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Small Heterodimer Partner Regulates Circadian Cytochromes p450 and Drug-Induced Hepatotoxicity.

ABSTRACT: The role of small heterodimer partner (SHP) in regulation of xenobiotic detoxification remains elusive. Here, we uncover a critical role for SHP in circadian regulation of cytochromes P450 (CYPs) and drug-induced hepatotoxicity. Methods: The mRNA and protein levels of CYPs in the livers of wild-type and SHP-/- mice were measured by quantitative real-time polymerase chain reaction and Western blotting, respectively. Regulation of CYP by SHP was investigated using luciferase reporter, mobility shift, chromatin immunoprecipitation, and/or co-immunoprecipitation assays. Results: The circadian rhythmicities of xenobiotic-detoxifying CYP mRNAs and proteins were disrupted in SHP-deficient mice. Of note, SHP ablation up-regulated Cyp2c38 and Cyp2c39, whereas it down-regulated all other CYP genes. Moreover, SHP regulated the expression of CYP genes through different mechanisms. SHP repressed Lrh-1/Hnf4α to down-regulate Cyp2c38, E4bp4 to up-regulate Cyp2a5, Dec2/HNF1α axis to up-regulate Cyp1a2, Cyp2e1 and Cyp3a11, and Rev-erbα to up-regulate Cyp2b10, Cyp4a10 and Cyp4a14. Furthermore, SHP ablation sensitized mice to theophylline (or mitoxantrone)-induced toxicity. Higher level of toxicity was correlated with down-regulated metabolism and clearance of theophylline (or mitoxantrone). In contrast, SHP ablation blunted the circadian rhythmicity of acetaminophen-induced hepatotoxicity and alleviated the toxicity by down-regulating Cyp2e1-mediated metabolism and reducing formation of the toxic metabolite. Toxicity alleviation by SHP ablation was also observed for aflatoxin B1 due to reduced formation of the toxic epoxide metabolite. Conclusion: SHP participates in circadian regulation of CYP enzymes, thereby impacting xenobiotic metabolism and drug-induced hepatotoxicity.

SUBMITTER: Zhang T

PROVIDER: S-EPMC6276094 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:This Account summarizes recent findings centered on the role that redox partner binding, allostery, and conformational dynamics plays in cytochrome P450 proton coupled electron transfer. P450s are one of Nature's largest enzyme families and it is not uncommon to find a P450 wherever substrate oxidation is required in the formation of essential molecules critical to the life of the organism or in xenobiotic detoxification. P450s can operate on a remarkably large range of substrates from the very small to the very large, yet the overall P450 three-dimensional structure is conserved. Given this conservation of structure, it is generally assumed that the basic catalytic mechanism is conserved. In nearly all P450s, the O2 O-O bond must be cleaved heterolytically enabling one oxygen atom, the distal oxygen, to depart as water and leave behind a heme iron-linked O atom as the powerful oxidant that is used to activate the nearby substrate. For this process to proceed efficiently, externally supplied electrons and protons are required. Two protons must be added to the departing O atom while an electron is transferred from a redox partner that typically contains either a Fe2S2 or FMN redox center. The paradigm P450 used to unravel the details of these mechanisms has been the bacterial CYP101A1 or P450cam. P450cam is specific for its own Fe2S2 redox partner, putidaredoxin or Pdx, and it has long been postulated that Pdx plays an effector/allosteric role by possibly switching P450cam to an active conformation. Crystal structures, spectroscopic data, and direct binding experiments of the P450cam-Pdx complex provide some answers. Pdx shifts the conformation of P450cam to a more open state, a transition that is postulated to trigger the proton relay network required for O2 activation. An essential part of this proton relay network is a highly conserved Asp (sometimes Glu) that is known to be critical for activity in a number of P450s. How this Asp and proton delivery networks are connected to redox partner binding is quite simple. In the closed state, this Asp is tied down by salt bridges, but these salt bridges are ruptured when Pdx binds, leaving the Asp free to serve its role in proton transfer. An alternative hypothesis suggests that a specific proton relay network is not really necessary. In this scenario, the Asp plays a structural role in the open/close transition and merely opening the active site access channel is sufficient to enable solvent protons in for O2 protonation. Experiments designed to test these various hypotheses have revealed some surprises in both P450cam and other bacterial P450s. Molecular dynamics and crystallography show that P450cam can undergo rather significant conformational gymnastics that result in a large restructuring of the active site requiring multiple cis/trans proline isomerizations. It also has been found that X-ray driven substrate hydroxylation is a useful tool for better understanding the role that the essential Asp and surrounding residues play in catalysis. Here we summarize these recent results which provide a much more dynamic picture of P450 catalysis.

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Small Heterodimer Partner Regulates Circadian Cytochromes p450 and Drug-Induced Hepatotoxicity.

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