G.P.38 The LGMD1D gene DNAJB6: Its role in sarcomeric protein maintenance and aggregation

Abstract

We have recently identified mutations in DNAJB6 as the cause of LGMD1D, and shown that DNAJB6 interacts with the chaperone-assisted selected autophagy machinery (CASA). CASA is important for maintenance of the Z-disk and protein turnover. Mutations in the CASA component BAG3 also cause myofibrillar myopathies. As DNAJB6 and BAG3 are part of the same complex a significant overlap in the affected molecular mechanisms in the disorders caused by mutations in DNAJB6 and BAG3 is expected. However, the molecular disease mechanisms are currently not well understood. To study the altered anti-aggregation effect of mutant DNAJB6, in particular the role of the acetylation state of DNAJB6 previously reported as important for its anti-aggregation activity, and the functional relationship between DNAJB6 and the CASA proteins, especially BAG3 and STUB1. The importance of DNAJB6 in protein turnover and anti-aggregation was studied in transiently transfected mammalian cells using co-expression of DNAJB6, other CASA proteins, and an aggregation prone protein (GFP-tagged poly-Q huntingtin). Aggregated proteins were filter trapped and visualised using antibodies. Soluble proteins were measured by western blotting. The anti-aggregation activity was calculated as the ratio of aggregated vs. soluble protein amounts. The effect of acetylation and deacetylation of DNAJB6 was studied using constructs simulating the acetylated or deacetylated states. Our studies indicate that the acetylation state is of less importance for the anti-aggregational capacity of DNAJB6. Our results also indicate a direct functional relationship between DNAJB6 and the CASA proteins BAG3 and STUB1 on the anti-aggregation activity. This could be of importance as altered function of the CASA complex caused by mutations in DNAJB6 and BAG3 would directly affect the maintenance of sarcomeric proteins.