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.

项目概要/摘要 过氧化物酶体是普遍存在的细胞器，整合到真核生物的基本代谢功能中 细胞内嘌呤的分解代谢、胆汁酸和醚磷脂的合成，以及β-和α-氧化等 长链脂肪酸（VLCFA）。过氧化物酶体的缺乏与多种疾病有关 状态，包括遗传性神经病理学、衰老、心脏病、癌症和糖尿病。它们的重要性是 过氧化物酶体生物合成障碍（PBD）的发生进一步强调了这一点；严重的幼儿期 通常是致命的，其特征是脂质代谢改变。过氧化物酶体丰度可 通过内质网 (ER) 的从头合成，受细胞代谢需求的调节。反过来， 当不再需要它们时，过氧化物酶体会通过一种选择性的自噬形式被降解，称为 pexophagy。最近的研究发现，过氧化物酶体和脂滴（LD，富含脂质的细胞器） 调节来自相同 ER 的中性脂质（例如 TAG 和甾醇酯）的储存和水解 子域。泛素-X 结构域 8 (UBXD8) 是 p97 AAA-ATPase 的 ER 嵌入接头。在 ER UBXD8 在 ER 相关降解 (ERAD) 以及脂肪酸和甾醇中具有重要功能 体内平衡。多个小组的工作，包括我们自己未发表的研究表明，UBXD8 调节 LD 的丰度。 在比较野生型和 UBXD8 无效细胞的蛋白质组的定量蛋白质组学研究中，我们发现 UBXD8 的缺失会降低多种过氧化物酶体蛋白的丰度。此外，从脂质组学 分析表明，与 UBXD8 敲除 (KO) 相比，VLCFA 增加，胆固醇减少 野生型（WT）细胞。有趣的是，据观察，PBD 患者会积累 VLCFA，并且持续存在 降低血浆胆固醇水平。我发现过氧化物酶体数量显着减少，并且 相对于野生型细胞，UBXD8 KO 细胞中过氧化物酶体大小增加。我们进一步能够挽救这个 通过用野生型 UBXD8 补充 UBXD8 KO 细胞来抑制异常的过氧化物酶体表型。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 的缺失会导致显着的 几种过氧化物酶体蛋白的水平较低。尽管 UBXD8 调节 ERAD 的机制已被很好地证实。 据了解，其在过氧化物酶体功能中的作用完全未知。拟议的工作将测试 假设 UBXD8 在内质网过氧化物酶体生物合成中发挥关键作用。提议的实验 将使用先进的显微镜和蛋白质组学技术来确定 UBXD8 在过氧化物酶体中的作用 稳态并检查 UBXD8 KO 在代谢相关细胞系中的影响。一个分子 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 疾病状态下过氧化物酶体功能的调节。