National Institute of Chemistry, Hajdrihova 19, Ljubljana, Slovenia.
National Institute of Chemistry, Hajdrihova 19, Ljubljana, Slovenia.
Biochim Biophys Acta Proteins Proteom. 2018 May-Jun;1866(5-6):602-607. doi: 10.1016/j.bbapap.2018.03.005. Epub 2018 Mar 18.
Cancer cells consume more glucose than normal human cells and convert most glucose into lactate. It has been proposed that deregulated glycolysis is triggered by the posttranslational modification of 85 kDa muscle-type 6-phosphofructo-1-kinase (PFK-M) which is cleaved by a specific protease to form shorter, highly active, feedback-inhibition-resistant PFK-M fragments.
To find the protease involved in PFK-M modification, analyses of the protease target sites on the human PFK-M enzyme yielding 45-47 kDa fragments were performed in silico. The results suggested that an enzyme in the kallikrein (KLK) family may be involved. Kallikreins can be self-activated in the cytosol and are often overexpressed in cancer cells. After incubating the internally quenched FRET peptide with a sequence characteristic of the target site, along with the active KLK6, the cleavage of the peptide was observed. The ability of KLK6 to cleave native PFK-M and form highly active citrate-resistant 45 kDa fragments was further confirmed by enzymatic tests and SDS-PAGE. A role of KLK6 in the posttranslational modification of native PFK-M was ultimately confirmed in vivo. A yeast strain that encoded native human PFK-M as the only PFK1 enzyme was additionally transformed with proKLK6 or KLK6 genes under the control of an inducible promoter. The transformants growth rate was found to increase after the induction of proKLK6 gene expression as compared to the strain with the native PFK-M enzyme.
KLK6 may be the key protease involved in the modification of PFK-M and trigger deregulated glycolytic flux in cancer cells.
癌细胞比正常人类细胞消耗更多的葡萄糖，并将大部分葡萄糖转化为乳酸。有人提出，85 kDa 肌型 6-磷酸果糖-1-激酶（PFK-M）的翻译后修饰触发了糖酵解的失调，该酶被特定的蛋白酶切割形成较短的、高度活跃的、反馈抑制抗性的 PFK-M 片段。
为了找到参与 PFK-M 修饰的蛋白酶，对产生 45-47 kDa 片段的人 PFK-M 酶的蛋白酶靶位进行了计算机分析。结果表明，激肽释放酶（KLK）家族中的一种酶可能参与其中。激肽释放酶可以在细胞质中自我激活，并且在癌细胞中常常过度表达。在用具有靶位特征的序列的内部猝灭 FRET 肽孵育后，观察到肽的切割。用 KLK6 酶孵育后，通过酶试验和 SDS-PAGE 进一步证实了 KLK6 切割天然 PFK-M 并形成高度活跃的柠檬酸抗性 45 kDa 片段的能力。KLK6 在天然 PFK-M 的翻译后修饰中的作用最终在体内得到了证实。酵母菌株，其编码天然人 PFK-M 作为唯一的 PFK1 酶，此外还被编码 proKLK6 或 KLK6 基因的质粒转化，这些基因受诱导型启动子的控制。与含有天然 PFK-M 酶的菌株相比，在诱导 proKLK6 基因表达后，转化体的生长速度增加。
KLK6 可能是参与 PFK-M 修饰的关键蛋白酶，并触发癌细胞中失调的糖酵解通量。
