A ROLE FOR AMYLOIDS IN FORCE-DEPENDENT ACTIVATION OF CELL ADHESION

淀粉样蛋白在细胞粘附的力依赖性激活中的作用

• 批准号: 8516835
• 负责人: PETER N LIPKE
• 金额: $ 3.87万
• 依托单位: BROOKLYN COLLEGE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8516835
• 关键词: Adherence; Adhesions; Adhesives; Amino Acids; Amyloid; Avidity; Bacterial Adhesins; Behavior; Binding; Bioinformatics; Biological Assay; Biological Models; Candida albicans; Cell Adhesion; Cell Adhesion Molecules; Cell Communication; Cell surface; Cells; Characteristics; Coagulation Process; Devices; Discrimination; Eukaryota; Eukaryotic Cell; Gene Family; Immune response; Infection; Intervention; Knowledge; Lead; Maintenance; Mammalian Cell; Mannose-Binding Lectins; Mediating; Membrane Proteins; Microbial Biofilms; Modeling; Molecular; Molecular Structure; Monitor; Neoplasm Metastasis; Outcome; Positioning Attribute; Property; Proteins; Publications; Reporter; Research; Role; Structure; System; Testing; Tissue Engineering; Variant; Work; Yeasts; amyloid formation; design; infectious disease treatment; isoleucylvaline; mutant; nanofabrication; nanoscale; novel; prevent; protein activation; sensor; small molecule; tool

DESCRIPTION (provided by applicant): Force-activated adherence, (the increase in cell-to-cell or cell-to-substrate binding after application of physical force) is common in many biomedical settings, including pathogenic and environmental biofilms, thrombogenesis, and mammalian cell adhesion. It is also a desirable property for nanofabrication of materials and flow-sensitive devices. However, we have little understanding of the molecular structures that underlie the phenomenon. We recently discovered functional amyloid-forming sequences in Candida albicans Als5p and other yeast cell adhesion proteins from many gene families. These amyloid sequences are required for formation of strong cell-to-cell adhesive bonds, and have an unusual composition being rich in Ile, Val, and Thr. The adhesins are force-activated, and the amyloid sequences mediate formation of "nanodomains" of adhesin molecules on the cell surface. Bioinformatics studies demonstrate similar sequences to be widespread among eukaryote cell adhesion molecules. Therefore, our objective in this proposal is to understand the molecular roles of amyloid sequences in force activation of fungal cell adhesins. Our central hypothesis is that these novel amyloid sequences cause force-sensitive clustering of adhesion molecules to form cell surface regions conferring strong adhesive interactions between cells. We have assembled the tools to carry out aims that will test 3 working hypotheses: 1) that Ile, Val, Thr-rich sequences are specific for force-dependent activation. We will assay the effects of substitutions of other amyloid-forming sequences on protein stability and activation. 2) That the sequence of Ile, Val, Thr-rich amyloids is less important than amino acid composition in the activity of the adhesins. Sequence variants will be tested in adhesion assays. 3) That the T domain of Als proteins acts as a force-sensitive folding switch, which unfolds to expose the amyloid sequence under extension force. The amyloid sequence in Als adhesins is in the highly conserved T domain. The wild- type sequence and amyloid-disrupted V326N mutant T domain will be embedded in other surface protein, including a GFP reporter and an adhesin constructed from mammalian mannose-specific lectins. Successful completion of this work will lead to basic understanding of the newly-discovered role of amyloids in force-responsive cell adhesion phenomena. Specific Aim 2 will generate a search criterion for discrimination between potentially functional force-sensitive amyloid assembly systems, and fortuitous sequences. Specific aim 3 will produce model systems for assay of role of amyloids in cell adhesion proteins in general. Knowledge generated in Aims 1 and 3 will lead to strategies for intervention in biofilm formation or other conditions in which it is desirable to prevent robust adhesion (desirable in treatment of infectious diseases or in metastasis). Conversely, the work will provide a new structural module and capability for regulating cell interactions in nanofabrication and tissue engineering.

描述（由申请人提供）：力激活粘附（施加物理力后细胞与细胞或细胞与基质结合的增加）在许多生物医学环境中很常见，包括病原体和环境生物膜、血栓形成和哺乳动物细胞粘附。它也是材料的纳米纤维和流动敏感器件的期望性质。然而，我们对这种现象背后的分子结构知之甚少。我们最近在白色念珠菌Als5p和其他来自许多基因家族的酵母细胞粘附蛋白中发现了功能性淀粉样蛋白形成序列。这些淀粉样蛋白序列是形成强的细胞间粘附键所必需的，并且具有富含Ile、瓦尔和Thr的不寻常的组成。粘附素是力激活的，淀粉样蛋白序列介导细胞表面粘附素分子的“纳米结构域”的形成。生物信息学研究表明，类似的序列广泛存在于真核生物细胞粘附分子中。因此，我们的目标是在这个建议是了解淀粉样蛋白序列的分子作用，在部队激活真菌细胞粘附素。我们的中心假设是，这些新的淀粉样蛋白序列引起粘附分子的力敏性聚集，形成细胞表面区域，赋予细胞之间强的粘附相互作用。我们已经组装了工具来实现将测试3个工作假设的目标：1）Ile、瓦尔、Thr富集序列对于力依赖性激活是特异性的。我们将分析其他淀粉样蛋白形成序列的取代对蛋白质稳定性和活化的影响。2)富含Ile、瓦尔、Thr的淀粉样蛋白的序列对粘附素活性的影响不如氨基酸组成的影响大。将在粘附试验中检测序列变体。3)Als蛋白的T结构域作为一个力敏感的折叠开关，在伸展力的作用下展开暴露淀粉样蛋白序列。Als粘附素中的淀粉样蛋白序列位于高度保守的T结构域中。野生型序列和淀粉样蛋白破坏的V326N突变T结构域将嵌入其他表面蛋白中，包括GFP报告基因和由哺乳动物甘露糖特异性凝集素构建的粘附素。这项工作的成功完成将导致对淀粉样蛋白在力响应细胞粘附现象中新发现的作用的基本理解。具体目标2将产生一个搜索标准之间的潜在功能的力敏感性淀粉样蛋白组装系统，和偶然序列的歧视。具体目标3将产生用于测定淀粉样蛋白在细胞粘附蛋白中的作用的模型系统。在目标1和3中产生的知识将导致用于干预生物膜形成或期望防止强粘附的其他状况（在治疗感染性疾病或转移中期望）的策略。相反，这项工作将提供一种新的结构模块和能力，用于调节纳米纤维和组织工程中的细胞相互作用。