Cellular and proteomic approaches to study the role of ACTG2 mutation-mediated misfolding and protein aggregation as druggable targets in the pathogenesis of Chronic Intestinal Pseudo-Obstruction

Gastrointestinal motility failure and chronic constipation, due to various and heterogeneous causes, have collectively been referred to as Chronic Intestinal Pseudo-Obstruction (CIPO) syndromes. A myogenic form of CIPO (also known as visceral myopathy-VSCM) is associated with g-enteric smooth muscle actin (ACTG2) variants, which mainly occur as de novo mutational events. Clinical treatment of VSCM is still limited, focusing primarily on symptom management rather than disease treatment: patients often undergo abdominal surgery and food replacement with life-long total parenteral nutrition. New therapeutic approaches are therefore needed to restore smooth muscle function and to improve patients’ quality of life. Insights into the molecular details of pathogenic mechanisms underlying VSCM and identification of drug targets are still lacking. Based on our preliminary results, we will test the hypothesis that the VSCM molecular pathogenesis is sustained by the persistence in cells of ACTG2 proteins unable to fold properly, thus triggering a protein stress response and the failure of cell clearance systems, i.e. ubiquitin-proteasome and autophagy. To this end, we will use, in patient and control cells, a powerful high-content imaging system to: i) perform a thorough cellular phenotyping with characterization of intracellular aggregates, ii) evaluate the proteasome and autophagy activity, iii) test, using appropriate molecules, the effects of suppression of both ACTG2 aggregates and expression of ACTG2 mutant alleles, iv) apply a proteomic approach of mass-spectrometry. This will allow us to test our working hypothesis and to identify new cellular and molecular targets to prospectively develop a possible therapeutic approach for VSCM.