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

Abstract

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.

The yapsin family of aspartyl proteases regulate glucose homeostasis in Candida glabrata

“…Although the mechanistic basis of CgYapsin-mediated regulation of glucose metabolism remains to be determined, it is possible that the glucose sensors, CgSnf3 and CgRgt2, in the plasma membrane are substrates of CgYapsins, with their J o u r n a l P r e -p r o o f processing potentially by CgYapsins generating a signal for activation of the glucose response pathway. In this context, it is worth noting that the plasma membrane flavodoxin-like protein CgPst2 has recently been shown to be a target of the CgYps1 protease (27). Furthermore, our preliminary results suggest that the proteolytic activity of CgYapsins is likely to be required for their role in glucose homeostasis, as ectopic expression of the catalytically-active CgYPS1…”

“…but not of the catalytically-inactive CgYPS1 D91A [carries alanine in place of the catalytic residue aspartic acid, and lacks proteolytic activity; (27)] could restore the deregulated expression of glucose sensing and transport genes [CgSNF3, CgHXT2/10 (I), CgHXT4 and CgHXT14] in the Cgyps1-11 mutant to wt-levels (Fig. S5).…”

“…The medium containing varied glucose concentration (0.01% to 5%) was prepared by adding the requisite amount of dextrose to yeast nitrogen base with ammonium sulphate for YNB medium, before autoclaving the medium. C. glabrata deletion strains, Cgsnf3 and Cgsnf3yps1-11, were generated using the homologous based strategy with nourseothricin resistance-conferring gene nat1 or hygromcyin resistanceconferring gene hph1 as the selection marker (27). For generation of the Cgsnf3yps1-11 strain that lacks 12 genes, CgSNF3 and CgYPS1-11, Cgyps1 mutant (CgYPS1 gene replaced hph1 gene) was used as the parent strain, and CgSNF3 and CgYPS2-11 genes were sequentially deleted from its genome.…”

“…The serial dilution spotting assay and human THP-1 macrophage infection assay was performed, as described previously (18,27).…”

Extracellular Matrix of Echinoderms

“…Aspartyl proteases represent a group of peptidases that cleave protein substrates using two aspartic acid residues located in their catalytic center [146]. One of proteases of this type is cathepsin D. It is a lysosomal enzyme, but it can also be localized in the extracellular space [147], where it can cleave aggrecan molecules [148].…”