{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T18:23:45Z","timestamp":1787163825029,"version":"build-2736575974"},"reference-count":30,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,28]],"date-time":"2025-12-28T00:00:00Z","timestamp":1766880000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Histatins are histidine-rich antimicrobial peptides present in human saliva, with histatin 5 (Hst5) demonstrating the most potent antifungal activity. Previous studies have linked the antifungal properties of histatins, particularly those against Candida species, to their ability to bind metal ions such as Cu(II) and Zn(II). While the antimicrobial activity of some histatins is well established, the impact of metal ion coordination on this activity remains an area of ongoing investigation. This study focuses on histatin 8 (Hst8), a less-explored member of the histatin family, and compares its metal-binding and antimicrobial properties to those of Hst5. Using isothermal titration microcalorimetry (ITC), we examined the interactions of Hst8 with Cu(II), Zn(II), and Ni(II) ions and evaluated its antimicrobial activity against Escherichia coli, Staphylococcus aureus and two Candida\u00a0albicans strains. Our findings revealed significant differences in copper and zinc binding between Hst5 and Hst8, with both peptides exhibiting distinct antifungal profiles. Interestingly, it has been shown that copper ions bind to Hst5 in a distinctly different manner than to Hst8. Hst5 exhibits two binding sites with dissociation constants (KDITC) of 0.2 \u00b5M and 14.8 \u00b5M, whereas Hst8 has only one set of binding sites with a KDITC of 12.3 \u00b5M. These results highlight the potential role of metal ion coordination in modulating the antimicrobial efficacy of histatins, providing further insight into their therapeutic potential.<\/jats:p>","DOI":"10.3390\/molecules31010110","type":"journal-article","created":{"date-parts":[[2025,12,28]],"date-time":"2025-12-28T23:54:36Z","timestamp":1766966076000},"page":"110","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Histatin 8 Interactions with Copper, Zinc, and Nickel Ions, and Its Antimicrobial Profile in Relation to Histatin 5"],"prefix":"10.3390","volume":"31","author":[{"given":"Justyna","family":"Soko\u0142owska","sequence":"first","affiliation":[{"name":"Institute of Health Sciences, University of Opole, Katowicka 68, 45-060 Opole, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joanna","family":"S\u0142owik","sequence":"additional","affiliation":[{"name":"Institute of Health Sciences, University of Opole, Katowicka 68, 45-060 Opole, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katarzyna","family":"Zam\u0142y\u0144ska","sequence":"additional","affiliation":[{"name":"Department of Genetics and Microbiology, Institute of Biological Sciences, Maria Curie-Sk\u0142odowska University, Akademicka Str. 19, 20-033 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3307-591X","authenticated-orcid":false,"given":"Jolanta","family":"Kutkowska","sequence":"additional","affiliation":[{"name":"Department of Genetics and Microbiology, Institute of Biological Sciences, Maria Curie-Sk\u0142odowska University, Akademicka Str. 19, 20-033 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8515-4428","authenticated-orcid":false,"given":"Pawe\u0142","family":"Lenartowicz","sequence":"additional","affiliation":[{"name":"Institute of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6346-7543","authenticated-orcid":false,"given":"Danuta","family":"Witkowska","sequence":"additional","affiliation":[{"name":"Institute of Health Sciences, University of Opole, Katowicka 68, 45-060 Opole, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1038\/s41573-019-0058-8","article-title":"Antimicrobial host defence peptides: Functions and clinical potential","volume":"19","author":"Mookherjee","year":"2020","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Cort\u00e9s, M., Olate, P., Rodriguez, R., Diaz, R., Mart\u00ednez, A., Hern\u00e1ndez, G., Sepulveda, N., Paz, E.A., and Qui\u00f1ones, J. (2025). Human Microbiome as an Immunoregulatory Axis: Mechanisms, Dysbiosis, and Therapeutic Modulation. Microorganisms, 13.","DOI":"10.3390\/microorganisms13092147"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Zolin, G.V.S., da Fonseca, F.H., Zambom, C.R., and Garrido, S.S. (2021). Histatin 5 metallopeptides and their potential against Candida albicans pathogenicity and drug resistance. Biomolecules, 11.","DOI":"10.3390\/biom11081209"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"9236","DOI":"10.1128\/JVI.00796-06","article-title":"Histatin 5-Derived Peptide with Improved Fungicidal Properties Enhances Human Immunodeficiency Virus Type 1 Replication by Promoting Viral Entry","volume":"80","author":"Groot","year":"2006","journal-title":"J. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2224","DOI":"10.1128\/AAC.41.10.2224","article-title":"Studies of the Mechanism of Human Salivary Histatin-5 Candidacidal Activity with Histatin-5 Variants and Azole-Sensitive And -Resistant Candida Species","volume":"41","author":"Tsai","year":"1997","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"851","DOI":"10.1128\/CDLI.6.6.851-855.1999","article-title":"Further characterization of human salivary anticandidal activities in a human immunodeficiency virus-positive cohort by use of microassays","volume":"6","author":"Lin","year":"1999","journal-title":"Clin. Diagn. Lab. Immunol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"mfad070","DOI":"10.1093\/mtomcs\/mfad070","article-title":"Histatin-5 interacts with cellular copper to promote antifungal activity against Candida albicans","volume":"15","author":"Campbell","year":"2023","journal-title":"Metallomics"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"4244","DOI":"10.1021\/acs.biochem.7b00348","article-title":"Specific Histidine Residues Confer Histatin Peptides with Copper-Dependent Activity Against Candida albicans","volume":"56","author":"Conklin","year":"2017","journal-title":"Biochemistry"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Norris, H.L., Kumar, R., Ong, C.Y., Xu, D., and Edgerton, M. (2020). Zinc binding by histatin 5 promotes fungicidal membrane disruption in C. albicans and C. glabrata. J. Fungi, 6.","DOI":"10.3390\/jof6030124"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1920","DOI":"10.1021\/acsinfecdis.2c00289","article-title":"Zinc Binding Inhibits Cellular Uptake and Antifungal Activity of Histatin-5 in Candida albicans","volume":"8","author":"Campbell","year":"2022","journal-title":"ACS Infect. Dis."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"8663","DOI":"10.1039\/c0dt00137f","article-title":"Competition between histamine-like and poly-imidazole coordination sites for Cu2+ and Zn2+ ions in zebra-fish peptide of prion-like protein","volume":"39","author":"Migliorini","year":"2010","journal-title":"Dalton Trans."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1016\/j.jinorgbio.2010.11.004","article-title":"Cu2+ and Ni2+ interactions with N-terminal fragments of Hpn and Hpn-like proteins from Helicobacter pylori: Unusual impact of poly-Gln sequence on the complex stability","volume":"105","author":"Witkowska","year":"2011","journal-title":"J. Inorg. Biochem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/j.jinorgbio.2011.11.012","article-title":"Coordination of Ni2+ and Cu2+ to metal ion binding domains of E. coli SlyD protein","volume":"107","author":"Witkowska","year":"2012","journal-title":"J. Inorg. Biochem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1007\/s10973-015-4660-7","article-title":"Investigations of copper(II) complexation by fragments of the FBP28 protein using isothermal titration (ITC) and differential scanning calorimetry (DSC)","volume":"121","author":"Makowska","year":"2015","journal-title":"J. Therm. Anal. Calorim."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.ymeth.2014.11.018","article-title":"Fitting two- and three-site binding models to isothermal titration calorimetric data","volume":"76","author":"Brautigam","year":"2015","journal-title":"Methods"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"7561","DOI":"10.1039\/D4DT00565A","article-title":"Impact of metal coordination and pH on the antimicrobial activity of histatin 5 and the products of its hydrolysis","volume":"53","author":"Kola","year":"2024","journal-title":"Dalton Trans."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1736","DOI":"10.1002\/1097-0231(20001015)14:19<1736::AID-RCM86>3.0.CO;2-2","article-title":"Evaluation of the metal binding properties of the histidine-rich antimicrobial peptides histatin 3 and 5 by electrospray ionization mass spectrometry","volume":"14","author":"Brewer","year":"2000","journal-title":"Rapid Commun. Mass Spectrom."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1016\/S0167-4838(00)00265-X","article-title":"Is salivary histatin 5 a metallopeptide?","volume":"1545","author":"Gusman","year":"2001","journal-title":"Biochim. Biophys."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1016\/S0014-5793(01)02157-3","article-title":"Zinc and copper bind to unique sites of histatin 5","volume":"491","author":"Grogan","year":"2001","journal-title":"FEBS Lett."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Cragnell, C., Staby, L., Lenton, S., Kragelund, B.B., and Skep\u00f6, M. (2019). Dynamical oligomerisation of histidine rich intrinsically disordered proteins is regulated through zinc-histidine interactions. Biomolecules, 9.","DOI":"10.3390\/biom9050168"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1111\/febs.12612","article-title":"Histatins: Salivary peptides with copper(II)- and zinc(II)-binding motifs\u2014Perspectives for biomedical applications","volume":"281","author":"Melino","year":"2014","journal-title":"FEBS J."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1220","DOI":"10.1016\/j.jinorgbio.2011.06.002","article-title":"Salivary histatin-5, a physiologically relevant ligand for Ni(II) ions","volume":"105","author":"Kurowska","year":"2011","journal-title":"J. Inorg. Biochem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"15145","DOI":"10.1074\/jbc.M600417200","article-title":"Copper-mediated amyloid-beta toxicity is associated with an intermolecular histidine bridge","volume":"281","author":"Smith","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"20466","DOI":"10.1074\/jbc.M100175200","article-title":"Alzheimer\u2019s Disease Amyloid-\u03b2 Binds Copper and Zinc to Generate an Allosterically Ordered Membrane-penetrating Structure Containing Superoxide Dismutase-like Subunits","volume":"276","author":"Curtain","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Zhang, R., Fan, X., Jiang, X., Zou, M., Xiao, H., and Wu, G. (2020). Multiple Mechanisms of the Synthesized Antimicrobial Peptide TS against Gram-Negative Bacteria for High Efficacy Antibacterial Action In Vivo. Molecules, 26.","DOI":"10.3390\/molecules26010060"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Du, H., Puri, S., McCall, A., Norris, H.L., Russo, T., and Edgerton, M. (2017). Human salivary protein Histatin 5 has potent bactericidal activity against ESKAPE pathogens. Front. Cell. Infect. Microbiol., 7.","DOI":"10.3389\/fcimb.2017.00041"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"85","DOI":"10.5539\/ijb.v5n2p85","article-title":"Investigating the Effects of Hydrophobicity and Charge on the Therapeutic Ability of the Antimicrobial Histatin 8 Peptide for Potential Use in Oral Applications","volume":"5","author":"Matheson","year":"2013","journal-title":"Int. J. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"104238","DOI":"10.1016\/j.micpath.2020.104238","article-title":"Antimicrobial peptides as a promising treatment option against Acinetobacter baumannii infections","volume":"146","author":"Neshani","year":"2020","journal-title":"Microb. Pathog."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Witkowska, D., Szebesczyk, A., W\u0105t\u0142y, J., Braczkowski, M., and Rowi\u0144ska-\u017byrek, M. (2021). A Comparative Study on Nickel Binding to Hpn-like Polypeptides from Two Helicobacter pylori Strains. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms222413210"},{"key":"ref_30","unstructured":"(2012). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically (Standard No. CLSI M07-A9). Available online: www.clsi.org."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/1\/110\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,28]],"date-time":"2025-12-28T23:55:23Z","timestamp":1766966123000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/1\/110"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,28]]},"references-count":30,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["molecules31010110"],"URL":"https:\/\/doi.org\/10.3390\/molecules31010110","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,28]]}}}