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，这在我们这个时代被认为是欧盟和西方世界的主要健康问题。代谢、食欲、性行为和疼痛的重要调节剂是肽激素α-黑素细胞刺激激素（α-MSH）和β-内啡肽（β-END）。这两种激素都是通过N-末端乙酰化（Nt-Ac）进行调节的。到目前为止，六种不同的N-末端乙酰转移酶（NatA-NatF）已在高等真核生物中被鉴定，负责人类80%的蛋白质的共翻译乙酰化。然而，负责修饰α-MSH和β-END的NAT（NatX）的身份仍然难以捉摸。NatX的鉴定将揭示肽激素在肥胖和其他几个重要的生理过程中的作用和调节，这些过程可能由翻译后Nt-Ac调节。此外，没有N-末端脱乙酰化的机制和N-末端脱乙酰酶（NDAC）已被确定。虽然，一些迹象表明存在NDAC的可能性，特别是β-END在乙酰化时以其生物学无活性形式储存，因此必须脱乙酰化才能变得有活性并释放。此外，最近的研究结果表明，Nt-Ac是动态的酿酒酵母在生长的改变。因此，本研究的主要目的是鉴定NatX，揭示不同裂解液中存在的脱乙酰酶活性，并鉴定潜在的NDAC候选物。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与肽的化学融合）或乙酰肽与光反应交联剂特异性结合并分离NatX和潜在的NDAC。此外，我将使用具有代谢标记的100% N-末端乙酰化肽的全局肽文库，并将其与具有潜在高脱乙酰酶活性的各种裂解物一起孵育。随后将采用HPLC和质谱法分析肽的脱乙酰基部分。通过将这些肽类似物与上述裂解物一起孵育，我将能够交联潜在的NDAC，分离它们并通过质谱法鉴定它们。总之，这些分析持有的承诺，显着提高我们的理解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。

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.