Background: The skeletal muscle Z-band has become a prime target for the discovery of novel genes implicated in muscular dystrophies. ZASPopathy, a prototype skeletal muscle Z-band disease, is caused by heterozygous mutations in the Z-band alternatively spliced PDZ motif protein (ZASP). The A165V mutation occurs in a highly conserved motif (ZM) expressed exclusively in skeletal muscle. PDZ proteins have been shown to undergo self associations to form multimers and interact with other PDZ proteins. However, self association of ZASP or its interactions with mutant ZASP are not explored yet.
Objectives: 1) To explore self-association of ZASP; 2) To investigate whether the A165V mutation affects oligomerization of ZASP; and 3) To determine the role of the ZM in ZASP oligomer formation.
Approach: Untagged or tagged ZASP, ZASP-A165V, and ZASP-ΔZM proteins (n=18) were transiently expressed in HEK293T cells. In vivo oligomerization was explored by chemical crosslinking as well as by co-immunoprecipitation.
Summary: 1) ZASP undergoes self-association to form multimers. Presence of the A165V mutation does not affect self-association of ZASP. 2) The mutant protein ZASP-A165V interacts with ZASP. 3) Exclusion of the ZM region, in which the A165V mutation occurs, did not affect ZASP multimer formation by chemical crosslinking (validation by co-immunoprecipitation is in progress).
Future Directions: 1) Identify interacting sites involved with ZASP self-association and its interaction with the mutant protein; 2) Assays designed to probe the endogenous level of ZASP interaction in live cells; 3) Analyze the binding efficiency as well as the kinetics of ZASP multimer formation; 4) Analyze the post-translational modifications to the interacting sites that modulate ZASP oligomerization; 5) Investigate the effects of the interaction between ZASP-A165V and ZASP on skeletal muscle structure and function in ZASPopathy.
Last updated March 20, 2013