Short-chain lipid peroxidation products form covalent adducts with pyruvate kinase and inhibit its activity in vitro and in breast cancer cells.
Short-chain lipid peroxidation products form covalent adducts with pyruvate kinase and inhibit its activity in vitro and in breast cancer cells.
复制标题
DOI：
10.1016/j.freeradbiomed.2019.05.028
复制
发表时间：
2019-11
影响因子：
7.4
通讯作者：
B. Sousa;Tanzim Ahmed;W. L. Dann;Jed Ashman;A. Guy;T. Durand;A. Pitt;C. Spickett
中科院分区：
文献类型：
--
作者：
B. Sousa;Tanzim Ahmed;W. L. Dann;Jed Ashman;A. Guy;T. Durand;A. Pitt;C. Spickett
丙酮酸激酶催化糖酵解的最后一步，并被认为有助于调节癌细胞中的有氧糖酵解。在体外和体内，半胱氨酸残留物的氧化可以抑制它，这与癌症和增殖组织中更有利于氧化的状态有关。这些条件也有利于脂质过氧化和形成亲电裂解产物，包括可以共价修饰蛋白质的短链醛。然而，到目前为止还没有关于它们与丙酮酸激酶相互作用的研究，所以我们研究了丙烯醛、丙二醛和4-羟基-2(E)-己烯醛(HHE)三种不同的醛对酶的结构和活性的影响。LC-MS/MS分析显示，每种醛都有独特的修饰图谱，但Cys152、Cys423和Cys474是最容易发生亲电修饰的残基。在此条件下的酶活性分析表明，丙烯醛是最强的抑制剂，当孵育时间大于2 h时，病理生理浓度对酶活性有显著影响。用醛处理MCF-7 细胞后，丙酮酸激酶活性的丧失与体外观察到的相似，且浓度低于导致细胞死亡所需的浓度，具有时间和剂量依赖性；在Cys152和Cys358上检测到丙烯醛加合物。Cys358和Cys474位于变构或活性中心或附近，在这些残基上形成加合物可能是低处理浓度下活性丧失的原因之一。本研究首次详细分析了C3和C6醛与丙酮酸激酶的构效关系，并提示氧化病因性疾病或吸烟等环境暴露所产生的反应性短链醛可能通过改变丙酮酸激酶活性而参与癌细胞的代谢变化。
Pyruvate kinase catalyses the last step in glycolysis and has been suggested to contribute to the regulation of aerobic glycolysis in cancer cells. It can be inhibited by oxidation of cysteine residuesin vitroandin vivo, which is relevant to the more pro-oxidant state in cancer and proliferating tissues. These conditions also favour lipid peroxidation and the formation of electrophilic fragmentation products, including short-chain aldehydes that can covalently modify proteins. However, as yet few studies have investigated their interactions with pyruvate kinase, so we investigated the effects of three different aldehydes, acrolein, malondialdehyde and 4-hydroxy-2(E)-hexenal (HHE), on the structure and activity of the enzyme. Analysis by LC-MS/MS showed unique modification profiles for each aldehyde, but Cys152, Cys423 and Cys474 were the residues most susceptible to electrophilic modification. Analysis of enzymatic activity under these conditions showed that acrolein was the strongest inhibitor, and at incubation times longer than 2 h, pathophysiological concentrations induced significant effects. Treatment of MCF-7 cells with the aldehydes caused similar losses of pyruvate kinase activity to those observedin vitro, and at lower concentrations than those required to cause cell death, with time and dose-dependent effects; acrolein adducts on Cys152 and Cys358 were detected. Cys358 and Cys474 are located at or near the allosteric or active sites, and formation of adducts on these residues probably contributes to loss of activity at low treatment concentrations. This study provides the first detailed analysis of the structure-activity relationship of C3 and C6 aldehydes with pyruvate kinase, and suggests that reactive short-chain aldehydes generated in diseases with an oxidative aetiology or from environmental exposure such as smoking could be involved in the metabolic alterations observed in cancer cells, through alteration of pyruvate kinase activity.