Yu Bess Yi Kun, Tossounian Maria-Armineh, Hristov Stefan Denchev, Lawrence Ryan, Arora Pallavi, Tsuchiya Yugo, Peak-Chew Sew Yeu, Filonenko Valeriy, Oxenford Sally, Angell Richard, Gouge Jerome, Skehel Mark, Gout Ivan
Department of Structural and Molecular Biology, University College London, London, WC1E 6BT, United Kingdom.
MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, CB2 0QH, United Kingdom.
Redox Biol. 2021 Aug;44:101978. doi: 10.1016/j.redox.2021.101978. Epub 2021 Apr 15.
The metastasis suppressor protein NME1 is an evolutionarily conserved and multifunctional enzyme that plays an important role in suppressing the invasion and metastasis of tumour cells. The nucleoside diphosphate kinase (NDPK) activity of NME1 is well recognized in balancing the intracellular pools of nucleotide diphosphates and triphosphates to regulate cytoskeletal rearrangement and cell motility, endocytosis, intracellular trafficking, and metastasis. In addition, NME1 was found to function as a protein-histidine kinase, 3'-5' exonuclease and geranyl/farnesyl pyrophosphate kinase. These diverse cellular functions are regulated at the level of expression, post-translational modifications, and regulatory interactions. The NDPK activity of NME1 has been shown to be inhibited in vitro and in vivo under oxidative stress, and the inhibitory effect mediated via redox-sensitive cysteine residues. In this study, affinity purification followed by mass spectrometric analysis revealed NME1 to be a major coenzyme A (CoA) binding protein in cultured cells and rat tissues. NME1 is also found covalently modified by CoA (CoAlation) at Cys109 in the CoAlome analysis of HEK293/Pank1β cells treated with the disulfide-stress inducer, diamide. Further analysis showed that recombinant NME1 is efficiently CoAlated in vitro and in cellular response to oxidising agents and metabolic stress. In vitro CoAlation of recombinant wild type NME1, but not the C109A mutant, results in the inhibition of its NDPK activity. Moreover, CoA also functions as a competitive inhibitor of the NME1 NDPK activity by binding non-covalently to the nucleotide binding site. Taken together, our data reveal metastasis suppressor protein NME1 as a novel binding partner of the key metabolic regulator CoA, which inhibits its nucleoside diphosphate kinase activity via non-covalent and covalent interactions.
转移抑制蛋白NME1是一种进化上保守的多功能酶，在抑制肿瘤细胞的侵袭和转移中发挥重要作用。NME1的核苷二磷酸激酶（NDPK）活性在平衡细胞内核苷酸二磷酸和三磷酸的池以调节细胞骨架重排和细胞运动、内吞作用、细胞内运输及转移方面已得到充分认识。此外，发现NME1还具有蛋白组氨酸激酶、3'-5'核酸外切酶和香叶基/法尼基焦磷酸激酶的功能。这些多样的细胞功能在表达水平、翻译后修饰及调节相互作用层面受到调控。已表明在氧化应激下，NME1的NDPK活性在体外和体内均受到抑制，且这种抑制作用是通过对氧化还原敏感的半胱氨酸残基介导的。在本研究中，亲和纯化后进行质谱分析显示，NME1是培养细胞和大鼠组织中的一种主要辅酶A（CoA）结合蛋白。在用二硫键应激诱导剂二酰胺处理的HEK293/Pank1β细胞的CoAlome分析中，还发现NME1在Cys109处被CoA共价修饰（CoAlation）。进一步分析表明，重组NME1在体外以及对氧化剂和代谢应激的细胞反应中能有效地进行CoAlation。重组野生型NME1而非C109A突变体的体外CoAlation会导致其NDPK活性受到抑制。此外，CoA还通过非共价结合到核苷酸结合位点而作为NME1 NDPK活性竞赛性抑制剂发挥作用。综上所述，我们的数据揭示转移抑制蛋白NME1是关键代谢调节因子CoA的一种新型结合伴侣，其通过非共价和共价相互作用抑制核苷二磷酸激酶活性。
