GTP-binding Motifs Research Articles

Comprehensive analyses of annexins in naked carp (Gymnocypris przewalskii) unveil their roles in saline-alkaline stress

Naked carp (Gymnocypris przewalskii) is a migratory fish endemic to the Qinghai Lake, the largest inland saline-alkaline lake in China. Based on our previous results of comparative transcriptome sequencing, we found that pathways related to regulation of actin cytoskeleton, plasma membrane damage, and inflammation-related signaling were significantly enriched during saline-alkaline stress, suggesting involvement of annexin (ANN) family proteins in stress adaption in naked carp. Herein, to explore the role of annexin family in naked carp under saline-alkaline stress, twenty GpANNs family members were identified at the genome-wide scale, and bioinformatics and expression analysis under saline-alkaline stress were performed. The predicted subcellular localization showed that most members of GpANNs were distributed in the cytoplasm, and the rest were localized in the cytoskeleton, nucleus, or mitochondria, respectively. GpANNs family proteins contained specific type II Ca2+-binding motifs, actin-binding motifs, GTP-binding motifs, etc. Phylogenetic tree analysis showed that the annexin homologues from naked carp, carp (Cyprinus carpio), zebrafish (Danio rerio), and chinese cavefish (Sinocyclocheilus rhinocerous) were divided into eight groups, i.e. ANN1 ∼ 6, ANN11 and ANN13. But homologues of ANN7 ∼ 10 groups were not found in these three fish species. Twenty-eight, nineteen and twenty-nine orthologous gene pairs were identified between naked carp and carp, zebrafish and chinese cavefish, respectively. Six paralogous gene pairs were identified in naked carp, all of which had a Ka/Ks ratio less than one, suggesting that the GpANNs family may have undergone purifying selection pressure during evolution. Expression of GpANN3 was significantly up-regulated in the kidney tissue under saline-alkaline stress, while GpANN2 was significantly down-regulated. Similar trends of protein expression were also observed by Western blot, suggesting involvement of GpANN3 and GpANN2 in stress response. Confocal microscopy revealed that GpANN2 was localized in both cytoplasm and nucleus, but GpANN3 was preferentially localized in the cytoplasm. Our study demonstrated the role of annexins in stress adaption, and provided the basic information required for further function analyses in naked carp.

Structural and functional investigation of mycobacterial HflX protein and its mutational hotspots annotation by in silico approach

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains a critical burden on global health. GTP binding proteins play a considerable role in the cell signaling processes of Mtb. The present manuscript elaborates important aspects of a mycobacterial gene Rv2725c (HflX), a probable GTP binding protein. The HflX protein contains four GTP binding motifs, which postulates its role in GTP binding activity. In silico assessment of the HflX protein was done by various computational tools. A homology modelling perspective has been employed to define the tertiary structure of the protein using the SWISS-MODEL server and further validated by the RAMPAGE server. In addition, secondary structure, protein-protein and protein-chemical interactions, phosphorylation site prediction, ligand binding sites, molecular docking and mutational investigations were performed to give more recognition to HflX. Mycobacterial HflX protein is predicted to be present in the cytoplasmic compartment, interacts with the ribosome in a GTP-dependent manner, and helps in translational processes. Hence, the present study provides a way to understand its normal functioning and basic biology, which can enhance the information for developing novel therapeutic targeting approaches for HflX protein.

A myxovirus resistance like protein involved in CgIFNLP mediated immune response of oyster Crassostrea gigas

The myxovirus resistance (Mx) proteins belong to interferon (IFN)-induced dynamin GTPase and play a pivotal role in the inhibition of replication of numerous viruses. In the present study, an Mx homologue (designated as CgMx1) was identified from oyster Crassostrea gigas. The open reading frame (ORF) of CgMx1 cDNA was of 1689 bp encoding a peptide of 562 amino acid residues. There was an N-terminal dynamin GTPase domain in the predicted peptide, which consisted of a tripartite GTP-binding motif (GDXXSGKS, DLPG and T/NKXD). The deduced amino acid sequence of CgMx1 shared 30–39% similarity with other Mx family members. And CgMx1 was clustered with Mx from H. discus, and then assigned into the invertebrate branch of the phylogenetic tree. The mRNA transcripts of CgMx1 were constitutively distributed in all the tested tissues, with the highest level in haemocytes (1342.45-fold of labial palps, p < 0.05). The mRNA expression of CgMx1 in haemocytes was significantly up-regulated to the highest level at 6 h (13.14-fold, p < 0.001) after poly (I:C) treatment and at 24 h (66.28-fold, p < 0.001) after recombinant IFN-like protein (rCgIFNLP) stimulation, respectively. CgMx1 protein was found to distribute in both the cytoplasm and nucleus of haemocytes. In the oysters with CgIFNLP and signal transducer and activator of transcription (CgSTAT) silenced by RNAi, the mRNA expression of CgMx1 decreased significantly in the haemocytes at 12 h after poly (I:C) stimulation, which was 0.02-fold and 0.04-fold of that in EGFP-RNAi oysters (p < 0.001), respectively. Meanwhile, EMSA assay revealed that CgSTAT was able to transactivate CgMx1 promoter through directly binding to its interferon-stimulated response element (ISRE) and gamma interferon activation site (GAS). The above results indicated that CgMx1 participated in the immune response of C. gigas through the signal pathway mediated by CgIFNLP and CgSTAT.

Mobility of Nucleostemin in Live Cells Is Specifically Related to Transcription Inhibition by Actinomycin D and GTP-Binding Motif.

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility.

Essential biochemical, biophysical and computational inputs on efficient functioning of Mycobacterium tuberculosis H37Rv FtsY

Mycobacterium tuberculosis (M. tuberculosis H37Rv) utilizes the signal recognition particle pathway (SRP pathway) system for secretion of various proteins from ribosomes to the extracellular surface which plays an important role in the machinery running inside the bacterium. This system comprises of three major components FtsY, FfH and 4.5S rRNA. This manuscript highlights essential factors responsible for the optimized enzymatic activity of FtsY. Kinetic parameters include Vmax and Km for the hydrolysis of GTP by ftsY which were 20.25±5.16 μM/min/mg and 39.95±7.7 μM respectively. kcat and catalytic efficiency of the reaction were 0.012±0.003 s−1 and 0.00047±0.0001 μM/s−1 respectively. These values were affected upon changing the standard conditions. Cations (Mg2+ and Mn2+) play important role in FtsY enzymatic activity as increasing Mg2+ decrease the activity. Mn2+on the other hand is required at higher concentration around 60 mM for carrying optimum GTPase activity. FtsY is hydrolyzing ATP and GDP as well and GDP acts as an inhibitor of the reaction. MD simulation shows effective binding and stabilization of the FtsY complexed structure with GTP, GDP and ATP. Mutational analysis was done at two important residues of GTP binding motif of FtsY, namely, GXXXXGK (K236) and DXXG (D367) and showed that these mutations significantly decrease FtsY GTPase activity. FtsY is comprised of α helices, but this structural pattern was shown to change with increasing concentrations of GTP and ATP which symbolize that these ligands cause significant conformational change by variating the secondary structure to transduce signals required by downstream effectors. This binding favors the functional stabilization of FtsY by destabilization of α-helix integrity. Revealing the hidden aspects of the functioning of FtsY might be an essential part for the understanding of the SRP pathway which is one of the important contributors of M. tuberculosis virulence.

Revelation of the Involvement of Rv1651c of Mycobacterium tuberculosis H 37 Rv in Carbohydrate Metabolism

Tuberculosis (TB) is the major cause of mortality across the world. About one-third of world population is affected by this fatal disease. Mycobacterium tuberculosis H 37 Rv ( M. tuberculosis ) which is a gram- positive bacterium is responsible for the cause of TB. M. tuberculosis is spreading its roots worldwide with the help of various survival mechanisms and making its cure more difficult. In the present study, we have made use of various in-silico tools to predict the properties of Rv1651c which is a member of the PE_PGRS protein family. This manuscript reveals some important aspects of Rv1651c as its function is still unknown. The major part of this study includes protein sequence retrieval, multiple sequence alignment, protein-protein interaction study, epitope prediction, localization, function prediction, structure prediction and its validation, ligand binding prediction and mutational analysis. This protein shows the presence of GTP-binding motifs such as DXXG and GXXXXGK. These motifs can be targeted to mutate the protein and thereby, decrease its stability. This protein also shows similarity with enzyme ribose-5-phosphate isomerase, which performs the function of interconversion of ribose-5-phosphate and ribulose-5-phosphate. This similarity proves to be of great importance as this protein has ribulose-5-phosphate as one of its predicted ligands. All these in-silico generated results of Rv1651c give a hint of it being involved in carbohydrate metabolism. Carbohydrate metabolism is an important process required for the production of energy molecules. Thus, this protein might be targeted to block the carbohydrate metabolism pathway. These prediction-based studies using computational approach might prove to be a successful step towards developing drugs against TB.

Labeo rohita Mx1 exhibits the critical structural motifs of the family of large GTPases of mammals and is activated by rhabdovirus vaccination and bacterial RNA stimulations

Myxovirus resistance (Mx) proteins belonging to the dynamin superfamily of high molecular weight GTPases exist in various isoforms and play crucial role in innate immunity. In addition to the isoforms, Mx1 also plays important role in exerting its anti-viral actions against a broad range of animal RNA viruses. In rohu (Labeo rohita), mx1 full-length cDNA sequence consists of 2440 nucleotides (nt) encoding 628 amino acids (aa) polypeptide of 71.289 kDa. Structurally, it belongs to the family of large GTPases with one DYNc domain (13-257aa) comprising of dynamin family motifs (LPRGSGIVTR) and the tripartite GTP-binding motifs (GDQSSGKS, DLPG and TKPD) at the N-terminal and one GED domain (537-628aa) at C-terminus. Rohu Mx1 is closely related to zebrafish Mx1 and is widely expressed in gill, liver, kidney, spleen and blood. In response to rhabdovirus vaccinations, poly I:C stimulation and bacterial infections, mx1 gene expression in rohu was significantly (p < 0.05) induced in majority of the tested organs/tissues. Stimulation of rohu gill cell line with bacterial RNA also induced mx1 gene expression. Together these data suggest the important role of Mx1 in innate immunity in rohu against wide spectrum of fish pathogens.

Characterization, Expression Pattern and Antiviral Activities of Mx Gene in Chinese Giant Salamander, Andrias davidianus.

Mx, Myxovirus resistance is an important interferon-stimulated protein that mediates antiviral responses. In this study, the expression and activities of Chinese giant salamander, Andrias davidianus Mx gene, AdMx, were investigated. The AdMx cDNA sequence contains an open reading frame (ORF) of 2112 nucleotides, encoding a putative protein of 703 aa. Meanwhile, AdMx possesses the conserved tripartite GTP binding motif and a dynamin family signature. qRT-PCR analysis revealed a broad expression of AdMx in vivo, with the highest expression levels in brain, kidney and spleen. The AdMx expression level in kidney, spleen and muscle significantly increased at 6 h after Chinese giant salamander iridovirus (GSIV) infection and peaked at 48 h, while that in muscle cell line (GSM) was not noticeably up-regulated until 72 h post infection. Additionally, a plasmid expressing AdMx was constructed and transfected into the Chinese giant salamander GSM cells. The virus load and gene copies in AdMx over-expressed cells were significantly reduced compared with those in the control cells. Moreover, compared to the control cells, a lower level of virus major capsid protein (MCP) synthesis in AdMx over-expressed cells was confirmed by Western blot. These results collectively suggest that Mx plays an important antiviral role in the immune responses against GSIV in Chinese giant salamander.

Gene characterization and expression pattern of Mx and Viperin genes in Dabry’s sturgeonAcipenser dabryanus

Mx and Viperin are important interferon‐stimulated genes that mediate the antiviral immune response. In this study, we cloned the Mx and viperin genes from Dabry's sturgeon (Acipenser dabryanus). The Mx cDNA sequence contained an open reading frame (ORF) of 1,449 nucleotides, encoding a putative protein of 392 aa, which is significantly shorter than other animal Mx proteins. Although the similarity and identity were low between sturgeon Mx and other animal Mx proteins, sturgeon Mx contains the conserved tripartite GTP binding motif and a dynamin family signature. The sturgeon Mx gene contains eight exons split by seven introns. The sturgeon viperin cDNA sequence contained an ORF of 1,047 bp encoding a putative protein of 349 aa, which is relatively well conserved among species. Sturgeon viperin proteins show 82% similarity with those of Xiphophorus maculatus platyfish and Poecilia formosa Amazon molly. The sturgeon viperin gene has a six exon/five intron structure with the same size of second, third, fourth, and fifth exons between different species. The expression of Mx and viperin was detectable in all tissues examined, with the highest expression in skin for Mx and in peripheral blood for viperin. After mock infection using polyinosine‐polycytidylic acid, Mx and viperin showed significantly upregulated expression in primary spleen leukocytes from 3 hr to 72 hr. Lipopolysaccharide could also induce their expression. These results suggested Mx and Viperin could play a vital antiviral role in the innate immune system of Dabry's sturgeon.

Myxovirus resistance protein A inhibits hepatitis C virus replication through JAK-STAT pathway activation.

The interferon-inducible dynamin-like GTPase myxovirus resistance protein A (MxA) exhibits activity against multiple viruses. However, its role in the life cycle of hepatitis C virus (HCV) is unclear, and the mechanisms underlying the anti-HCV activity of MxA require further investigation. In this study, we demonstrated that exogenous MxA expression in the Huh7 and Huh7.5.1 hepatoma cell lines significantly decreased the levels of HCV RNA and core proteins, whereas MxA knockdown exerted the opposite effect. MxA-mediated inhibition of HCV replication was found to involve the JAK-STAT pathway: STAT1 phosphorylation and the expression of IFN-stimulated genes (ISGs) such as guanylate-binding protein 1 and 2'-5'-oligoadenylate synthetase 1 were augmented by MxA overexpression and reduced by endogenous MxA silencing. Treatment with the JAK inhibitor ruxolitinib abrogated the MxA-mediated suppression of HCV replication and activation of the JAK-STAT pathway. Additionally, transfection with an MxA mutant with disrupted GTP-binding consensus motifs abrogated activation of the JAK-STAT pathway and resistance to HCV replication. This study shows that MxA inhibits HCV replication by activating the JAK-STAT signaling pathway through a mechanism involving its GTPase function.

Cloning and structural characterization of juvenile hormone diol kinase in Spodoptera litura.

Juvenile hormone (JH) is one of key insect hormones that regulate metamorphosis. Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH metabolism and catalyzes JH diol to form a polar end product, JH diol phosphate that has no JH activity. In this study, a JHDK cDNA was cloned from Spodoptera litura and the structure and expression of the gene was characterized. The cDNA was 714 base pairs in length and encoded a protein of 183 amino acids with a molecular mass of 21 kDa and a pI of 4.55. Based on the structure three putative calcium binding motifs and GTP-binding motifs were predicted in the protein. Modeling of the 3-D structure showed that the protein consisted of eight α-helixes linked with loops, with no β-sheets. The gene was expressed in the epidermis, fat body and midgut of 5th and 6th instar larvae. The expression level in the epidermis was lower than in the fat body and midgut. The gene was expressed at higher levels at the early stages than in the later stages of 5th and 6th instar midgut and fat body. The results suggest that this gene may be involved in the regulation of the JH titer in larvae of S. litura. This article is protected by copyright. All rights reserved.

Cloning and characterization of ADP-ribosylation factor 1b from the olive flounder Paralichthys olivaceus

Small GTPases are well known as one of the signal transduction factors of immune systems. The ADP-ribosylation factors (ARFs) can be classified into three groups based on the peptide sequence, protein molecular weight, gene structure, and phylogenetic analysis. ARF1 recruits coat proteins to the Golgi membranes when it is bound to GTP. The class I duplicated ARF gene was cloned and characterized from the olive flounder (Paralichthys olivaceus) for this study. PoARF1b contains the GTP-binding motif and the switch 1 and 2 regions. PoARF1b and PoARF1b mutants were transfected into a Hirame natural embryo cell to determine the distribution of its GDP/GTP-bound state; consequently, it was confirmed that PoARF1b associates with the Golgi body when it is in a GTP-binding form. The results of the qPCR-described PoARF1b were expressed for all of the P. olivaceus tissues. The authors plan to study the gene expression patterns of PoARF1b in terms of immunity challenges.

Biochemical characterization of purified mammalian ARL13B protein indicates that it is an atypical GTPase and ARL3 guanine nucleotide exchange factor (GEF)

Primary cilia play central roles in signaling during metazoan development. Several key regulators of ciliogenesis and ciliary signaling are mutated in humans, resulting in a number of ciliopathies, including Joubert syndrome (JS). ARL13B is a ciliary GTPase with at least three missense mutations identified in JS patients. ARL13B is a member of the ADP ribosylation factor family of regulatory GTPases, but is atypical in having a non-homologous, C-terminal domain of ∼20 kDa and at least one key residue difference in the consensus GTP-binding motifs. For these reasons, and to establish a solid biochemical basis on which to begin to model its actions in cells and animals, we developed preparations of purified, recombinant, murine Arl13b protein. We report results from assays for solution-based nucleotide binding, intrinsic and GTPase-activating protein-stimulated GTPase, and ARL3 guanine nucleotide exchange factor activities. Biochemical analyses of three human missense mutations found in JS and of two consensus GTPase motifs reinforce the atypical properties of this regulatory GTPase. We also discovered that murine Arl13b is a substrate for casein kinase 2, a contaminant in our preparation from human embryonic kidney cells. This activity, and the ability of casein kinase 2 to use GTP as a phosphate donor, may be a source of differences between our data and previously published results. These results provide a solid framework for further research into ARL13B on which to develop models for the actions of this clinically important cell regulator.

Involvement of three annexin genes in the ripening of strawberry fruit regulated by phytohormone and calcium signal transduction.

Three annexin genes may be involved in the ripening progress of strawberry fruit. Phytohormones and calcium regulate the expressions of three annexin genes during strawberry fruit ripening. Plant annexins are multi-functional membrane- and Ca(2+)-binding proteins that are involved in various developmental progresses and stress responses. Three annexins FaAnn5a, FaAnn5b and FaAnn8 cDNA obtained from strawberry fruit encode amino acid sequences of approximately 35 kDa containing four annexin repeats, Ca(2+)-binding site, GTP-binding motif, peroxidase residue, and conserved amino acid residues of tryptophan, arginine and cysteine. During fruit development, the transcript levels of FaAnn5a and FaAnn5b increased while FaAnn5b declined after 3/4R stage. The expression patterns of annexins suggested their potential roles in strawberry fruit development and ripening. Expressions of annexin genes were also highly correlated with hormone levels. In addition, exogenous abscisic acid (ABA) enhanced the expressions of FaAnn5a and FaAnn8 while exogenous auxin (IAA) retarded it. However, both ABA and IAA promoted the transcript levels of FaAnn5b, indicating the independent regulation of annexins in fruit likely due to multi-functions of their large family. The responses of annexin genes to exogenous ABA and IAA inhibitors verified the involvement of annexins in plant hormone signaling. Besides, calcium restrained the expressions of FaAnn5s (FaAnn5a and FaAnn5b) but promoted the expression of FaAnn8. Effects of calcium and ethylene glycol tetraacetic acid (EGTA) on the transcript levels of annexins confirmed that calcium likely mediated hormone signal transduction pathways, which helped to elucidate the mechanism of calcium in fruit ripening. Therefore, FaAnn5s and FaAnn8 might be involved in plant hormones' regulation in the development and ripening of strawberry fruit through calcium signaling in the downstream.

Modeling and structural analysis of human Guanine nucleotide-binding protein-like 3,nucleostemin.

Human GNL3 (nucleostemin) is a recently discovered nucleolar protein with pivotal functions in maintaining genomic integrity and determining cell fates of various normal and cancerous stem cells. Recent reports suggest that targeting this GTP-binding protein may have therapeutic value in cancer. Although, sequence analyzing revealed that nucleostemin (NS) comprises 5 permuted GTP-binding motifs, a crystal structure for this protein is missing at Protein Data Bank (PDB). Obviously, any attempt for predicting of NS structure can further our knowledge on its functional sites and subsequently designing molecular inhibitors. Herein, we used bioinformatics tools and could model 262 amino acids of NS (132-393 aa). Initial models were built by MODELLER, refined with Scwrl4 program, and validated with ProsA and Jcsc databases as well as PSVS software. Then, the best quality model was chosen for motif and domain analyzing by Pfam, PROSITE and PRINTS. The final model was visualized by vmd program. This predicted model may pave the way for next studies regarding ligand binding states and interaction sites as well as screening of databases for potential inhibitors.

Interferon inducible antiviral MxA is inversely associated with prostate cancer and regulates cell cycle, invasion and Docetaxel induced apoptosis.

The interferon inducible Myxovirus (influenza virus) resistance A (MxA) is considered as a key mediator of the interferon-induced antiviral response. Mx proteins contain the typical GTP-binding motif and show significant homology to dynamin family of GTPases. Strong interaction of MxA with tubulin suggests that Mx proteins could be involved in mitosis. Studies have shown that MxA inhibit tumor motility/metastasis and virus induced apoptosis. However, the clear association between MxA expression and cancer remains unknown. Meta-analysis suggested that MxA expression was inversely correlated with prostate cancer (PCa). In this study, we demonstrate the expression MxA in PCa and its functional significance on the cancer phenotype. The expression of MxA protein in prostate cancer was examined by immuno-histochemistry. MxA was knocked down (shMxA) or over-expressed (pMxA) in DU145 or LNCaP PCa cell lines respectively. These cell lines were used to study proliferation, apoptosis, invasion, migration, and anchorage independent growth. Co-localization of MxA with tubulin was performed by immuno-cytochemistry following Docetaxel treatment. The expression of MxA protein was significantly decreased in PCa as compared to the normal tissues. DU145 cells lacking MxA (DU145 + chMxA) showed significant increase in proliferation, associated with decreased expression of CDKN1A and B. Increased migration, anchorage independent growth in DU145 + shMxA cells was associated with increased MMP13 expression. Tubulin organization was also dependent on MxA expression. Tubulin polymerizing agents such as Docetaxel was less effective in promoting apoptosis in cells lacking MxA due to altered tubulin organization. Gain of MxA expression in LNCaP cells (LNCaP + pMxA) resulted in cell cycle arrest that was associated with increased expression of CDKN1A. MxA expression was also down-regulated by dihydrotestosterone in LNCaP cells. MxA expression is inversely correlated with prostate cancer. Down-regulation of MxA in LNCaP cells by DHT suggests that MxA could play a significant role in disease progression. Loss of MxA expression results in increased metastasis and decreased sensitivity to Docetaxel suggesting that MxA expression could determine the outcome of chemo-therapeutic treatment. Additional studies will be required to fully establish the cross-talk between androgen receptor-IFN pathway in regulating MxA expression in the normal prostate and prostate cancer. Prostate 75:266-279, 2015. © 2014 Wiley Periodicals, Inc.

Discovery of Widespread GTP-Binding Motifs in Genomic DNA and RNA

Biological RNAs that bind small molecules have been implicated in a variety of regulatory and catalytic processes. Inspired by these examples, we used in vitro selection to search a pool of genome-encoded RNA fragments for naturally occurring GTP aptamers. Several aptamer classes were identified, including one (the "G motif") with a G-quadruplex structure. Further analysis revealed that most RNA and DNA G-quadruplexes bind GTP. The G motif is abundant in eukaryotes, and the human genome contains ~75,000 examples with dissociation constants comparable to the GTP concentration of a eukaryotic cell (~300 μM). G-quadruplexes play roles in diverse cellular processes, and our findings raise the possibility that GTP may play a role in the function of these elements. Consistent with this possibility, the sequence requirements of several classes of regulatory G-quadruplexes parallel those of GTP binding.

The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii

One of the most abundantly IFN-γ-induced protein families in different cell types is the 65-kDa guanylate-binding protein family that is recruited to the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to Toxoplasma gondii. The wild type protein exhibits low affinities to guanine nucleotides, self-assembles upon GTP binding, forming tetramers in the activated state, and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing multiparameter fluorescence image spectroscopy (MFIS) using a Homo-FRET assay shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase domain. The consistent results indicate that GTP binding, self-assembly, and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase domain regulates efficient recruitment to T. gondii in response to IFN-γ.

DrosophilaGTPase Nucleostemin 2 Changes Cellular Distribution during Larval Development and the GTP-Binding Motif Is Essential to Nucleoplasmic Localization

Nucleostemin (NS), a nucleolar guanosine triphosphate (GTP)-binding protein, plays significant roles in cell cycle progression and ribosomal biogenesis. Drosophila Nucleostemin 2 (NS2), a member of the Drosophila NS family, regulates early eye development and is essential to cell survival in vivo, but the underlying mechanisms have yet to be clarified. Biochemical analysis using the recombinant NS2 protein indicated that NS2 has GTPase activity. Immunohistochemistry revealed that NS2 changes in subcellular locus from the nucleolus to the nucleoplasm during larval development, and that a mutation in the ATP/GTP-binding site motif A (p-loop) prevents nuclear localization of NS2 and results in cytoplasmic distribution. Furthermore, downregulation of NS2 altered the rRNA proportions between the nucleus and the cytoplasm. These results suggest that NS2 at least requires GTP to import into the nucleoplasm.

The Protein Interaction Network of the Human Transcription Machinery Reveals a Role for the Conserved GTPase RPAP4/GPN1 and Microtubule Assembly in Nuclear Import and Biogenesis of RNA Polymerase II

RNA polymerase II (RNAPII), the 12-subunit enzyme that synthesizes all mRNAs and several non-coding RNAs in eukaryotes, plays a central role in cell function. Although multiple proteins are known to regulate the activity of RNAPII during transcription, little is known about the machinery that controls the fate of the enzyme before or after transcription. We used systematic protein affinity purification coupled to mass spectrometry (AP-MS) to characterize the high resolution network of protein interactions of RNAPII in the soluble fraction of human cell extracts. Our analysis revealed that many components of this network participate in RNAPII biogenesis. We show here that RNAPII-associated protein 4 (RPAP4/GPN1) shuttles between the nucleus and the cytoplasm and regulates nuclear import of POLR2A/RPB1 and POLR2B/RPB2, the two largest subunits of RNAPII. RPAP4/GPN1 is a member of a newly discovered GTPase family that contains a unique and highly conserved GPN loop motif that we show is essential, in conjunction with its GTP-binding motifs, for nuclear localization of POLR2A/RPB1 in a process that also requires microtubule assembly. A model for RNAPII biogenesis is presented.