Determining the Role of p97 Adaptor UBXD8 in Peroxisome Function

确定 p97 适配器​​ UBXD8 在过氧化物酶体功能中的作用

• 批准号: 10534586
• 负责人: Iris Montes
• 金额: $ 4.25万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-16 至 2025-08-15
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10534586
• 关键词: ATP phosphohydrolase; Adaptor Signaling Protein; Adipocytes; Aging; Autophagocytosis; Bile Acids; Biochemical; Biogenesis; Biological Assay; Catabolism; Cell Line; Cells; Cholesterol; Complement; Confocal Microscopy; Development; Diabetes Mellitus; Disease; Endoplasmic Reticulum; Enzymes; Eukaryotic Cell; Exposure to; Fatty Acids; Feedback; Future; Heart Diseases; Homeostasis; Hydrolysis; Image; Inherited; Knock-out; Knockout Mice; Knowledge; Lead; Life Cycle Stages; Lipids; Malignant Neoplasms; Mammalian Cell; Measures; Mediating; Metabolic; Metabolic Diseases; Microscopy; Microsomes; Molecular; Null Lymphocytes; Organelles; Pathology; Pathway interactions; Patients; Phenotype; Phospholipid Ethers; Physiologic pulse; Plasma; Play; Positioning Attribute; Process; Proteins; Proteome; Proteomics; Purines; Quality Control; Research Personnel; Role; Signal Pathway; Site; System; Techniques; Testing; Ubiquitin; Very Long Chain Fatty Acid; Vesicle; Work; Yeasts; career; cellular targeting; early childhood; experience; experimental study; lipid biosynthesis; lipid mediator; lipid metabolism; lipidomics; neuropathology; novel; oxidation; peroxisome; sensor; species difference; sterol ester; sterol homeostasis; therapeutic target

PROJECT SUMMARY/ ABSTRACT Peroxisomes are ubiquitous organelles that are integrated into essential metabolic functions of eukaryotic cells such as purine catabolism, bile acid and ether phospholipid synthesis, as well as β- and α- oxidation of very long chain fatty acids (VLCFA). Deficiencies in peroxisomes have been associated with a variety of disease states, including inherited neuropathologies, aging, heart disease, cancer, and diabetes. Their importance is further underscored by the occurrence of peroxisome biogenesis disorders (PBD); serious early childhood pathologies that are often fatal and characterized by altered lipid metabolism. Peroxisomes abundance can be modulated by cellular metabolic demand via de novo synthesis at the Endoplasmic Reticulum (ER). Conversely, when they are no longer needed, peroxisomes are degraded via a selective form of autophagy known as pexophagy. Recent studies have found that peroxisomes and lipid droplets (LDs, lipid rich organelles that regulate the storage and hydrolysis of neutral lipids such as TAG and sterol esters), arise from the same ER sub-domains. The Ubiquitin-X domain 8 (UBXD8) is an ER-embedded adaptor to the p97 AAA-ATPase. At the ER UBXD8 has essential functions in ER-associated degradation (ERAD) as well as fatty acid and sterol homeostasis. Work from several groups, including our own unpublished studies indicate that UBXD8 regulates the abundance of LDs. In quantitative proteomic studies comparing the proteomes of wildtype and UBXD8 null cells, we find that loss of UBXD8 decreases the abundance of numerous peroxisomal proteins. Furthermore, from lipidomics analysis we identified an increase in VLCFAs and a decrease in cholesterol in UBXD8 knockout (KO) compared to wildtype (WT) cells. Interestingly, it is observed PBD patients accumulate VLCFAs and have consistently reduced cholesterol plasma levels. I have identified a significant decrease in peroxisome number and an increase in peroxisome size in UBXD8 KO cells relative to wildtype cells. We were further able to rescue this aberrant peroxisome phenotype by complementing UBXD8 KO cells with wildtype UBXD8. Additionally, consistent with our proteomics analyses, we found that loss of UBXD8 in different cells results in significantly lower levels of several peroxisomal proteins. Although the mechanism by which UBXD8 regulates ERAD is well understood, its role in peroxisome function is completely unknown. The proposed work will test the hypothesis that UBXD8 plays a critical role in peroxisome biogenesis at the ER. Proposed experiments will use advanced microscopy and proteomics techniques to ascertain the role of UBXD8 in peroxisome homeostasis and examine the effects of UBXD8 KO in a metabolically relevant cell line. A molecular understanding of the mechanisms and signaling pathways controlling peroxisome abundance may allow for modulation of peroxisome function during disease states.

项目摘要/摘要 过氧化物体是无处不在的细胞器，它们整合到真核的必需代谢功能中 细胞，例如纯分解代谢，胆汁酸和乙醚磷脂合成，以及非常非常的β-和α-氧化 长链脂肪酸（VLCFA）。过氧化物体的缺陷与多种疾病有关 国家，包括遗传神经病理学，衰老，心脏病，癌症和糖尿病。他们的重要性是 过氧化物组生物发生障碍（PBD）进一步强调了；严重的幼儿 通常致命的病理，其特征是脂质代谢改变。过氧化物体丰度可以是 通过内塑性网状（ER）通过从头合成的细胞代谢需求调节。反过来， 如果不再需要它们，则通过称为自噬的选择性形式降解过氧人理，称为 pexophagy。最近的研究发现，过氧化物体和脂质液滴（LDS，富含脂质的细胞器， 调节中性脂质（例如TAG和固醇酯）的储存和水解）来自相同的ER 子域。泛素-X域8（UBXD8）是p97 AAA-ATPase的ER插入适配器。在 ER UBXD8在ER相关降解（ERAD）以及脂肪酸和固醇中具有重要功能 稳态。来自几个小组的工作，包括我们自己未发表的研究，表明UBXD8调节 LDS的抽象。 在比较野生型和UBXD8无效细胞蛋白质组织的定量蛋白质组学研究中，我们发现 UBXD8的损失减少了许多过氧化物体蛋白的抽象。此外，来自脂肪态 分析我们确定了VLCFA的增加，UBXD8敲除（KO）中的胆固醇降低 到野生型（WT）细胞。有趣的是，观察到PBD患者积累了VLCFA，并且始终如一 胆固醇血浆水平降低。我已经确定过氧组小数和一个显着下降 相对于野生型细胞，UBXD8 KO细胞中过氧体大小的增加。我们进一步能够营救这个 通过使用WildType UBXD8完成UBXD8 KO细胞，异常的过氧表型。此外， 与我们的蛋白质组学分析一致，我们发现不同细胞中UBXD8的损失显着导致 几种过氧蛋白的较低水平。虽然UBXD8调节ERAD的机制很好 理解，其在过氧组合功能中的作用是完全未知的。拟议的工作将测试 假设UBXD8在ER的过氧化物体生物发生中起关键作用。提出的实验 将使用先进的显微镜和蛋白质组学技术来确定UBXD8在过氧化物中的作用 稳态并检查UBXD8 KO在代谢相关的细胞系中的影响。分子 了解控制过氧体丰度的机制和信号通路可能允许 在疾病状态下过氧化物功能的调节。