Mechanism of targeting of Peroxisome-Mitochondria localizing proteins

过氧化物酶体-线粒体定位蛋白的靶向机制

• 批准号: RGPIN-2020-05865
• 负责人: Kim, Peter
• 金额: $ 3.64万
• 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=744134
• 关键词: Mechanism; targeting; Peroxisome; Mitochondria; localizing

Peroxisomes and mitochondria are synergistic metabolic organelles sharing several functions from lipid metabolism to innate immune response. This is partly made possible by the sharing of genes that regulate their morphology and signaling pathways. Although only a handful has been discovered, the close relationship of the two organelles suggests there are likely more of these dual peroxisome-mitochondria proteins (dPMPs). Therefore, identifying novel dPMPs may elucidate new roles of these organelles in cellular processes. Over the last decade, my NSERC-funded research program has focused on understanding the molecular mechanisms of peroxisome biogenesis. We discovered that the peroxisomal biogenesis factor PEX16 is initially targeted to the ER where it recruits other peroxisomal membrane proteins that traffic subsequently to peroxisomes. We now propose to build upon our findings to study how dPMPs are targeted to peroxisomes. dPMPs are a functionally diverse group of membrane proteins that localize to both peroxisomes and mitochondria. However, how these membrane proteins are capable of targeting to both organelles that possess distinct protein import machinery is not known. Our main objective is to understand how dPMPs are targeted to peroxisomes. Peroxisomal membrane proteins (PMPs) can be imported by two distinct pathways, directly to peroxisomes or indirectly via the ER. We have recently shown that the peroxisomal biogenesis factor, PEX16, is required for the indirect pathway as it can recruit PMPs to the ER. In our unpublished work, we have found that PEX16 can take on multiple topologies on the ER including two-transmembrane and four-transmembrane proteins. We have also found that PEX16 is necessary and sufficient to import at least one dPMP called USP30 to the ER. Therefore, we hypothesis that PEX16 mediates the targeting of non-classic PMPs such as dPMPs by importing them first to the ER. The objectives of this research are to: 1. Determine whether dual localized proteins (dPMPs) are imported into peroxisomes by PEX16; 2. Identify the targeting signal of dPMPs for peroxisome targeting; 3. Determine the protein structure of PEX16 using cryoEM. To accomplish these objectives, we will use a combination of live-cell super-resolution microscopy with biochemical and molecular biology techniques. We will also employ the latest cryo-electron microscopy to elucidate the protein structure of PEX16. Protein targeting is essential for proper cellular function. Our proposed study is at the forefront of peroxisome protein targeting research and will provide detailed insights into how this understudied organelle recruits their proteins. Moreover, the knowledge gained from our NSERC funded research can be applied by the wider biological community to bring about broader understanding of the peroxisomes and their roles in a vast array of physiological processes.

过氧化物酶体和线粒体是协同代谢的细胞器，从脂质代谢到先天免疫应答，它们具有多种功能。这在一定程度上是因为它们共享了调控其形态和信号通路的基因。虽然只发现了少数，但这两种细胞器的密切关系表明可能有更多的这种双重过氧化物酶体-线粒体蛋白（dPMP）。因此，识别新的dPMPs可能阐明这些细胞器在细胞过程中的新作用。 在过去的十年里，我的NSERC资助的研究计划一直专注于了解过氧化物酶体生物合成的分子机制。我们发现过氧化物酶体生物合成因子PEX 16最初靶向ER，在ER中它招募随后运输到过氧化物酶体的其他过氧化物酶体膜蛋白。我们现在建议建立在我们的研究结果，研究如何dPMP针对过氧化物酶体。dPMP是定位于过氧化物酶体和线粒体两者的功能多样的膜蛋白组。然而，这些膜蛋白如何能够靶向具有不同蛋白质输入机制的两种细胞器尚不清楚。我们的主要目标是了解dPMP如何靶向过氧化物酶体。过氧化物酶体膜蛋白（PMP）可以通过两种不同的途径输入，直接进入过氧化物酶体或间接通过ER。我们最近发现过氧化物酶体生物合成因子PEX 16是间接途径所必需的，因为它可以将PMP募集到ER。在我们未发表的工作中，我们发现PEX 16可以在ER上呈现多种拓扑结构，包括两个跨膜和四个跨膜蛋白。我们还发现，PEX 16是必要的，足以将至少一种称为USP 30的dPMP导入ER。因此，我们假设PEX 16通过首先将它们导入ER来介导非经典PMP如dPMP的靶向。本研究的目的是：1。确定双定位蛋白（dPMPs）是否通过PEX 16输入到过氧化物酶体中; 2.识别dPMP的过氧化物酶体靶向信号; 3.使用cryoEM确定PEX 16的蛋白质结构。为了实现这些目标，我们将使用活细胞超分辨率显微镜与生物化学和分子生物学技术相结合。我们还将采用最新的冷冻电子显微镜来阐明PEX 16的蛋白质结构。 蛋白质靶向对于正常的细胞功能至关重要。我们提出的研究是在过氧化物酶体蛋白靶向研究的最前沿，并将提供详细的见解，了解这个未充分研究的细胞器如何招募他们的蛋白质。此外，从我们的NSERC资助的研究中获得的知识可以被更广泛的生物界应用，以更广泛地了解过氧化物酶体及其在各种生理过程中的作用。