It is well established that, in the last decades, invasive mycoses have developed alarming drug resistance attitudes, making fungal pathogens extremely dangerous for the patients’ subsistence. Candida albicans is one of the most diffused opportunistic pathogenic yeasts, known to be a commensal organism of the human body, but also an extremely dangerous threat in immunocompromised individuals, which can develop severe, life-threatening candidemia [1]. Several lines of evidence confirm that the ability of pathogenic microorganisms, such as C. albicans, to assimilate metal nutrients from the host environment is a fundamental aspect of infection. As a consequence, studies concerning the characterization of the host/pathogen interface and the mechanism of metal uptake and transport in fungal species can provide crucial information and a base to identify new pharmacological targets for the treatment and diagnosis of mycoses [2-4]. Zinc and copper are crucial for the virulence and survival of fungal pathogens in humans, since they are indispensable in the expression of several metalloproteins and enzymes. The acquisition processes of these metals by pathogens is extremely challenging: the concentration of free Zn(II) is normally as low as subnanomolar [5], and Cu(II), that is an endogenous metal, can widely compete with Zn(II) for the same binding sites. As a consequence, both the uptake and safe storage of these metals must be strictly controlled [6]. We recently focused our attention on Zn(II) and Cu(II) binding behaviour towards C4YJH2 (UniProt Knowledgebase [7]), a protein sequence of 199 amino acid residues, found in the genome of C. albicans (Fig. 1). C4YJH2 is suggested to be involved in metal transport processes, since it shares high percentage of identity with several putative Zn(II) transporters and proteins involved in metal homeostasis. This sequence is of remarkable interest, since it has a significant high number of histidine and serine residues, especially in its C-terminal domain, which was confirmed to have a role as metal binding site [8]. The present work represents an in-depth analysis of the metal binding domain of C4YJH2: the protected peptide Ac-GSDHSGDSK-NH2, encompassing residues 148-156 of the protein, is considered, along with its following analogues: Ac-GSDHSGASK-NH2, Ac-GADHAGDAK-NH2, Ac- GSDH-NH2, Ac-HSGD-NH2. The preliminary results presented here concern the stoichiometry and thermodynamics of complex-formation of the above reported ligands with Zn(II) and Cu(II), investigated by means of potentiometric titrations; the hypothesized geometry of the formed species is also discussed, on the basis of UV-Vis spectrophotometric data at variable pH. The comparison between the behaviour of the different analogues helps to shed light on the role of some residues of the sequence (such as Ser and Asp), not directly involved in complexation but influencing the complex geometry and contributing to its stability. The study on the two short peptides, Ac-GSDH-NH2 and Ac-HSGD-NH2, gives information about the attitude of Cu(II) to form complexes which involve amide nitrogens in the amine-terminus or carboxylic-terminus direction. References: [1] T. Kourkoumpetis, D. Manolakaki, G. C. Velmahos, Y. C. Chang, H. B. Alam, M. M. De Moya, E. A. Sailhamer, E. Mylonakis, Virulence 2010, 1, 359–366. [2] M. Blatzer, J.-P. Latge´, Curr. Opin. Microbiol. 2017, 40, 152–159. [3] M. I. Hood, E. P. Skaar, Nat. Rev. Microbiol. 2012, 10, 525. [4] E. R. Ballou, D. Wilson, Curr. Opin. Microbiol. 2016, 32, 128–134. [5] I. Bremner, P. M. May, in Zinc in Human Biology, C. F. Mills, Springer, London, 1989, pp. 95– 108. [6] A. N. Besold, B. A. Gilston, J. N. Radin, C. Ramsoomair, E.M. Culbertson, C. X. Li, B. P.Cormack,W. J. Chazin, T. E. Kehl-Fie, V. C. Culotta, Infect. Immun. 2018, 86, e00779-17. [7] The_UniProt_Consortium, Nucleic Acids Res. 2017, 45, D158–D169. [8] D. Bellotti, D. Łoboda, M. Rowińska-Żyrek, M. Remelli, New J. Chem. 2018, in press, DOI: 10.1039/c8nj00533h
An in-depth analysis of the metal binding domain in a putative Zn(II) transporter of Candida albicans
Denise BELLOTTI
;Maurizio REMELLI
2018
Abstract
It is well established that, in the last decades, invasive mycoses have developed alarming drug resistance attitudes, making fungal pathogens extremely dangerous for the patients’ subsistence. Candida albicans is one of the most diffused opportunistic pathogenic yeasts, known to be a commensal organism of the human body, but also an extremely dangerous threat in immunocompromised individuals, which can develop severe, life-threatening candidemia [1]. Several lines of evidence confirm that the ability of pathogenic microorganisms, such as C. albicans, to assimilate metal nutrients from the host environment is a fundamental aspect of infection. As a consequence, studies concerning the characterization of the host/pathogen interface and the mechanism of metal uptake and transport in fungal species can provide crucial information and a base to identify new pharmacological targets for the treatment and diagnosis of mycoses [2-4]. Zinc and copper are crucial for the virulence and survival of fungal pathogens in humans, since they are indispensable in the expression of several metalloproteins and enzymes. The acquisition processes of these metals by pathogens is extremely challenging: the concentration of free Zn(II) is normally as low as subnanomolar [5], and Cu(II), that is an endogenous metal, can widely compete with Zn(II) for the same binding sites. As a consequence, both the uptake and safe storage of these metals must be strictly controlled [6]. We recently focused our attention on Zn(II) and Cu(II) binding behaviour towards C4YJH2 (UniProt Knowledgebase [7]), a protein sequence of 199 amino acid residues, found in the genome of C. albicans (Fig. 1). C4YJH2 is suggested to be involved in metal transport processes, since it shares high percentage of identity with several putative Zn(II) transporters and proteins involved in metal homeostasis. This sequence is of remarkable interest, since it has a significant high number of histidine and serine residues, especially in its C-terminal domain, which was confirmed to have a role as metal binding site [8]. The present work represents an in-depth analysis of the metal binding domain of C4YJH2: the protected peptide Ac-GSDHSGDSK-NH2, encompassing residues 148-156 of the protein, is considered, along with its following analogues: Ac-GSDHSGASK-NH2, Ac-GADHAGDAK-NH2, Ac- GSDH-NH2, Ac-HSGD-NH2. The preliminary results presented here concern the stoichiometry and thermodynamics of complex-formation of the above reported ligands with Zn(II) and Cu(II), investigated by means of potentiometric titrations; the hypothesized geometry of the formed species is also discussed, on the basis of UV-Vis spectrophotometric data at variable pH. The comparison between the behaviour of the different analogues helps to shed light on the role of some residues of the sequence (such as Ser and Asp), not directly involved in complexation but influencing the complex geometry and contributing to its stability. The study on the two short peptides, Ac-GSDH-NH2 and Ac-HSGD-NH2, gives information about the attitude of Cu(II) to form complexes which involve amide nitrogens in the amine-terminus or carboxylic-terminus direction. References: [1] T. Kourkoumpetis, D. Manolakaki, G. C. Velmahos, Y. C. Chang, H. B. Alam, M. M. De Moya, E. A. Sailhamer, E. Mylonakis, Virulence 2010, 1, 359–366. [2] M. Blatzer, J.-P. Latge´, Curr. Opin. Microbiol. 2017, 40, 152–159. [3] M. I. Hood, E. P. Skaar, Nat. Rev. Microbiol. 2012, 10, 525. [4] E. R. Ballou, D. Wilson, Curr. Opin. Microbiol. 2016, 32, 128–134. [5] I. Bremner, P. M. May, in Zinc in Human Biology, C. F. Mills, Springer, London, 1989, pp. 95– 108. [6] A. N. Besold, B. A. Gilston, J. N. Radin, C. Ramsoomair, E.M. Culbertson, C. X. Li, B. P.Cormack,W. J. Chazin, T. E. Kehl-Fie, V. C. Culotta, Infect. Immun. 2018, 86, e00779-17. [7] The_UniProt_Consortium, Nucleic Acids Res. 2017, 45, D158–D169. [8] D. Bellotti, D. Łoboda, M. Rowińska-Żyrek, M. Remelli, New J. Chem. 2018, in press, DOI: 10.1039/c8nj00533hI documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
