Understanding the roles of intrinsically disordered proteins in stress granules

了解内在无序蛋白质在应激颗粒中的作用

• 批准号: RGPIN-2022-04849
• 负责人: YOUN, JIYOUNG
• 金额: $ 2.04万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761255
• 关键词: Understanding; roles; intrinsically; disordered; proteins

Living organisms are constantly challenged by stressful conditions. To cope, their cells have evolved to utilize specialized subcellular compartments, called membraneless organelles, that rapidly respond to cellular needs by regulating biological processes when stressed. These include stress granules (SGs), which are found in all eukaryotic organisms. Stress stops cells from producing new proteins, and SGs form in the cytoplasm when the proteins and RNAs involved in the process form discrete droplets, much like oil does in water. I previously used a technique called BioID to predict ~140 proteins present in SGs and a related organelle, the processing body (PB; Youn et al. 2018 Mol. Cell). I later curated a comprehensive inventory of SGs and PBs, defining ~300 SG-resident proteins in mammalian cells (Youn et al. 2019 Mol. Cell). Complementing these proteomics studies, advanced microscopy studies have shed light on overall SG organization. Several studies suggest that SGs comprise two phases: a stable core and a labile shell. However, these studies have only examined a few well-known SG proteins, and overall SG organization remains poorly understood. SGs are highly enriched for proteins with intrinsically disordered regions, which may play crucial roles in their organization. These proteins can generate large numbers of interactions, and this high valency is required for membraneless organelle formation. In the mammalian SG proteome, proline rich coiled-coil (PRRC)2A and PRRC2B are prime candidates for high valency interactions because of their largely disordered regions and associations with many SG proteins (Youn et al. 2018 Mol. Cell). To investigate their roles in SG organization, we performed PRRC2A and PRRC2B BioID in human cells during stress and observed distinct interaction profiles during SG assembly. Immunofluorescence-based microscopy also revealed that PRRC2A solely resides in SGs, whereas PRRC2B dually localizes to SGs and a nuclear membraneless organelle. Based on this, we hypothesize that PRRC2A and PRRC2B reside in distinct SG regions and play specific roles in SG organization and interplay with other membraneless organelles. To test this, we will: 1)Determine the distinct SG regions occupied by PRRC2A and PRRC2B 2)Investigate the role of PRRC2A in recruiting other proteins to SGs 3)Elucidate the function of PRRC2B's dual localization to SGs and a distinct nuclear membraneless organelle Our preliminary work sets the stage to elucidate the divergent roles of PRRC2A and PRRC2B in SG organization, function, and interplay with other membraneless organelles. Spearheaded by two graduate students, our findings will provide a better understanding of SGs, knowledge that will be applicable to all eukaryotic systems.

生物体不断受到压力条件的挑战。为了科普，它们的细胞已经进化到利用专门的亚细胞区室，称为无膜细胞器，在压力下通过调节生物过程迅速响应细胞需求。这些包括在所有真核生物中发现的应激颗粒（SG）。压力会阻止细胞产生新的蛋白质，当参与该过程的蛋白质和RNA形成离散的液滴时，细胞质中就会形成SGs，就像油在水中一样。我之前使用了一种名为BioID的技术来预测SGs和相关细胞器（加工体）中存在的约140种蛋白质（PB; Youn et al. 2018 Mol. Cell）。我后来策划了一个全面的SG和PB清单，定义了哺乳动物细胞中约300种SG驻留蛋白（Youn et al. 2019 Mol.细胞）。补充这些蛋白质组学研究，先进的显微镜研究揭示了整个SG组织。一些研究表明，SGs包括两个阶段：一个稳定的核心和一个不稳定的外壳。然而，这些研究只研究了一些众所周知的SG蛋白，整体SG组织仍然知之甚少。SG高度富集具有内在无序区域的蛋白质，这可能在其组织中起关键作用。这些蛋白质可以产生大量的相互作用，而这种高价是无膜细胞器形成所必需的。在哺乳动物SG蛋白质组中，富含脯氨酸的卷曲螺旋（PRRC）2A和PRRC 2B是高价相互作用的主要候选者，因为它们的区域很大程度上是无序的，并且与许多SG蛋白质相关（Youn et al. 2018 Mol. Cell）。为了研究它们在SG组织中的作用，我们在应激期间在人类细胞中进行PRRC 2A和PRRC 2B BioID，并在SG组装期间观察到不同的相互作用特征。基于免疫荧光的显微镜也显示PRRC 2A仅存在于SG中，而PRRC 2B双重定位于SG和无核膜细胞器。基于此，我们假设PRRC 2A和PRRC 2B位于不同的SG区域，在SG组织中发挥特定作用，并与其他无膜细胞器相互作用。为了测试这一点，我们将：1）确定PRRC 2A和PRRC 2B所占据的不同SG区域2）研究PRRC 2A在招募其他蛋白质到SG中的作用3）阐明PRRC 2B在SG和不同的无核膜细胞器中的双重定位的功能我们的初步工作为阐明PRRC 2A和PRRC 2B在SG组织、功能和与其他无膜细胞器的相互作用中的不同作用奠定了基础。由两名研究生带头，我们的研究结果将提供更好的了解SGs，知识将适用于所有真核系统。