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.
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The Children's Hospital Research Foundation, Cincinnati, Ohio 45229-3039, USA.
Busald
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Grabowski
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D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000502:physiology*; D015870:Gene Expression / Q000502:physiology; D050778:NFATC Transcription Factors; D050779:Nuclear Factor 90 Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000502:physiology*; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; C429643:ILF3 protein, human; D050778:NFATC Transcription Factors; D050779:Nuclear Factor 90 Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D001616:beta-Galactosidase