Synthesis and antiproliferative activity of novel limonene derivatives with a substituted thiourea moiety

A series of R-(+)-limonene derivatives bearing a substituted thiourea moiety (3-13) and five S-methyl analogs (14-18) were synthesized and evaluated for their in vitro antiproliferative activity against human cancer cell lines. Compounds bearing aromatic substituents (3-6) exhibit cytotastic activity in the full panel of cell lines tested, with GI50 values in the range of 2.5 to 24 µmol L-1. Compounds 3, 10, 12 and 16 were the most active with GI50 values in the range of 0.41 to 3.0 µmol L-1, against different cell lines.No presente trabalho descrevemos a síntese e a avaliação da atividade antiproliferativa, frente a linhagens de células tumorais humanas, de derivados do R-(+)-limoneno (3-18) contendo uma unidade tiouréia substituída. Os derivados com substituintes arílicos (3-6) exibiram atividade citostática frente a todas linhagens testadas, com inibição de 50% do crescimento celular (GI50) em concentrações na faixa de 2,5 a 24 µmol L-1. Os compostos 3, 10, 12 e 16 foram os mais ativos, com GI50 na faixa de 0,41 a 3,0 mmol L-1, frente a diferentes linhagens celulares.954960Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Mitochondrial O-GlcNAc Transferase (mOGT) Regulates Mitochondrial Structure, Function, and Survival in HeLa Cells

O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH: ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survivals iRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined thatmOGTis probably involved in the glycosylation of a restricted set of mitochondrial targets. Weidentified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, includ-ing leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics

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Dual knockdown of MK-STYX and PTPMT1 is sufficient to resensitize MK-STYX knockdown cells to chemotherapeutic treatment.

<p>(A) HeLa cells were plated on an electrode-containing plate and viability was measured using the xCELLigence system. Cells were transfected for 30 hours and incubated in the presence of 50 nM paclitaxel or a vehicle-only control for an additional 24 hours. Mean cell viability values following both drug (dark gray bars) and vehicle (light gray bars) treatments are shown. Bars represent standard deviation of at least triplicate measurements. Unpaired, two-tailed t-tests: **p≤0.01; ***p≤0.001. (B–E) HeLa cells were transfected with control (B–D, gray circles), MK-STYX (B, blue circles), PTPMT1 (C, green circles), or MK-STYX+PTPMT1 siRNAs (D, red circles) for 30 hours before being treated with a dose response of paclitaxel for an additional 24 hours. Mean EC₅₀ values were determined for each condition using GraphPad PRISM (plotted in E; indicated on each plot). Bars represent standard deviation of four measurements per condition. Unpaired, two-tailed t-tests: ***p≤0.001.</p

MK-STYX knockdown fails to protect cells from the loss of viability caused by PTPMT1 depletion.

<p>(<b>A–B</b>) HeLa cells were transfected with a pool of two unique MK-STYX siRNAs (A), a pool of two unique PTPMT1 siRNAs (B), or a non-targeting control siRNA (A and B) for 30 hours. Relative knockdown was assessed via RT-PCR. Values represent mean mRNA levels normalized to control siRNA-treated cells using HPRT1 as a reference gene. Error bars represent standard deviation. (<b>C</b>) HeLa cells were transfected with control, MK-STYX, PTPMT1, or MK-STYX + PTPMT1 siRNAs for 24 hours before cellular viability was assayed in real-time using a xCELLigence plate reader.</p

MK-STYX interacts with PTPMT1.

<p>(A) TAP-tagged MK-STYX was expressed and immunopurified from HeLa cells. Endogenous interaction partners were identified by LC-MS/MS and were bioinformatically filtered against a large control dataset (∼350 non-MK-STYX TAP tag experiments) to identify unique and significant interactions with MK-STYX. The gene name of each protein identified is listed in order of significance (the most significant hits listed at the top). A p value (demonstrating the significance of each interaction partner), and interactor score (calculated based on the p value, number of replicates in which the interactor was identified, uniqueness of the MK-STYX interaction relative to the control interactions, and MASCOT scores and total coverage of the peptides) is shown for each interactor (lower p-value and/or higher interactor score correlates with a stronger molecular interaction with MK-STYX). (<b>B</b>) The mitochondrial localization of top MK-STYX interaction partners is shown (gray rectangle: bait; white circle: interaction partner; line: interaction). (<b>C</b>) V5-MK-STYX and FLAG-PTPMT1 (wildtype) or FLAG-PTPMT1 C132S (a catalytically inactive mutant) were transfected into 293FT cells and FLAG immunoprecipitated. Input lysates or immunoprecipitates were immunoblotted with the indicated antibodies. The * indicates light chain of the antibody used to immunoprecipitate the protein.</p