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Excess Nitric Oxide Activates TRPV1-Ca(2+)-Calpain Signaling and Promotes PEST-dependent Degradation of Liver X Receptor ?.

ABSTRACT: Excess nitric oxide (NO) deregulates cholesterol metabolism in macrophage foam cells, yet the underlying molecular mechanism is incompletely understood. To investigate the mechanism, we found that in macrophages, treatment with NO donors S-nitroso-N-acetyl-D,L-penicillamine (SNAP) or diethylenetriamine/nitric oxide induced LXR? degradation and reduced the expression of the downstream target of LXR?, ATP-binding cassette transporter A1 (ABCA1), and cholesterol efflux. In addition, SNAP induced calcium (Ca(2+)) influx into cells, increased calpain activity and promoted the formation of calpain-LXR? complex. Pharmacological inhibition of calpain activity reversed the SNAP-induced degradation of LXR?, down-regulation of ABCA1 and impairment of cholesterol efflux in macrophages. SNAP increased the formation of calpain-LXR? complex in a Pro-Glu-Ser-Thr (PEST) motif-dependent manner. Truncation of the PEST motif in LXR? abolished the calpain-dependent proteolysis. Removal of extracellular Ca(2+) by EGTA or pharmacological inhibition of TRPV1 channel activity diminished SNAP-induced increase in intracellular Ca(2+), calpain activation, LXR? degradation, ABCA1 down-regulation and impaired cholesterol efflux. In conclusion, excess NO may activate calpain via TRPV1-Ca(2+) signaling and promote the recognition of calpain in the PEST motif of LXR?, thereby leading to degradation of LXR? and, ultimately, downregulated ABCA1 expression and impaired ABCA1-dependent cholesterol efflux in macrophages.

SUBMITTER: Zhao JF

PROVIDER: S-EPMC4679395 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Excess Nitric Oxide Activates TRPV1-Ca(2+)-Calpain Signaling and Promotes PEST-dependent Degradation of Liver X Receptor α.

Zhao Jin-Feng JF Shyue Song-Kun SK Lee Tzong-Shyuan TS

International journal of biological sciences 20160101 1

Excess nitric oxide (NO) deregulates cholesterol metabolism in macrophage foam cells, yet the underlying molecular mechanism is incompletely understood. To investigate the mechanism, we found that in macrophages, treatment with NO donors S-nitroso-N-acetyl-D,L-penicillamine (SNAP) or diethylenetriamine/nitric oxide induced LXRα degradation and reduced the expression of the downstream target of LXRα, ATP-binding cassette transporter A1 (ABCA1), and cholesterol efflux. In addition, SNAP induced ca ...[more]

PMID: 26722214

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Project description:Human cytochrome P450 (P450) CYP2B6 undergoes nitric oxide (NO)-dependent proteasomal degradation in response to the NO donor dipropylenetriamine NONOate (DPTA) and biologic NO in HeLa and HuH7 cell lines. CYP2B6 is also downregulated by NO in primary human hepatocytes. We hypothesized that NO or derivative reactive nitrogen species may generate adducts of tyrosine and/or cysteine residues, causing CYP2B6 downregulation, and selected Tyr and Cys residues for mutation based on predicted solvent accessibility. CYP2B6V5-Y317A, -Y380A, and -Y190A mutant proteins expressed in HuH7 cells were less sensitive than wild-type (WT) enzyme to degradation evoked by DPTA, suggesting that these tyrosines are targets for NO-dependent downregulation. The Y317A or Y380A mutants did not show increases in high molecular mass (HMM) species after treatment with DPTA or bortezomib + DPTA, in contrast to the WT enzyme. Carbon monoxide-releasing molecule 2 treatment caused rapid suppression of 2B6 enzyme activity, significant HMM species generation, and ubiquitination of CYP2B6 protein but did not stimulate CYP2B6 degradation. The CYP2B6 inhibitor 4-(4-chlorophenyl)imidazole blocked NO-dependent CYP2B6 degradation, suggesting that NO access to the active site is important. Molecular dynamics simulations predicted that tyrosine nitrations of CYP2B6 would cause significant destabilizing perturbations of secondary structure and remove correlated motions likely required for enzyme function. We propose that cumulative nitrations of Y190, Y317, and Y380 by reactive nitrogen species cause destabilization of CYP2B6, which may act synergistically with heme nitrosylation to target the enzyme for degradation. SIGNIFICANCE STATEMENT: This work provides novel insight into the mechanisms by which nitric oxide, which is produced in hepatocytes in response to inflammation, triggers the ubiquitin-dependent proteasomal degradation of the cytochrome P450 (P450) enzyme CYP2B6. Our data demonstrate that both nitration of specific tyrosine residues and interaction of nitric oxide (NO) with the P450 heme are necessary for NO to trigger ubiquitination and protein degradation.

Dataset Information

Excess Nitric Oxide Activates TRPV1-Ca(2+)-Calpain Signaling and Promotes PEST-dependent Degradation of Liver X Receptor ?.

Publications

Excess Nitric Oxide Activates TRPV1-Ca(2+)-Calpain Signaling and Promotes PEST-dependent Degradation of Liver X Receptor α.

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