Mechanism of dominant-negative telomerase function.
Human telomerase uses its integral core components, hTR and hTERT, to maintain telomeres in many cell types. Expression of a dominant-negative mutant of the catalytic subunit of telomerase, DN-hTERT, has been shown to cause telomere shortening and ultimately cell death in a number of tumor-derived cell lines. However, the mechanism of dominant-negative hTERT function and its fate inside the cell are still unknown. In order to understand the effect of the dominant-negative on wild-type hTERT, each was fused with GFP and expressed in telomerase-positive cells. GFP-DN-hTERT expression resulted in cytoplasmic exportation and degradation via ubiquitination. Co-expression of wild-type GFP-hTERT with an untagged DN-hTERT resulted in decreased wild-type hTERT levels, export to the cytoplasm, and increased ubiquitination, suggesting that DN-hTERT complexes with wild-type hTERT to induce cytoplasmic localization. Based on the cytoplasmic degradation, we propose two new mechanisms of dominant-negative hTERT, employing the theory of interactive dimerization. First, the heterodimer of DN-hTERT with wild-type hTERT is exported to the cytoplasm for ubiquitin-mediated protein degradation, and second, the heterodimer may be degraded at a faster rate than the wild-type hTERT homodimer. Understanding mechanisms of telomerase degradation will guide future drug design to target sites on telomerase important for catalytic activity and protein stability.
Nguyen
Binh N
BN
Department of Pathology, Medical College of Virginia at Virginia Commonwealth University, Richmond, VA, USA.
Elmore
Lynne W
LW
Holt
Shawn E
SE
D017209:Apoptosis; D020134:Catalytic Domain; D006801:Humans; D009154:Mutation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D019098:Telomerase / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D016615:Telomere / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination
D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D019098:Telomerase