Molecular mechanisms of Wnt and mechanical signaling through β-catenin

Wnt 的分子机制和通过 β-catenin 的机械信号传导

基本信息

批准号：
10382116
负责人：
William I Weis
金额：
$ 2.21万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-07 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10382116
关键词：
APC gene Actins Address Adherens Junction Adult Affect Area Binding Biochemical Cadherins Cell Fate Control Cell Nucleus Cell-Cell Adhesion Cells Complex Cysteine-Rich Domain Cytoplasmic Protein Defect Development Dsh protein E-Cadherin Embryonic Development Epithelial Family Frizzled Domain Genes Goals Growth Factor Homeostasis Intercellular Junctions Knowledge Ligands Link Maintenance Malignant Neoplasms Mechanics Microfilaments Molecular Molecular Conformation Neoplasms Pathway interactions Phosphorylation Phosphotransferases Process Property Proteins Role Scaffolding Protein Signal Transduction Signaling Protein Solid Specific qualifier value Structure Tight Junctions Tissues Vinculin WNT Signaling Pathway Wnt proteins afadin alpha catenin base beta catenin biophysical techniques extracellular mechanical force multicatalytic endopeptidase complex novel strategies protein B receptor recruit tissue regeneration transmission process zonula occludens-1 protein

项目摘要

The development and homeostasis of solid tissues depends upon biochemical and mechanical signals that control cell fate and the resulting organization of cells in the tissue. The conserved protein β-catenin is a key effector of signals from both the Wnt family of secreted growth factors that specify cell fate during embryogenesis and tissue renewal in the adult, and mechanical force transmitted through cell-cell junctions in multicellular tissues. We hypothesize that mechanical force transmitted through β-catenin links Wnt signaling and cell-cell adhesion, and our overall goal is to understand the molecular mechanisms underlying these dual roles of β-catenin. Our strategy is to use biochemical, structural and biophysical methods to address critical knowledge gaps in these areas. In the absence of Wnts, the β-catenin is bound in a “destruction complex” that includes the proteins Axin and Adenomatous Polyposis Coli (APC), and kinases that phosphorylate β-catenin; phosphorylation leads to ubiquitylation and destruction of β-catenin by the proteasome. Wnt binding to the receptors Frizzled (Fzd) and LRP5/6 enables Fzd to recruit the cytoplasmic protein Dishevelled (Dvl), which in turn binds to Axin and thereby recruits the destruction complex to the activated receptor complex. This leads to phosphorylation of the LRP5/6 intracellular domain, which inhibits β-catenin destruction; the stabilized β-catenin enters the nucleus and activates target genes. We will address critical mechanistic aspects of this pathway that are not understood: 1) how secreted ligands “activate” the Fzd-Dvl interaction needed for β-catenin stabilization through interaction with the extracellular cysteine-rich domain of Fzd and LRP5/6; 2) how activated Dvl recruits Axin to turn off β-catenin destruction; 3) how the β-catenin destruction complex forms and interacts with the ubiquitylation/proteosomal machinery; 4) the essential role of APC in β-catenin destruction. Force transmission through cell-cell adherens junctions (AJ) requires a complex of E-cadherin, β-catenin, and α-catenin, which binds to actin filaments and forms a minimal force-sensing unit. Tension on cadherins can release β-catenin and cause its translocation to the nucleus independent of, but synergized by, Wnt signaling. Understanding such tension-triggered release of β-catenin requires understanding how force is transmitted through the AJ complex. α-Catenin additionally has a central role in organizing epithelial tissues based on its interactions with vinculin, Epithelial Protein Lost in Neoplasm (EPLIN), the tight junction (TJ) protein Zonula Occludens (ZO)-1, and afadin, all of which bind actin and recruit other scaffolding and signaling proteins. We will study: 1) The force-dependent conformational landscape and force responsivness of α-catenin alone and bound to its partners, including how β-catenin modifies α-catenin force responsiveness; 2) How αE-catenin conformation and force transmission properties are affected by its binding to its other junctional partners.

实体组织的发育和稳态取决于生化和机械信号保守的蛋白质β-连环蛋白是控制细胞命运和由此产生的组织细胞的关键。来自 Wnt 分泌生长因子家族信号的效应器，这些因子在生长过程中指定细胞命运成人的胚胎发生和组织更新，以及通过细胞-细胞连接传递的机械力在多细胞组织中，我们对抗通过 β-连环蛋白链接 Wnt 传递的机械力。信号传导和细胞间粘附，我们的总体目标是了解潜在的分子机制我们的策略是利用生物化学、结构和生物物理方法来发挥β-连环蛋白的这些双重作用。解决这些领域的关键知识差距。在没有 Wnt 的情况下，β-连环蛋白会结合在包含 Axin 蛋白的“破坏复合物”中和腺瘤性结肠息肉病 (APC)，以及磷酸化 β-连环蛋白的激酶；通过与受体 Frizzled (Fzd) 结合的蛋白酶体对 β-catenin 进行泛素化和破坏。 LRP5/6 使 Fzd 能够募集细胞质蛋白 Disheveled (Dvl)，该蛋白反过来又与 Axin 和将破坏复合物招募到激活的受体复合物上，从而导致其磷酸化。 LRP5/6 胞内结构域，抑制 β-catenin 的破坏；我们将解决该途径中不存在的关键机制方面。了解：1) 分泌配体如何“激活”β-连环蛋白稳定所需的 Fzd-Dvl 相互作用通过与 Fzd 和 LRP5/6 的胞外富含半胱氨酸结构域相互作用 2) 如何激活 Dvl；招募 Axin 来关闭 β-连环蛋白破坏；3) β-连环蛋白破坏复合物如何形成和相互作用泛素化/蛋白体机制；4) APC 在 β-连环蛋白破坏中的重要作用。通过细胞与细胞粘附连接 (AJ) 的力传递需要 E-钙粘蛋白、β-连环蛋白、 α-连环蛋白与肌动蛋白丝结合并形成钙粘蛋白的最小力感应单元。可以释放 β-catenin 并导致其易位至细胞核，独立于 Wnt，但与 Wnt 协同作用了解这种由张力触发的 β-连环蛋白释放需要了解力是如何产生的。通过 AJ 复合物传播的α-连环蛋白在组织上皮组织中也发挥着核心作用。基于其与纽蛋白、肿瘤中丢失的上皮蛋白 (EPLIN)、紧密连接 (TJ) 的相互作用蛋白质闭塞带 (ZO)-1 和 afadin，所有这些都与肌动蛋白结合并招募其他支架和我们将研究：1）力依赖性构象景观和力。单独的 α-连环蛋白和与其伙伴结合的响应性，包括 β-连环蛋白如何修饰 α-连环蛋白力响应性；2) αE-连环蛋白构象和力传递特性如何受其影响与其其他连接伙伴具有约束力。