Polyamine Metabolism in Leishmania

描述（由申请人提供）：结合分子生物学、遗传学、生物化学和免疫细胞化学的工具，这个竞争性的延续申请提供了利什曼原虫多胺途径关键酶的跨学科解剖。该提案将重点关注利什曼原虫的精氨酸酶（ARG）、agmatinase （AGM）、鸟氨酸脱羧酶（ODC）、s -腺苷蛋氨酸脱羧酶（ADOMETDC）和亚精胺合成酶（SYN），它们是利什曼原虫多胺和多胺前体酶的全部补体。虽然我们之前的研究是针对L. donovani进行的，但这些研究将主要针对M379 L. mexicana进行，这是一种可以作为promastigotes维持的菌株，也可以作为无性系和体内无性系，从而使我们的发现能够在更相关的生理框架中进行考虑。我们分离了L. mexicana编码这5种蛋白的dna，对推测的全长LmARG和LmAGM PCR产物进行了测序，并从L. donovani中克隆并鉴定了LdODC、LdADOMETDC和LdADOMETDC的全长。此外，我们还生成了ldodc、LdADOMETDC和ldspdsvn基因敲除、大量可补充的LdSPDSYN蛋白，以及针对ldodc、LdADOMETDC和LdSPDSYN的单特异性抗血清。这些分子、细胞、生化和免疫试剂是三个特定目标的基石。多组分特异性目标I主要研究LmARG和LmAGM。将对LmARG和LmAGM基因组克隆进行测序和定位。为了评估基因功能，我们将通过靶向基因替换创建lmarg和A/magm零突变体，并在两个生命周期阶段描述所产生的表型。LmARG和LmAGM将在大肠杆菌中过表达，并纯化重组蛋白用于生化研究和提高抗体。然后通过免疫细胞化学和细胞分离方案定位LmARG和LmAGM。ODC、ADOMETDC和SPDSYN的细胞内位置也将被评估。特异性目的II涉及重建L. mexicana细胞系中的odc、adometdc和spdsyn敲除，以明确确定多胺基因功能是否对长期无马鞭毛体增殖至关重要，从而确定多胺途径是否为治疗利什曼病提供了有效的治疗靶点。通过LdSPDSYN的定点诱变和突变体LdSPDSYN蛋白的生化分析，将首次确定SPDSYN酶中保守的酸性和芳香氨基酸在催化活性和底物结合中的作用。

DESCRIPTION (provided by the applicant): Amalgamating tools of molecular biology, genetics, biochemistry, and immunocytochemistry, this competing continuation application offers an interdisciplinary dissection of key enzymes of the polyamine pathway of Leishmania. The proposal will focus on arginase (ARG), agmatinase (AGM), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (ADOMETDC), and spermidine synthase (SYN), the entire complement of polyamine and polyamine precursor enzymes in Leishmania. Although our previous investigations were conducted with L. donovani, these studies will be performed primarily with M379 L. mexicana, a strain that can be maintained as promastigotes and both axenic and in vivo amastigotes, thereby enabling our findings to be considered in a more relevant physiological framework. We have isolated L. mexicana DNAs encoding all five of these proteins, sequenced putative full length LmARG and LmAGM PCR products, and cloned and characterized full length LdODC, LdADOMETDC, and LdADOMETDC from L. donovani. In addition, we have generated ldodc, 4ldadometdc, and ldspdsvn knockouts, large and replenishable quantities of LdSPDSYN protein, and mono-specific antisera against LdODC, LdADOMETDC, and LdSPDSYN. These molecular, cellular, biochemical, and immunological reagents are the cornerstone of the three Specific Aims. Multicomponent Specific Aim I focuses on LmARG and LmAGM. LmARG and LmAGM genomic clones will be sequenced and mapped. To assess gene function, we will create lmarg and A/magm null mutants by targeted gene replacement and characterize the resultant phenotypes within both life cycle stages. LmARG and LmAGM will be over-expressed in E. coli, and recombinant protein purified for biochemical studies and raising antibodies. LmARG and LmAGM will then be localized by immunocytochemistry and cell fractionation protocols. The intracellular locations of ODC, ADOMETDC, and SPDSYN will also be assessed. Specific Aim II involves the reconstruction of the odc, adometdc, and spdsyn knockouts in L. mexicana line in order to definitively determine whether polyamine gene function is essential for long term amastigote proliferation, and thus, whether the polyamine pathway offers valid therapeutic targets for treating leishmaniasis Finally, the role of conserved acidic and aromatic amino acids among SPDSYN enzymes in catalytic activity and substrate binding will be defined for the first time by site-directed mutagenesis of LdSPDSYN and biochemical analysis of the mutant ldspdsyn proteins.