P.144 - Turnover studies on DNAJB6 and the CASA pathway proteins

Abstract

Limb-girdle muscular dystrophy type 1D (LGMD1D) results from dominant mutations in the co-chaperone DNAJB6. The mutations confer toxic properties to the short cytoplasmic isoform DNAJB6b, and slow down the turnover of mutant DNAJB6, as well as that of the co-expressed wild-type protein. We have previously shown that DNAJB6 interacts with the chaperone-assisted selective autophagy (CASA) system. This macroautophagy pathway, important for muscle maintenance, depends on the co-chaperone BAG3, together with HSPA8 (Hsc70) and HSPB8 (Hsp22). Interestingly, overexpression of BAG3 increases DNAJB6b toxicity in zebrafish – an effect not seen with the BAG3 myopathy mutant P209L – suggesting an active role for BAG3 in LGMD1D pathogenesis. The role of altered DNAJB6 turnover in the pathogenesis of LGMD1D, and its relationship with the CASA proteins is unknown. BAG3 could increase the toxicity of mutant DNAJB6b by aggravating its turnover block. Alternatively, if BAG3 and HSPB8 lie downstream of DNAJB6b mutations, also their turnover rates could be affected. To explore these possibilities, we have carried out cell-culture based protein turnover studies. In our setup, proteins of interest are expressed in a tetracycline-inducible system, and their turnover is followed after tetracycline removal. Coexpression or knockdown allows identification of modulating effects of other proteins. Unexpectedly, while wild-type BAG3 had little effect, BAG3 P209L blocked the turnover of both wild-type and mutant DNAJB6b. This correlated with a shift of DNAJB6b to the insoluble fraction, suggesting that mutant BAG3 may sequester DNAJB6b into an insoluble pool that is resistant to normal degradation. Our results reinforce a functional interaction between DNAJB6 and BAG3, provide a possible explanation for the different effects of wild-type and P209L BAG3 on DNAJB6 toxicity, and place DNAJB6 downstream of the BAG3 P209L mutation, potentially linking the pathomechanisms of LGMD1D and BAG3 myopathy.