NatF contributes to an evolutionary shift in protein N-terminal acetylation and is important for normal chromosome segregation.
NatF contributes to an evolutionary shift in protein N-terminal acetylation and is important for normal chromosome segregation.
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DOI：
10.1371/journal.pgen.1002169
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发表时间：
2011-07
期刊：
影响因子：
4.5
通讯作者：
Arnesen T
中科院分区：
文献类型：
--
作者：
Van Damme P;Hole K;Pimenta-Marques A;Helsens K;Vandekerckhove J;Martinho RG;Gevaert K;Arnesen T
n端乙酰化（N-Ac）是一种丰富的真核蛋白修饰。蛋白质组学显示，从低级真核生物到高级真核生物，N-Ac的发生显著增加，但目前缺乏解释其潜在分子机制的证据。我们首先分析了蛋白质n -末端及其乙酰化程度，表明底物的进化不是N-Ac进化转变的主要原因。此外，我们研究了高等真核生物中n端乙酰转移酶（NATs）的存在。纯化的重组人与果蝇同源物对一种新的NAT候选物进行了体外肽库乙酰化分析。这为其靶向Met-Lys-和其他met起始蛋白n -末端的NAT活性提供了证据，该酶被命名为Naa60p，其活性为NatF。通过在酵母中异位表达人Naa60p，并进行n端COFRADIC分析，研究了其体内活性。hNaa60p乙酰化了不同的met起始酵母蛋白n端，并增加了乙酰化水平，从而改变了酵母体内的乙酰化模式，使其向高等真核生物的乙酰化模式转变。此外，其在人类细胞中的活性通过hNAA60的过表达和敲低以及n端COFRADIC得到验证。在果蝇细胞中证实了NatF的细胞影响，其中NAA60敲低诱导染色体分离缺陷。总之，我们的研究揭示了一种新的主要蛋白质修饰剂，有助于N-Ac的进化，NATs之间的冗余，以及正常染色体分离的重要调节因子。随着NatF的表征，共翻译N-Ac机制似乎是完整的，因为真核生物中所有主要的底物群都被考虑在内。小的化学基团通常附着在蛋白质上，以控制它们的活性、定位和稳定性。一种丰富的蛋白质修饰是n端乙酰化，其中n端乙酰转移酶（NAT）催化乙酰基转移到蛋白质的n端氨基酸上。当从低级真核生物到高级真核生物时，n端乙酰化的发生显著增加。我们在这里证明，这部分是因为高等真核生物独特地表达NatF，一种能够乙酰化一大群蛋白质n -末端的酶，包括那些先前发现在高等真核生物中显示出增加的n -乙酰化潜力的酶。因此，目前的研究可能已经确定了真核生物机制中负责蛋白质共翻译n -乙酰化的最后一个主要组成部分。所有真核生物的蛋白质都是从蛋氨酸开始的，当第二个氨基酸很小时，蛋氨酸被共翻译切割。随后，NatA可能将这些新暴露的n端乙酰化。有趣的是，NatF也有可能作用于这些类型的n末端蛋氨酸没有被切割。在细胞水平上，我们进一步发现NatF在细胞分裂过程中对正常染色体分离至关重要。
N-terminal acetylation (N-Ac) is a highly abundant eukaryotic protein modification. Proteomics revealed a significant increase in the occurrence of N-Ac from lower to higher eukaryotes, but evidence explaining the underlying molecular mechanism(s) is currently lacking. We first analysed protein N-termini and their acetylation degrees, suggesting that evolution of substrates is not a major cause for the evolutionary shift in N-Ac. Further, we investigated the presence of putative N-terminal acetyltransferases (NATs) in higher eukaryotes. The purified recombinant human and Drosophila homologues of a novel NAT candidate was subjected to in vitro peptide library acetylation assays. This provided evidence for its NAT activity targeting Met-Lys- and other Met-starting protein N-termini, and the enzyme was termed Naa60p and its activity NatF. Its in vivo activity was investigated by ectopically expressing human Naa60p in yeast followed by N-terminal COFRADIC analyses. hNaa60p acetylated distinct Met-starting yeast protein N-termini and increased general acetylation levels, thereby altering yeast in vivo acetylation patterns towards those of higher eukaryotes. Further, its activity in human cells was verified by overexpression and knockdown of hNAA60 followed by N-terminal COFRADIC. NatF's cellular impact was demonstrated in Drosophila cells where NAA60 knockdown induced chromosomal segregation defects. In summary, our study revealed a novel major protein modifier contributing to the evolution of N-Ac, redundancy among NATs, and an essential regulator of normal chromosome segregation. With the characterization of NatF, the co-translational N-Ac machinery appears complete since all the major substrate groups in eukaryotes are accounted for. Small chemical groups are commonly attached to proteins in order to control their activity, localization, and stability. An abundant protein modification is N-terminal acetylation, in which an N-terminal acetyltransferase (NAT) catalyzes the transfer of an acetyl group to the very N-terminal amino acid of the protein. When going from lower to higher eukaryotes there is a significant increase in the occurrence of N-terminal acetylation. We demonstrate here that this is partly because higher eukaryotes uniquely express NatF, an enzyme capable of acetylating a large group of protein N-termini including those previously found to display an increased N-acetylation potential in higher eukaryotes. Thus, the current study has possibly identified the last major component of the eukaryotic machinery responsible for co-translational N-acetylation of proteins. All eukaryotic proteins start with methionine, which is co-translationally cleaved when the second amino acid is small. Thereafter, NatA may acetylate these newly exposed N-termini. Interestingly, NatF also has the potential to act on these types of N-termini where the methionine was not cleaved. At the cellular level, we further found that NatF is essential for normal chromosome segregation during cell division.
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