Ablation of Kex2 activity enhances proIAPP proteotoxicity in yeast

Abstract

Pancreatic deposition of Islet Amyloid PolyPeptide (IAPP) is a histopathological hallmark of type 2 diabetes. Inadequate processing of immature IAPP by proprotein convertases (PCs) leads to the accumulation of unprocessed forms, which in turn favors increased aggregate formation in pancreatic β-cells. Kexin 2 (Kex2) of Saccharomyces cerevisiae is the prototype of eukaryotic PCs family, but to date no direct correlations between Kex2 activity and preproIAPP processing in yeast have been reported. In this study we aimed to address the possible role of Kex2 on IAPP maturation as a tool to investigate the contribution of impaired processing towards IAPP proteototoxicity. Genetically modified S. cerevisiae models lacking the KEX2 gene and carrying different chimeric fusions of human IAPP linked to GFP were used. The cytotoxic effects of Kex2 ablation were assessed by means of growth curve analysis and cell viability assays using flow cytometry. IAPP protein profile was evaluated by immunoblotting assays using an anti-IAPP antibody. Intracellular IAPP aggregates were monitored by confocal fluorescence microscopy. Data showed that kex2 mutants exhibit growth defects, potentiated by preproIAPP-GFP, proIAPP-GFP and mature IAPP-GFP expression with an increased cytotoxicity for proIAPP-GFP. Notwithstanding, Kex2 absence does not seem to affect IAPP protein pattern nor the frequency/distribution of intracellular IAPP aggregates in yeast. Our findings suggest that Kex2 is not essential for IAPP processing in yeast, at least under the conditions tested. Keywords: Amylin, Islet Amyloid Polypeptide (IAPP), Kexin 2, Prohormone processing, Saccharomyces cerevisiae