Developmental analysis of SELCT proteins

描述(申请人提供)：保守的Notch信号通路对脊椎动物和无脊椎动物发育过程中细胞命运的指定和侧向抑制至关重要。Notch受体结合细胞外配体(如Delta、Serrate和Lag-2)，其中包含一个保守的DSL结构域，这对受体的激活至关重要。我们已经在线虫中发现了一个新的Notch配体家族，并在脊椎动物中发现了同源物。与经典的Notch配体不同，这些蛋白质缺乏保守的DSL结构域，但它们与经典的Notch配体共享一个保守的序列基序。这项提议的第一个目的是详细审查这些新的假定配体之一的功能。SEL-14是正常发育所必需的，初步研究表明它激活了Notch信号转导。SEL-14在Notch信号中的作用将利用遗传学、谱系分析和免疫组织化学在分子和细胞水平上进行研究。线虫SEL-14同源物的发育作用也将通过缺失等位基因的鉴定、表达分析、突变表型的表征和遗传分析来解决。第二个目的是验证SEL-14直接与Notch受体相互作用并与经典的DSL配体合作激活受体的假设。这一模型由体内研究提出，并将通过双杂交研究和组织培养实验在体外进行测试。我们还将研究以前未描述的保守序列基序的作用，该基序仅在SEL-14、线虫同源物和经典的Notch配体中存在。结构/功能研究将解决这个保守的基序在SEL-14和经典的Notch配体中的功能。鉴于Notch信号在发育、干细胞维持、癌症和记忆中的重要作用，了解Notch配体至关重要。Notch信号和早老素过程中的缺陷会导致许多疾病。三角区被认为与脊柱肋骨发育不良有关。NotchS和Jaggedl的突变分别导致CADASIL和Alagille综合征。这些主要是与中风和痴呆症相关的遗传性疾病。Notch和APR不仅像早老素一样共享共同的处理途径，而且最近的研究表明，Notch和APR可能都像早老素一样对阿尔茨海默病的病理起作用。所有这些疾病都知之甚少，也没有有效的治疗方法。我们预计，了解Notch信号的配体调节将是开发这些和其他疾病治疗的第一步。

DESCRIPTION (provided by applicant): The conserved Notch signaling pathway is critical for cell fate specification and lateral inhibition during development in vertebrates and invertebrates. Notch receptors bind extracellular ligands (e.g. Delta, Serrate and LAG-2) containing a conserved DSL domain that is critical for receptor activation. We have identified a new family of putative Notch ligands in C. elegans with homologs in vertebrates. Unlike canonical Notch ligands, these proteins lack the conserved DSL domain but they share a conserved sequence motif with classical Notch ligands. The first aim of this proposal examines in detail the function of one of these new putative ligands. SEL-14 is required for normal development and preliminary studies suggest that it activates Notch signaling. The role of SEL-14 in Notch signaling will be examined at a molecular and cellular level using genetics, lineage analysis and immunohistochemistry. The developmental role of C. elegans SEL-14 homologs will also be addressed by identification of deletion alleles, expression analysis, characterization of mutant phenotypes, and genetic analysis. The second aim tests the hypothesis that SEL-14 directly interacts with Notch receptors and cooperates with classical DSL ligands in receptor activation. This model suggested by in vivo studies and will be tested in vitro using two-hybrid studies and tissue culture experiments. We will also examine the role of a previously uncharacterized, conserved sequence motif that is found only in SEL-14, C. elegans homologs and classical Notch ligands. Structure/function studies will address the function of this conserved motif in SEL-14 and in classical Notch ligands. Understanding Notch ligands is critical given the essential role of Notch signaling in development, stem cell maintenance, cancer and memory. Defects in Notch signaling and presenilin processing cause numerous diseases. Deltas has been implicated in spondylocostal dysostosis. Mutations in NotchS and Jaggedl cause CADASIL and Alagille syndromes, respectively. These are dominantly inherited disorders associated with stroke and dementia. Notch and APR not only share common processing pathways like presenilins, but recent studies suggest that Notch and APR may both contribute to Alzheimer's disease pathology like presenilins. All of these disorders are poorly understood and no effective treatment is available. We anticipate that understanding ligand regulation of Notch signaling will be a first step in the development of therapies for these and other disorders.