Deciphering molecular functions of the “multitalented” scaffold protein USH1G/SANS in primary cilia and the nucleus

解读初级纤毛和细胞核中“多才多艺”支架蛋白 USH1G/SANS 的分子功能

• 批准号: 521147274
• 负责人: Professor Dr. Uwe Wolfrum
• 金额: --
• 依托单位: Institut für Molekulare Physiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/521147274?language=en
• 关键词: Deciphering; molecular; functions; multitalented; scaffold

SANS (scaffold protein containing ankyrin repeats and SAM domain) is encoded by USH1G gene and serves as a central scaffold molecule of the protein interactome related to the human Usher syndrome (USH), the most common form of heritetery combined deaf-blindness. Although defects in USH1G/SANS predominately lead to dysfunctions in the eye and the inner ear in patients, it is almost ubiquitously expressed. Previous studies have demonstrated that the scaffold protein participates in diverse cell modules and processes, such as the assembly of mechanosensitive signaling complexes, primary ciliogenesis, intracellular transport, endocytosis, and pre-mRNA splicing. Here, we will focus on molecular interactions and the dynamics of processes related to SANS in primary cilia and the nucleus. In our project, we aim to unravel the molecular function of USH1G/SANS in primary cilia and the nucleus by integrative approaches, combining different methods from biochemistry, molecular cell biology and biophysics, as well as molecular modeling. We will decipher the interactions with ligand proteins and their binding kinetics and properties to SANS, monitor the molecular dynamics of MLOs and between them in the living cell, and the LLPS properties in the MLOs of both cell organelles, namely the primary ciliary base and the nuclear speckles or Cajal bodies in the nucleus. We have the following specific objectives: (1) Unraveling the physico-chemical basis for multivalent protein binding to SANS. (2) Elucidation the function of SANS for the molecular dynamics in the nucleus and primary cilia. (3) Evaluation of SANS-dependent molecular interconnections between nucleus and primary cilia. (4) Deciphering the role of SANS in the formation of polymer condensates by LLPS. With our experimental approaches and the in-silico modeling we aim to deliver a comprehensive view of the molecular mechanisms underlying the role of SANS in protein-protein interaction and LLPS. We expect not only novel insights into common mechanisms and the dynamics and interplay of LLPS, but also further understanding of the regulation of the spliceosome and ciliogenesis and /or deciliation. In addition, prospective results will elucidate the link between nuclear and ciliary MLOs. Moreover, we expect to pinpoint to what extent alterations in the formation of LLPS and in the “interMLOar” exchange are disease-relevant, underlying the pathophysiology of human Usher syndrome.

SANS（包含锚蛋白重复序列​​和 SAM 结构域的支架蛋白）由 USH1G 基因编码，作为与人类 Usher 综合征 (USH) 相关的蛋白质相互作用组的中心支架分子，USH 是遗传性聋盲症最常见的形式。尽管 USH1G/SANS 缺陷主要导致患者眼睛和内耳功能障碍，但它几乎无处不在。先前的研究表明支架蛋白参与多种细胞模块和过程，例如机械敏感信号复合物的组装、初级纤毛发生、细胞内运输、内吞作用和前mRNA剪接。在这里，我们将重点关注初级纤毛和细胞核中与 SANS 相关的分子相互作用和过程动力学。在我们的项目中，我们的目标是通过综合方法，结合生物化学、分子细胞生物学和生物物理学以及分子建模的不同方法，揭示USH1G/SANS在初级纤毛和细胞核中的分子功能。我们将破译与配体蛋白的相互作用及其与 SANS 的结合动力学和特性，监测活细胞中 MLO 及其之间的分子动力学，以及两种细胞器（即初级纤毛基底和细胞核中的核斑点或卡哈尔小体）的 MLO 中的 LLPS 特性。我们有以下具体目标：（1）揭示多价蛋白与 SANS 结合的物理化学基础。 (2)阐明SANS对于细胞核和初级纤毛分子动力学的功能。 (3) 细胞核和初级纤毛之间SANS依赖性分子互连的评估。 (4) 破译SANS在LLPS形成聚合物缩合物中的作用。通过我们的实验方法和计算机模拟，我们的目标是全面了解 SANS 在蛋白质-蛋白质相互作用和 LLPS 中作用的分子机制。我们不仅期望对 LLPS 的常见机制以及动力学和相互作用有新的见解，而且还期望进一步了解剪接体和纤毛发生和/或纤毛的调节。此外，前瞻性结果将阐明核 MLO 和纤毛 MLO 之间的联系。此外，我们希望查明 LLPS 形成和“interMLOar”交换的改变在多大程度上与疾病相关，是人类亚瑟综合征病理生理学的基础。