TYPE XVIII COLLAGEN/ENDOSTATIN FUNCTION IN C ELEGANS

线虫中的 XVIII 型胶原蛋白/内皮抑素功能

• 批准号: 6378052
• 负责人: JAMES M KRAMER
• 金额: $ 23.15万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-04 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6378052
• 关键词: Caenorhabditis elegans; alleles; angiogenesis; apoptosis; cell migration; collagen; cornea; gene induction /repression; gene mutation; laboratory rat; protein isoforms; protein structure function; proteoglycan; recombinant proteins

The goals of this research are to determine the normal functions of type XVIII collagen and its endostatin domain, and to elucidate the molecular mechanisms by which they act. Endostatin has strong potential for use as an anti-angiogenic tumor therapy, however very little is known about the normal functions of it, or type XVIII collagen, from which it is derived. A type XVIII collagen homologue, cle-1, has been identified in the nematode C. elegans and the advantages of this model system will be exploited to analyze its function in development and to identify other molecules with which it may interact. Mutations in cle-1 can cause defects in cell and axon guidance, and may block apoptotic death of certain cells. Ectopic expression of the endostatin domain of cle-1 causes guidance defects and apoptotic death of particular cells. Whether mammalian and C. elegans endostatin domains are functionally equivalent will be tested by comparing the effects of recombinant proteins on migration and apoptosis of human endothelial cells, and on neovascularization of rat corneas. Human endostatin sequences will be expressed in C. elegans to determine if they can rescue cle-1 mutations and cause the expected ectopic phenotypes. The identities of specific cells affected by cle-1 mutations and ectopic expression will be determined using GFP markers and lineage analyses. By controlling the timing and location of ectopic endostatin expression, requirements for it to affect particular cells will be examined. Antibodies will be generated to determine the localization of the three CLE-1 isoforms and to assess how they are affected in mutants. The antibodies will also be used to analyze release of the endostatin domain from CLE-1 and other characteristics of the protein. Additional cle-1 mutations will be generated and characterized. Mutations that enhance or suppress cle-1 mutant or ectopic expression phenotypes will be generated and the genes that they affect identified. These genes may encode molecules that interact with type XVIII collagen and/or endostatin, e.g. other matrix proteins or receptors, or are necessary for their normal function, e.g. signaling pathway. Further studies of these genes may identify the mechanisms by which type XVIII collagen and endostatin exert their effects. The results of these studies may suggest possible side effects of endostatin treatment, that should be carefully monitored, and potential means to augment its effectiveness.

本研究的目的是确定XVIII型胶原及其内皮抑制素结构域的正常功能，并阐明其作用的分子机制。 内皮抑素具有很强的抗血管生成肿瘤治疗的潜力，但很少有人知道它的正常功能，或XVIII型胶原，它是从它的衍生。 已在线虫C中鉴定出XVIII型胶原蛋白同源物cle-1。elegans和这个模型系统的优势将被用来分析其在发育中的功能，并确定其他分子，它可能与相互作用。 cle-1的突变可导致细胞和轴突导向的缺陷，并可阻断某些细胞的凋亡性死亡。 cle-1的内皮抑制素结构域的异位表达导致特定细胞的引导缺陷和凋亡性死亡。 无论是哺乳动物还是C.将通过比较重组蛋白对人内皮细胞的迁移和凋亡以及对大鼠角膜的新血管形成的影响来测试elegans内皮抑制素结构域在功能上是等同的。 人内皮抑制素序列将在C. elegans以确定它们是否可以拯救CLE-1突变并引起预期的异位表型。 受cle-1突变和异位表达影响的特定细胞的身份将使用GFP标记和谱系分析来确定。 通过控制异位内皮抑制素表达的时间和位置，将检查其影响特定细胞的要求。 将产生抗体以确定三种CLE-1同种型的定位并评估它们在突变体中如何受到影响。 这些抗体还将用于分析内皮抑制素结构域从CLE-1的释放和蛋白质的其他特征。 将产生和表征另外的cle-1突变。 将产生增强或抑制cle-1突变体或异位表达表型的突变，并鉴定它们影响的基因。 这些基因可以编码与XVIII型胶原和/或内皮抑制素相互作用的分子，例如其他基质蛋白或受体，或者是其正常功能所必需的，例如信号传导途径。对这些基因的进一步研究可以确定XVIII型胶原和内皮抑素发挥作用的机制。这些研究的结果可能表明内皮抑制素治疗可能的副作用，应仔细监测，并可能增加其有效性。