RESUMO
The ability to target DNA specifically at any given position within the genome allows many intriguing possibilities and has inspired scientists for decades. Early gene-targeting efforts exploited chemicals or DNA oligonucleotides to interfere with the DNA at a given location in order to inactivate a gene or to correct mutations. We here describe an example towards correcting a genetic mutation underlying Pompe's disease using a nucleotide-fused nuclease (TFO-MunI). In addition to the promise of gene correction, scientists soon realized that genes could be inactivated or even re-activated without inducing potentially harmful DNA damage by targeting transcriptional modulators to a particular gene. However, it proved difficult to fuse protein effector domains to the first generation of programmable DNA-binding agents. The engineering of gene-targeting proteins (zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs)) circumvented this problem. The disadvantage of protein-based gene targeting is that a fusion protein needs to be engineered for every locus. The recent introduction of CRISPR/Cas offers a flexible approach to target a (fusion) protein to the locus of interest using cheap designer RNA molecules. Many research groups now exploit this platform and the first human clinical trials have been initiated: CRISPR/Cas has kicked off a new era of gene targeting and is revolutionizing biomedical sciences.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.
Assuntos
DNA/química , Marcação de Genes , RNA/química , HumanosRESUMO
The introduction of mutation D119N (or its homolog) in the NKxD nucleotide binding motif of various Ras-like proteins produces constitutively activated or dominant-negative effects, depending on the system and assay. Here we show that Ras(D119N) has an inhibitory effect at a cell-specific concentration in PC12 and NIH 3T3 cells. Biochemical data strongly suggest that the predominant effect of mutation D119N in Ras-a strong decrease in nucleotide affinity-enables this mutant (i) to sequester its guanine nucleotide exchange factor, as well as (ii) to rapidly bind GTP, independent of the regulatory action of the exchange factor. Since mutation D119N does not affect the interaction between Ras and effector molecules, the latter effect causes Ras(D119N) to act as an activated Ras protein at concentrations higher than that of the exchange factor. In comparison, Ras(S17N), which also shows a strongly decreased nucleotide affinity, does not bind to effector molecules. These results point to two important prerequisites of dominant-negative Ras mutants: an increased relative affinity of the mutated Ras for the exchange factor over that for the nucleotide and an inability to interact with the effector or effectors. Remarkably, the introduction of a second, partial-loss-of-function, mutation turns Ras(D119N) into a strong dominant-negative mutant even at high concentrations, as demonstrated by the inhibitory effects of Ras(E37G/D119N) on nerve growth factor-mediated neurite outgrowth in PC12 cells and Ras(T35S/D119N) on fetal calf serum-mediated DNA synthesis in NIH 3T3 cells. Interpretations of these results are discussed.
Assuntos
Mutação Puntual , Proteínas ras/genética , Células 3T3 , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Ciclo Celular/metabolismo , Primers do DNA/genética , Regulação da Expressão Gênica , Genes Dominantes , Camundongos , Células PC12 , Ratos , Proteínas ras/metabolismo , ras-GRF1RESUMO
Ral is a ubiquitously expressed Ras-like small GTPase which is abundantly present in human platelets. The biological function of Ral and the signaling pathway in which Ral is involved are largely unknown. Here we describe a novel method to measure Ral activation utilizing the Ral binding domain of the putative Ral effector RLIP76 as an activation-specific probe. With this assay we investigated the signaling pathway that leads to Ral activation in human platelets. We found that Ral is rapidly activated after stimulation with various platelet agonists, including alpha-thrombin. In contrast, the platelet antagonist prostaglandin I2 inhibited alpha-thrombin-induced Ral activation. Activation of Ral by alpha-thrombin could be inhibited by depletion of intracellular Ca2+, whereas the induction of intracellular Ca2+ resulted in the activation of Ral. Our results show that Ral can be activated by extracellular stimuli. Furthermore, we show that increased levels of intracellular Ca2+ are sufficient for Ral activation in platelets. This activation mechanism correlates with the activation mechanism of the small GTPase Rap1, a putative upstream regulator of Ral guanine nucleotide exchange factors.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Plaquetas/enzimologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase , Ativação Plaquetária/fisiologia , Cálcio/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ativação Enzimática , Epoprostenol/farmacologia , Guanosina Trifosfato/metabolismo , Humanos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Ligação Proteica , Transdução de Sinais , Trombina/farmacologia , Proteínas ral de Ligação ao GTP , Proteínas rap de Ligação ao GTPRESUMO
Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand cytokine known for its cytotoxic activity against malignantly transformed cells. TRAIL induces cell death through binding to death receptors DR4 and DR5. The inhibitory decoy receptors (DcR1 and DcR2) co-expressed with death receptor 4 (DR4)/DR5 on the same cell can block the transmission of the apoptotic signal. Here, we show that DcRs also regulate TRAIL sensitivity at a supracellular level and thus represent a mechanism by which the microenvironment can diminish tumour TRAIL sensitivity. Mathematical modelling and layered or spheroid stroma-extracellular matrix-tumour cultures were used to model the tumour microenvironment. By engineering TRAIL to escape binding by DcRs, we found that DcRs do not only act in a cell-autonomous or cis-regulatory manner, but also exert trans-cellular regulation originating from stromal cells and affect tumour cells, highlighting the potent inhibitory effect of DcRs in the tumour tissue and the necessity of selective targeting of the two death-inducing TRAIL receptors to maximise efficacy.
Assuntos
Membro 10c de Receptores do Fator de Necrose Tumoral/metabolismo , Células Estromais/patologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Linhagem Celular Tumoral , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Membro 10c de Receptores do Fator de Necrose Tumoral/genética , Células Estromais/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores Chamariz do Fator de Necrose Tumoral/genéticaRESUMO
A catalytically active fragment of the Rap-specific guanine-nucleotide exchange factor C3G was expressed in E coli. It was purified and its interaction with GTP-binding proteins was investigated using fluorescence spectroscopy. C3G stimulates GDP dissociation from Rap1, but not from Rap2, neither from Bud1, which is believed to be the yeast homologue of Rap1 nor from all other proteins of the human Ras-subfamily. Like the corresponding fragment from CDC25Mm, the increase in the GDP dissociation rate is linear with increasing concentration of Rap1A x GDP up to 100 microM, indicating an apparent K(M) higher than 100 microM. Unlike the Ras-CDC25Mm system, the Rap1A(S17N) mutant does not inhibit the C3G-activated guanine nucleotide dissociation from wild-type Rap1A in vitro. These data suggest that Rap1A(S17N) is unlikely to titrate away C3G in vivo, the proposed mechanism by which S17N-mutants exert their dominant negative effects.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Escherichia coli/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Humanos , Mutação , Fragmentos de Peptídeos/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas/genética , Proteínas rap de Ligação ao GTP , Fatores ras de Troca de Nucleotídeo Guanina , ras-GRF1RESUMO
The D119N mutation of p21ras was prepared by site-directed mutagenesis. Its nucleotide binding properties were investigated using fluorescently labelled guanosine and xanthosine nucleotides. Its affinity for guanosine nucleotides is severely reduced, with a concomitant increase in the affinity for xanthosine nucleotides, which leads to an almost complete reversal of base specificity. The protein is a GTPase as well as a XTPase and the hydrolysis reaction can be efficiently stimulated by GAP. Dissociation of XDP from the mutant is stimulated by the guanine nucleotide exchange factor Cdc25Mm in a similar manner to that of GDP from wildtype. The interaction of the mutant with the effector domain of c-Raf kinase or Ral-GEF is normal. In microinjection experiments in PC12 and NIH3T3 cells the protein behaves as an oncogenic mutant due to its high dissociation rate for GDP. However, when the protein is loaded with XDP before microinjection the onset of the oncogenic signal can be efficiently retarded. Thus, the protein behaves initially as wildtype and later as an oncogenic protein.
Assuntos
Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ribonucleotídeos/metabolismo , Células 3T3 , Animais , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Camundongos , Microinjeções , Dados de Sequência Molecular , Mutação , Fosfoproteínas Fosfatases/metabolismo , Sensibilidade e Especificidade , ras-GRF1RESUMO
The small GTPase Rap 1A is a close relative of Ras that, when overexpressed, is able to revert oncogenic transformation induced by active Ras. We screened a mouse embryonic cDNA library using the yeast two-hybrid system and isolated the cDNA of a novel Rap 1A-interacting protein. The open reading frame encodes for an 84 kDa protein with a Cdc25-homology domain which shares approximately 30% identity with Ral guanine nucleotide dissociation stimulator (RalGDS) and RalGDS-like (Rg1). The C-terminal region reveals a striking conservation of sequences with the Ras-binding domain of RalGDS. We designated this protein Rlf, for RalGDS-like factor. In the yeast system, Rlf interacts with Rap 1A, H-Ras and R-Ras, but not with Rac and Rho. In addition, we found that Rlf interacts with Rap 1Aval12 but not with Rap 1AAsn17. In vitro binding studies revealed that a C-terminally located 91 amino acid region of Rlf is sufficient for direct association with the GTP-bound form of Ras and Rap 1A. The observed dissociation constants are 0.6 microM and 0.4 microM, respectively. No significant association with Ras-GDP or Rap 1A-GDP could be detected. These binding characteristics indicate that Rlf is a putative effector for Ras and Rap 1A.
Assuntos
DNA Complementar/genética , DNA Complementar/isolamento & purificação , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/isolamento & purificação , Fatores de Troca do Nucleotídeo Guanina , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Proteínas ras/genética , Proteínas ras/isolamento & purificação , ras-GRF1 , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Clonagem Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo , Fator ral de Troca do Nucleotídeo Guanina , Proteínas rap de Ligação ao GTP , Proteínas ras/metabolismoRESUMO
Mutagenesis was carried out in the N-terminal domain of elongation factor Tu (EF-Tu) to characterize the structure-function relationships of this model GTP binding protein with respect to stability, the interaction with GTP and GDP, and the catalytic activity. The substitutions were introduced in elements around the guanine nucleotide binding site or in the loops defining this site, in the intact molecule or in the isolated N-terminal domain (G domain). The double substitution Val88----Asp and Leu121----Lys, two residues situated on two vicinal alpha-helices, influences the basic activities of the truncated factor to a limited extent, probably via long-range interactions, and induces a destabilisation of the G domain structure. The functional alterations brought about by substitutions on the consensus sequences 18-24 and 80-83 highlight the importance of these residues for the interaction with GTP/GDP and the GTPase activity. Mutations concerning residues interacting with the guanine base lead to proteins in large part insoluble and inactive. In one case, the mutated protein (EF-TuAsn135----Asp) inhibited the growth of the host cell. This demonstrates the crucial role of the base specificity for the active conformation of EF-Tu. The obtained results are discussed in the light of the three-dimensional structure of EF-Tu.
Assuntos
Mutagênese Sítio-Dirigida , Fator Tu de Elongação de Peptídeos/metabolismo , Sequência de Aminoácidos , Ácido Aspártico , Sítios de Ligação , Gráficos por Computador , Escherichia coli/genética , Escherichia coli/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Leucina , Lisina , Modelos Moleculares , Dados de Sequência Molecular , Fator Tu de Elongação de Peptídeos/genética , Conformação Proteica , Termodinâmica , Ureia/farmacologia , ValinaRESUMO
Epac1 is a Rap-specific guanine-nucleotide exchange factor (GEF) which is activated by the binding of cAMP to a cyclic nucleotide monophosphate (cNMP)-binding domain. We investigated the equilibrium and dynamics of the interaction of cAMP and Epac1 using a newly designed fluorescence analogue of cAMP, 8-MABA-cAMP. We observed that the interaction of cAMP, measured by competition with 8-MABA-cAMP, with an isolated cNMP binding domain of Epac1 has an overall equilibrium constant (Kd) of 4 microM and that the kinetics of the interaction are highly dynamic. The binding properties of cAMP are apparently not affected when the catalytic domain is present, despite the fact that binding of cAMP results in activation of Epac1. This indicates that for the activation process, no appreciable binding energy is required. However, when bound to Rap1b, the apparent Kd of Epac to cAMP was about fivefold lower, suggesting that substrate interaction stabilizes cAMP binding. Since the fluorescent analogues used here were either less able or unable to induce activation of Epac1, we concluded that the binding of nucleotide to Epac and the activation of GEF activity are uncoupled processes and that thus appropriate cAMP analogues can be used as inhibitors of the Epac1-mediated signal transduction pathway of Rap.
Assuntos
AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sítios de Ligação , AMP Cíclico/análogos & derivados , Fluorescência , Produtos do Gene vpr/metabolismo , Vetores Genéticos , Glutationa Transferase/metabolismo , Marcação por Isótopo , Cinética , Estrutura MolecularRESUMO
The role of two residues within the catalytic domain of CDC25Mm, a mouse ras-specific guanine nucleotide exchange factor (GEF), was investigated by site-directed mutagenesis. The function of the mutant proteins was tested in vivo in both a Saccharomyces cerevisiae cdc25 complementation assay and in a mammalian fos-luciferase assay, and in in vitro assays on human and yeast Ras proteins. Mutants CDC25Mm(E1048K) and CDC25Mm(S1122V) were shown to be (partly) inactive proteins, similar to their yeast homologs. Mutant CDC25Mm(S1122A) showed higher nucleotide exchange activity than the wild type protein on the basis of both in vitro and in vivo assays. Thus, alanine and valine substitutions at position 1122 within the GEF catalytic domain originate mutations with opposite biological properties, indicating an important role for position 1122 in GEF function.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Células 3T3 , Animais , Células CHO , Domínio Catalítico , Proteínas de Ciclo Celular/genética , Cricetinae , Genes fos , Nucleotídeos de Guanina/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Fosfoproteínas Fosfatases/genética , Saccharomyces cerevisiae , Transfecção , Proteínas ras/metabolismo , ras-GRF1RESUMO
We have modified elongation factor Tu (EF-Tu) from Escherichia coli via mutagenesis of its encoding tufA gene to study its function-structure relationships. The isolation of the N-terminal half molecule of EF-Tu (G domain) has facilitated the analysis of the basic EF-Tu activities, since the G domain binds the substrate GTP/GDP, catalyzes the GTP hydrolysis and is not exposed to the allosteric constraints of the intact molecule. So far, the best studied region has been the guanine nucleotide-binding pocket defined by the consensus elements typical for the GTP-binding proteins. In this area most substitutions were carried out in the G domain and were found to influence GTP hydrolysis. In particular, the mutation VG20 (in both G domain and EF-Tu) decreases this activity and enhances the GDP to GTP exchange; PT82 induces autophosphorylation of Thr82 and HG84 strongly affects the GTPase without altering the interaction with the substrate. SD173, a residue interacting with (O)6 of the guanine, abolishes the GTP and GDP binding activity. Substitution of residues Gln114 and Glu117, located in the proximity of the GTP binding pocket, influences respectively the GTPase and the stability of the G domain, whereas the double replacement VD88/LK121, located on alpha-helices bordering the GTP-binding pocket, moderately reduces the stability of the G domain without greatly affecting GTPase and interaction with GTP(GDP). Concerning the effect of ligands, EF-TuVG20 supports a lower poly(Phe) synthesis but is more accurate than wild-type EF-Tu, probably due to a longer pausing on the ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fator Tu de Elongação de Peptídeos/genética , Sítios de Ligação , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/fisiologia , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/fisiologia , Relação Estrutura-AtividadeRESUMO
Actinomycetes have the genetic capability to synthesize many different biologically active secondary metabolites and of these compounds, antibiotics predominate in therapeutic and commercial importance. Intensive research often centres on the use of molecular techniques to investigate the physiology and genetics of antibiotic biosynthesis with a view to improving production. The isolation of clones of Streptomyces hygroscopicus, the producer of geldanamycin, which synthesizes geldanamycin in S. lividans, is reported. Molecular approaches using genes for elongation factors (tuf) were used in attempts to increase the fermentation yield of kirromycin, whilst probes for aphD and sph, genes for streptomycin phosphotransferases, were used to gather information on streptomycin genes in soil. Actinomycete populations in soil and earthworms may help in developing a strategy for discovering additional antimicrobials in soil. The relationship of proline metabolism to the secondary metabolite undecylprodigiosin and the carbon regulation of spiramycin biosynthesis in S. ambofaciens is also reported.
Assuntos
Actinomycetales/metabolismo , Antibacterianos/biossíntese , Streptomyces/metabolismo , Tobramicina/biossíntese , Resistência Microbiana a Medicamentos , Glicerol/farmacologia , Técnicas In Vitro , Lactamas Macrocíclicas , Prodigiosina/análogos & derivados , Prodigiosina/biossíntese , Piridonas/metabolismo , Espiramicina/biossíntese , Streptomyces/efeitos dos fármacosRESUMO
The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.
Assuntos
Antineoplásicos/química , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/química , Substituição de Aminoácidos , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Biologia Computacional , Feminino , Humanos , Cinética , Mutação , Neoplasias Ovarianas/tratamento farmacológico , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaAssuntos
Proteínas de Ciclo Celular/metabolismo , Nucleotídeos de Guanina/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Escherichia coli/genética , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Nucleotídeos de Guanina/síntese química , Nucleotídeos de Guanina/química , Técnicas In Vitro , Cinética , Camundongos , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/isolamento & purificação , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , ortoaminobenzoatos/síntese química , ortoaminobenzoatos/química , ortoaminobenzoatos/metabolismo , Fatores ras de Troca de Nucleotídeo Guanina , ras-GRF1RESUMO
Mutation of His84, a residue situated in one of the loops forming the guanine nucleotide binding pocket, was introduced in the G domain, the isolated N-terminal half molecule of bacterial elongation factor Tu (EF-Tu), in order to investigate the role of this residue on the basic activities of EF-Tu: the interaction with GDP and GTP and the hydrolysis of GTP. Substitution of His84 by Gly reduces the GTPase activity of the G domain to 5%; this activity can still be stimulated by raising the KCl concentration as the activity of wild-type G domain or the intact molecule. Since the affinities of the mutant protein for GDP and GTP are essentially the same as those of the wild-type G domain, His84 is apparently not involved in the binding of the substrates. Calculations of the change in free energy of activation of the GTPase reaction following substitution of His84 by Gly point to the disruption of a weak hydrogen bond, involved in the catalytic reaction. This probably concerns an interaction via a water molecule. The possible mechanism underlying the GTPase reaction is discussed in light of the three-dimensional structure of EF-Tu, taking into account the situation of Ha-ras p21.
Assuntos
Fatores de Elongação Ligados a GTP Fosfo-Hidrolases/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Fator Tu de Elongação de Peptídeos/fisiologia , Sítios de Ligação , Cátions Monovalentes/farmacologia , Clonagem Molecular , Gráficos por Computador , Análise Mutacional de DNA , Escherichia coli/fisiologia , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Histidina/química , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
The influence of the ionic environment on the intrinsic GTPase activity and the guanine-nucleotide interaction of Ha-ras protein p21 were studied in various experimental conditions and compared with the behaviour of elongation factor (EF) Tu. To this purpose, nucleotide-free p21 was prepared, which is much more stable than by any other reported method. Specific differences between p21 and EF-Tu were found in the action of divalent anions which strongly enhance the dissociation rate of p21.GDP without affecting that of EF-Tu. Unlike EF-Tu, the GTPase activity of p21 is only slightly dependent on the presence and concentration of monovalent cations. The concentrations of Mg2+ influencing the dissociation rate of the p21.GDP complex are much higher than for the intrinsic GTPase activity, an effect also observed for EF-Tu. These results point to two distinct roles of Mg2+: as a conformational regulator of the interaction with the substrate and as a key element for the hydrolysis of GTP. The GTPase activity of p21 is not affected by changes in pH over the range 6-9.2, different from that of EF-Tu. However, stabilization by kirromycin confers a pH independence to the GTPase of EF-Tu in the pH range 6.5-10, suggesting that the bell-shaped behaviour of this activity in the absence of the antibiotic is due to denaturation. This implies similar properties in the catalytic mechanism of these two guanine-nucleotide-binding proteins.
Assuntos
Íons , Fator Tu de Elongação de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Cloreto de Amônio/farmacologia , Sulfato de Amônio/farmacologia , Ânions , Cátions Bivalentes , Cátions Monovalentes , Fatores de Elongação Ligados a GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Difosfato/farmacologia , Guanosina Trifosfato/metabolismo , Concentração de Íons de Hidrogênio , Cloreto de Magnésio/farmacologiaRESUMO
Coat protein-protected fragments of alfalfa mosaic virus RNA (AlMV-RNA) and tobacco streak virus RNA (TSV-RNA), which were isolated as described [D. Zuidema, M. F. A. Bierhuizen, B. J. C. Cornelissen, J. F. Bol, and E. M. J. Jaspars (1983)Virology, 125, 361-369], were tested for their ability to rebind AlMV coat protein in the presence of an excess of Escherichia coli tRNA by means of a nitrocellulose filter retention assay. In order to obtain the minimum requirements for coat protein binding, a 3'-terminal binding site and several internal binding sites were isolated and fragmented by mild alkali treatment so that various lengths of a particular binding site were present in the mixture to be tested for rebinding capacity. All fragments which originated from the Wend of AlMV-RNA 1 and could bind AlMV coat protein have in common the sequence 5'-CUCAUGCUA-3'. However, this sequence alone is not sufficient to bind viral coat protein. Either an extension by at least 27 nucleotides of this oligomer to the right or an extension by 45 nucleotides (or possibly less) to the left is necessary for AlMV coat protein binding. Also, smaller extensions simultaneously occurring at both sides are sufficient. The smallest fragment which still has binding capacity for viral coat protein is 23 nucleotides long and originates from an internal site of RNA 1. All bound fragments have two common features: the occurrence of AUG(C) twice in the sequence and the potential ability to form a stable secondary structure. A striking observation was that 3'-terminal fragments of TSV-RNAs 1 and 2 rebind AlMV coat protein with low efficiency (about 27 and 37%, respectively), whereas a 3'-terminal fragment of TSV-RNA 3 rebinds AlMV coat protein with an efficiency of about 71%.
RESUMO
The inability of the S17N mutant of Rap1A to sequester the catalytic domain of the Rap guanine nucleotide exchange factor C3G (van den Berghe, N., Cool, R. H., Horn, G., and Wittinghofer, A. (1997) Oncogene 15, 845-850) prompted us to study possible fundamental differences in the way Rap1 interacts with C3G compared with the interaction of Ras with the catalytic domain of the mouse Ras guanine nucleotide exchange factor Cdc25(Mm). A variety of mutants in both Ras and Rap1A were designed, and both the C3G and Cdc25(Mm) catalyzed release of guanine nucleotide from these mutants was studied. In addition, we could identify regions in Rap2A that are responsible for the lack of recognition by C3G and induce high C3G activity by replacement of these residues with the corresponding Rap1A residues. The different Ras and Rap mutants showed that many residues were equally important for both C3G and Cdc25(Mm), suggesting that they interact similarly with their substrates. However, several residues were also identified to be important for the exchange reaction with only C3G (Leu70) or only Cdc25(Mm) (Gln61 and Tyr40). These results are discussed in the light of the structure of the Ras-Sos complex and suggest that some important differences in the interaction of Rap1 with C3G and Ras with Cdc25(Mm) indeed exist and that marker residues have been identified for the different structural requirements.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas/metabolismo , Proteínas ras/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Fatores de Troca do Nucleotídeo Guanina , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Proteínas/química , Proteínas/genética , Homologia de Sequência de Aminoácidos , Proteínas rap de Ligação ao GTP , Fatores ras de Troca de Nucleotídeo Guanina , Proteínas ras/química , Proteínas ras/genéticaRESUMO
In order to elucidate the components of the oxygen sensory complex in HepG2 cells which regulates the production of erythropoietin, we have microinjected recombinant variants of the human small GTP-binding protein hRac1 and measured their effects on the production of reactive oxygen species (ROS) by the dihydrorhodamine-123 technique. The dominant-negative mutant hRac1(T17N) inhibits the NADH-stimulated production of ROS in HepG2 cells, whereas the constitutively activated hRac1(G12V) leads to an increase in intracellular ROS concentration. Reverse transcriptase PCR analysis showed that the hRac1, but not the hRac2, gene is expressed in HepG2 cells. These results demonstrate that hRac1, and not hRac2, is involved in the regulation of ROS production in HepG2 cells and suggest that hRac1 specifically functions in the non-phagocytic NAD(P)H oxidase complex.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Peróxido de Hidrogênio/metabolismo , Proteínas de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Linfócitos/metabolismo , Microinjeções , NAD/farmacologia , NADH NADPH Oxirredutases/metabolismo , Proteínas de Neoplasias/fisiologia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Células Tumorais Cultivadas , Proteínas rac de Ligação ao GTPRESUMO
Mutation of Pro82 into Thr, a residue situated in the second element (D80CPG83) of the consensus sequence proposed to interact with GTP/GDP in GTP-binding proteins was introduced via site-directed mutagenesis in the isolated guanine nucleotide-binding domain (G domain) of elongation factor Tu. G domainPT82 displays virtually no GTPase activity. As a major change, the apparent inhibition of the GTPase reaction is associated with the appearance of autophosphorylating activity, as in ras product p21 in the case of mutation Ala59----Thr, corresponding to 82 in elongation factor Tu. Dependence of this reaction on mono- and divalent cation concentration and on pH is essentially the same as for the GTPase of wild-type G domain. The autokinase reaction follows an apparent first order rate, suggesting an intermolecular mechanism. Analysis of amino acid and peptide composition of the 32P-labeled G domainPT82, as well as Edman degradation of the tryptic peptide containing the covalently bound 32P, shows that Thr82 is the phosphorylated residue. Taken together, these results point out that Thr82 is in close proximity to the gamma-phosphate of GTP, as in the case of Thr59 in p21. These results are in agreement with the observations derived from x-ray diffraction analysis that the tertiary structure of the GTP-binding domain of elongation factor Tu and that of p21 are similar.