In the last two decades, the incidence of drug resistant invasive fungi has increased dramatically, in both immunosuppressed and non- immunosuppressed patients [1]. New resistance mechanisms emerge, making common infectious diseases untreatable. Pathogens resistant to standard forms of treatment are becoming a serious threat and novel, effective treatments and ways to specifically deliver them to drug resistant invasive mycoses are being actively sought. One of the biggest obstacles in finding effective, pathogen-specific therapeutics that will not cause severe side-effects in patients arises from the fact that fungi share essential metabolic pathways with humans, many more than with bacteria (both fungi and humans are eukaryotes). In order to design a highly specific antifungal drug, it is crucial to understand and aim at differences in the metabolism of humans and pathogens. Although fungus-selective targets are scarce, there is at least one significant difference between the fungal and mammalian cells: the transport system of zinc. Zinc, the second most abundant transition metal in living organisms (after iron) [2], is also a crucial survival and virulence factor for pathogens. It is present in superoxide dismutases (SODs), central enzymes in bacteria and fungi associated with the detoxification of ROS generated by host cells during host-pathogen interactions [3]. Zinc-binding metalloproteases are involved in pathogen invasion; they are secreted by distinct species of pathogenic fungi such as the ADAM metalloproteinases or deuterolysin [4]. Both the uptake and safe storage of zinc in vacuoles must be strictly controlled. COT1 is a transmembrane protein composed of 199 amino acid residues, located in the vacuolar, mitochondrial and cell membrane. Its main function is the transport of cobalt and zinc ions, and its structure remains unsolved [5]. We used Phyre2 [6] to simulate and analyze the predicted, highly probable structure. It occurs that the most probable Zn(II) binding sites of COT1 are located at the C-terminal region, in the extracellular/cytoplasmic part of the protein (Figure 1). In this work, we try to understand the thermodynamics of Zn(II) binding to COT1. We focus on the FHEHGHSHSHGSGGGGGGSDHSGDSKSHSHSHSHS sequence (residues 131-165) from the C-terminal region of COT1, since it is quite well conserved among zincbinding proteins from fungal species (Fig. 2). We use mass spectrometry to understand the stoichiometry and potentiometric titrations to measure the affinity of binding. In order to establish which part of this sequence is a better Zn(II) binder, we work on three separate peptides: Ac-FHEHGHSHSHGSGGGGGG-NH2, Ac-SHSHSHSHS-NH2 and AcFHEHGHSHSHGSGGGGGGSDHSGDSKSHSHSHSHS -NH2. References: [1] P. Carver et al., Ann. Pharmacother. 2015, 49825-49837. [2] M. Vasak, D. Hasler, Curr. Opin. Chem. Biol. 2000, 4, 177-189. [3] C. Hwang et al., Microbiology 2002, 148, 3705-3711. [4] I. Yike, Mycopathologia 2011, 171, 299-315. [5] D. Conklin, J. McMaster, M. Culbertson, C. Kung, Mol. Cell Biol. 1992, 12, 3678-3688. [6] L. A. Kelley et al., Nature Protocols 2015, 10, 845-858.

COT1 mediated zinc transport in Candida albicans – searching for the metal binding site

Denise BELLOTTI;Maurizio REMELLI
2017

Abstract

In the last two decades, the incidence of drug resistant invasive fungi has increased dramatically, in both immunosuppressed and non- immunosuppressed patients [1]. New resistance mechanisms emerge, making common infectious diseases untreatable. Pathogens resistant to standard forms of treatment are becoming a serious threat and novel, effective treatments and ways to specifically deliver them to drug resistant invasive mycoses are being actively sought. One of the biggest obstacles in finding effective, pathogen-specific therapeutics that will not cause severe side-effects in patients arises from the fact that fungi share essential metabolic pathways with humans, many more than with bacteria (both fungi and humans are eukaryotes). In order to design a highly specific antifungal drug, it is crucial to understand and aim at differences in the metabolism of humans and pathogens. Although fungus-selective targets are scarce, there is at least one significant difference between the fungal and mammalian cells: the transport system of zinc. Zinc, the second most abundant transition metal in living organisms (after iron) [2], is also a crucial survival and virulence factor for pathogens. It is present in superoxide dismutases (SODs), central enzymes in bacteria and fungi associated with the detoxification of ROS generated by host cells during host-pathogen interactions [3]. Zinc-binding metalloproteases are involved in pathogen invasion; they are secreted by distinct species of pathogenic fungi such as the ADAM metalloproteinases or deuterolysin [4]. Both the uptake and safe storage of zinc in vacuoles must be strictly controlled. COT1 is a transmembrane protein composed of 199 amino acid residues, located in the vacuolar, mitochondrial and cell membrane. Its main function is the transport of cobalt and zinc ions, and its structure remains unsolved [5]. We used Phyre2 [6] to simulate and analyze the predicted, highly probable structure. It occurs that the most probable Zn(II) binding sites of COT1 are located at the C-terminal region, in the extracellular/cytoplasmic part of the protein (Figure 1). In this work, we try to understand the thermodynamics of Zn(II) binding to COT1. We focus on the FHEHGHSHSHGSGGGGGGSDHSGDSKSHSHSHSHS sequence (residues 131-165) from the C-terminal region of COT1, since it is quite well conserved among zincbinding proteins from fungal species (Fig. 2). We use mass spectrometry to understand the stoichiometry and potentiometric titrations to measure the affinity of binding. In order to establish which part of this sequence is a better Zn(II) binder, we work on three separate peptides: Ac-FHEHGHSHSHGSGGGGGG-NH2, Ac-SHSHSHSHS-NH2 and AcFHEHGHSHSHGSGGGGGGSDHSGDSKSHSHSHSHS -NH2. References: [1] P. Carver et al., Ann. Pharmacother. 2015, 49825-49837. [2] M. Vasak, D. Hasler, Curr. Opin. Chem. Biol. 2000, 4, 177-189. [3] C. Hwang et al., Microbiology 2002, 148, 3705-3711. [4] I. Yike, Mycopathologia 2011, 171, 299-315. [5] D. Conklin, J. McMaster, M. Culbertson, C. Kung, Mol. Cell Biol. 1992, 12, 3678-3688. [6] L. A. Kelley et al., Nature Protocols 2015, 10, 845-858.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2480167
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