Lin Xiukun, Liu Jun, Maley Frank, Chu Edward
Department of Medicine and Pharmacology, Yale Cancer Center, Yale University School of Medicine and VA Connecticut Healthcare System, New Haven, CT 06520, USA.
Nucleic Acids Res. 2003 Aug 15;31(16):4882-7. doi: 10.1093/nar/gkg678.
The role of cysteine sulfhydryl residues on the RNA binding activity of human thymidylate synthase (TS) was investigated by mutating each cysteine residue on human TS to a corresponding alanine residue. Enzymatic activities of TS:C43A and TS:C210A mutant proteins were nearly identical to wild-type TS, while TS:C180A and TS:C199A mutants expressed >80% of wild-type enzyme activity. In contrast, TS:C195A was completely inactive. Mutant proteins, TS:C195A, TS:C199A and TS:C210A, retained RNA binding activity to nearly the same degree as wild-type human TS. RNA binding activity of TS:C43A was reduced by 30% when compared to wild-type TS, while TS:C180A was completely devoid of RNA binding activity. In vitro translation studies confirmed that mutant proteins TS:C43A, TS:C195A, TS:C199A and TS:C210A, significantly repressed human TS mRNA translation, while TS:C180A was unable to do so. To confirm the in vivo significance of the cysteine sulfhydryl residue, mutant proteins TS:C180A and TS:C195A were each expressed in human colon cancer HCT-C18:TS(-) cells that expressed a functionally inactive TS. A recombinant luciferase reporter gene under the control of a TS-response element was co-transfected into these same cells, and luciferase activity increased in the presence of the TS:C195A mutant TS protein to a level similar to that observed upon expression of wild-type TS protein. In contrast, luciferase activity remained unchanged in cells expressing the TS:C180A mutant protein. Taken together, these findings identify Cys-180 as a critical residue for the in vitro and in vivo translational regulatory effects of human TS.
通过将人胸苷酸合成酶（TS）上的每个半胱氨酸残基突变为相应的丙氨酸残基，研究了半胱氨酸巯基残基对人TS RNA结合活性的作用。TS:C43A和TS:C210A突变蛋白的酶活性与野生型TS几乎相同，而TS:C180A和TS:C199A突变体表达的酶活性>野生型酶活性的80%。相比之下，TS:C195A完全无活性。突变蛋白TS:C195A、TS:C199A和TS:C210A保留的RNA结合活性与野生型人TS几乎相同。与野生型TS相比，TS:C43A的RNA结合活性降低了30%，而TS:C180A完全没有RNA结合活性。体外翻译研究证实，突变蛋白TS:C43A、TS:C195A、TS:C199A和TS:C210A显著抑制人TS mRNA翻译，而TS:C180A则无法做到这一点。为了证实半胱氨酸巯基残基在体内的重要性，分别在表达功能失活TS的人结肠癌HCT-C18:TS(-)细胞中表达突变蛋白TS:C180A和TS:C195A。将受TS反应元件控制的重组荧光素酶报告基因共转染到这些相同的细胞中，在存在TS:C195A突变TS蛋白的情况下，荧光素酶活性增加到与野生型TS蛋白表达时观察到的水平相似。相比之下，在表达TS:C180A突变蛋白的细胞中，荧光素酶活性保持不变。综上所述，这些发现确定Cys-180是人类TS体外和体内翻译调节作用的关键残基。
