RESUMEN

Rab proteins are a large family of GTP-binding proteins that regulate cellular membrane traffic and organelle identity. Rab proteins cycle between association with membranes and binding to RabGDI. Bound on membranes, each Rab has a very specific cellular location and it is this remarkable degree of specificity with which Rab GTPases recognize distinct subsets of intracellular membranes that forms the basis of their ability to act as key cellular regulators, determining the recruitment of downstream effectors to the correct membrane at the correct time. The molecular mechanisms controlling Rab localization remain poorly understood. Here, we present a fluorescence-based assay to investigate Rab GTPase membrane extraction and delivery by RabGDI. Using EGFP-Rab fusion proteins the amount of Rab:GDI complex obtained by GDI extraction of Rab proteins from HEK293 membranes could be determined, enabling control of complex concentration. Subsequent partitioning of the Rab GTPases into vesicles made up of artificial binary lipid mixtures showed for the first time, that the composition of the target membrane plays a key role in the localization of Rab proteins by sensing the stored curvature elastic energy in the membrane.

Asunto(s)

Membrana Celular/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Membrana Celular/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Lípidos de la Membrana/genética , Unión Proteica/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab5/genética

RESUMEN

3-(3-Pyridyl)-2-hydroxy-2-phosphonopropanoic acid (3-PEHPC, 1) is a phosphonocarboxylate (PC) analogue of 2-(3-pyridyl)-1-hydroxyethylidenebis(phosphonic acid) (risedronic acid, 2), an osteoporosis drug that decreases bone resorption by inhibiting farnesyl pyrophosphate synthase (FPPS) in osteoclasts, preventing protein prenylation. 1 has lower bone affinity than 2 and weakly inhibits Rab geranylgeranyl transferase (RGGT), selectively preventing prenylation of Rab GTPases. We report here the synthesis and biological studies of 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid (3-IPEHPC, 3), the PC analogue of minodronic acid 4. Like 1, 3 selectively inhibited Rab11 vs. Rap 1A prenylation in J774 cells, and decreased cell viability, but was 33-60x more active in these assays. After resolving 3 by chiral HPLC (>98% ee), we found that (+)-3-E1 was much more potent than (-)-3-E2 in an isolated RGGT inhibition assay, approximately 17x more potent (LED 3 microM) than (-)-3-E2 in inhibiting Rab prenylation in J774 cells and >26x more active in the cell viability assay. The enantiomers of 1 exhibited a 4-fold or smaller potency difference in the RGGT and prenylation inhibition assays.

Asunto(s)

Transferasas Alquil y Aril/antagonistas & inhibidores , Lactatos/farmacología , Organofosfonatos/farmacología , Compuestos Organofosforados/farmacología , Conservadores de la Densidad Ósea/síntesis química , Conservadores de la Densidad Ósea/farmacología , Resorción Ósea/tratamiento farmacológico , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Ácido Etidrónico/análogos & derivados , Ácido Etidrónico/farmacología , Ácido Etidrónico/uso terapéutico , Humanos , Lactatos/síntesis química , Lactatos/uso terapéutico , Organofosfonatos/síntesis química , Organofosfonatos/uso terapéutico , Compuestos Organofosforados/síntesis química , Compuestos Organofosforados/uso terapéutico , Osteoporosis/tratamiento farmacológico , Prenilación de Proteína/efectos de los fármacos , Ácido Risedrónico , Estereoisomerismo , Proteínas de Unión al GTP rab

Asunto(s)

Proteínas Recombinantes/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular , Colorantes Fluorescentes/química , Humanos , Lovastatina/análogos & derivados , Lovastatina/farmacología , Prenilación de Proteína , Coloración y Etiquetado , Proteínas de Unión al GTP rab/metabolismo

RESUMEN

PURPOSE: Mutations of the CHM gene underlie the X-linked chorioretinal degeneration choroideremia (CHM). The affected gene product, Rab Escort Protein (REP)1, mediates the posttranslational prenyl modification of Rab GTPases. In patients with CHM, the related REP2 partially compensates for the loss of function of REP1. The objective of this investigation was to study the natural history of disease in a zebrafish model of CHM. METHODS: Zebrafish chm(-/-) were bred and subjected to extensive histologic analysis and TUNEL assays, and cellular extracts were used for immunoblot and in vitro prenylation assays. A detailed evolutionary analysis was performed on the REP family. RESULTS: The retina of chm(-/-) zebrafish develops normally for the first 4 days postfertilization (dpf) but that catastrophic multilayer degeneration synchronous with severe multisystem disease follows. Mean survival time is 4.8 dpf. At the onset of generalized disease, a significant reduction in rep expression levels and activity, with unprenylated rabs accumulating in the cytosol was demonstrated. Extensive bioinformatic analysis of the REP family of proteins revealed a single rep isoform in fish and other nonmammalian vertebrates and invertebrates that is similar to mammalian REP1. CONCLUSIONS: REP1 appears to be the ancestral gene in the family, whereas the intronless REP2 gene is restricted to the mammalian lineage. The results of this study propose that in chm(-/-) zebrafish, maternally derived rep allows initial successful development of the embryo, but its gradual loss leads to multisystem disease and invariably to lethality. In its current form, the chm(-/-) zebrafish has limited usefulness.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/genética , Coroideremia/genética , Pérdida del Embrión/genética , Embrión no Mamífero/patología , Genes Letales , Retina/embriología , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Apoptosis , Coroideremia/patología , Modelos Animales de Enfermedad , Desarrollo Embrionario/genética , Evolución Molecular , Immunoblotting , Etiquetado Corte-Fin in Situ , Fenotipo , Células Fotorreceptoras de Vertebrados/patología , Prenilación de Proteína , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Fracciones Subcelulares , Pez Cebra/embriología , Proteínas de Pez Cebra , Proteínas de Unión al GTP rab/metabolismo

RESUMEN

Rab geranylgeranyl transferase (RGGT) catalyzes the post-translational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((+)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC50 and Ki.(+)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (+)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by only a single GG addition. The presence of (+)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases.

Asunto(s)

Transferasas Alquil y Aril/antagonistas & inhibidores , Difosfonatos/farmacología , Inhibidores Enzimáticos/farmacología , Fosfatos de Poliisoprenilo/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Piridinas/farmacología , Proteínas de Unión al GTP rab/metabolismo , Transferasas Alquil y Aril/metabolismo , Secuencias de Aminoácidos/fisiología , Animales , Sitios de Unión/fisiología , Línea Celular , Perros , Humanos , Estructura Terciaria de Proteína/fisiología

RESUMEN

Prenylation (or geranylgeranylation) of Rab GTPases is catalysed by RGGT (Rab geranylgeranyl transferase) and requires REP (Rab escort protein). In the classical pathway, REP associates first with unprenylated Rab, which is then prenylated by RGGT. In the alternative pathway, REP associates first with RGGT; this complex then binds and prenylates Rab proteins. In the present paper we show that REP mutants defective in RGGT binding (REP1 F282L and REP1 F282L/V290F) are unable to compete with wild-type REP in the prenylation reaction in vitro. When over-expressed in cells, REP wild-type and mutants are unable to form stable cytosolic complexes with endogenous unprenylated Rabs. These results suggest that the alternative pathway may predominate in vivo. We also extend previous suggestions that GGPP (geranylgeranyl pyrophosphate) acts as an allosteric regulator of the prenylation reaction. We observed that REP-RGGT complexes are formed in vivo and are unstable in the absence of intracellular GGPP. RGGT increases the ability of REP to extract endogenous prenylated Rabs from membranes in vitro by stabilizing a soluble REP-RGGT-Rab-GG (geranylgeranylated Rab) complex. This effect is regulated by GGPP, which promotes the dissociation of RGGT and REP-Rab-GG to allow delivery of prenylated Rabs to membranes.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transferasas Alquil y Aril/metabolismo , Fosfatos de Poliisoprenilo/farmacología , Prenilación/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Regulación Alostérica , Animales , Células Cultivadas , Humanos , Cinética , Complejos Multienzimáticos/efectos de los fármacos , Complejos Multienzimáticos/metabolismo , Mutación , Proteínas Recombinantes/metabolismo , Spodoptera , Proteína de Unión al GTP rab3A/metabolismo

RESUMEN

Rab GTPases regulate discrete steps in vesicular transport pathways. Rabs require activation by specific guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP. Rab27a controls motility and regulated exocytosis of secretory granules and related organelles. In melanocytes, Rab27a regulates peripheral transport of mature melanosomes by recruiting melanophilin and myosin Va. Here, we studied the activation of Rab27a in melanocytes. We identify Rab3GEP, previously isolated as a GEF for Rab3a, as the non-redundant Rab27a GEF. Similar to Rab27a-deficient ashen melanocytes, Rab3GEP-depleted cells show both clustering of melanosomes in the perinuclear area and loss of the Rab27a effector Mlph. Consistent with a role as an activator, levels of Rab27a-GTP are decreased in cells lacking Rab3GEP. Recombinant Rab3GEP exhibits guanine nucleotide exchange activity against Rab27a and Rab27b in vitro, in addition to its previously documented activity against Rab3. Our results indicate promiscuity in Rab GEF action and suggest that members of related but functionally distinct Rab subfamilies can be controlled by common activators.

Asunto(s)

Guanosina Difosfato/química , Guanosina Trifosfato/química , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Animales , Línea Celular , Humanos , Melanocitos/metabolismo , Melanosomas/metabolismo , Ratones , Modelos Biológicos , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Proteínas rab27 de Unión a GTP

RESUMEN

Isoprenylcysteine carboxyl methyltransferase (Icmt) catalyzes the methylation of the C-terminal prenylcysteine found on prenylated proteins. Numerous studies have shown that the methylation step is important for the correct localization and function of many prenylated proteins, most notably GTPases in the Ras superfamily. We recently reported identification of a small molecule derived from an indole core as a potent, cell-active inhibitor of Icmt whose potency was increased upon preincubation with the enzyme [Winter-Vann, A. M., Baron, R. A., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102 (12), 4336-41]. In the study presented here, we performed a kinetic characterization of this time-dependent inhibition of Icmt by 2-[5-(3-methylphenyl)-1-octyl-1H-indol-3-yl]acetamide (cysmethynil). These analyses revealed that cysmethynil is a competitive inhibitor with respect to the isoprenylated cysteine substrate and a noncompetitive inhibitor with respect to AdoMet, the methyl donor in the reaction. The Ki of cysmethynil for Icmt, which represents the dissociation constant of the initial complex with the enzyme, was 2.39 +/- 0.02 microM, and the Ki*, which is the overall dissociation constant of the inhibitor for the final complex, was 0.14 +/- 0.01 microM. The first-order rate constant for the conversion of the initial enzyme-inhibitor complex to the final high-affinity complex was 0.87 +/- 0.06 min-1, and that for the reverse process was 0.053 +/- 0.003 min-1; the latter rate constant corresponds to a half-life for the high-affinity complex of 15 min. Structure-activity relationships of a number of closely related indole compounds revealed that the hydrophobicity of the substituent on the nitrogen of the indole core was responsible for the manifestation of time-dependent inhibition. These findings markedly enhance our understanding of the mechanism of inhibition of Icmt by this indole class of compounds and should facilitate ongoing efforts to assess the potential of targeting this enzyme in anticancer drug design.

Asunto(s)

Proteína Metiltransferasas/antagonistas & inhibidores , Endopeptidasas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Indoles/química , Indoles/farmacología , Cinética , Proteína Metiltransferasas/metabolismo , Prenilación de Proteína , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo

RESUMEN

Many key regulatory proteins, including members of the Ras family of GTPases, are modified at their C terminus by a process termed prenylation. This processing is initiated by the addition of an isoprenoid lipid, and the proteins are further modified by a proteolytic event and methylation of the C-terminal prenylcysteine. Although the biological consequences of prenylation have been characterized extensively, the contributions of prenylcysteine methylation to the functions of the modified proteins are not well understood. This reaction is catalyzed by the enzyme isoprenylcysteine carboxyl methyltransferase (Icmt). Recent genetic disruption studies have provided strong evidence that blocking Icmt activity has profound consequences on oncogenic transformation. Here, we report the identification of a selective small-molecule inhibitor of Icmt, 2-[5-(3-methylphenyl)-1-octyl-1H-indol-3-yl]acetamide (cysmethynil). Cysmethynil treatment results in inhibition of cell growth in an Icmt-dependent fashion, demonstrating mechanism-based activity of the compound. Treatment of cancer cells with cysmethynil results in mislocalization of Ras and impaired epidermal growth factor signaling. In a human colon cancer cell line, cysmethynil treatment blocks anchorage-independent growth, and this effect is reversed by overexpression of Icmt. These findings provide a compelling rationale for development of Icmt inhibitors as another approach to anticancer drug development.

Asunto(s)

Acetamidas/farmacología , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Proteína Metiltransferasas/antagonistas & inhibidores , Acetamidas/química , Animales , Antineoplásicos/química , División Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Perros , Inhibidores Enzimáticos/química , Humanos , Ratones , Fenotipo , Proteína Metiltransferasas/deficiencia , Proteína Metiltransferasas/genética , Proteína Metiltransferasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas ras/metabolismo

RESUMEN

BACKGROUND: Isoprenylcysteine carboxyl methyltransferase (Icmt) is the third of three enzymes that posttranslationally modify proteins that contain C-terminal CaaX motifs. The processing of CaaX proteins through this so-called prenylation pathway via a route initiated by addition of an isoprenoid lipid is required for both membrane targeting and function of the proteins. The involvement of many CaaX proteins such as Ras GTPases in oncogenesis and other aberrant proliferative disorders has led to the targeting of the enzymes involved in their processing for therapeutic development, necessitating a detailed understanding of the mechanisms of the enzymes. RESULTS: In this study, we have investigated the kinetic mechanism of recombinant human Icmt. In the reaction catalyzed by Icmt, S-adenosyl-L-methionine (AdoMet) provides the methyl group that is transferred to the second substrate, the C-terminal isoprenylated cysteine residue of a CaaX protein, thereby generating a C-terminal prenylcysteine methyl ester on the protein. To facilitate the kinetic analysis of Icmt, we synthesized a new small molecule substrate of the enzyme, biotin-S-farnesyl-L-cysteine (BFC). Initial kinetic analysis of Icmt suggested a sequential mechanism for the enzyme that was further analyzed using a dead end competitive inhibitor, S-farnesylthioacetic acid (FTA). Inhibition by FTA was competitive with respect to BFC and uncompetitive with respect to AdoMet, indicating an ordered mechanism with SAM binding first. To investigate the order of product dissociation, product inhibition studies were undertaken with S-adenosyl-L-homocysteine (AdoHcy) and the N-acetyl-S-farnesyl-L-cysteine methylester (AFCME). This analysis indicated that AdoHcy is a competitive inhibitor with respect to AdoMet, while AFCME shows a noncompetitive inhibition with respect to BFC and a mixed-type inhibition with respect to AdoMet. These studies established that AdoHcy is the final product released, and that BFC and AFCME bind to different forms of the enzyme. CONCLUSIONS: These studies establish that catalysis by human Icmt proceeds through an ordered sequential mechanism and provide a kinetic framework for analysis of specific inhibitors of this key enzyme.

Asunto(s)

Proteína Metiltransferasas/análisis , Proteína Metiltransferasas/farmacocinética , Animales , Línea Celular , Humanos , Cinética , Unión Proteica/fisiología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/farmacocinética