Interaction analysis of Hsc70 and CSPbeta proteins during the acrosome reaction in human sperm

BELLAVITA LUCAS; DURÁN NÉSTOR ; FLORES MONTERO KARINA JOHANA; DI LELLA SANTIAGO ; RUETE, MARÍA CELESTE

Mendoza

Congreso; Reunión anual Edición LVIII - SAIB 2022; 2022

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

The fertilization of the ovule by the sperm gives rise to a new individual. In this process,the sperm executes a regulated exocytosis called acrosome reaction (AR), which ismediated by evolutionarily conserved proteins. Molecular chaperones participate inprotein complex association. Cysteine String Protein (CSP) and Heat shock cognate 70kDa (Hsc70) are chaperones that contribute to exocytosis interacting with the SNAREcomplex. In a previous work of our lab, we showed that recombinant non-palmitoylatedCSPβ inhibited the AR triggered by calcium. But, the mechanism of this interaction hasyet to be fully explained in human sperm, so we have focused on these chaperones’interplay. On the other hand, it has been reported that a loop in the CSP protein, calledHPD loop, is crucial for the interaction and functionality of the Hsc70-CSP complex. This work aimed at studying the interaction between Hsc70 and CSPβ, both in vitro andin silico, and proposes a mechanism to explain the AR inhibition by the recombinantCSPβ. Using dot blot and functional assays, we demonstrated that a CSP mutant(D45A) cannot bind Hsc70 and does not affect the AR stimulated with calcium. Basedon already reported complexes in other species, we have performed a model for theHsc70-CSPβ complex and further molecular dynamics simulations to understandcritical determinants for the interaction between both proteins at an atomistic level. Ourresults suggest a possible mechanism to explain the inhibition of AR in which therecombinant non-palmitoylated CSPβ sequesters endogenous Hsc70, restricting itsfunction. These results contribute to the importance of adequately forming this complexand offer insight into the implication of impairing proper acrosome reaction duringfertilization. A discussion on implications in exocytosis regulation and further openquestions will be presented.