GTPase domains of ras p21 oncogene protein and elongation factor Tu: analysis of three-dimensional structures, sequence families, and functional sites.

Valencia, Alfonso; Kjeldgaard, Morten; Pai, E.F.; Sander, Chris

doi:10.1073/pnas.88.12.5443

Cited by 65 publications

(38 citation statements)

References 41 publications

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“…In particular, the B and G motifs are positioned together in space so as to mediate binding with the phosphates and guanine base, respectively, and contribute to guanosine triphosphatase function. 24,25 The importance of the corresponding elements in the NS4B NBM (Fig. 5A) was revealed by the dramatic impairment in HCV replication that results from point mutations in the NS4B B-(D228L) and G-(F211A) motifs (to be published elsewhere).…”

Section: Ns4b Transforms Nih 3t3 Cells Independently Ofmentioning

confidence: 99%

The nucleotide binding motif of hepatitis C virus NS4B can mediate cellular transformation and tumor formation without Ha-ras co-transfection

et al. 2007

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Hepatitis C virus (HCV) is an important cause of chronic liver disease and is complicated by hepatocellular carcinoma (HCC). Mechanisms whereby the virus promotes cellular transformation are poorly understood. We hypothesized that the guanosine triphosphatase activity encoded in the HCV NS4B protein's nucleotide binding motif (NBM) might play a role in the transformation process. Here we report that NS4B can transform NIH-3T3 cells, leading to tumor formation in vivo. This transformation was independent of co-transfection with activated Haras. Detailed analyses of NS4B mutants revealed that this transforming activity could be progressively inhibited and completely abrogated by increasing genetic impairment of the NS4B nucleotide binding motif. Conclusion: NS4B has in vitro and in vivo tumorigenic potential, and the NS4B transforming activity is indeed mediated by its NBM. Moreover, our results suggest that pharmacological inhibition of the latter might inhibit not only HCV replication but also the associated HCC. (HEPATOLOGY 2008;47:827-835.) C hronic infection with the hepatitis C virus (HCV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The incidence of HCC and the mortality rate associated with it are increasing dramatically. 1,2 Although chronic inflammation, fibrosis, and liver cell proliferation are considered the major factors contributing to the development of HCC, accumulating data support direct viral effects as well. 3 HCV is a positive single-stranded RNA virus. Its 9.6-kb genome encodes a single ϳ3000-amino-acid polyprotein, which is proteolytically processed into structural proteins, which are components of the mature virus, and nonstructural proteins, which are involved in replicating the viral genome. 4 Several viral proteins including NS3, NS5A, and core have been implicated in cellular transformation. 5-8 NS4B, a 27-kDa membrane protein, has been similarly shown by Park et al 9 to transform NIH 3T3 cells. 9 Transformation, however, only occurred when NS4B was co-transfected with the activated Ha-ras gene. The mechanism by which NS4B mediates its transformation potential remains unknown.We have recently reported the identification of a nucleotide-binding motif (NBM) within NS4B and shown that this motif mediates both binding and hydrolysis of guanosine triphosphate (GTP) and HCV RNA replication. 10 The NBMs of human oncogenes, such as ras, mediate their malignant transformation. 11,12 We thus hypothesized that the NBM of NS4B may similarly mediate its role in transformation. Here we report that NS4B of the Con 1 isolate transforms NIH3T3 cells independently of exogenous Ha-ras, the transformed cells lead to tumor formation in nude mice, and the NBM of NS4B mediates the latter's role in transformation, with exciting implications for novel therapies. Materials and MethodsPlasmids. Standard recombinant DNA technology was used to construct and purify all plasmids. All regions

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Section: Ns4b Transforms Nih 3t3 Cells Independently Ofmentioning

confidence: 99%

The nucleotide binding motif of hepatitis C virus NS4B can mediate cellular transformation and tumor formation without Ha-ras co-transfection

et al. 2007

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“…High-resolution crystal structures reveal that although these motifs are short and the amino acid sequences between them are generally not conserved, they are embedded within very similar tertiary structures even in highly divergent proteins. For example, the GTP-binding domains of ras, EF-Tu, and transducin-ax, which are only 16 to 18% identical in their primary sequences, are virtually superimposable (57,98). The four conserved motifs cradle the bound GTP molecule and serve to position critical residues for guanine ring and phosphate binding and 1--y phosphate bond cleavage (57,62,69).…”

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confidence: 99%

“…3 by dynamin, is the presence of threonine in place of asparagine at a conserved position (248 in S. pombe Srp54) in the G-4 motif (reviewed in references 2 and 14). The amide functional group of E-116, the corresponding residue in Ras, stabilizes the guanine binding pocket through hydrogen bonds to the main chain at-amino group of V-14 in G-1 (98), and mutations at this position weaken the affinity of the enzyme for nucleotide (reviewed in reference 65). Restoration of the GTPase superfamily consensus in Srp54 produces cold sensitivity (Table 2; Fig.…”

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confidence: 99%

The Srp54 GTPase is essential for protein export in the fission yeast Schizosaccharomyces pombe.

Althoff¹,

Stevens²,

Wise³

1994

Mol. Cell. Biol.

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Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein required for targeting a subset of presecretory proteins to the endoplasmic reticulum (ER) The signal recognition particle (SRP) serves as a biological adaptor between the cytoplasmic machinery for protein synthesis and the translocation apparatus of the endoplasmic reticulum (ER) (for reviews, see references 31, 59, 70, and 82). SRP recognizes ribosomes translating proteins destined for export from the cytoplasm via the ER by virtue of their hydrophobic signal sequences, usually located at the amino terminus. Association of SRP with the ribosome transiently halts elongation of the nascent polypeptide at natural translational pause sites (107), preventing synthesis of the completed protein in the cytoplasm. Upon reaching the cytoplasmic face of the ER, the SRP-ribosome-nascent chain complex first interacts with a heterodimeric membrane protein known as the SRP receptor (32,42,95) (11,37,46,48,74,92,93 (35,39,63,66,73).Comparative sequence analysis, together with the results of partial proteolysis of the mammalian homolog, indicates that the Srp54 protein consists of two structurally distinct domains (75, 109). The carboxyl-terminal M domain, so named because it is rich in methionine (11), binds directly to SRP RNA (75, 109) and can be photochemically cross-linked to a signal sequence (38,47,109). The amino-terminal region is designated the G domain on the basis of the presence of the four consensus motifs that define the GTPase superfamily (11, 74), which includes small ras-related proteins, a variety of transla-7839 on May 12, 2018 by guest

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“…However, partial proteolysis, a technique used to detect changes in protein structure (5), with five different proteases has failed to reveal any major conformational change in CodY induced by interaction with GTP (P. Joseph, L. D. Handke, and A. L. Sonenshein, unpublished results). In addition, in members of the GTPase superfamily of proteins, the side chain from a highly conserved aspartic acid residue in the G4 motif (NXXD) forms hydrogen bonds with the guanine base (4,33,45). In CodY, this position is occupied by a leucine residue (31).…”

Section: Discussionmentioning

confidence: 99%

Interaction ofBacillus subtilisCodY with GTP

Handke¹,

2008

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Many of the adaptive mechanisms that allow Bacillus subtilis to adjust to changes in nutrient availability are controlled by CodY. Binding of CodY to its target genes is stimulated by interaction with its effectors, GTP and the branched-chain amino acids (BCAAs). Upon nutrient limitation, intracellular pools of these effectors are depleted and CodY can no longer repress genes required for adaptation. In vitro studies reported here explored in more detail the interaction of CodY with GTP. DNase I footprinting experiments indicated that CodY has an affinity for GTP in the millimolar range. Further, CodY was shown to interact specifically with GTP and dGTP; no other naturally occurring nucleotides that were tested, including ppGpp and pppGpp, resulted in DNA protection. Two nonhydrolyzable analogs of GTP were fully able to activate CodY binding to target DNA, demonstrating that GTP hydrolysis is not necessary for CodY-dependent regulation. GTP and the BCAAs were shown to act additively to increase the affinity of CodY for DNA; increased protection was observed in DNase I footprinting experiments when both effectors were present, compared to either effector alone, and in in vitro transcription reactions, transcriptional repression by CodY was stronger in the presence of both GTP and BCAAs than of BCAAs alone. Thus, interaction of CodY with GTP is specific and results in increased affinity for its target genes. This increase in affinity is independent of GTP hydrolysis and is augmented in the presence of BCAAs.The ability to adapt to changes in nutrient availability is central to bacterial colonization of diverse habitats. When nutrients are limiting, the gram-positive bacterium Bacillus subtilis employs several adaptive strategies. It may migrate to a more favorable environment; secrete enzymes to degrade macromolecules; induce the expression of transport systems for amino acids, peptides, DNA, and other potential nutrients; and activate intracellular catabolic pathways. In addition, the cell may synthesize antibiotics to eliminate nearby competitors. If these adaptive strategies fail to restore conditions suitable for growth, B. subtilis can differentiate to form a dormant spore.CodY, a highly conserved regulatory protein in gram-positive bacteria (8,20,23,29,43,44), is one of the factors that control the expression of these adaptive mechanisms. CodY was initially identified in B. subtilis as a regulator of the dipeptide permease (dpp) operon (25, 34, 38-40), but we now know that the B. subtilis CodY regulon is large, encompassing nearly 200 genes (1,7,10,16,27,46). Many of these genes encode proteins that mediate the adaptive responses cited above. In addition, CodY has been proved to repress branched-chain amino acid biosynthesis (36) and activate the expression of the acetate kinase gene (35). CodY also participates in repression of the initiation of sporulation; unlike wild-type cells, those of B. subtilis codY mutant strains sporulate readily in a nutrientrich medium (12,15,31).CodY exists primarily as a dimer...

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GTPase domains of ras p21 oncogene protein and elongation factor Tu: analysis of three-dimensional structures, sequence families, and functional sites.

Cited by 65 publications

References 41 publications

The nucleotide binding motif of hepatitis C virus NS4B can mediate cellular transformation and tumor formation without Ha-ras co-transfection

The nucleotide binding motif of hepatitis C virus NS4B can mediate cellular transformation and tumor formation without Ha-ras co-transfection

The Srp54 GTPase is essential for protein export in the fission yeast Schizosaccharomyces pombe.

Interaction ofBacillus subtilisCodY with GTP

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