Oligomerization of ZASP, a Myofibrillar Myopathy Gene Product, In Vitro and In Vivo (P1.058)

OBJECTIVE: 1) To explore self-association of ZASP; 2) To identify oligomerization domains of ZASP; and 3) To investigate the role of ZASP oligomerization in normal skeletal muscle and in zaspopathy BACKGROUND: Zaspopathy, a prototype myofibrillar myopathy, is caused by heterozygous mutations in ZASP, a Z-disc associated PDZ-LIM protein. The long and short ZASP isoforms are defined by the presence or absence of LIM domains, respectively. The mutated sZM domain is common to both isoforms. ZASP interacts with skeletal actin filaments via the sZM domain (abstract submitted).The PDZ-LIM proteins are regulated by intramolecular association. However, oligomerization of ZASP has not yet been explored. DESIGN/METHODS: ZASP oligomerization was investigated with native gel filtration of purified ZASP proteins as well as co-immunoprecipitation and chemical crosslinking assays in transfected cells and mouse and patient skeletal muscle. The domains involved in dimerization were identified with yeast two-hybrid assays. Effects of dimerization on ZASP-actin interaction will be examined by modified co-sedimentation assays. Future studies will explore the effects of post-translational modification on ZASP dimerization. RESULTS: The long ZASP isoforms self-associate, whereas the short isoform does not. The long and short ZASP isoforms can interact with each other. Wild type and mutant ZASP form homodimers and heterodimers. The sZM domain is not involved in oligomerization. The LIM domains of ZASP dimerize and interact with the short ZASP isoform. ZASP oligomerization is inhibited by reducing agents (DTT and BME). CONCLUSIONS: Wild-type and mutant ZASP form homodimers and heterodimers in zaspopathy, an autosomal dominant disease. Self-association of ZASP is specific to the long isoform that is required for the integrity of the Z-discs in skeletal muscle. The sensitivity to reducing agents suggests that the conserved cysteine residues in the LIM domains are likely involved in ZASP dimerization. Study Supported by: Disclosure: Dr. Brubaker has nothing to disclose. Dr. Ohman has nothing to disclose. Dr. Chow has nothing to disclose. Dr. Lin has nothing to disclose. Dr. Mankodi has nothing to disclose.