Mesoscopic clusters of ICA512-RESP18HD

PAMELA TOLEDO; DIEGO S. VAZQUEZ; ALEJO R. GIANOTTI; MARIO R. ERMÁCORA

CABA

Congreso; XLIX Reunión Anual de la Sociedad Argentina de Biofísica; 2021

SAB

ICA512 is a protein tyrosine phosphatase enriched in secretory granules (SGs) of the pancreatic β-cells and other neuroendocrine cells. In this work, we focused on the N-terminal extracellular domain of ICA512 called RESP18HD (glucocorticoid-responsive regulated endocrine-specific protein 18 homology domain) and on its condense properties. In previous studies, we discovered that RESP18HD displays an in vitro protein condensing activity at close to neutral pH, i.e. in the pH range found in the early secretory pathway in cells. Remarkably, this condensing activity is exerted towards insulin, which in the presence of isolated RESP18HD aggregates at pH 6.8. Now we could demonstrate that behaviour extends to proinsulin, the insulin precursor, which is present in early stages of the SG secretion. Also we found that the (pro)insulin condensing activity of RESP18HD, evidenced by turbidimetry assay, increases dramatically in reducing conditions, suggesting a potential thioredoxin-like activity of RESP18HD. In this regard, a FITC-labeled insulin reagent shows a change in the fluorescence signal in presence of RESP18HD and DTT according to the turbidimetry assays. Our main hypothesis is that ICA512-RESP18HD aggregation properties are related to a liquid phase separation known as mesoscopic clustering - the formation of nanometer-sized soluble conglomerates - leading to the formation of RESP18HD larger amorphous aggregates, which could contribute to the sorting of proteins from the endoplasmic reticulum to the Golgi in the secretory pathway.