Hepatic cytochrome P450 reductase-null mice reveal a second microsomal reductase for squalene monooxygenase

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Abstract

Squalene monooxygenase is a microsomal enzyme that catalyzes the conversion of squalene to 2,3(s)-oxidosqualene, the immediate precursor to lanosterol in the cholesterol biosynthesis pathway. Unlike other flavoprotein monooxygenases that obtain electrons directly from NAD(P)H, squalene monooxygenase requires a redox partner, and for many years it has been assumed that NADPH-cytochrome P450 reductase is this requisite redox partner. However, our studies with hepatic cytochrome P450-reductase-null mice have revealed a second microsomal reductase for squalene monooxygenase. Inhibition studies with antibody to P450 reductase indicate that this second reductase supports up to 40% of the monooxygenase activity that is obtained with microsomes from normal mice. Studies carried out with hepatocytes from CPR-null mice demonstrate that this second reductase is active in whole cells and leads to the accumulation of 24-dihydrolanosterol; this lanosterol metabolite also accumulates in the livers of CPR-null mice, indicating that cholesterol synthesis is blocked at lanosterol demethylase, a cytochrome P450.

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Animals

CPR liver-specific knockout mice (Alb-Cre+/−/Cprlox/lox) were obtained from Drs. Xinxin Ding and Jun Gu at the School of Public Health, State University of New York, Albany. These animals were generated as described [11] by crossing mice expressing liver-specific Cre under control of the albumin promoter (Alb-Cre) [13] with mice bearing a conditional CPR allele (Cprlox/lox) [14]. Littermates lacking the Alb-Cre transgene (Alb-Cre−/−/Cprlox/lox) were designated as wild-type. Two to four month

The cytochrome b5 pathway does not support squalene monooxygenase in mouse liver microsomes

Gene deletion studies revealed that CPR is not essential to ergosterol synthesis in yeast [16], and subsequent studies demonstrated that the three enzymes thought to be dependent on CPR, squalene monooxygenase, lanosterol 14α-demethylase (CYP51) and sterol Δ22-desaturase (CYP61), could be supported efficiently by the microsomal NADH-dependent cytochrome b5 electron transport pathway [8]. To determine if the b5 pathway was similarly effective in mammalian cells, we monitored 2,3-oxidosqualene

Discussion

The selective deletion of hepatic cytochrome P450 reductase results in a marked decrease in plasma cholesterol levels even while lipids accumulate in the liver [[11], [12], and present data]. This contrasting effect on lipids reflects the essential role of CPR in both the synthesis and degradation of cholesterol. While it was anticipated that cholesterol synthesis would be interrupted by the loss of CPR, it was not anticipated that squalene monooxygenase activity would remain partially intact

Acknowledgments

We thank Drs. Xinxin Ding and Jun Gu, Wadsworth Center, New York State Department of Health, for providing the mice and liver tissue used in these studies. Financial support for the generation and maintenance of this mouse line was provided in part by United States Public Health Service Grant ES07462 (to X. Ding) from the NIEHS, National Institutes of Health.

References (32)

Cited by (15)

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    2020, Progress in Lipid Research
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    The electrons required for this process are transferred from NADPH to an SM-bound FAD molecule via an external NADPH-cytochrome P450 reductase, classifying SM as a group E flavin-dependent monooxygenase [96,97]. However, the initial in vitro studies confirming this requirement were challenged by the maintenance of SM activity in hepatic cytochrome P450 reductase-null mice, likely due to the presence of an unidentified microsomal reductase [98]. Several candidate reductases have been proposed but are yet to be verified [99].

  • Neurobehavioral abnormalities in a brain-specific NADPH-cytochrome P450 reductase knockout mouse model

    2012, Neuroscience
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    In the Cyp46a1-null mice, the loss of CYP46A1-mediated cholesterol degradation was compensated by active suppression of cholesterol synthesis, which led to altered homeostasis of metabolic intermediates that are critical for hippocampal long-term potentiation, and learning and memory (Russell et al., 2009). In the brain-Cpr-null mice, Cpr deletion would lead to suppression of the functions of two CPR-dependent enzymes in the cholesterol biosynthesis pathway, lanosterol 14α-demethylase (CYP51) and squalene monooxygenase (Weng et al., 2005; Li and Porter, 2007); the resultant changes in intermediate metabolites, such as 24,25-dihydrolanosterol, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, are expected to differ, at least to some extent, from those occurring in the Cyp46a1-null mice. Further studies to clarify the impact of CPR loss on the cholesterol synthesis pathway in the brain are needed in order to understand the behavioral consequences.

  • Potential biological functions of cytochrome P450 reductase-dependent enzymes in small intestine: Novel link to expression of major histocompatibility complex class II genes

    2012, Journal of Biological Chemistry
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    We hypothesized that the induction of expression of multiple genes involved in cholesterol synthesis in the enterocytes of the IE-Cpr-null mice was in response to a blockage of de novo cholesterol biosynthesis that would result from a functional loss of the POR-dependent enzyme CYP51 (9, 10). To confirm the absence of cholesterol synthesis in the enterocytes of the IE-Cpr-null mice, we measured the levels of 24-DHL (a metabolite of the cholesterol precursor, lanosterol), which has been reported to accumulate in the liver of the liver-Cpr-null mice (18). As shown in Fig. 1, 24-DHL, which was readily analyzed as its trimethylsilyl derivative (24-DHL-TMS) by using GC/MS, was not detected in enterocytes from WT mice, but it was abundant in enterocytes of IE-Cpr-null mice.

  • 7-Dehydrocholesterol reductase activity is independent of cytochrome P450 reductase

    2011, Journal of Steroid Biochemistry and Molecular Biology
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    A subsequent study with rat liver microsomes [4] presented several lines of evidence that DHCR7 required NADPH-cytochrome P450 reductase (POR) for activity; this included a marked inhibition of DHCR7 activity in microsomes by addition of antibody to POR, as well as a reconstitution of DHCR7 activity by addition of purified POR to protease-treated microsomes and to partially purified DHCR7 preparations. Although NADPH-cytochrome P450 reductase is more widely recognized for its role in the reduction of cytochrome P450 in the oxidation of xenobiotics and steroids, POR is required for cholesterol synthesis, where it is the requisite electron donor to lanosterol demethylase (CYP51A1) [5] and the principal, but not exclusive, electron donor to squalene monooxygenase [6]. POR is ubiquitously expressed in mammalian cells and also in yeast.

  • Defining the in vivo role for cytochrome b<inf>5</inf> in cytochrome P450 function through the conditional hepatic deletion of microsomal cytochrome b<inf>5</inf>

    2008, Journal of Biological Chemistry
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    In order to confirm that this enzyme was not induced in the HBN mice, and potentially compensating for the loss of microsomal cytochrome b5, we obtained antiserum to NAD(P)H cytochrome b5 oxidoreductase; immunoblotting liver samples from wild-type and HBN mice found no difference in expression levels (data not shown). Li and Porter (48) have also described a second microsomal reductase, which is active with squalene monooxygenase and apparently capable of supporting up to 40% of the activity of this enzyme, although the identity of this reductase and its interactions with other enzymes remain unclear. In addition, due to its role in fatty acid desaturation, it is possible that changes in the phospholipid bilayer, as observed in stearoyl-CoA-desaturase null mice (49), are responsible for the observed effects of cytochrome b5 on P450 metabolism.

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