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

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底物结合的增加）在许多生物医学环境中很常见，包括致病性和环境生物膜，血栓形成和哺乳动物细胞粘附。这也是纳米材料和流动敏感器件的理想特性。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al5p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大的细胞间粘附键所必需的，并且具有富含Ile， Val和Thr的不寻常的组成。黏附素是力激活的，淀粉样蛋白序列介导黏附素分子在细胞表面“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粘附分子中广泛存在。因此，我们的目标是了解淀粉样蛋白序列在真菌细胞粘附素的力激活中的分子作用。我们的中心假设是，这些新的淀粉样蛋白序列导致粘附分子的力敏感集群形成细胞表面区域，赋予细胞之间的强粘附相互作用。我们已经组装了工具来执行将测试3个工作假设的目标：1)Ile, Val， Thr-rich序列对力依赖性激活是特定的。我们将分析替代其他淀粉样蛋白形成序列对蛋白质稳定性和激活的影响。2)在黏附素活性中，富含Ile、Val、thr3的淀粉样蛋白的序列不如氨基酸组成重要。序列变异将在粘附试验中进行测试。3) Als蛋白的T结构域是一个力敏感的折叠开关，在拉伸力作用下展开，使淀粉样蛋白序列暴露出来。Als黏附蛋白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突变体T结构域将嵌入到其他表面蛋白中，包括GFP报告蛋白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性细胞粘附现象中的新发现的作用的基本理解。特异性目标2将产生一个搜索标准，以区分潜在的功能力敏感淀粉样蛋白组装系统和偶然序列。具体目标3将产生模型系统，以测定淀粉样蛋白在细胞粘附蛋白中的作用。在目标1和目标3中产生的知识将导致干预生物膜形成或其他情况的策略，在这些情况下，需要防止强大的粘附（在感染性疾病或转移的治疗中需要）。相反，这项工作将为纳米制造和组织工程中调节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.