An aspartyl protease-mediated cleavage regulates structure and function of a flavodoxin-like protein and aids oxidative stress survival

A family of eleven glycosylphosphatidylinositol-anchored aspartyl proteases, commonly referred to as CgYapsins, regulate a myriad of cellular processes in the pathogenic yeast Candida glabrata, but their protein targets are largely unknown. Here, using the immunoprecipitation-mass spectrometry approach, we identify the flavodoxin-like protein (Fld-LP), CgPst2, to be an interactor of one of the aspartyl protease CgYps1. We also report the presence of four Fld-LPs in C. glabrata, which are required for survival in kidneys in the murine model of systemic candidiasis. We further demonstrated that of four Fld-LPs, CgPst2 was solely required for menadione detoxification. CgPst2 was found to form homo-oligomers, and contribute to cellular NADH:quinone oxidoreductase activity. CgYps1 cleaved CgPst2 at the C-terminus, and this cleavage was pivotal to oligomerization, activity and function of CgPst2. The arginine-174 residue in CgPst2 was essential for CgYps1-mediated cleavage, with alanine substitution of the arginine-174 residue also leading to elevated activity and oligomerization of CgPst2. Finally, we demonstrate that menadione treatment led to increased CgPst2 and CgYps1 protein levels, diminished CgYps1-CgPst2 interaction, and enhanced CgPst2 cleavage and activity, thereby implicating CgYps1 in activating CgPst2. Altogether, our findings of proteolytic cleavage as a key regulatory determinant of CgPst2, which belongs to the family of highly conserved, electron-carrier flavodoxin-fold-containing proteins, constituting cellular oxidative stress defense system in diverse organisms, unveil a hidden regulatory layer of environmental stress response mechanisms.
Author summary Fungal bloodstream infections in immunodeficient patients are a serious clinical problem. Infections caused by the opportunistic fungal pathogen Candida glabrata are difficult to treat owing to its low susceptibility to widely used azole antifungals and emerging co-resistance to azole and echinocandin drugs. Despite encountering oxidative stress in host macrophages, C. glabrata is able to replicate intracellularly. Here, we show for the first time that CgYps1 aspartyl protease-mediated cleavage regulates the structural state, activity and functions of a flavodoxin-like protein CgPst2, which belongs to the family of highly conserved electron-carrier proteins that are pivotal to oxidative stress response in organisms ranging from bacteria to humans. We also show that CgPst2 is required for oxidative stress survival in vitro, and is sufficient to rescue, in vivo, the attenuated survival of a C. glabrata mutant lacking four flavodoxin-like proteins. Further, our data suggest that CgYps1 protease-mediated cleavage of CgPst2 is a dynamic event which may be coupled closely with intracellular quinone accumulation and detoxification. This strategy may aid C. glabrata establish successful infections in the human host. While advancing our understanding of fungal pathogenesis, our findings also provide insights into hitherto unknown regulatory mechanisms for flavodoxin-like fold-containing proteins.