Human Gpn1 purified from bacteria binds guanine nucleotides and hydrolyzes GTP as a protein dimer stabilized by its C-terminal tail

González‐González, Rogelio; Guerra-Moreno, José A.; Cristóbal‐Mondragón, Gema R.; Romero, Violeta; Peña-Gómez, Sonia G.; Montero-Morán, Gabriela M.; Lara-González, Samuel; Hernández‐Arana, Andrés; Fernández‐Velasco, D. Alejandro; Calera, Mónica R.; Sánchez‐Olea, Roberto

doi:10.1016/j.pep.2017.01.009

Cited by 3 publications

(6 citation statements)

References 20 publications

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“…To test the effect of W132D and M227D mutations on Gpn1 structure and function, we purified both W132D and M227D HisGpn1 mutants as recombinant proteins expressed in bacteria, and compared their ability to hydrolyze GTP with that of wild‐type HisGpn1. W132D and M227D HisGpn1 mutants were successfully purified with the same method as wild‐type HisGpn1 , first by affinity chromatography and then by size in a gel filtration column. W132D and M227D HisGpn1 mutants formed mainly homodimers (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The single GPN protein present in Archaea is able to form homodimers , which is likely enough to support the assembly of the RNA polymerase in these organisms. Interestingly, although recombinant human Gpn1 is also purified as a homodimer , the available experimental evidence does not support Gpn2 homodimer formation . How eukaryotic cells regulate the ability of the GPN proteins to form homo‐ versus heterodimers, as well as the kind of heterodimer, that is, Gpn1–Gpn2, Gpn2–Gpn3or Gpn1‐Gpn3, is an interesting question that needs to be further investigated if we want to truly understand the role played by this family of essential GTPases in RNAPII assembly.…”

Section: Discussionmentioning

confidence: 99%

“…The intrinsic GTPase activity of recombinant HisGpn1 purified from bacteria was determined semiquantitatively by luminescence in enzymatic assays using the GTPase‐Glo assay kit (Promega, Madison, WI, USA), as previously described . Briefly, the GTPase reaction was carried out at a protein concentration of 5 μM of the different HisGpn1 versions, including the wild‐type and the M227D or W132D HisGpn1 mutants, 5 μM GTP in the kit′s GTPase/GAP buffer containing 1 m m DTT.…”

Section: Methodsmentioning

confidence: 99%

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FRET‐based analysis and molecular modeling of the human GPN‐loop GTPases 1 and 3 heterodimer unveils a dominant‐negative protein complex

et al. 2019

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GPN‐loop GTPases 1 and 3 are required for RNA polymerase II (RNAPII) nuclear import. Gpn1 and Gpn3 display some sequence similarity, physically associate, and their protein expression levels are mutually dependent in human cells. We performed here Fluorescence Resonance Energy Transfer (FRET), molecular modeling, and cell biology experiments to understand, and eventually disrupt, human Gpn1–Gpn3 interaction in live HEK293‐AD cells. Transiently expressed EYFP‐Gpn1 and Gpn3‐CFP generated a strong FRET signal, indicative of a very close proximity, in the cytoplasm of HEK293‐AD cells. Molecular modeling of the human Gpn1–Gpn3 heterodimer based on the crystallographic structure of Npa3, the Saccharomyces cerevisiae Gpn1 ortholog, revealed that human Gpn1 and Gpn3 associate through a large interaction surface formed by internal α‐helix 7, insertion 2, and the GPN‐loop from each protein. In site‐directed mutagenesis experiments of interface residues, we identified the W132D and M227D EYFP‐Gpn1 mutants as defective to produce a FRET signal when coexpressed with Gpn3‐CFP. Simultaneous but not individual expression of Gpn1 and Gpn3, with either or both proteins fused to EYFP, retained RNAPII in the cytoplasm and markedly inhibited global transcription in HEK293‐AD cells. Interestingly, the W132D and M227D Gpn1 mutants that showed an impaired ability to interact with Gpn3 by FRET were also unable to delocalize RNAPII in this assay, indicating that an intact Gpn1–Gpn3 interaction is required to display the dominant‐negative effect on endogenous Gpn1/Gpn3 function we described here. Altogether, our results suggest that a Gpn1–Gpn3 strong interaction is critical for their cellular function.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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FRET‐based analysis and molecular modeling of the human GPN‐loop GTPases 1 and 3 heterodimer unveils a dominant‐negative protein complex

et al. 2019

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“…There have been identified a 24 kDa GTP-binding protein in etiolated seedlings of oat (Avena sativa) by using western blot analysis using anti-G antibodies, GTP-binding assay and ADP ribosylation with cholera toxin. This protein is smaller than any known heterotrimeric G protein subunits (Chen et al, 1998;González-González et al, 2017) identified two small GTP-binding proteins (28 and 30 kDa) in rice coleoptile using anti-G antibodies and a filter GTP-binding assay. Recognized aleurone protoplasts of 22 and 24 kDa in barley by using anti-ras antibodies.…”

Section: Detection Of Gtp-binding Proteinsmentioning

confidence: 96%

Era-like GTP protein gene expression in rice

et al. 2022

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The mutations are genetic changes in the genome sequences and have a significant role in biotechnology, genetics, and molecular biology even to find out the genome sequences of a cell DNA along with the viral RNA sequencing. The mutations are the alterations in DNA that may be natural or spontaneous and induced due to biochemical reactions or radiations which damage cell DNA. There is another cause of mutations which is known as transposons or jumping genes which can change their position in the genome during meiosis or DNA replication. The transposable elements can induce by self in the genome due to cellular and molecular mechanisms including hypermutation which caused the localization of transposable elements to move within the genome. The use of induced mutations for studying the mutagenesis in crop plants is very common as well as a promising method for screening crop plants with new and enhanced traits for the improvement of yield and production. The utilization of insertional mutations through transposons or jumping genes usually generates stable mutant alleles which are mostly tagged for the presence or absence of jumping genes or transposable elements. The transposable elements may be used for the identification of mutated genes in crop plants and even for the stable insertion of transposable elements in mutated crop plants. The guanine nucleotide-binding (GTP) proteins have an important role in inducing tolerance in rice plants to combat abiotic stress conditions.

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“…Genes encoding the human GPN2 protein (hGPN2) were obtained from the human cDNA library Open Biosystems, cloned together with a group of six histidines (hexahistidines) into the vector pET28-DL3, and inserted into E. coli bacteria following the methodology proposed by Gonzalez [40].…”

Section: Construction Of the Expression Vector For Hgpn2mentioning

confidence: 99%

Development of a Methodology to Adapt an Equilibrium Buffer/Wash Applied to the Purification of hGPN2 Protein Expressed in Escherichia coli Using an IMAC Immobilized Metal Affinity Chromatography System

et al. 2022

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Protein purification is a complex and non-standardized process; the fact that proteins have different structural types making it difficult to create a standard methodology to obtain them in a pure, soluble, and homogeneous form. The present study shows the selective development of a buffer suitable for proteins of interest that allows high concentrations of hGPN2 protein to be obtained with low polydispersion and high homogeneity and purity. By taking the different reagents used in the construction of different buffers as a basis and performing purifications using different additives in different concentrations to determine the optimal amounts, the developed process helps to minimize the bonds, maintain solubility, release the proteins present in inclusion bodies, and provide an adequate environment for obtaining high concentrations of pure protein. GPN proteins are of unknown function, have not been purified in high concentrations, and have been found as part of the RNA polymerase assembly; if they are not expressed, the cell dies, and overexpression of certain GPN proteins has been linked to decreased survival in patients with invasive ductal carcinoma breast cancer types ER+ and HER2+. The results of the present study show that the use of the buffer developed for recombinant hGPN2 protein expressed in Escherichia coli could be manipulated in order to isolate the protein in a totally pure form and without the use of protease inhibitor tablets. The resulting homogeneity and low polydispersion was corroborated by studies carried out using dynamic dispersion analysis. Thanks to these properties, it can be used for crystallography or structural genomics studies.

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Human Gpn1 purified from bacteria binds guanine nucleotides and hydrolyzes GTP as a protein dimer stabilized by its C-terminal tail

Cited by 3 publications

References 20 publications

FRET‐based analysis and molecular modeling of the human GPN‐loop GTPases 1 and 3 heterodimer unveils a dominant‐negative protein complex

FRET‐based analysis and molecular modeling of the human GPN‐loop GTPases 1 and 3 heterodimer unveils a dominant‐negative protein complex

Era-like GTP protein gene expression in rice

Development of a Methodology to Adapt an Equilibrium Buffer/Wash Applied to the Purification of hGPN2 Protein Expressed in Escherichia coli Using an IMAC Immobilized Metal Affinity Chromatography System

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