{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T05:01:02Z","timestamp":1790226062680,"version":"4.1.0"},"reference-count":51,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T00:00:00Z","timestamp":1790035200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Medical University of Bialystok","award":["B.SUB.26.351\/01"],"award-info":[{"award-number":["B.SUB.26.351\/01"]}],"id":[{"id":"https:\/\/ror.org\/00y4ya841","id-type":"ROR","asserted-by":"crossref"}]},{"award":["B.SUB.26.351\/01"],"award-info":[{"award-number":["B.SUB.26.351\/01"]}],"id":[{"id":"https:\/\/ror.org\/00y4ya841","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Prolidase (PEPD) is a metalloproteinase that hydrolyzes imidodipeptides containing proline or hydroxyproline at the C-terminus and also participates in the non-catalytic regulation of cellular stress responses and p53 availability. Here, we characterized population-level responses associated with transient CRISPR\/Cas9-directed targeting of PEPD in heterogeneous MCF7 breast cancer cultures using three independent sgRNAs. PEPD enzymatic activity was quantified in three independent biological experiments, whereas PEPD and p53\/apoptosis-related proteins were examined by representative Western blotting and exploratory immunofluorescence microscopy. All three PEPD-targeting constructs were associated with reduced population-average PEPD activity, and representative immunoblots showed directionally concordant reductions in PEPD protein signal. Representative protein analyses additionally revealed a qualitative pattern involving p53, MDM2, cleaved caspase-9, cleaved caspase-7, and cleaved PARP-1. These protein observations were not supported by independent biological replication suitable for inferential analysis and are therefore interpreted descriptively. Editing of the endogenous PEPD locus was not directly confirmed or quantified by genomic analysis. During prolonged puromycin selection, the persistence of adherent PEPD-targeted cells decreased under the conditions tested; however, this experiment was qualitative and was not designed as a formal viability or rescue assay. Collectively, the data demonstrate a replicated population-level reduction in PEPD enzymatic activity following PEPD-directed CRISPR\/Cas9 perturbation and identify an associated exploratory p53\/MDM2- and apoptosis-related protein pattern. A contribution of Cas9-mediated DNA-damage signaling to the latter cannot be excluded.<\/jats:p>","DOI":"10.3390\/molecules31193360","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T08:17:54Z","timestamp":1790065074000},"page":"3360","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["CRISPR\/Cas9-Directed PEPD Targeting Is Associated with Reduced PEPD Activity and an Exploratory p53- and Apoptosis-Related Protein Pattern in MCF7 Breast Cancer Cells"],"prefix":"10.3390","volume":"31","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0225-9740","authenticated-orcid":false,"given":"Magda","family":"Chalecka","sequence":"first","affiliation":[{"name":"Department of Medicinal Chemistry, Faculty of Pharmacy, Medical University of Bialystok, Mickiewicza 2D, 15-222 Bialystok, Poland"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-4052-203X","authenticated-orcid":false,"given":"Dominika","family":"Stencel","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia in Katowice, Jagiello\u0144ska 4, 41-200 Sosnowiec, Poland"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"formal-analysis","vocabulary":"credit"}]},{"given":"Jerzy","family":"Palka","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, Faculty of Pharmacy, Medical University of Bialystok, Mickiewicza 2D, 15-222 Bialystok, Poland"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4475-873X","authenticated-orcid":false,"given":"Arkadiusz","family":"Surazynski","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, Faculty of Pharmacy, Medical University of Bialystok, Mickiewicza 2D, 15-222 Bialystok, Poland"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/j.biochi.2020.09.017","article-title":"Prolidase\u2014A Protein with Many Faces","volume":"183","author":"Wilk","year":"2021","journal-title":"Biochimie"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"731","DOI":"10.1007\/s00726-008-0051-8","article-title":"Prolidase-Dependent Regulation of Collagen Biosynthesis","volume":"35","author":"Surazynski","year":"2008","journal-title":"Amino Acids"},{"key":"ref_3","first-page":"497","article-title":"Platelet-Rich Plasma Counteracts Interleukin-1 Induced Inhibition of Collagen Biosynthesis through Recovery of Impaired \u03921-Integrin Signaling and Prolidase Activity in Human Skin Fibroblasts","volume":"79","author":"Guszczyn","year":"2022","journal-title":"Acta Pol. 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