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 过氧化物酶体是一种普遍存在的细胞器，整合到真核生物的基本代谢功能中。 细胞中的嘌呤分解代谢、胆汁酸和乙醚磷脂的合成以及β-和α-氧化的非常 长链脂肪酸(VLCFA)。过氧化物体缺陷与多种疾病有关。 这些疾病包括遗传性神经病变、衰老、心脏病、癌症和糖尿病。它们的重要性在于 过氧化物酶体生物发生障碍(PBD)的发生进一步强调了这一点；严重的幼儿 通常是致命的、以脂类代谢改变为特征的病理。过氧化物体的丰度可以 通过内质网(ER)的从头合成来调节细胞的代谢需求。相反， 当不再需要它们时，过氧化物体通过一种选择性的自噬形式降解，称为 食欲不化。最近的研究发现，过氧化物体和脂滴(LD，富含脂质的细胞器 调节中性脂类的储存和水解，如TAG和甾醇酯)，产生于相同的ER 子域。泛素-X结构域8(UBXD8)是p97 AAA-ATPase的内质网嵌合接头。在 内质网UBXD8在内质网相关降解(ERAD)以及脂肪酸和甾醇中具有重要作用 动态平衡。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的丰富性。 在比较野生型和UBXD8缺失细胞的蛋白质组的定量蛋白质组学研究中，我们发现 UBXD8的缺失降低了许多过氧化体蛋白的丰度。此外，从脂质组学来看 分析发现，与UBXD8基因敲除(KO)相比，UBXD8基因敲除(KO)中VLCFAs增加，胆固醇降低 到野生型(WT)单元格。有趣的是，据观察，PBD患者积累了VLCFA，并一致地 降低血浆胆固醇水平。我发现了过氧化物酶体数的显著下降和一种 与野生型细胞相比，UBXD8 KO细胞中的过氧化物体大小增加。我们进一步能够挽救这一点 用野生型UBXD8补充UBXD8 KO细胞的异常过氧酶体表型。另外， 与我们的蛋白质组学分析一致，我们发现UBXD8在不同细胞中的丢失导致显著的 较低水平的几种过氧化物体蛋白。尽管UBXD8调节ERAD的机制很好 了解到，它在过氧化物体功能中的作用是完全未知的。拟议中的工作将测试 假设UBXD8在内质网中的过氧化物体生物发生中起关键作用。建议的实验 将使用先进的显微镜和蛋白质组学技术来确定UBXD8在过氧化酶体中的作用 并检测UBXD8 KO在代谢相关细胞系中的作用。一种分子 了解控制过氧化物酶体丰度的机制和信号通路可能有助于 疾病状态下过氧酶体功能的调节。