| Summary: | 碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 100 === Thymidylate kinase catalyzes the phosphrylation of dTMP to form dTDP. It is a critical step for dTTP synthesis in either de novo pathway or salvage pathway. Previously, our laboratory found that TMPK knockdown is not lethal for cell growth, but the combination of doxorubicin administration and TMPK knockdown leads to synthetic lethality. In addition, TMPK can be recruited to the DNA damage site for DNA repair and an effective TMPK inhibitor (YMU1) is identified for chemosensitization. In this study, I used the yeast system to assess three questions that have been evoked from the previous studies. First, if there is a TMPK isoform in mammalian cells to support growth when the major TMPK expression has been silenced. Second, can the yeast system be used for TMPK inhibitor in vivo screening? Third, is there any TMPK specific interacting protein to influence its subcellular distribution?
In this study, expression of human TMPK effectively rescues the growth of cdc8 temperature sensitive yeast strain. In contrast, a putative mitochondrial TMPK, Loc129607, does not improve the growth. It indicates that the protein coded by Loc129607 does not give sufficient TMPK activity for growth. I also generated a strain of cdc8 temperature sensitive yeast deleted of ERG6 to improve cell permeability for drug screening at non-permissive temperature (30℃) and proved that YMU1 treatment can inhibit the growth of cdc8 temperature sensitive yeast expressing human TMPK. A number of YMU1 related compounds were also tested and their effects on growth inhibition on cdc8 temperature sensitive yeast were found to correlate with their in vitro TMPK inhibition ability. Lastly, I used a yeast 2-hybrid system to screen TMPK interaction protein. Five proteins are identified to be potential candidates as hTMPK interacting protein. One of the 5 candidates is UMP-CMP kinase 1(CMPK1). By co-expression of Flag-CMPK1 and TMPK-GFP and co- immunoprecipitation experiment, a small fraction of TMPK-GFP was found to interact with CMPK1.
In conclusion, three different aspects of research in human TMPK by using the yeast system were developed in this study.
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