Protein-folding can go wrong in vivo and in vitro, with significant consequences for the living organism and the pharmaceutical industry, respectively. Here we propose a design principle for small-peptide-based protein-specific folding modifiers. The principle is based on constructing a “xenonucleus”, which is a prefolded peptide that mimics the folding nucleus of a protein. Using stopped-flow kinetics, NMR spectroscopy, Förster resonance energy transfer, single-molecule force measurements, and molecular dynamics simulations, we demonstrate that a xenonucleus can make the refolding of ubiquitin faster by 33 ± 5%, while variants of the same peptide have little or no effect. Our approach provides a novel method for constructing specific, genetically encodable folding catalysts for suitable proteins that have a well-defined contiguous folding nucleus.

16Protein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and 17 the pharmaceutical industry, respectively. Here we propose a general design principle for constructing 18 small peptide-based protein-specific folding modifiers. We construct a 'xenonucleus', which is a pre-19folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the 20 folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy 21 transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the 22 ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the 23 refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for 24 constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined 25 contiguous folding nucleus. 26 27 flow kinetics, time resolved Forster Resonance Energy transfer, NMR, and coarse grain simulation 70 studies show that kinetics of ubiquitin folding can indeed be made faster by a suitable xenonucleus 71 interacting at the appropriate site. 72 73 Results: 74 75Refolding kinetics: Ubiquitin has been widely studied for its thermodynamic and mechanical stability. 76 Previous mutational studies on Ubiquitin, especially using the F45W mutant, has established 77 tryptophan fluorescence as a valuable probe for studying its folding. Here, we measured the refolding 78 kinetics of Ubiquitin (F45W) in presence and in absence of a 19 residue nucleus mimic. It has the amino 79 acid sequence C-MQIFVKTLTGKTITLEV-C, which is the same as residues 1 to 17 of Ubiquitin, except for 80 the terminal cysteines. It has a disulphide bridge between the termini, and will henceforth be called 81 the 'stapled xenonucleus'. We use a three syringe stopped-flow fluorescence instrument (SFM300, 82Biologic, see Methods) to change the GdnHCl concentration from 4.25 M to a series of lower 83 concentrations in less than 6 ms. We measure the change in fluorescence intensity of the Trp residue 84as a function of time as a reporter for the progress of folding (or unfolding). The unfolded state of 85 ubiquitin F45W has a higher quantum yield and has an emission maximum at 360 nm, but when it 86 goes to the folded form, the emission blue-shifts to 340 nm. The quantum yield of fluorescence also 87 goes down (it is speculated that a backbone carboxyl oxygen may be quenching the fluorescence) 57,58 . 88Hence, as folding proceeds, the overall fluorescence signal at 360 nm decreases. We have plotted the 89 change in the fluorescence intensity as a function of time for ubiquitin F45W, with (Figure 1 (A), red) 90 and without (Figure 1 (A), blue) the xenonucleus. The data are fitted with a two-component 91 exponential decay function: 92 102 Refolding kinetics as a function of the xenonucleus concentration: We also perform the refolding 103 experiment as a function of the concentration of the xenonucleus peptide...

Edited by James SiedowThe phosphoinositide 3-kinase (PI3K), which phosphorylates phosphatidylinositol and produces PI3P, has been implicated in protein trafficking, intracellular survival, and virulence in the pathogenic yeast Candida glabrata. Here, we demonstrate PI3-kinase (CgVps34) to be essential for maintenance of cellular iron homeostasis. We examine how CgVps34 regulates the fundamental process of iron acquisition, and underscore its function in vesicular trafficking as a central determinant. RNA sequencing analysis revealed iron homeostasis genes to be differentially expressed upon CgVps34 disruption. Consistently, the Cgvps34⌬ mutant displayed growth attenuation in low-and high-iron media, increased intracellular iron content, elevated mitochondrial aconitase activity, impaired biofilm formation, and extenuated mouse organ colonization potential. Furthermore, we demonstrate for the first time that C. glabrata cells respond to iron limitation by expressing the iron permease CgFtr1 primarily on the cell membrane, and to iron excess via internalization of the plasma membrane-localized CgFtr1 to the vacuole. Our data show that CgVps34 is essential for the latter process. We also report that macrophage-internalized C. glabrata cells express CgFtr1 on the cell membrane indicative of an iron-restricted macrophage internal milieu, and Cgvps34⌬ cells display better survival in iron-enriched medium-cultured macrophages. Overall, our data reveal the centrality of PI3K signaling in iron metabolism and host colonization.

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.