Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element–binding protein family of transcription factors. ATF5 regulates stress responses and cell survival, proliferation, and differentiation and also plays a role in viral infections, cancer, diabetes, schizophrenia, and the olfactory system. Moreover, it was found to also have a critical cell cycle–dependent structural function at the centrosome. However, the mechanism that controls the localization of ATF5 at the centrosome is unclear. Here we report that ATF5 is small ubiquitin-like modifier (SUMO) 2/3–modified at a conserved SUMO-targeting consensus site in various types of mammalian cells. We found that SUMOylation of ATF5 is elevated in the G₁ phase of the cell cycle and diminished in the G₂/M phase. ATF5 SUMOylation disrupted the interaction of ATF5 with several centrosomal proteins and dislodged ATF5 from the centrosome at the end of the M phase. Of note, blockade of ATF5 SUMOylation deregulated the centrosome cycle, impeded ATF5 translocation from the centrosome, and caused genomic instability and G₂/M arrest in HeLa cells. Our results indicate that ATF5 SUMOylation is an essential mechanism that regulates ATF5 localization and function at the centrosome.

In this study, to better understand the mechanisms of the profound impact of alcohol consumption on drug pharmacokinetics, efficacy, and toxicity, we characterized the alcohol-induced changes in the ensemble of drug-metabolizing enzymes and transporters (DMETs) in the human liver by performing global proteomic analysis of human liver microsomes from 94 donors. DMET protein levels were analyzed concerning alcohol consumption, smoking history, and sex using non-parametric tests, which were further strengthened by correlational analysis. To this end, we used a provisional index of alcohol exposure formulated based on the relative abundances of four marker proteins best correlating with the level of alcohol consumption. Alcohol-induced changes in the cytochrome P450 pool include significant increases in CYP2E1, CYP2B6, CYP2J2, and NADPH-cytochrome P450 reductase levels and the lowering of CYP1A2, CYP2C8, CYP2C9, CYP4A11, and cytochrome b₅. Changes in UDP-glucuronosyltransferase (UGT) abundances comprise elevated UGT1A6, UGT1A9, and UGT2A1, and reduced UGT1A3, UGT1A4, UGT2B7, UGT2B10, and UGT2B15 levels. Tobacco smokers showed elevated CYP1A2, UGT1A6, and UGT2B4 and reduced FMO3, FMO4, and FMO5 levels, while in females, CYP1A2, UGT2B17, and UGT2B15 levels were lower, and UGT2A3 and STS were higher compared to males. The alcohol-induced changes in the DMET ensemble at the protein level reported herein provide deep insights into how alcohol impacts drug and xenobiotic metabolism.

In a search for a reliable, inexpensive, and versatile technique for high-throughput kinetic assays of drug metabolism, we elected to rehire an old-school approach based on the determination of formaldehyde (FA) formed in cytochrome P450-dependent demethylation reactions. After evaluating several fluorometric techniques for FA detection, we chose the method based on the Hantzsch reaction with acetoacetanilide as the most sensitive, robust, and adaptable to high-throughput implementation. Here we provide a detailed protocol for using our new technique for automatized assays of cytochrome P450-dependent drug demethylations and discuss its applicability for high-throughput scanning of drug metabolism pathways in the human liver. To probe our method further, we applied it to re-evaluating the pathways of metabolism of ketamine, a dissociative anesthetic and potent antidepressant increasingly used in the treatment of alcohol withdrawal syndrome. Probing the kinetic parameters of ketamine demethylation by ten major cytochrome P450 (CYP) enzymes, we demonstrate that in addition to CYP2B6 and CYP3A enzymes, which were initially recognized as the primary metabolizers of ketamine, an important role is also played by CYP2C19 and CYP2D6. At the same time, the involvement of CYP2C9 suggested in the previous reports was deemed insignificant.

Cellular stress response mechanisms normally function to enhance survival and allow for cells to return to homeostasis following an adverse event. Cancer cells often co-opt these same mechanisms as a means to evade apoptosis and mitigate a state of constant cellular stress. Activating transcription factor 5 (ATF5) is upregulated under diverse stress conditions and is overexpressed in a variety of cancers. It was demonstrated ATF5 is a survival factor in transformed, but not normal cells. However, the regulation of ATF5 is not fully understood. The purpose of the present study was to investigate miRNA regulation at the 3’ untranslated region (UTR) of ATF5, with the goal of demonstrating a reversal of the upregulation of ATF5 induced under diverse cellular stress in cancer cells. A multifactorial approach using in silico analysis was employed to identify miRNAs 433-3p, 520b-3p, and 129-5p as potential regulators of ATF5, based on their predicted binding sites over the span of the ATF5 3’ UTR. Luciferase reporter assay data validated all three miRNA candidates by demonstrating direct binding to the target ATF5 3’. However, functional studies revealed miR-520b-3p as the sole candidate able to reverse the upregulation of ATF5 protein under diverse cellular stress. Additionally, miR-520b-3p levels were inversely related to ATF5 mRNA under endoplasmic reticulum stress and amino acid deprivation. This is the first evidence that regulation at the 3’ UTR is involved in modulating ATF5 levels under cellular stress and suggests an important role for miRNA-520b-3p in the regulation of ATF5.