Structural and Functional Studies of Invertebrate Classical Cadherins

描述(由申请人提供)： 本研究的目的是利用X射线结晶学的结构测定和结构导向功能实验来确定果蝇神经(DN)-钙粘蛋白这一重要的细胞间黏附蛋白的分子功能基础。DN-钙粘附素在果蝇视觉系统的模式形成中起着重要作用。在项目的后期阶段，一旦确定了合适的dN-钙粘蛋白结构，我们将使用强大的果蝇遗传分析方法来探索dN-钙粘蛋白在体内的作用机制。果蝇基因组编码3种经典钙粘附素，它们都在眼睛中表达。这些钙粘附素中只有两个在黏附中起作用，但只有dN-钙黏附素的黏附活性是光感受器传入-靶点选择所必需的。尽管在脊椎动物中有多达5种不同的经典钙粘附素参与类似的功能，但12种不同的dN-钙粘附素亚型是通过选择性剪接在果蝇中编码的，这表明dN-钙粘附素可能包含多种角色。脊椎动物经典钙粘附素通过其N末端的结合机制在细胞间黏附中发挥作用。出现在并列细胞上的脊椎动物经典钙粘附素分子通过其N末端(3-链)的互换结合形成粘附性二聚体。这种链交换是通过将来自一个配对钙粘蛋白的交换链中高度保守的色氨酸残基插入到来自相对细胞的配对分子中保守的疏水口袋中来锚定的。虽然这种相互作用中的关键残基在脊椎动物中高度保守，但脊椎动物和无脊椎动物经典钙粘附素的比对表明，这些残基在无脊椎动物分子中不是保守的。这表明，无脊椎动物钙粘附素，包括dN-钙粘附素，可能通过不同的机制在粘连中发挥作用。此外，脊椎动物和无脊椎动物的经典钙粘附素在细胞外结构域的总数和结构上存在显著差异：成熟脊椎动物经典钙粘附素含有5个细胞外钙粘附素结构域(EC1-5)，而dN-钙粘附素包含10到15个(取决于剪接形式)，一些EC结构域似乎与脊椎动物的EC结构域明显背离。像脊椎动物钙粘蛋白一样，dN-钙粘蛋白可能需要一种类似呋喃的蛋白水解酶来处理，以获得其成熟形式。 公共卫生相关性：钙粘附素的功能和功能障碍的知识适用于人类健康的多个方面，因为钙粘附素在脊椎动物几乎所有固体组织的发育过程中发挥着作用。腭裂41、皮肤疾病42和青少年黄斑营养不良最终导致失明(HJMD)43可由人类经典钙粘素基因突变引起。事实上，对dN-钙粘附素的研究在视觉系统发育和疾病方面特别相关。

DESCRIPTION (provided by applicant): The goal of the research proposed here is to use structure determination by X-ray crystallography and structure-guided functional experiments to determine the molecular basis of function for Drosophila neural (DN)-cadherin, an important intercellular adhesion protein. DN-cadherin plays an important role in patterning the Drosophila visual system. At later stages in the project, once appropriate DN-cadherin structures have been determined, we will employ the powerful methods of Drosophila genetic analysis to probe the mechanism of DN-cadherin function in vivo. The Drosophila genome encodes 3 classical cadherins, all of which are expressed in the eye. Only two of these cadherins function in adhesion, but it is only the adhesive activity of DN-cadherin is required for photoreceptor afferent-target selection. Although up to 5 distinct classical cadherins have been implicated in analogous functions in vertebrates, 12 distinct DN-cadherin isoforms are encoded by alternative splicing in the fly, suggesting the possibility that DN-cadherin may subsume multiple roles. Vertebrate classical cadherins are known to function in intercellular adhesion through binding at their extreme N-termini through a well-characterized binding mechanism. Vertebrate classical cadherin molecules presented on juxtaposed cells form an adhesive dimer by the reciprocal binding, or "swapping", of their Nterminal (3-strands. This strand-exchange is anchored by the insertion of highly conserved tryptophan residues from the exchanged strand from one partner cadherin into a conserved hydrophobic pocket in the partner molecule from the apposed cell. While key residues in this interaction are highly conserved among vertebrate species, alignments of vertebrate and invertebrate classical cadherins show that these residues are not conserved in the invertebrate molecules. This suggests that invertebrate cadherins, including DN-cadherin, are likely to function in adhesion by distinct mechanisms. Moreover, vertebrate and invertebrate classical cadherins are strikingly different in the overall number and architecture of their extracellular domains: mature vertebrate classical cadherins contain five extracelluar cadherin (EC) domains (EC1-5), whereas DN-cadherin contains between 10 and 15 (depending on splice form), and some EC domains appear to be significantly diverged from vertebrate EC domains. Like vertebrate cadherins, DN-cadherin may require proteolytic processing by a furin-like protease to achieve its mature form. PUBLIC HEALTH RELEVANCE: Knowledge of cadherin function and dysfunction is applicable to multiple facets of human health because of the role played by cadherins in the development of almost all solid tissues in vertebrate organisms. Cleft palate41, skin disorders42, and juvenile macular dystrophy eventually leading to blindness (HJMD)43 can be caused by mutations in human classical cadherin genes. Indeed, the study of DN-cadherin is particularly relevant in visual system development and disease.