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: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740880
• 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 In our unpublished work, we have found that PEX16 can take on multiple topologies on the ER including two-transmembrane and four-transmembrane proteins.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的代谢细胞器，具有从脂质代谢到先天免疫反应的多种功能。通过调节其形态和信号通路的基因的共享，这部分使它成为可能。尽管只发现了少数几个，但两个细胞器的密切关系表明，这些双重过氧化物酶体单聚合物蛋白（DPMP）可能会有更多。因此，识别新型DPMP可以阐明这些细胞器在细胞过程中的新作用。 在过去的十年中，我由NSERC资助的研究计划集中在理解过氧化物酶体生物发生的分子机制上。我们发现，过氧化物酶体生物发生因子PEX16最初针对的是ER，在该ER招募其他过氧化物酶体膜蛋白，后者随后交通过氧化物酶体。现在，我们建议以我们的发现为基础，以研究DPMP如何针对过氧化物酶体。 DPMP是一组功能多样的膜蛋白，可定位于过氧化物酶体和线粒体。但是，这些膜蛋白如何能够靶向具有不同蛋白质进口机械的两个细胞器。我们的主要目标是了解DPMP的目标是过氧化物酶体的。过氧化物酶体膜蛋白（PMP）可以通过两种不同的途径直接导入过氧化物酶体或间接通过ER导入。我们最近表明，间接途径需要过氧化物酶体生物发生因子PEX16，因为它可以募集PMP到ER。在我们未发表的工作中，我们发现PEX16可以在ER上进行多个拓扑结构，包括两种跨膜和四跨膜蛋白。我们还发现，PEX16是必要且足够的，足以将至少一个DPMP导入ER，称为USP30。因此，我们假设PEX16通过首先将其导入ER来介导非经典PMP（例如DPMP）的靶向。这项研究的目标是：1。确定是否通过PEX16将双重局部蛋白（DPMP）导入过氧化物酶体； 2。确定过氧化物酶体靶向DPMP的靶向信号； 3。使用冷冻确定PEX16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化和分子生物学技术的组合。我们还将采用最新的冷冻电子显微镜来阐明PEX16的蛋白质结构。 蛋白质靶向对于适当的细胞功能至关重要。我们提出的研究位于靶向研究的过氧化物酶体蛋白质的最前沿，并将提供有关该研究细胞器如何募集其蛋白质的详细见解。此外，更广泛的生物界可以应用我们的NSERC资助研究所获得的知识，以使对过氧化物酶体及其在各种生理过程中的作用更广泛地了解。