TRIM32 is the gene responsible for Limb Girdle Muscular Dystrophy type 2H and Sarcotubular Myopathy. The TRIM32 gene product is an E3 ubiquitin ligase for which many substrates have been reported. To unravel its function in muscular dystrophy, it is important to understand how TRIM32 pleiotropy is achieved. TRIM32 ability to homo-interact, thus offering several RING moieties to E2 binding, and to interact with different E2 enzymes can underlie TRIM32 potential to form different complex combinations. As E3-E2 pairs determine the type of ubiquitin modification, this may result in the amplification of TRIM32 spectrum of action in regulating the fate of several targets and implication in diverse pathological conditions.
The objective of this project is to thoroughly define TRIM32 ubiquitin E3 ligase activity by assessing the specific TRIM32-E2 complexes and the ubiquitin chains formed for the control of specific muscular targets, combining biochemical, biophysical and cell biology approaches.
ESR3 will investigate TRIM32 role and biochemistry, self-interaction requirements, and potential to catalyse different ubiquitin chain topologies, dissecting in vitro its E3 activity with different E2 enzyme combinations. These features will be then validated on TRIM32 natural substrates in normal and pathological cellular systems. C2C12 cells induced to differentiate into myofibers and fibroblasts from LGMD2H patients differentiated towards a muscular phenotype by MyoD transduction will be employed for these studies. The accomplishment of the proposed experiments will elucidate the ability of TRIM32 to use different partners within the ubiquitination machinery to produce specific ubiquitin modifications of muscular substrates. These data will be important for the design of muscular dystrophy therapies without interfering with non-muscular pathways.