RESUMO
Sequential processing of H-Ras by protein farnesyl transferase (FTase), Ras converting enzyme (Rce1), and protein-S-isoprenylcysteine O-methyltransferase (Icmt) to give H-Ras C-terminal farnesyl-S-cysteine methyl ester is required for appropriate H-Ras membrane localization and function, including activation of the mitogen-activated protein kinase (MAPK) cascade. We employed a Xenopus laevis oocyte whole-cell model system to examine whether anilinogeranyl diphosphate analogues of similar shape and size, but with a hydrophobicity different from that of the FTase substrate farnesyl diphosphate (FPP), could ablate biological function of H-Ras. Analysis of oocyte maturation kinetics following microinjection of in vitro analogue-modified H-Ras into isoprenoid-depleted oocytes revealed that analogues with a hydrophobicity near that of FPP supported H-Ras biological function, while the analogues p-nitroanilinogeranyl diphosphate (p-NO2-AGPP), p-cyanoanilinogeranyl diphosphate (p-CN-AGPP), and isoxazolaminogeranyl diphosphate (Isox-GPP) with hydrophobicities 2-5 orders of magnitude lower than that of FPP did not. We found that although H-Ras modified with FPP analogues p-NO2-AGPP, p-CN-AGPP, and Isox-GPP was an efficient substrate for C-terminal postprenylation processing by Rce1 and Icmt, co-injection of H-Ras with analogues p-NO2-AGPP, p-CN-AGPP, or Isox-GPP could not activate MAPK. We propose that H-Ras biological function requires a minimum lipophilicity of the prenyl group to allow important interactions downstream of the C-terminal processed H-Ras protein. The hydrophilic FPP analogues p-NO2-AGPP, p-CN-AGPP, and Isox-GPP are H-Ras function inhibitors (RFIs) and serve as lead compounds for a unique class of potential anticancer therapeutics.
Assuntos
Proteína Oncogênica p21(ras)/antagonistas & inibidores , Proteína Oncogênica p21(ras)/fisiologia , Fosfatos de Poli-Isoprenil/farmacologia , Prenilação de Proteína/efeitos dos fármacos , Sesquiterpenos/farmacologia , Alquil e Aril Transferases/metabolismo , Animais , Catálise , Endopeptidases/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Metaloendopeptidases , Proteína Oncogênica p21(ras)/metabolismo , Oócitos/metabolismo , Fosfatos de Poli-Isoprenil/síntese química , Pró-Proteína Convertases , Proteínas Metiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/síntese química , Xenopus laevisRESUMO
As part of an effort to identify substrate analogs suitable for helping to resolve structural features important for terpene synthases, the inhibition of 5-epi-aristolochene biosynthesis from farnesyl diphosphate (FPP) by the tobacco 5-epi-aristolochene synthase incubated with anilinogeranyl diphosphate (AGPP) was examined. The apparent noncompetitive nature of the inhibition supported further assessment of how AGPP might be bound to crystallographic forms of the enzyme. Surprisingly, the bound form of the inhibitor appeared to have undergone a cyclization event consistent with the native mechanism associated with FPP catalysis. Biocatalytic formation of a novel 13-membered macrocyclic paracyclophane alkaloid was confirmed by high-resolution GC-MS and NMR analysis. This work provides insights into new biosynthetic means for generating novel, functionally diversified, medium-sized terpene alkaloids.
Assuntos
Alcaloides/metabolismo , Alquil e Aril Transferases/metabolismo , Compostos Macrocíclicos/metabolismo , Nicotiana/enzimologia , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos/metabolismo , Alcaloides/química , Alquil e Aril Transferases/química , Compostos Macrocíclicos/química , Modelos Moleculares , Fosfatos de Poli-Isoprenil/química , Sesquiterpenos/química , Terpenos/química , Terpenos/metabolismo , Nicotiana/química , Nicotiana/metabolismoRESUMO
Proteases are one of the largest and best-characterized families of enzymes in the human proteome. Unfortunately, the understanding of protease function in the context of complex proteolytic cascades remains in its infancy. One major reason for this gap in understanding is the lack of technologies that allow direct assessment of protease activity. We report here an optimized solid-phase synthesis protocol that allows rapid generation of activity-based probes (ABPs) targeting a range of cysteine protease families. These reagents selectively form covalent bonds with the active-site thiol of a cysteine protease, allowing direct biochemical profiling of protease activities in complex proteomes. We present a number of probes containing either a single amino acid or an extended peptide sequence that target caspases, legumains, gingipains and cathepsins. Biochemical studies using these reagents highlight their overall utility and provide insight into the biochemical functions of members of these protease families.
Assuntos
Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/química , Cetonas/química , Sondas Moleculares/química , Animais , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Humanos , Cetonas/síntese química , Técnicas de Sonda Molecular , Sondas Moleculares/síntese química , Especificidade por SubstratoRESUMO
Three isoprenoid diphosphate analogues of farnesyl diphosphate (FPP) where the diphosphate has been replaced by methylene diphosphonate and the negative charges masked by frangible pivaloyloxymethyl (POM) esters were prepared. Farnesyl methylenediphosphonate is a sub-micromolar substrate for protein farnesyl transferase. The tripivaloyloxymethyl esters of isoprenoid methylenediphosphonate have significantly increased lipophilicity and may act as important farnesyl diphosphate prodrugs.
Assuntos
Alquil e Aril Transferases/metabolismo , Fosfatos de Poli-Isoprenil/síntese química , Pró-Fármacos/síntese química , Farnesiltranstransferase , Fosfatos de Poli-Isoprenil/química , Pró-Fármacos/química , Sesquiterpenos , Solubilidade , Relação Estrutura-Atividade , Terpenos/síntese químicaRESUMO
Farnesylation is a posttranslational lipid modification in which a 15-carbon farnesyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized analogues (3-6) of farnesyl pyrophosphate (FPP) to probe the range of modifications possible to the FPP skeleton which allow for efficient transfer by FTase. Photoaffinity analogues of FPP (5, 6) were prepared by substituting perfluorophenyl azide functional groups for the omega-terminal isoprene of FPP. Substituted anilines replace the omega-terminal isoprene in analogues 3 and 4. Compounds 3-5 were prepared by reductive amination of the appropriate anilines with 8-oxo-geranyl acetate, followed by ester hydrolysis, chlorination, and pyrophosphorylation. Additional substitution of three methylenes for the beta-isoprene of FPP gave photoprobe 6 in nine steps. Preparation of the analogues required TiCl(4)-mediated imine formation prior to NaBH(OAc)(3) reduction for anilines with a pK(a) < 1. The azide moiety was not affected by Ph(3)PCl(2) conversion of allylic alcohols 13-16 into corresponding chlorides 17-20. Analogues 3-6 are efficiently transferred to target N-dansyl-GCVLS peptide substrate by mammalian FTase. Comparison of analogue structures and kinetics of transfer to those of FPP reveals that ring fluorination and para substituents have little effect on the affinity of the analogue pyrophosphate for FTase and its transfer efficiency. These results are also supported with models of the analogue binding modes in the active site of FTase. The transferable azide photoprobe 5 photoinactivates FTase. Transferable analogues 5 and 6 allow the formation of appropriately posttranslationally modified photoreactive peptide probes of isoprene function.