Studies on human myosin V interactome

Myosins are motor proteins with ATPase activity and processivity in actin filaments. The Myosin Class V presents, in humans, three paralogous genes, MYO5A, MYO5B and MYO5C, which encode the dimeric proteins MyoVa, MyoVb and MyoVc, respectively. These motor proteins are responsible for the transport of vesicles, organelles, nucleic acids and proteins, which is fundamental for exocytosis, membrane recycling and cell polarization; processes that are closely related to skin pigmentation, synaptic plasticity and cellular differentiation. Most of the cargoes transported by MyoVs bind to their globular tail domain, located at the C-terminal region of the protein, usually in the presence of one or more adaptor proteins. Moreover, the globular tail domain contains a phosphorylation site whose function is not yet well described. Evidence indicates that it may act as a further level of MyoVa regulation for the choice of cargoes, or also to indicate when MyoVa should release these cargoes. In this context, through the yeast two-hybrid technique, we sought to expand on the knowledge of the MyoVa interactome mainly related to the phosphorylation event. From the use of a double mutant phospho-mimetic was identified four new putative molecular partners: Spermine Synthase; Tandem C2 Nuclear Protein; WD Repeat-Containing Protein 48; and Cold Shock Domain-Containing Protein E1. Two of these interactions - Spermine Synthase (which had its interaction confirmed in vitro) and Tandem C2 Nuclear Protein - were studied by immunocytochemistry; and these proteins showed a punctual and discrete pattern of colocalization with MyoVa in the cytoplasm, and phenotypic changes were observed when the MYO5A gene was silenced by siRNA. Unexpectedly none of the proteins identified, validated and cured by the yeast two-hybrid method showed a preference for interaction by the phospho-mimetic form in vitro, which did not allow to advance in the better understanding of the role of phosphorylation. In parallel, another novel interaction between MyoVc and Rab3A was the subject of biophysical and cellular studies, which originated from the cooperation of our research group with Prof. Fukuda from Tohoku University, suggesting a potential new biological role for MyoVc that is still poorly studied compared to MyoVa (AU)