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）：几种与膜甾醇相互作用或抑制其合成的药物可有效清除真菌感染，但许多这些疗法不能清除肺孢子虫。 P. carinii 通常合成不同的 delta7 C28 和 C29 24-烷基甾醇和一些罕见的 24-烷基羊毛甾醇衍生物，但主要的真菌甾醇麦角甾醇不在其中。几种肺孢子菌 24-烷基甾醇并不具有任何其他临床重要微生物的特征。目前，关于卡氏疟原虫甾醇生物合成存在混乱，因为从头合成的成分尚未与被清除的成分和被卡氏疟原虫酶清除然后代谢的成分区分开。需要直接的生化实验来解决信息的缺乏。该项目的目标是鉴定卡氏假单胞菌合成的甾醇并阐明其形成的主要途径。要测试的假设是：(A) 乙酸甲羟戊酸前羊毛甾醇途径是有功能的，(B) 主要后羊毛甾醇途径在甾醇侧链的 C-24 去甲基化之前经历甾醇侧链 C-24 的烷基化。甾醇核，(C) P. carinii 甾醇 delta24(28) 还原酶催化在 C-24 处形成 β 构型的甲基和乙基(D) 羊毛甾醇核的代谢首先涉及去除 C-4 处的两个甲基之一，然后在 C-14 处进行去甲基化，然后再次在 C-4 处进行去甲基化； C-14 处的去甲基化先于 C-8 至 C-7 处的双键异构化。该项目的具体目标是：(1) 使用放射性标记前体证明从乙酸盐到羊毛甾醇的从头途径，(2) 使用重组酶确定羊毛甾醇代谢速率，并使用带有放射性标记前体的 P. carinii 匀浆阐明分支途径和已知阻断甾醇合成中特定反应的抑制剂，(3) 分析 P. carinii 重组甾醇 delta24(28) 还原酶的立体化学特征通过核磁共振波谱分析产物，以及(4)确定甾醇核代谢的反应顺序。由于肺孢子菌肺炎仍然是全世界艾滋病患者的主要机会性感染，并且由于各种耐药感染性微生物不断出现，因此需要开发更多种类的能够清除感染的药物。为了改善治疗和公共卫生，更好地了解这种真菌的性质至关重要。由于甾醇生物合成反应是抗真菌药物开发的极好目标，因此鉴定肺孢子虫实际合成的甾醇并了解该病原体如何产生这些重要的膜成分非常重要。