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Understanding the Biology of Chronic Ulcers: Histological and Molecular Basis...

了解慢性溃疡的生物学：组织学和分子基础......

基本信息

批准号：
7645672
负责人：
HAROLD BREM
金额：
$ 4.99万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7645672
关键词：
Adherens Junction Adhesions Adhesives Attention Binding Biochemical Biological Assay Biology C cadherin Cadherins Calcium ion Categories Cell Adhesion Cell surface Cells Chinese Hamster Ovary Cell Chronic Collaborations Coupled Crystallization Crystallography Dermatology Desmosomes Eligibility Determination Extracellular Domain Family Feasibility Studies Fusion Protein Expression Goals Green Fluorescent Proteins Guidelines Human In Vitro Integral Membrane Protein Intercalated Cell Intercellular Junctions Internal Ribosome Entry Site Light Mammalian Cell Mediating Molecular Molecular Biology Phase Principal Investigator Production Property Proteins Qualifying Regulation Research Research Personnel Resolution Site Skin Specificity Structural Biologist Structure System Testing Tryptophan Ulcer Universities Work base desmocollin desmoglein expression vector extracellular interest mutant preference programs promoter

项目摘要

The molecular mechanism of cell adhesion by classical cadherins is now understood at an atomic level of detail. However, the basis of desmosomal cadherin function has not yet been determined. This Pilot and Feasibility project is focused on determining the first high-resolution structures of desmosomal cadherins in order to determine the detailed molecular.basis of their cell adhesive function. The cadherin family is characterized by their extracellular cadherin repeat domains (EC1-EC5). It is known that the adhesive function of the cadherin extracellular region is dependent upon the binding of calcium ions between each of these repeat domains which leads to the rigidification of the entire region, however, the specific interfaces and interactions responsible for adhesion are not clearly understood. Some light was shed on this issue recently when the crystal structure of the extracellular domain of C-cadherin was solved by our group. This structure revealed that binding between C-cadherins on opposing cell surfaces most likely occurs when a conserved tryptophan (Trp2) residue of a C-cadherin on one cell intercalates into a hydrophobic pocket, containing highly conserved R-A-L residues, of a C-cadherin on an opposing cell. Further, it was noted that C-cadherins on the same cell surface may interact via a similar mechanism to promote molecular clustering thereby allowing for enhanced adhesion between cells. Both the Trp2 residue and the hydrophobic binding pocket are conserved in desmosomal cadherins, therefore, it seems likely that desmogleins and desmocollins may interact via this same mechanism. However, to date no crystal structure for any of the desmosomal cadherins has been solved. This led us to hypothesize: What is the structure of the adhesive interface of a desmosomal cadherin? How do the adhesive interfaces of desmosomal cadherins interact to contribute to the formation of desmosomes? In order to gain some understanding about the structure of the extracellular domain of desmosomal cadherins, we have embarked on a collaborative effort with Dr. Angela Christiano in the Department of Dermatology at Columbia University to crystallize the extracellular domain of human desmogleins. The goal of this Pilot and Feasibility study is to understand the detailed molecular mechanisms of cadherin function in desmosome intercellular junctions. Dr. Shapiro qualifies under eligibility Category #2 in the Guidelines as an Established Investigator with no previous work in research related to the SDRC. He is an internationally renowned structural biologist who has resolved the structure of many classical cadherins. In this proposal, he will turn his attention to the desmosomal cadherins, central players in skin biology. Dr. Shapiro's P&F study utilizes Cores C and D.

经典钙粘蛋白细胞粘附的分子机制现已在原子水平上得到了解细节。然而，桥粒钙粘蛋白功能的基础尚未确定。这个试点和可行性项目的重点是确定桥粒钙粘蛋白的第一个高分辨率结构以确定其细胞粘附功能的详细分子基础。钙粘蛋白家族是其特征在于细胞外钙粘蛋白重复结构域 (EC1-EC5)。据了解，粘合剂钙粘蛋白胞外区的功能取决于每个钙粘蛋白之间的结合这些重复域导致整个区域的刚性化，但是，特定的界面和导致粘附的相互作用尚不清楚。关于这个问题的一些启示最近我们课题组解决了C-cadherin胞外域的晶体结构。这结构表明，相对细胞表面上的 C-钙粘蛋白之间的结合最有可能发生在以下情况：一个细胞上 C-钙粘蛋白的保守色氨酸 (Trp2) 残基插入疏水袋中，含有相对细胞上 C-钙粘蛋白的高度保守的 R-A-L 残基。此外，有人指出同一细胞表面上的 C-钙粘蛋白可能通过类似的机制相互作用以促进分子聚集从而增强细胞之间的粘附力。 Trp2 残基和疏水结合口袋在桥粒钙粘蛋白中是保守的，因此，桥粒糖蛋白和桥粒胶蛋白可能通过相同的机制相互作用。然而，迄今为止，还没有任何晶体结构桥粒钙粘蛋白已得到解决。这使我们做出假设：粘合剂的结构是什么？桥粒钙粘蛋白的界面？桥粒钙粘蛋白的粘附界面如何相互作用有助于桥粒的形成？为了对结构有一定的了解桥粒钙粘蛋白的细胞外结构域，我们已开始与 Angela 博士合作哥伦比亚大学皮肤科的 Christiano 结晶了细胞外结构域人类桥粒糖蛋白。这项试点和可行性研究的目标是了解详细的分子钙粘蛋白在桥粒细胞间连接中的功能机制。 Shapiro 博士符合《指南》中资格类别#2 作为资深研究者的资格，并且没有之前从事与 SDRC 相关的研究工作。他是国际知名的结构生物学家解析了许多经典钙粘蛋白的结构。在这项提议中，他将把注意力转向桥粒钙粘蛋白，皮肤生物学的核心参与者。 Shapiro 博士的 P&F 研究利用了核心 C 和 D。