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The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity.

ABSTRACT: The activity of the TRPM7 channel is negatively regulated by intracellular Mg2+. We previously reported that oxidative stress enhances the inhibition of TRPM7 by intracellular Mg2+. Here, we aimed to clarify the mechanism underlying TRPM7 inhibition by hydrogen peroxide (H2O2). Site-directed mutagenesis of full-length TRPM7 revealed that none of the cysteines other than C1809 and C1813 within the zinc-binding motif of the TRPM7 kinase domain were involved in the H2O2-induced TRPM7 inhibition. Mutation of C1809 or C1813 prevented expression of full-length TRPM7 on the plasma membrane. We therefore developed an assay to functionally reconstitute full-length TRPM7 by coexpressing the TRPM7 channel domain (M7cd) and the TRPM7 kinase domain (M7kd) as separate proteins in HEK293 cells. When M7cd was expressed alone, the current was inhibited by intracellular Mg2+ more strongly than that of full-length TRPM7 and was insensitive to oxidative stress. Coexpression of M7cd and M7kd attenuated the inhibition by intracellular Mg2+ and restored sensitivity to oxidative stress, indicating successful reconstitution of a full-length TRPM7-like current. We observed a similar effect when M7cd was coexpressed with the kinase-inactive mutant M7kd-K1645R, suggesting that the kinase activity is not essential for the reconstitution. However, coexpression of M7cd and M7kd carrying a mutation at either C1809 or C1813 failed to restore the full-length TRPM7-like current. No reconstitution was observed when using M7kd carrying a mutation at H1750 and H1807, which are involved in the zinc-binding motif formation with C1809 and C1813. These data suggest that the zinc-binding motif is essential for the intracellular Mg2+-dependent regulation of the TRPM7 channel activity by its kinase domain and that the cysteines in the zinc-binding motif play a role in the oxidative stress response of TRPM7.

SUBMITTER: Inoue H

PROVIDER: S-EPMC8129778 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Oxidative stress has emerged as an essential factor in the pathogenesis of intestinal ischemia/reperfusion (I/R) injury. The adaptor protein p66Shc is a key regulator of reactive oxygen species (ROS) generation and a mediator of I/R damage in the intestine, but the upstream mechanisms that directly regulate p66Shc expression during intestinal I/R remain largely unknown. Recent studies have suggested that noncoding RNAs, such as circular RNAs (circRNAs), are important players in physiological and pathological processes based on their versatile regulatory roles in gene expression. The aim of this study was to elucidate the contribution of p66Shc to oxidative damage in intestinal I/R and to investigate the regulation of p66Shc by circRNA sponges. Methods: Intestinal I/R was induced in mice via superior mesenteric artery (SMA) occlusion. A miR-339-5p agomir or circ-protein kinase C beta (PRKCB) siRNA was injected intravenously before I/R challenge. In addition, Caco-2 cells were subjected to hypoxia/reoxygenation (H/R) in vitro to simulate an in vivo I/R model. Results: In vitro, p66Shc deficiency significantly reduced H/R-induced ROS overproduction by attenuating mitochondrial superoxide anion (O2 -) levels, suppressing NADPH oxidase activity and enhancing antioxidant enzyme expression. Moreover, miR-339-5p was identified to directly regulate p66Shc expression in the intestine. Furthermore, we found that a circRNA transcribed from the PRKCB gene, named circ-PRKCB, acted as an endogenous miR-339-5p sponge to regulate p66Shc expression. circ-PRKCB silencing or miR-339-5p overexpression significantly downregulated p66Shc expression and attenuated oxidative stress levels and I/R injury in vivo and in vitro. Notably, the increased circ-PRKCB levels and decreased miR-339-5p levels associated with murine intestinal I/R were consistent with those in patients with intestinal infarction. Conclusions: Our findings reveal a crucial role for the circ-PRKCB/miR-339-5p/p66Shc signaling pathway in regulating oxidative stress in the I/R intestine. This pathway may be a potential therapeutic target for intestinal I/R injury.

Project description:Objective: To determine the oxidative stress during cycles of chemotherapy in patients after surgery for colorectal cancer, with or without oral zinc supplementation. Subjects: Twenty four adults from both genders participated in this study. All patients underwent stage II, III or IV colorectal cancer surgical resection and were starting chemotherapy in HCFMRP- USP. Patients were randomized into two groups. The first one (QTx-Zn Group, n=10) received 70 mg/d of zinc orally and the second one received placebo (QTx-Placebo Group, n=14) for 16 weeks. The study also included 30 healthy volunteers matched for age, gender and socioeconomic status, who received 70 mg/d of zinc supplement (Control-Zn Group, n=21) or placebo (Control-Placebo Group, n=9) for 16 weeks. Methods: The questionnaires about dietary intake (semiquantitative food frequency and food record), fatigue and quality of life (FACIT-F) and questionnaires that assess the side effects of chemotherapy (CTCAE) were evaluated. Anthropometry and bioelectrical impedance measurements were made. Blood collection was performed before the 1st, 2nd, 3rd and 4th cycles of chemotherapy (median duration of 21 days among cicles). Routine laboratory tests, vitamin E and markers anti and pro-oxidants (MDA, SOD, GPx and isoprostane) ere determined. The control group underwent the same procedures, except for chemotherapy. A longitudinal linear mixed effects model was adjusted for each of the variables of interest. The models were fitted using PROC MIXED of SAS version 9 (SAS, CARY, NC, USA). To analyze the association of categorical variables in the different items of the CTCAE, the investigators used the Fisher exact test. Results: The oral zinc supplementation was sufficient to increase plasma levels of zinc and did not alter food intake, body composition and routine laboratory evaluation of patients undergoing chemotherapy for colorectal cancer. Compared with QTx-Placebo Group, QTx-Zn Group showed lower prevalence of complaint on the salivary gland (17 vs. 75%). Fatigue (43 ± 6 vs. 36 ± 13) and quality of life (126 ± 160 vs. 116 ± 27) has become worst in the period between the 1st and 4th cycles of QTx in QTx-Placebo Group. When compared with QTx-Placebo Group, QTx-Zn Group had higher values of SOD before the 1st (2297 ± 503 vs. 1604 ± 352 USOD/g Hb), 2nd (2037 ± 515 vs. 1712 ± 417 USOD/g Hb) and 4th (2202 ± 323 vs. 1821 ± 360 USOD/g Hb) cycles of QTx. GPx values decreased in QTx-Zn Group before the 3rd cycle of QTx (48.5 ± 7.0 vs. 54.3 ± 2.3 mol NADPH/min/gHb). Conclusions: These data suggest that zinc supplementation reduces complaints related to the change in salivary gland, preserving the quality of life and preventing the worsening of fatigue. The increase in SOD can be attributed to zinc supplementation per se, whereas this mineral is a cofactor that endogenous antioxidant enzyme. The highest activity of SOD increases the production of H2O2, whose detoxification involves the participation of GPx, justifying its reduction. There were no changes in plasma levels of vitamin E, MDA and isoprostane during the study period. Considering the values of MDA and isoprostane, the data indicate that regardless of zinc supplementation, the lipid peroxidation of the cell membrane was unchanged during chemotherapy.

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The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity.

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