Sterol Metabolism in Pneumocystis

肺孢子虫中的甾醇代谢

• 批准号: 7176177
• 负责人: EDNA SAYOMI KANESHIRO
• 金额: $ 31.1万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-10 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7176177
• 关键词: Acetates; Acquired Immunodeficiency Syndrome; Address; Adverse effects; Alkylation; Anabolism; Animals; Biochemical; Budgets; C14 isotope; Carbon; Cell Nucleus; Cells; Characteristics; Cholesterol; Confusion; Cytochrome P450; Data; Desmosterol; Development; Disease; Drug Delivery Systems; Drug resistance; Environment; Enzyme Kinetics; Enzymes; Erg4p; Ergosterol; Excision; Goals; Hand; Human; Human Resources; Immunologic Deficiency Syndromes; Infection; Iron binding capacity measurement; Isomerase; Lanosterol; Lipids; Literature; Measures; Membrane; Metabolic; Metabolism; Methionine; Methods; Methyltransferase; Microbe; Molds; Mycoses; NMR Spectroscopy; Nature; Numbers; Object Attachment; Opportunistic Infections; Organism; Oxidoreductase; Parasites; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacotherapy; Plants; Pneumocystis; Pneumocystis carinii; Pneumocystis carinii Pneumonia; Positioning Attribute; Public Health; Publications; Published Comment; Radiolabeled; Rate; Rattus; Reaction; Reader; Recombinant Proteins; Recombinants; Relative (related person); Research; Research Design; Scheme; Side; Squalene; Standards of Weights and Measures; Sterol Biosynthesis Pathway; Sterols; Suggestion; System; Technology; Testing; Text; Today; Work; Yeasts; base; chemotherapy; cost; demethylation; ebericol; enzyme activity; experience; fungus; improved; inhibitor/antagonist; insight; methyl group; mevalonate; pathogen; preference; radiotracer; research study; zymosterol

DESCRIPTION (provided by applicant): Several drugs that interact with membrane sterols or inhibit their syntheses are effective in clearing fungal infections but Pneumocystis is not cleared by many of these therapies. P. carinii normally synthesizes distinct delta7 C28 and C29 24-alkylsterols and some rare 24-alkyllanosterol derivatives, but the major fungal sterol ergosterol is not among them. Several Pneumocystis 24-alkylsterols are not characteristic of any other clinically important microbe. Currently, there is confusion with respect to P. carinii sterol biosynthesis because the components that are synthesized de novo have not been distinguished from those that are scavenged and those that are scavenged and then metabolized by P. carinii enzymes. Direct biochemical experiments are required to address this lack of information. The goals of this project are to identify sterols synthesized by P. carinii and to elucidate the major pathways by which they are formed. The hypotheses to be tested are: (A) the acetate-mevalonate pre-lanosterol pathway is functional, (B) the major post-lanosterol pathway goes through alkylation at C-24 of the sterol side chain prior to demethylation of C-14 of the sterol nucleus, (C) the P. carinii sterol delta24(28) reductase catalyzes the formation of methyl and ethyl groups in the beta configuration at C-24 of the sterol side chain, and (D) metabolism of the lanosterol nucleus first involves removal of one of two methyl groups at C-4, followed by demethylation at C-14, and then again at C-4; demethylation at C-14 precedes double bond isomerization at C-8 to C-7. The specific aims of this project are to: (1) demonstrate the de novo pathway from acetate to lanosterol using radiolabeled precursors, (2) determine the rates of lanosterol metabolism using recombinant enzymes, and elucidate branched pathways using P. carinii homogenates with radiolabeled precursors and inhibitors known to block specific reactions in sterol synthesis, (3) analyze the stereochemical features of the P. carinii recombinant sterol delta24(28) reductase products by nuclear magnetic resonance spectroscopy, and (4) determine the sequence of reactions in sterol nucleus metabolism. Because Pneumocystis pneumonia remains a major opportunistic infection in AIDS patients worldwide, and because various drug resistant infectious microbes are emerging, there is a need to develop a greater variety of drugs that can clear the infection. To improve therapy and public health, it is critical to better understand the nature of this fungus. Since reactions in sterol biosynthesis are excellent targets for the development of antimycotic drugs, it is important to identify the sterols actually synthesized by Pneumocystis and to understand how this pathogen produces these vital membrane components.

描述（由申请人提供）：几种与膜固醇相互作用或抑制其合成的药物可有效清除真菌感染，但许多疗法没有清除肺炎囊肿。 P. carinii通常合成不同的Delta7 C28和C29 24-烷基替补醇和一些罕见的24-烷基糖衍生物，但其中主要的真菌固醇含量不在其中。几种肺炎藻24-烷基替代不是任何其他临床上重要的微生物的特征。目前，关于carinii固醇生物合成的混淆，因为从头合成的成分尚未与被清除的成分区分开，而那些被清除的成分被清除，然后被carinii酶进行了代谢。需要直接的生化实验来解决这种缺乏信息。该项目的目标是识别由P. carinii合成的固醇，并阐明形成它们的主要途径。 The hypotheses to be tested are: (A) the acetate-mevalonate pre-lanosterol pathway is functional, (B) the major post-lanosterol pathway goes through alkylation at C-24 of the sterol side chain prior to demethylation of C-14 of the sterol nucleus, (C) the P. carinii sterol delta24(28) reductase catalyzes the formation of methyl and ethyl groups in the固醇侧链的C-24的β构型和（d）羊毛醇核的代谢首先涉及在C-4时去除两个甲基之一，然后在C-14处脱甲基化，然后再次在C-4处进行脱甲基化。 C-14的去甲基化在C-8至C-7的双键异构化之前。 The specific aims of this project are to: (1) demonstrate the de novo pathway from acetate to lanosterol using radiolabeled precursors, (2) determine the rates of lanosterol metabolism using recombinant enzymes, and elucidate branched pathways using P. carinii homogenates with radiolabeled precursors and inhibitors known to block specific reactions in sterol synthesis, (3) analyze the stereochemical Carinii重组固醇Delta24（28）通过核磁共振光谱的还原酶产物的特征，（4）确定了固醇核代谢中反应的序列。由于肺炎藻肺炎仍然是全球艾滋病患者的主要机会感染，并且由于各种耐药性感染性微生物正在出现，因此需要开发更多可以清除感染的药物。为了改善治疗和公共卫生，至关重要的是要更好地了解这种真菌的性质。由于固醇生物合成中的反应是抗虫质药物开发的极好靶标，因此鉴定实际上由肺结核合成的固醇并了解该病原体如何产生这些重要的膜成分很重要。