumulation of their specific ubiquitin conjugates in H9c2 cells Third HSPB1 knockdown directly increased the levels of all polyubiquitin conjugates And fourth the selective attenuation of 464delCT expression by HSPB1 over expression was abrogated by the proteasome inhibition We conclude that such selective interactions between CryAB mutants and HSPB1 overexpression might have important implications for the clinical manifestations and potential treatment Published by Elsevier Ltd