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

描述(由申请人提供)：力激活黏附(施加物理力量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黏附。它也是纳米制造材料和流动敏感器件的理想特性。然而，我们对这一现象背后的分子结构知之甚少。我们最近在白色念珠菌Als5p和其他酵母细胞黏附蛋白中发现了许多基因家族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大的细胞间粘附键所必需的，并且具有一种不寻常的组成，富含ILE、Val和Thr。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，类似的序列在真核细胞黏附分子中广泛存在。因此，我们在这项建议中的目标是了解淀粉样蛋白序列在真菌细胞粘附素强制激活中的分子作用。我们的中心假设是，这些新的淀粉样蛋白序列导致黏附分子的力敏感聚集，形成细胞表面区，赋予细胞之间强烈的黏附相互作用。我们已经组装了工具来实现AIMS，这些工具将检验三个工作假说：1)Ile、Val、Thr丰富的序列是力依赖激活所特有的。我们将分析其他淀粉样蛋白形成序列的替换对蛋白质稳定性和激活的影响。2)富含Ile、Val、Thr的淀粉样蛋白的序列在粘附素活性中的重要性不如氨基酸组成。序列变异体将在粘附性试验中进行测试。3)Als蛋白的T结构域作为力敏感的折叠开关，在拉伸压力下展开，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。野生型序列和淀粉样蛋白破坏的V326N突变T结构域将嵌入到其他表面蛋白中，包括GFP报告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使人们对新发现的淀粉样蛋白在力反应细胞黏附现象中的作用有基本的了解。特定目标2将产生一个搜索标准，用于区分潜在的功能性力量敏感的淀粉样蛋白组装系统和偶然序列。特殊目标3将生产模型系统，用于分析淀粉样蛋白在细胞黏附蛋白中的作用。目标1和目标3中产生的知识将导致制定干预生物膜形成或其他情况的战略，在这些情况下，需要防止牢固的粘连(在治疗传染病或转移时是可取的)。相反，这项工作将为调节纳米制造和组织工程中的细胞相互作用提供新的结构模块和能力。

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.