In Vitro Assays to Characterize Inhibitors of the Activation of Small G Proteins by Their Guanine Nucleotide Exchange Factors

Zeeh, Jean-Christophe; Antonny, Bruno; Cherfils, Jacqueline; Zeghouf, Mahel

doi:10.1016/s0076-6879(07)38004-x

Cited by 8 publications

(18 citation statements)

References 48 publications

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“…The protein was essentially bound to GTP as estimated by tryptophan fluorescence. 31 Depending on the protein preparation, cleavage of the initiator methionine was found to be between 88% and 100%, as measured by Edman degradation or mass spectrometry (see Fig. 3A), thus making it …”

Section: Resultsmentioning

confidence: 99%

“…The nucleotide content of purified recombinant Arf6 was assessed by monitoring the change in tryptophan fluorescence (λ exc = 298nm, λ em = 340 nm) following addition of GDP or GTP in the presence of EDTA (5mM), which takes advantage of the large difference in fluorescence between the GDP-and GTP-bound forms of Arf proteins. 31 No fluorescence change was observed upon addition of 100 μM GTP, whereas there was a large fluorescence decrease upon addition of 100 μM GDP, indicating that bacteriallyexpressed Arf6 is mostly GTP-bound. Arf6•GDP was obtained by incubating Arf6•GTP (0.1-0.5 mM) in the presence of 5 mM GDP in 50 mM TRIS-HCl, pH 8.0, 5mM EDTA, 100 mM NaCl, 1 mM DTT for 40 min at 37°C.…”

Section: Disclosure Of Potential Conflicts Of Interestmentioning

confidence: 97%

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High yield production of myristoylated Arf6 small GTPase by recombinant N-myristoyl transferase

et al. 2013

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Section: Resultsmentioning

confidence: 99%

Section: Disclosure Of Potential Conflicts Of Interestmentioning

confidence: 97%

High yield production of myristoylated Arf6 small GTPase by recombinant N-myristoyl transferase

et al. 2013

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“…Several human diseases involve G-protein up regulation [4]. Specific inhibition of GEF action has thus attracted significant attention, with the G-protein-GEF interface itself as a potential therapeutic target [3], [5]–[8].…”

Section: Introductionmentioning

confidence: 99%

A Computational Study of a Recreated G Protein-GEF Reaction Intermediate Competent for Nucleotide Exchange: Fate of the Mg Ion

Hamida-Rebaï

Robert

2010

PLoS ONE

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Small G-proteins of the superfamily Ras function as molecular switches, interacting with different cellular partners according to their activation state. G-protein activation involves the dissociation of bound GDP and its replacement by GTP, in an exchange reaction that is accelerated and regulated in the cell by guanine-nucleotide exchange factors (GEFs). Large conformational changes accompany the exchange reaction, and our understanding of the mechanism is correspondingly incomplete. However, much knowledge has been derived from structural studies of blocked or inactive mutant GEFs, which presumably closely represent intermediates in the exchange reaction and yet which are by design incompetent for carrying out the nucleotide exchange reaction. In this study we have used comparative modelling to recreate an exchange-competent form of a late, pre-GDP-ejection intermediate species in Arf1, a well-characterized small G-protein. We extensively characterized three distinct models of this intermediate using molecular dynamics simulations, allowing us to address ambiguities related to the mutant structural studies. We observed in particular the unfavorable nature of Mg associated forms of the complex and the establishment of closer Arf1-GEF contacts in its absence. The results of this study shed light on GEF-mediated activation of this small G protein and on predicting the fate of the Mg ion at a critical point in the exchange reaction. The structural models themselves furnish additional targets for interfacial inhibitor design, a promising direction for exploring potentially druggable targets with high biological specificity.

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“…The exchange factors have also been treated as enzymes. 14 , 22 - 27 Here, we discuss the analysis of the exchange factors as enzymes as a tractable approach, which may also be used to elucidate fundamental kinetic properties of the GEF-GTPase pairs. The GEFs are appropriately considered enzymes: they accelerate the conversion of G•GDP to G•GTP and are not consumed by the reaction.…”

Section: Introductionmentioning

confidence: 99%

“…The advantages of considering the exchange factor as an enzyme is that the relationship to the generation of the biologically relevant product G•GTP is obvious and the experimental approach is tractable for all G protein/exchange factors as far as we are aware. We also discuss some pitfalls of the approach and encourage readers to refer to Goody 14 for a discussion of pitfalls of kinetic experiments and Zeeh et al 22 for additional explanation of the enzyme formalism for exchange factors. We believe that the approach considering the GEFs as enzymes make quantitative characterizations of GEFs, and comparisons between them, accessible to a wide number of researchers and will provide results that are readily interpreted in terms of the regulated biological process.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Analysis of Guanine Nucleotide Exchange Factors (GEFs) as Enzymes

Randazzo¹,

Jian²,

Chen³

et al. 2013

Cellular Logistics

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The proteins that possess guanine nucleotide exchange factor (GEF) activity, which include about ~800 G protein coupled receptors (GPCRs),1 15 Arf GEFs,2 81 Rho GEFs,3 8 Ras GEFs,4 and others for other families of GTPases,5 catalyze the exchange of GTP for GDP on all regulatory guanine nucleotide binding proteins. Despite their importance as catalysts, relatively few exchange factors (we are aware of only eight for ras superfamily members) have been rigorously characterized kinetically.5–13 In some cases, kinetic analysis has been simplistic leading to erroneous conclusions about mechanism (as discussed in a recent review14). In this paper, we compare two approaches for determining the kinetic properties of exchange factors: (i) examining individual equilibria, and; (ii) analyzing the exchange factors as enzymes. Each approach, when thoughtfully used,14,15 provides important mechanistic information about the exchange factors. The analysis as enzymes is described in further detail. With the focus on the production of the biologically relevant guanine nucleotide binding protein complexed with GTP (G•GTP), we believe it is conceptually simpler to connect the kinetic properties to cellular effects. Further, the experiments are often more tractable than those used to analyze the equilibrium system and, therefore, more widely accessible to scientists interested in the function of exchange factors.

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In Vitro Assays to Characterize Inhibitors of the Activation of Small G Proteins by Their Guanine Nucleotide Exchange Factors

Cited by 8 publications

References 48 publications

High yield production of myristoylated Arf6 small GTPase by recombinant N-myristoyl transferase

High yield production of myristoylated Arf6 small GTPase by recombinant N-myristoyl transferase

A Computational Study of a Recreated G Protein-GEF Reaction Intermediate Competent for Nucleotide Exchange: Fate of the Mg Ion

Quantitative Analysis of Guanine Nucleotide Exchange Factors (GEFs) as Enzymes

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