Determining the Role of p97 Adaptor UBXD8 in Peroxisome Function

项目总结/摘要 过氧化物酶体是一种普遍存在的细胞器，参与真核生物的代谢功能。 细胞，如嘌呤catenase，胆汁酸和醚磷脂的合成，以及β-和α-氧化的非常 长链脂肪酸（VLCFA）。过氧化物酶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 过氧化物酶体生物合成障碍（PBD）的发生进一步强调;严重的幼儿期 通常是致命的并且以脂质代谢改变为特征的病理。过氧化物酶体丰度可以是 通过内质网（ER）处的从头合成由细胞代谢需求调节。相反地， 当它们不再需要时，过氧化物酶体通过一种选择性的自噬形式降解， 食人花最近的研究发现，过氧化物酶体和脂滴（LD，富含脂质的细胞器， 调节中性脂质如TAG和甾醇酯的储存和水解），由相同的ER产生 子域。泛素-X结构域8（UBXD 8）是一个ER嵌入的p97 AAA-ATP酶衔接子。在 ER UBXD 8在ER相关降解（ERAD）以及脂肪酸和甾醇中具有重要功能 体内平衡几个小组的工作，包括我们自己未发表的研究表明，UBXD 8调节 大量的LD。 在比较野生型和UBXD 8缺失细胞蛋白质组的定量蛋白质组学研究中，我们发现， UBXD 8的缺失降低了许多过氧化物酶体蛋白的丰度。此外，从脂质组学 分析中，我们发现UBXD 8敲除（KO）组VLCFA增加，胆固醇降低， 野生型（WT）细胞。有趣的是，观察到PBD患者积累VLCFA，并且持续地 降低血浆胆固醇水平。我发现过氧化物酶体数量显著减少， 相对于野生型细胞，UBXD 8 KO细胞中过氧化物酶体大小增加。我们还能拯救这个 通过用野生型UBXD 8补充UBXD 8 KO细胞来诱导异常过氧化物酶体表型。此外，本发明还 与我们的蛋白质组学分析一致，我们发现不同细胞中UBXD 8的缺失导致了显著的 几种过氧化物酶体蛋白水平较低。尽管UBXD 8调节ERAD的机制很好地解释了ERAD的机制。 尽管如此，它在过氧化物酶体功能中的作用仍然完全未知。拟议的工作将测试 假设UBXD 8在ER的过氧化物酶体生物合成中起关键作用。拟议实验 将使用先进的显微镜和蛋白质组学技术来确定UBXD 8在过氧化物酶体中的作用， 体内平衡和检查UBXD 8 KO在代谢相关细胞系中的作用。分子 了解控制过氧化物酶体丰度的机制和信号通路， 疾病状态期间过氧化物酶体功能的调节。

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 Work from several groups, including our own unpublished studies indicate that UBXD8 regulates the abundance of LDs.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.