Amino-terminal acetylation of proteins in mammalian biology and disease

项目总结 尽管科学界在阐明许多基因的功能方面取得了实质性进展， 仍有许多未知之处，特别是关于引入到蛋白质中的辅酶和后蛋白的多样性 翻译修饰语。一种这样的修饰是氨基(或N-)末端乙酰化(NTA)，它是 对哺乳动物N-末端组的研究很少，报道很少。蛋白质发生乙酰化 蛋白质中的赖氨酸残基(赖氨酸乙酰化或N-ε-乙酰化)和蛋白质的N-末端 (NT-乙酰化或N--乙酰化)。蛋白质NT-乙酰化是最常见的修饰之一 真核蛋白质，由N-末端乙酰转移酶(NAT)执行。的击倒表型 细胞培养中的人类NAT表明，NTA蛋白是人类细胞维持的必要修饰 但缺乏对功能的洞察力和哺乳动物体内模型。对一般角色的理解 因为NTA仍然难以捉摸，只有少数几个NTA影响蛋白质功能、复合体形成、 活跃性或稳定性是已知的。我的实验室发现并表征了第一种遗传性疾病 蛋白质的NTA，涉及X连锁基因NAA10的错义突变；我们将这种罕见的疾病命名为奥格登 综合症(OS)，以纪念我们确认为OS的第一个家庭居住的家乡(犹他州奥格登)。这个 受影响的男孩有明显的头面部异常，低眼压，全球发育迟缓， 隐睾症、心脏畸形和心脏增大。然后我们和其他人找到了十几个家庭 在这个途径中有重叠的表型和NAA10的额外突变；我们最近也报道了 编码NAA10结合伙伴的NAA15基因的从头突变与先天性心脏病有关 和/或神经发育。这一发现与心脏异常的范围和 OS(现在更广泛地被称为NAA10相关疾病)中出现的神经发育迟缓。作为我们的一部分 专注于NTA的机械解剖，我们正在对患者进行详细的代谢特征- 衍生的诱导多能干细胞系。在本仪器补充申请中，我们要求 购买一台安捷伦海马机，这是可靠、准确和常规的黄金标准 可用于评估糖酵解率、能量消耗(例如，细胞激活， 增殖、分化)、三磷酸腺苷实时产生率、底物氧化、 线粒体功能、细胞死亡和一般细胞动态平衡。这一工具将使我们能够扩展 NAA10和NAA15相关疾病的分子生物学和病理生理学研究 和NTA途径，作为理解NTA在哺乳动物生物学中作用的持续努力的一部分。这些 研究将是揭示NTA在人类健康和疾病中的作用的关键一步，就像NTA一直以来的那样 与癌症进展和神经退行性疾病有关，包括帕金森氏症、阿尔茨海默氏症和 亨廷顿氏病。

PROJECT SUMMARY Although the scientific community has made substantial progress in elucidating the function of many genes, much remains unknown, particularly concerning the diversity introduced into proteins with co- and post- translational modifications. One such modification is amino- (or N-) terminal acetylation (NTA), which is considerably understudied, with very few reports on the mammalian N-terminome. Protein acetylation occurs both at lysine residues within proteins (lysine acetylation or N-ε-acetylation) and at the N-terminus of proteins (Nt-acetylation or N--acetylation). Protein Nt-acetylation is among the most common modifications of eukaryotic proteins and is carried out by N-terminal acetyltransferases (NATs). The knockdown phenotypes of human NATs in cell culture suggest that protein NTA is an essential modification in human cells to maintain proliferation, but functional insights and mammalian in vivo models are lacking. Understanding of a general role for NTA remains elusive, and only a few examples in which NTA affects protein function, complex formation, activity, or stability are known. My laboratory discovered and characterized the first genetic disease coupled to NTA of proteins, involving a missense mutation in the X-linked gene NAA10; we named this rare disease Ogden syndrome (OS) in honor of the hometown (Ogden, Utah), where the first family we identified with OS lived. The affected boys have a distinct combination of craniofacial anomalies, hypotonia, global developmental delays, cryptorchidism, cardiac anomalies, and cardiomegaly. We and others then found more than a dozen families with overlapping phenotypes with additional mutations in NAA10 in this pathway; we also reported recently that de novo mutations in NAA15, encoding a binding partner for NAA10, are involved in congenital heart defects and/or neurodevelopment. This finding is consistent with the range of cardiac anomalies and neurodevelopmental delays seen in OS (now more broadly known as NAA10-related disorders). As part of our focus on the mechanistic dissection of NTA, we are undertaking detailed metabolic characterization of patient- derived induced pluripotent stem cell lines. In this instrument supplement application, we are requesting purchase of an Agilent Seahorse machine, which is the gold standard for reliable, accurate, and routine measurements that can be applied to assess glycolytic rates, energy expenditure (e.g., cellular activation, proliferation, differentiation), adenosine triphosphate production rate in real-time, substrate oxidation, mitochondrial function, cell death, and general cellular homeostasis. This instrument will enable us to expand our study of the molecular biology and pathophysiology associated with NAA10- and NAA15-related disorders and the NTA pathway, as part of a sustained effort to understand the role of NTA in mammalian biology. These studies will be a critical step toward revealing the role of NTA in human health and disease, as NTA has been linked to cancer progression and neurodegenerative diseases, including Parkinson’s, Alzheimer’s, and Huntington’s diseases.