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Ubiquitin-RAS peptide extensions as substrates for farnesyl-protein transferase and carboxymethyltransferase.

ABSTRACT: Using oligonucleotide-mediated 'loop-in' mutagenesis strategies in M13, a heat-inducible ubiquitin (Ub) gene was extended by sequences coding for the C-terminal 11 amino acids of Ha-RAS. The resulting gene was transformed into AR13 and production of the Ub-peptide extension was induced by heat treatment. After one-step purification, the fusion protein (Ub-cRAS) was used as a substrate for farnesyl-protein transferase. Ub-cRAS was farnesylated on incubation in Xenopus egg extract or rabbit reticulocyte lysate. In contrast, when serine was substituted for the last cysteine in the RAS extension, transfer of the [3H]farnesyl group from [3H] farnesyl pyrophosphate to the modified Ub-cRAS was not observed. Farnesylation of Ub-cRAS permitted us to develop an easy membrane-binding assay for farnesyl-protein transferase enzyme activity. Using this assay, we partially purified the enzyme from rabbit reticulocyte lysate. We also detected methylation of the farnesylated Ub-cRAS terminus in Xenopus egg extract.

SUBMITTER: Yoo Y

PROVIDER: S-EPMC1132743 | biostudies-other | 1992 Jul

REPOSITORIES: biostudies-other

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Farnesyl diphosphate analogues with omega-bioorthogonal azide and alkyne functional groups for protein farnesyl transferase-catalyzed ligation reactions.

Project description:Eleven farnesyl diphosphate analogues, which contained omega-azide or alkyne substituents suitable for bioorthogonal Staudinger and Huisgen [3 + 2] cycloaddition coupling reactions, were synthesized. The analogues were evaluated as substrates for the alkylation of peptide cosubstrates by yeast protein farnesyl transferase. Five of the diphosphates were good alternative substrates for farnesyl diphosphate (FPP). Steady-state kinetic constants were measured for the active compounds, and the products were characterized by HPLC and LC-MS. Two of the analogues gave steady-state kinetic parameters (kcat and Km) very similar to those of the natural substrate.

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The effect of the farnesyl protein transferase inhibitor SCH66336 on isoprenylation and signalling by the prostacyclin receptor.

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O-GlcNAc transferase recognizes protein substrates using an asparagine ladder in the TPR superhelix

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2018-02-26 | GSE107911 | GEO

Protein isoprenylation regulates osteogenic differentiation of mesenchymal stem cells: effect of alendronate, and farnesyl and geranylgeranyl transferase inhibitors.

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| S-EPMC4784888 | biostudies-literature

"Depupylation" of prokaryotic ubiquitin-like protein from mycobacterial proteasome substrates.

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| S-EPMC2939144 | biostudies-literature

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