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: 8642194
• 负责人: PETER N LIPKE
• 金额: $ 34.17万
• 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/8642194
• 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）：在许多生物医学环境中，包括致病性和环境生物膜，血栓形成和哺乳动物的细胞粘附在许多生物医学环境中，强制激活的依从性（在施用物理力后的细胞对细胞或细胞间结合的增加）很常见。它也是对材料和流动敏感设备的纳米制造的理想特性。但是，我们对现象基础的分子结构几乎没有理解。我们最近发现了来自许多基因家族的白色念珠菌ALS5P和其他酵母细胞粘附蛋白中的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细胞到细胞粘附键所必需的，并且具有不寻常的组成在Ile，val和thr中。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“纳米构胺”的形成。生物信息学研究表明，在真核生物细胞粘附分子中相似的序列相似。因此，我们在该建议中的目标是了解真菌细胞粘附力的力激活中淀粉样蛋白序列的分子作用。我们的中心假设是，这些新型淀粉样蛋白序列会导致粘附分子的力敏感聚类，以形成细胞表面区域，从而赋予细胞之间强烈的粘附相互作用。我们已经组装了执行将测试3个工作假设的工具：1）Ile，Val，富含THR的序列是针对力依赖性激活的。我们将测定其他淀粉样蛋白序列对蛋白质稳定性和激活的取代的影响。 2）在粘合剂的活性中，ILE，Val，富含THR的淀粉样蛋白的序列不如氨基酸组成。序列变体将在粘附测定中进行测试。 3）ALS蛋白的T结构域充当了力敏感的折叠开关，该开关在延伸力下展开以暴露淀粉样蛋白序列。 ALS粘附素中的淀粉样蛋白序列在高度保守的T结构域中。野生型序列和淀粉样蛋白干扰的V326N突变体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拉染蛋白构建的粘附素。这项工作的成功完成将导致对淀粉样蛋白在力反应性细胞粘附现象中新发现的作用的基本理解。特定的目标2将产生搜索标准，以区分潜在功能敏感的淀粉样蛋白组装系统和偶性序列。特定的目标3通常会产生模型系统，以测定淀粉样蛋白在细胞粘附蛋白中的作用。目标1和3中产生的知识将导致干预生物膜形成的策略，或者希望在其中可以防止稳健粘附的其他条件（在治疗传染病或转移方面需要）。相反，这项工作将提供一个新的结构模块和能力，以调节纳米制造和组织工程中的细胞相互作用。