Reconstitution of TCP80/NF90 translation inhibition activity in insect cells.
Acid beta-glucosidase (GCase) is the enzyme deficient in Gaucher disease, an inherited metabolic prototype for enzyme and gene therapy. An 80-kDa mammalian cytoplasmic protein (TCP80/NF90) was discovered to interact with the GCase mRNA coding region and inhibit its translation in vitro and ex vivo. Human TCP80/NF90 is identical to NF90, an IL-2 enhancer protein, and MPP4, an M-phase phosphoprotein. The interaction of recombinant TCP80/NF90 with GCase mRNA was evaluated using the baculovirus/Sf9 insect cell system since these cells lack this protein. Purified recombinant and isolated mammalian cytoplasmic TCP80/NF90 had identical functions including binding of coding regions of selected RNAs and inhibition of their in vitro translation. Individual baculoviruses containing the human TCP80/NF90 cDNA (vSf9/TCP80) and GCase cDNA (vSf9/GCase) were used to co-infect Sf9 cells. The presence of preformed TCP80/NF90 significantly (>87%) inhibited wild-type GCase mRNA translation in these cells, but baculovirus containing a mutant GCase did not. Sf9 cells co-infected with vSf9/TCP80 showed a major reduction of GCase RNA polysome association. These results show that the multifunctional protein, TCP80/NF90, can function in vivo as a translation inhibitory protein and include alterations of mRNA binding to polysomes as a component of its mechanism of action.[1]References
- Reconstitution of TCP80/NF90 translation inhibition activity in insect cells. Xu, Y.H., Busald, C., Grabowski, G.A. Mol. Genet. Metab. (2000) [Pubmed]
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