Unravelling the regulation of beta-endorphin and other peptide hormones by N-terminal acetylation and deacetylation

肥胖是几种代谢紊乱之一，在我们这个时代，它被认为是欧盟和西方世界的一个主要健康问题。新陈代谢、食欲、性行为和疼痛的重要调节因素是多肽荷尔蒙α-黑素细胞刺激素(α-MSH)和β-内啡肽(β-END)。这两种激素都受N-末端乙酰化(NT-Ac)的翻译后调节。到目前为止，在高等真核生物中已经发现了六种不同的N-末端乙酰基转移酶(NatA-NatF)，它们负责人类80%的蛋白质的共翻译乙酰化。然而，负责α-MSH和β-END修饰的NAT(NatX)的身份仍然难以捉摸。NatX的鉴定将揭开多肽激素在肥胖和其他几个可能由翻译后NT-Ac调控的重要生理过程中的作用和调节。此外，还没有确定N-末端脱乙酰基和NO-N-末端脱乙酰基酶(NDAC)的机制。然而，一些迹象表明NDAC(S)的存在，特别是β-END在乙酰化时以其生物不活跃的形式储存，因此必须去乙酰化才能变得活跃和释放。此外，最近的发现表明，在酿酒酵母中，NT-ac在生长变化时是动态的。因此，这个项目的主要目标是鉴定NatX以及揭示各种裂解物中脱乙酰酶活性的存在，并确定潜在的NDAC候选对象。为此，我将使用最近开发的独特的双底物类似物(乙酰辅酶A化学融合到多肽)或带有光反应交联剂的乙酰多肽来特异地结合和分离NatX和潜在的NDAC。此外，我将使用一个带有代谢性标记的100%N-末端乙酰化肽的全球肽库，并将其与具有潜在高脱乙酰酶活性的各种裂解物孵育。这之后将是高效液相色谱和质谱学方法，我将用来分析多肽的脱乙酰化部分。通过将这些肽类似物与上述裂解物孵育，我将能够使潜在的NDAC交联，分离它们并通过质谱学鉴定它们。总之，这些分析有望显著提高我们对NT-ac及其在许多重要的分子和(病理)生理过程中的调节功能的理解。

Obesity is one of several metabolic disorders, which is considered in our time as a major health issue in the EU and the Western world. Important regulators for metabolism, appetite, sexual behaviour and pain are the peptide hormones alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin (beta-END). Both hormones are regulated post-translationally by N-terminal acetylation (Nt-Ac). To date, six different N-terminal acetyltransferases (NatA-NatF) have been characterized in higher eukaryotes, responsible for co-translational acetylation of 80% of all proteins in humans. Nevertheless, the identity of the NAT (NatX) responsible for the modification of alpha-MSH and beta-END remains elusive. Identification of NatX would unravel the role and regulation of peptide hormones in obesity and several other important physiological processes that may be regulated by post-translational Nt-Ac. Furthermore, no mechanism of N-terminal deacetylation and no N-terminal deacetylase (NDAC) have been identified. Although, several indications render the possibility of the existence of NDAC(s), especially the fact that beta-END is stored in its biologically inactive form when acetylated, and thus has to be deacetylated to become active and be released. Further, recent findings demonstrate that Nt-Ac is dynamic in Saccharomyces cerevisiae upon alterations in growth. Hence, the major objectives of this project are to identify NatX as well as to reveal the existence of deacetylase activity in various lysates and to identify potential candidates for NDACs.For this purpose I will use recently developed unique bisubstrate analogues (acetyl coenzyme A chemically fused to peptides) or acetyl-peptides with photo-reactive crosslinkers to specifically bind and isolate NatX and potential NDACs. Further, I will use a global peptide library with metabolically labelled 100% N-terminal acetylated peptides and incubate it with various lysates that have a potentially high deacetylase activity. This will be followed by HPLC and mass spectrometry approaches with which I will analyse the peptides for a deacetylated moiety. By incubating these peptide analogues with the above mentioned lysates, I will be able to crosslink potential NDACs, isolate them and identify them by mass spectrometry. Altogether, these analyses hold the promise to significantly enhance our understanding of Nt-Ac and its regulatory function in so many important molecular and (patho-) physiological processes.