Altered Matrix-Cells and Intermolecular Interactions

改变的基质细胞和分子间相互作用

• 批准号: 6663707
• 负责人: ANDRZEJ FERTALA
• 金额: $ 31.87万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-20 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6663707
• 关键词: animal tissue; biological signal transduction; chondrocytes; collagen; connective tissue development; extracellular matrix; intermolecular interaction; molecular assembly /self assembly; mutant; procollagen; protein engineering; protein localization; protein structure; protein structure function; recombinant proteins; site directed mutagenesis; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): This proposal will exploit a well-controlled experimental system that involves recombinant Collagen II and chondrocytes to study how mutations in fibrillar collagens influence the behavior of cells, change their spatial arrangement, and influence the assembly of the extracellular matrix in affected tissues. The effect of mutations on the interaction of mutant collagens with other macromolecules will also be analyzed. The present application is based on the following observations: (i) attachment and motility of chondrocytes depends on their interaction with specific domains of Collagen II, (ii) density of Collagen II fibrils, organization of hypertrophic chondrocytes, and morphology of chondrocytes secretory cysternae in mice expressing the mutated human COL2A1 gene are altered because of the Cys for Arg-alpha -519 substitution, and (iii) the Cys for Arg-alpha l -519 substitution in Collagen II alters its interaction with normal Collagen IX The overall goal of this proposal is to test hypotheses that mutations in fibrillar collagens affect not only extracellular but also affect the intracellular interaction of mutant proteins with other macromolecules, alter the structure of the extracellular matrix, and change the spatial arrangement of cells. To test these hypotheses, a cell-matrix experimental system, which exploits chondrocytes and recombinant Collagen II will be employed. To eliminate the influence of normal endogenous Collagen II on the behavior of cells, the existing transgenic mice with an inactivated Col2al gene will be exploited to obtain chondrocytes that do not produce procollagen II but express other cartilaginous macromolecules. These chondrocytes will be seeded onto engineered three-dimensional nanofibrillar scaffolds coated with mutated recombinant Collagen II. In another series of experiments, the mouse chondrocytes that do not express Col2al but express the normal human C0L2A1 will be transfected with mutant DNA constructs to express procollagen II that contains mutant chains in addition to normal ones. The cells that express the mutant protein will be analyzed for their ability to assemble a cartilaginous extracellular matrix in a long-term suspension culture. Moreover, the interaction of single mutant Collagen a-chains that resemble intracellular, non-folded Collagen, with other macromolecules will be studied. The Specific Aims are: (1) To determine how extracellular signals from mutant fibrillar collagens deposited in extracellular matrix influence the behavior and spatial organization of cells. (2) To analyze the density and distribution of Collagen fibrils assembled from mutant procollagen secreted and processed by cells. (3) To study intra- and extracellular interactions of mutant fibrillar collaqen with other molecules.

描述（由申请人提供）：该提案将利用一个良好控制的实验系统，该系统涉及重组胶原II和软骨细胞，以研究纤维状胶原的突变如何影响细胞的行为，改变其空间排列，并影响受影响组织中细胞外基质的组装。还将分析突变对突变胶原蛋白与其他大分子相互作用的影响。本申请基于以下观察：（i）软骨细胞的附着和运动性取决于它们与II型胶原蛋白的特定结构域的相互作用，（ii）表达突变的人COL 2A 1基因的小鼠中II型胶原蛋白原纤维的密度、肥大软骨细胞的组织化和软骨细胞分泌性囊泡的形态由于Cys取代Arg-α-519而改变，和（iii）胶原蛋白II中Arg-α 1 - 519的Cys取代改变了其与正常胶原蛋白IX的相互作用。该建议的总体目标是检验以下假设：纤维状胶原蛋白中的突变不仅影响细胞外而且影响突变蛋白与其它大分子的细胞内相互作用，改变细胞外基质的结构，并改变细胞的空间排列。为了验证这些假设，将采用利用软骨细胞和重组胶原蛋白II的细胞基质实验系统。为了消除正常内源性II型胶原对细胞行为的影响，将利用现有的具有失活的Col 2al基因的转基因小鼠来获得不产生II型前胶原但表达其他软骨大分子的软骨细胞。这些软骨细胞将被接种到涂有突变的重组胶原蛋白II的工程化三维纳米纤维支架上。在另一系列实验中，将不表达Col 2a 1但表达正常人C 0 L2 A1的小鼠软骨细胞用突变DNA构建体转染，以表达除正常链外还含有突变链的原胶原II。将分析表达突变蛋白的细胞在长期悬浮培养物中组装软骨细胞外基质的能力。此外，还将研究类似于细胞内非折叠胶原蛋白的单一突变胶原蛋白a链与其他大分子的相互作用。具体目标是：（1）确定来自沉积在细胞外基质中的突变纤维状胶原的细胞外信号如何影响细胞的行为和空间组织。(2)分析细胞分泌和加工的突变型前胶原组装成胶原纤维的密度和分布。(3)研究突变的纤维胶原蛋白与其他分子的细胞内和细胞外相互作用。