Does protein sequenceC4YJH2 participate in Candida albicans metal homeostasis? A focus on Zn(II) and Cu(II) binding behaviour

Invasive mycoses have significantly increased over the last decades and their associated mortality rate still remains a global concern. The necessity of new antifungal agents is undeniable, since current therapeutic treatments are not completely effective due to the development of drug resistance, host toxicity and undesirable side effects that limit their use in medical practice [1]. Nevertheless, studies concerning the molecular mechanisms of fungal resistance and virulence have identified some novel potential targets for antifungal drugs. For instance, the assimilation of metal nutrients from the host environment is a fundamental aspect of infections and deeper insights into the mechanism of metal uptake and transport in fungal species can provide crucial information to design new antifungal drugs for the treatment and diagnosis of mycoses [2-4]. C4YJH2 (UniProt Knowledgebase [5]) is a protein sequence of 199 amino acid residues, found in the genome of Candida albicans, with the potential ability to chelate metal ions. Zn(II) and Cu(II) are both endogenous metals involved in several biological processes and they can widely compete for the same binding sites; both the uptake and safe storage of these metals must be strictly controlled in order to assure the host and pathogen subsistence [6]. Recently, we studied the C4YJH2 amino acid sequences between residues 131-148 and 157-165, located in the carboxyl-terminal region which is evolutionarily well-conserved and shares high percentages of identity with other metal binding proteins from different fungal species. This sequence is of remarkable interest, since it contains a significant high number of histidine and serine residues and it has been confirmed to have a role in metals coordination [7]. In order to better characterize the metal binding domain, we decided to deepen our investigation focusing our attention on further peptide sequences belonging to the unstructured domain of C4YJH2. The fragment Ac-GSDHSGDSK-NH2 (148-156 residues) has been considered, along with its following analogues: Ac-GSDHSGASK-NH2, Ac-GADHAGDAKNH2, Ac-GSDH-NH2, Ac-HSGD-NH2. The experiments have been performed in aqueous solution, at 25 °C and I = 0.1 M (KCl). Information about stoichiometry and thermodynamic parameters of complex-formation were obtained by means of potentiometry and mass spectrometry; the hypothesized geometry of the formed species is also discussed, on the basis of several spectroscopic data at variable pH (Fig. 1). References: [1] Campoy, S.; Adrio, J.L., Antifungals. Biochem. Pharmacol. 2017, 133, 86-96. [2] Blatzer, M.;Latge´, J.P., Metal-homeostasis in the pathobiology of the opportunistic human fungal pathogen Aspergillus fumigatus. Curr. Opin. Microbiol. 2017, 40, 152– 159. [3] Hood, M.I.; Skaar, E.P.,Nutritional immunity: transition metals at the pathogen-host interface. Nat. Rev. Microbiol. 2012, 10(8), 525-537. [4] Ballou, E.R.; Wilson, D.,The roles of zinc and copper sensing in fungal pathogenesis. Curr. Opin. Microbiol. 2016, 32, 128–134. [5] The_UniProt_Consortium, UniProt: the universal protein knowledgebase. Nucleic Acids Res. 2017, 45, D158–D169. [6] Besold, A.N.; Gilston, B.A.; Radin, J.N.; Ramsoomair, C.; Culbertson, E.M.; Li, C.X.; Cormack, B.P.; Chazin, W.J.; Kehl-Fie, T.E.; Culotta, V.C., Role of Calprotectin in Withholding Zinc and Copper from Candida albicans. Infect. Immun. 2018, 86, e00779- 17. [7] Bellotti, D.; Łoboda, D.; Rowińska-Żyrek, M.; Remelli, M.,Investigation on the metal binding sites of a putative Zn(II) transporter in opportunistic yeast species Candida albicans. New J. Chem. 2018, 42, 8123-8130.