At the site of microbial infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments in which both the pathogen and host cells must survive. Currently, whether hypoxia adaptation is an important virulence attribute of opportunistic pathogenic molds is unknown. Here we report the characterization of a sterol-regulatory element binding protein, SrbA, in the opportunistic pathogenic mold, Aspergillus fumigatus. Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment and consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA). Transcriptional profiling revealed 87 genes that are affected by loss of SrbA function. Annotation of these genes implicated SrbA in maintaining sterol biosynthesis and hyphal morphology. Further examination of the SrbA null mutant consequently revealed that SrbA plays a critical role in ergosterol biosynthesis, resistance to the azole class of antifungal drugs, and in maintenance of cell polarity in A. fumigatus. Significantly, the SrbA null mutant was highly susceptible to fluconazole and voriconazole. Thus, these findings present a new function of SREBP proteins in filamentous fungi, and demonstrate for the first time that hypoxia adaptation is likely an important virulence attribute of pathogenic molds.

Cytochrome P450 enzymes are heme-dependent monoxygenases that play a central role in human physiology. Despite the numerous physiological processes that P450 enzymes impact, the electron donors P450 oxidoreductase and cytochrome b5 are the only proteins known to interact with and modulate the activity of ER microsomal P450s. Here, we report that Dap1/PGRMC1 is required for ER P450 function in yeast and humans. We show that S. pombe Dap1 is a hemoprotein that binds and positively regulates Cyp51A1 and Cyp61A1, two P450s required for sterol biosynthesis. Similarly, loss of human PGRMC1 reduces activity of Cyp51A1, blocking cholesterol synthesis and increasing production of toxic sterol intermediates. PGRMC1 stably binds Cyp51A1 and human P450s from three additional families including Cyp3A4, which metabolizes pharmaceutical compounds. These findings demonstrate that PGRMC1 is required for P450 activity and suggest that interindividual variation in PGRMC1 function may impact multiple biochemical pathways and drug metabolism.

We have cloned the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene. The sterol methyl oxidase performs the first of three enzymic steps required to remove the two C-4 methyl groups leading to cholesterol (animal), ergosterol (fungal), and stigmasterol (plant) biosynthesis. An ergosterol auxotroph, erg25, which fails to demethylate and concomitantly accumulates 4,4-dimethylzymosterol, was isolated after mutagenesis. A complementing clone consisting of a 1.35-kb Dra I fragment encoded a 309-amino acid polypeptide (calculated molecular mass, 36.48 kDa). The amino acid sequence shows a C-terminal endoplasmic reticulum retrieval signal KKXX and three histidine-rich clusters found in eukaryotic membrane desaturases and in a bacterial alkane hydroxylase and xylene monooxygenase. The sterol profile of an ERG25 disruptant was consistent with the erg25 allele obtained by mutagenesis.In the synthesis of sterols, required components of eukaryotic membranes, an initial sterol (lanosterol in animals and fungi and cycloartenol in plants) undergoes three demethylations prior to formation of the end product sterol. The first demethylation occurs directly with lanosterol or cycloartenol and results in removal of the C-14 methyl group. The fungal demethylation is performed by the product of the ERG11 gene (Fig.

ABSTRACT:Clinical outcomes of vincristine therapy, both neurotoxicity and efficacy, are unpredictable, and the reported pharmacokinetics of vincristine have considerable interindividual variability. In vitro and in vivo data support a dominant role for CYP3A enzymes in the elimination of vincristine. Consequently, genetic polymorphisms in cytochrome P450 (P450) expression may contribute to the interindividual variability in clinical response, but the contributions of individual P450s and the primary pathways of vincristine metabolism have not been defined. In the present study, vincristine was incubated with a library of cDNA-expressed P450s, and the major oxidative metabolites were identified. CYP3A4 and CYP3A5 were the only P450s to support substantial loss of parent drug and formation of the previously unidentified, major metabolite (M1). The structure of M1, arising as a result of an oxidative cleavage of the piperidine ring of the dihydro-hydroxycatharanthine unit of vincristine, was conclusively established after conversion to suitable derivatives followed by spectroscopic analysis, and a new pathway for vincristine metabolism is proposed. CYP3A5 was more efficient in catalyzing the formation of M1 compared with CYP3A4 (9-to 14-fold higher intrinsic clearance for CYP3A5). The formation of M1 was stimulated (3-fold) by the presence of coexpressed cytochrome b 5 , but the relative efficiencies of M1 formation by CYP3A4 and CYP3A5 were unaffected. Our findings demonstrate that in contrast to most CYP3A biotransformations, the oxidation of vincristine is considerably more efficient with CYP3A5 than with CYP3A4. We conclude that common genetic polymorphisms in CYP3A5 expression may contribute to the interindividual variability in the systemic elimination of vincristine.

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