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Ras Protein (ras + protein)
Selected AbstractsEnhanced Ras activity preserves dendritic size and extension as well as synaptic contacts of neurons after functional deprivation in synRas miceEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2008A. Alpár Abstract The monomeric GTP-binding protein p21Ras has been repeatedly implicated in neuronal stability and plastic changes of the adult nervous system. Recently, we have shown that expression of constitutively active Ras protein in transgenic synRas mice results in a significant increase in the dendritic size and complexity of differentiated pyramidal neurons as well as in increased synaptic connectivity. In the present study, we examined the organization of the vibrissae-barrel cortex in synRas mice and the effects of enhanced Ras activity on deprivation-induced dendritic reorganization after vibrissectomy. The results demonstrate a significant increase in vibrissae-barrel sizes and proportional spacing between barrels in synRas mice, suggesting that the neuronal target specificity of thalamocortical terminals is preserved. Accordingly, the arrangement of double bouquet cells at the borders of barrel columns ensuring functional distinctness is unchanged. Partial vibrissectomy is followed by significant dendritic regression of corresponding pyramidal neurons in the barrel cortex of wild-type mice, which, however, could not be observed in synRas mice. The results provide the first evidence for a role of Ras in preserving neuronal structure after functional deprivation in vivo. [source] Secondary structure of lipidated Ras bound to a lipid bilayerFEBS JOURNAL, Issue 23 2008Jörn Güldenhaupt Ras proteins are small guanine nucleotide binding proteins that regulate many cellular processes, including growth control. They undergo distinct post-translational lipid modifications that are required for appropriate targeting to membranes. This, in turn, is critical for Ras biological function. However, most in vitro studies have been conducted on nonlipidated truncated forms of Ras proteins. Here, for the first time, attenuated total reflectance-FTIR studies of lipid-modified membrane-bound N-Ras are performed, and compared with nonlipidated truncated Ras in solution. For these studies, lipidated N-Ras was prepared by linking a farnesylated and hexadecylated N-Ras lipopeptide to a truncated N-Ras protein (residues 1,181). It was then bound to a 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine bilayer tethered on an attenuated total reflectance crystal. The structurally sensitive amide I absorbance band in the IR was detected and analysed to determine the secondary structure of the protein. The NMR three-dimensional structure of truncated Ras was used to calibrate the contributions of the different secondary structural elements to the amide I absorbance band of truncated Ras. Using this novel approach, the correct decomposition was selected from several possible solutions. The same parameter set was then used for the membrane-bound lipidated Ras, and provided a reliable decomposition for the membrane-bound form in comparison with truncated Ras. This comparison indicates that the secondary structure of membrane-bound Ras is similar to that determined for the nonlipidated truncated Ras protein for the highly conserved G-domain. This result validates the multitude of investigations of truncated Ras without anchor in vitro. The novel attenuated total reflectance approach opens the way for detailed studies of the interaction network of the membrane-bound Ras protein. [source] Inactivation of RASSF2A by promoter methylation correlates with lymph node metastasis in nasopharyngeal carcinomaINTERNATIONAL JOURNAL OF CANCER, Issue 1 2007Zhe Zhang Abstract RASSF2 can bind directly to K-Ras and function as a negative effector of Ras protein. RASSF2A is the only isoform of RASSF2 that contains CpG islands in its promoter and it has been reported to be inactivated by its promoter methylation in several human cancers. In the present study, we investigated the correlation of RASSF2A expression with its promoter methylation in nasopharyngeal carcinoma (NPC). Expression of RASSF2A was down-regulated in 80% (4/5) of NPC cell lines. Decreased RASSF2A expression was also observed in NPC primary tumors compared with normal nasopharyngeal epithelia. Promoter methylation of RASSF2A could be detected in all the RASSF2A -silenced cell lines (4/5) of the NPC cell lines and 50.9% (27/53) of primary tumors, but not in any of the normal epithelia. RASSF2A -methylated cases showed a significantly lower level of RASSF2A expression than unmethylated cases. Loss of RASSF2A expression can be greatly restored by the methyltransferase inhibitor 5-aza-dC in NPC cell lines. In addition, patients with methylated RASSF2A presented a higher frequency of lymph node metastasis (p < 0.05). Ectopic expression of RASSF2A in RASSF2A -silenced and -methylated NPC cell line CNE2 shows that RASSF2A could inhibit cell cycle progression, colony formation and cell migration, which provided further evidence that RASSF2A is a candidate tumor suppressor gene. In conclusion, RASSF2A, a candidate tumor suppressor gene (TSG), is frequently inactivated by its promoter methylation and this aberrant methylation correlates with lymph node metastasis in NPC. © 2006 Wiley-Liss, Inc. [source] Significance of the Grb2 and Son of Sevenless (Sos) Proteins in Human Bladder Cancer Cell LinesIUBMB LIFE, Issue 4 2000Takafumi Watanabe Abstract The epidermal growth factor (EGF) receptor has been suggested to have an important role in tumor initiation and progression of human bladder cancers. Grb2 protein, which is the downstream effector of the EGF receptor, acts as an adaptor protein between the EGF receptor and the Ras guanine-nucleotide exchange factor, son of sevenless (Sos) protein. Sos protein regulates the action of Ras protein by promoting the exchange of GDP for GTP . However, the significance of Grb2 and Sos proteins, which is related to EGF-triggered Ras activation, has not been elucidated in human bladder cancer. The aim of the present study is to clarify the significance of these proteins in human bladder cancer cell lines. In the present study, we used four human bladder cancer cell lines (T24, KU-7, UMUC-2, UMUC-6) and two kinds of cultured normal urothelial cells (HMKU-1, HMKU-2) isolated from patients with no malignancy. We examined the expression of EGF receptor, Grb2, and Sos proteins in these cells by Western blot analysis. Furthermore, the bladder cancer cell lines were subjected to sequence analysis to identify a point mutation in the c-H-ras gene at codon 12. There was no marked difference in the expression of the EGF receptor between human bladder cancer cell lines and cultured normal urothelial cells. On the other hand, expression of Grb2 and Sos proteins was substantially increased in all human bladder cancer cell lines examined in comparison with cultured normal urothelial cells, whether codon 12 of H-ras was mutated or not. These results suggest that the amplification of both Grb2 and SOS proteins plays an important role in the carcinogenesis of human bladder cancer. [source] Shock Wave Application Enhances Pertussis Toxin Protein-Sensitive Bone Formation of Segmental Femoral Defect in Rats,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2003Yeung-Jen Chen Abstract Extracorporeal shock waves (ESWs) elicit a dose-dependent effect on the healing of segmental femoral defects in rats. After ESW treatment, the segmental defect underwent progressive mesenchymal aggregation, endochondral ossification, and hard callus formation. Along with the intensive bone formation, there was a persistent increase in TGF-,1 and BMP-2 expression. Pretreatment with pertussis toxin reduced ESW-promoted callus formation and gap healing, which presumably suggests that Gi proteins mediate osteogenic signaling. Introduction: Extracorporeal shock waves (ESWs) have previously been used to promote bone repair. In our previous report, we found that ESWs promoted osteogenic differentiation of mesenchymal cells through membrane perturbation and activation of Ras protein. In this report, we show that ESWs elicit a dose-dependent effect on the healing of segmental defects and that Gi proteins play an important role in mediating ESW stimulation. Materials and Methods: Rats with segmental femoral defects were subjected to ESW treatment at different energy flux densities (EFD) and impulses. Bone mass (mineral density and calcium content), osteogenic activities (bone alkaline phosphatase activity and osteocalcin content), and immunohistochemistry were assessed. Results: An optimal ESW energy (500 impulses at 0.16 mJ/mm2 EFD) stimulated complete bone healing without complications. ESW-augmented healing was characterized by significant increases (p < 0.01) in callus size, bone mineral density, and bone tissue formation. With exposure to ESW, alkaline phosphatase activity and osteocalcin production in calluses were found to be significantly enhanced (p < 0.05). After ESW treatment, the histological changes we noted included progressive mesenchymal aggregation, endochondral ossification, and hard callus formation. Intensive bone formation was associated with a persistent increase in transforming growth factor-beta 1 (TGF-,1) and bone morphogenetic protein-2 (BMP-2) expression, suggesting both growth factors were active in ESW-promoted bone formation. We also found that pertussis toxin, an inhibitor of membrane-bound Gi proteins, significantly reduced (p < 0.01) ESW promotion of callus formation and fracture healing. Conclusion: ESW treatments enhanced bone formation and the healing of segmental femoral defects in rats. It also seems likely that TGF-,1 and BMP-2 are important osteogenic factors for ESW promotion of fracture healing, presumably through Gi protein-mediated osteogenic signaling. [source] Ras family genes: An interesting link between cell cycle and cancerJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2002M. Macaluso Ras genes are evolutionary conserved and codify for a monomeric G protein binding GTP (active form) or GDP (inactive form). The ras genes are ubiquitously expressed although mRNA analysis suggests different level expression in tissue. Mutations in each ras gene frequently were found in different tumors, suggesting their involvement in the development of specific neoplasia. These mutations lead to a constitutive active and potentially oncogenic protein that could cause a deregulation of cell cycle. Ras protein moderates cellular responses at several mitogens and/or differentiation factors and at external stimuli. These stimuli activate a series of signal transduction pathways that either can be independent or interconnected at different points. Recent observations begin to clarify the complex relationship between Ras activation, apoptosis, and cellular proliferation. A greater understanding of these processes would help to identify the factors directly responsible for cell cycle deregulation in several tumors, moreover it would help the design of specific therapeutic strategies, for the control on the proliferation of neoplastic cells. We summarize here current knowledge of ras genes family: structural and functional characteristics of Ras proteins and their links with cell cycle and cancer. © 2002 Wiley-Liss, Inc. [source] Statin-induced apoptosis linked with membrane farnesylated Ras small G protein depletion, rather than geranylated Rho proteinJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 11 2005Sumio Matzno Rhabdomyolysis is a severe adverse effect of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins). This myopathy is strongly enhanced by the combination with statins and fibrates, another hypolipidaemic agent. We have evaluated the initial step of statin-induced apoptosis by the detection of membrane flip-flop using flow cytometric analysis. L6 rat myoblasts were treated with various statins (atorvastatin (3 ,m), cerivastatin (3 ,m), fluvastatin (3 ,m), pravastatin (3 mm), or simvastatin (3 ,m)) for 2, 4 or 6 h followed by reacting with FITC-conjugated annexin V for the detection of initial apoptosis signal (flip-flop). Various statin-treated myoblasts were significantly stained with FITC-annexin V at 6 h, whereas they were not detected at 2 h. Moreover, immunoblot analysis indicated that when the cells were treated with cerivastatin (3 ,m), membrane-associated Ras protein was activated and detached until 6 h, resulting in cell death through the consequent activation of caspase-8. On the other hand, since cytosolic Ras activation did not activate, there is still an unknown mechanism in statin-related Ras depletion. In conclusion, statin-induced apoptosis in muscular tissue was directly initiated by the farnesyl-anchored Ras protein depletion from cell membrane with subsequent apoptosis. [source] Site-Specific Functionalization of Proteins by Organopalladium Reactions,CHEMBIOCHEM, Issue 2 2007Koichiro Kodama Abstract A new carbon,carbon bond has been regioselectively introduced into a target position (position 32 or 174) of the Ras protein by two types of organopalladium reactions (Mizoroki,Heck and Sonogashira reactions). Reaction conditions were screened by using a model peptide, and the stability of the Ras protein under the reaction conditions was examined by using the wild-type Ras protein. Finally, the iF,Ras proteins containing a 4-iodo- L -phenylalanine residue were subjected to organopalladium reactions with vinylated or propargylated biotin. Site-specific biotinylations of the Ras protein were confirmed by Western blot and LC-MS/MS. [source] A New Protein Engineering Approach Combining Chemistry and Biology, Part I; Site-Specific Incorporation of 4-Iodo- L -phenylalanine in vitro by Using Misacylated Suppressor tRNAPheCHEMBIOCHEM, Issue 10 2006Koichiro Kodama Abstract An Escherichia coli suppressor tRNAPhe (tRNAPheCUA) was misacylated with 4-iodo- L -phenylalanine by using the A294G phenylalanyl,tRNA synthetase mutant (G294-PheRS) from E. coli at a high magnesium-ion concentration. The preacylated tRNA was added to an E. coli cell-free system and a Ras protein that contained the 4-iodo- L -phenylalanine residue at a specific target position was synthesized. Site-specific incorporation of 4-iodo- L -phenylalanine was confirmed by using LC,MS/MS. Free tRNAPheCUA was not aminoacylated by aminoacyl,tRNA synthetases (aaRSs) present in the E. coli cell-free system. Our approach will find wide application in protein engineering since an aryl iodide tag on proteins can be used for site-specific functionalization of proteins. [source] Regioselective Carbon,Carbon Bond Formation in Proteins with Palladium Catalysis; New Protein Chemistry by Organometallic ChemistryCHEMBIOCHEM, Issue 1 2006Koichiro Kodama Abstract Palladium-catalyzed reactions have contributed to the advancement of many areas of organic chemistry, in particular, the synthesis of organic compounds such as natural products and polymeric materials. In this study, we have used a Mizoroki,Heck reaction for site-specific carbon,carbon bond formation in the Ras protein. This was performed by the following two steps: 1) the His6 -fused Ras protein containing 4-iodo- L -phenylalanine at position 32 (iF32-Ras-His) was prepared by genetic engineering and 2) the aryl iodide group on the iF32-Ras-His was coupled with vinylated biotin in the presence of a palladium catalyst. The biotinylation was confirmed by Western blotting and liquid chromatography,mass spectrometry (LC-MS). The regioselectivity of the Mizoroki,Heck reaction was furthermore confirmed by LC-MS/MS analysis. However, in addition to the biotinylated product (bF32-Ras-His), a dehalogenated product (F32-Ras-His) was detected by LC-MS/MS. This dehalogenation resulted from the undesired termination of the Mizoroki,Heck reaction due to steric and electrostatic hindrance around residue 32. The biotinylated Ras showed binding activity for the Ras-binding domain as its downstream target, Raf-1, with no sign of decomposition. This study is the first report of an application of organometallic chemistry in protein chemistry. [source] Secondary structure of lipidated Ras bound to a lipid bilayerFEBS JOURNAL, Issue 23 2008Jörn Güldenhaupt Ras proteins are small guanine nucleotide binding proteins that regulate many cellular processes, including growth control. They undergo distinct post-translational lipid modifications that are required for appropriate targeting to membranes. This, in turn, is critical for Ras biological function. However, most in vitro studies have been conducted on nonlipidated truncated forms of Ras proteins. Here, for the first time, attenuated total reflectance-FTIR studies of lipid-modified membrane-bound N-Ras are performed, and compared with nonlipidated truncated Ras in solution. For these studies, lipidated N-Ras was prepared by linking a farnesylated and hexadecylated N-Ras lipopeptide to a truncated N-Ras protein (residues 1,181). It was then bound to a 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine bilayer tethered on an attenuated total reflectance crystal. The structurally sensitive amide I absorbance band in the IR was detected and analysed to determine the secondary structure of the protein. The NMR three-dimensional structure of truncated Ras was used to calibrate the contributions of the different secondary structural elements to the amide I absorbance band of truncated Ras. Using this novel approach, the correct decomposition was selected from several possible solutions. The same parameter set was then used for the membrane-bound lipidated Ras, and provided a reliable decomposition for the membrane-bound form in comparison with truncated Ras. This comparison indicates that the secondary structure of membrane-bound Ras is similar to that determined for the nonlipidated truncated Ras protein for the highly conserved G-domain. This result validates the multitude of investigations of truncated Ras without anchor in vitro. The novel attenuated total reflectance approach opens the way for detailed studies of the interaction network of the membrane-bound Ras protein. [source] Differential actions of p60c-Src and Lck kinases on the Ras regulators p120-GAP and GDP/GTP exchange factor CDC25MmFEBS JOURNAL, Issue 11 2001Carmela Giglione It is known that the human Ras GTPase activating protein (GAP) p120-GAP can be phosphorylated by different members of the Src kinase family and recently phosphorylation of the GDP/GTP exchange factor (GEF) CDC25Mm/GRF1 by proteins of the Src kinase family has been revealed in vivo[Kiyono, M., Kaziro, Y. & Satoh, T. (2000) J. Biol. Chem.275, 5441,5446]. As it still remains unclear how these phosphorylations can influence the Ras pathway we have analyzed the ability of p60c-Src and Lck to phosphorylate these two Ras regulators and have compared the activity of the phosphorylated and unphosphorylated forms. Both kinases were found to phosphorylate full-length or truncated forms of GAP and GEF. The use of the catalytic domain of p60c-Src showed that its SH3/SH2 domains are not required for the interaction and the phosphorylation of both regulators. Remarkably, the phosphorylations by the two kinases were accompanied by different functional effects. The phosphorylation of p120-GAP by p60c-Src inhibited its ability to stimulate the Ha-Ras-GTPase activity, whereas phosphorylation by Lck did not display any effect. A different picture became evident with CDC25Mm; phosphorylation by Lck increased its capacity to stimulate the GDP/GTP exchange on Ha-Ras, whereas its phosphorylation by p60c-Src was ineffective. Our results suggest that phosphorylation by p60c-Src and Lck is a selective process that can modulate the activity of p120-GAP and CDC25Mm towards Ras proteins. [source] Sustained activation of M-Ras induced by nerve growth factor is essential for neuronal differentiation of PC12 cellsGENES TO CELLS, Issue 9 2006Peng Sun Neuronal differentiation in PC12 cells induced by nerve growth factor (NGF) requires sustained activation of ERK/MAP kinase pathway (Raf,MEK,ERK cascade). Although classical Ras (H-Ras, K-Ras, and N-Ras) activated by NGF signaling induces activation of ERK pathway, the activation is transient and not sufficient for PC12 cell differentiation. Instead, it has been widely accepted that NGF signaling-mediated Rap1 activation causes sustained activation of ERK pathway. There has been no direct evidence, however, that Rap1 participates in neuronal differentiation. Here we show that NGF signaling induces sustained activation of M-Ras and subsequent sustained activation of ERK pathway and the transcription factor CREB leading to PC12 cell differentiation. Exogenously expressed constitutively active mutant of M-Ras caused neurite outgrowth in PC12 cells and activating phosphorylation of ERK, whereas activated Rap1 did not. Knockdown of endogenous M-Ras by small interfering RNAs as well as the expression of a dominant,negative mutant of M-Ras interfered with NGF-induced neuritogenesis. Since MEK inhibitors prevented M-Ras-induced neurite outgrowth, ERK pathway participates in this differentiation pathway. Furthermore, M-Ras brought about ERK pathway-mediated activating phosphorylation of CREB and the CREB-mediated transcription. In addition, a dominant,negative mutant of CREB inhibited M-Ras-induced neuritogenesis. Taken together, NGF-induced PC12 cell differentiation requires M-Ras,ERK pathway-mediated activation of CREB. M-Ras was predominantly expressed in the hippocampus and cerebellum of mouse brain and in the gray matter of the spinal cord. All these properties of M-Ras were apparently indistinguishable from those of H-Ras. However, NGF stimulation caused transient activation of classical Ras proteins but sustained activation of M-Ras as well as sustained activating phosphorylation of ERK and CREB. Therefore, M-Ras is essential for neuronal differentiation in PC12 cells by inducing sustained activation of ERK pathway. [source] Mutation rate of MAP2K4/MKK4 in breast carcinoma ,,HUMAN MUTATION, Issue 1 2002Gloria H. Su Abstract The stress-activated protein kinase (SAPK) pathways represent phosphorylation cascades that convey pro-apoptotic signals. The relevant inputs include Ras proteins as well as exposure of cells to ultraviolet light, tumor-necrosis factor, and other stress-related inputs. The mitogen-activated protein kinase kinase (MAPKK) homolog MAP2K4 (MKK4, SEK, JNKK1) is a centrally-placed mediator of the SAPK pathways. MAP2K4 mutations or homozygous deletions are reported in about 5% of a wide variety of tumor types. The exception is breast cancer, where genetic inactivation in 3 of 22 (15%) cell lines had suggested that the mutational involvement of MAP2K4 might be accentuated in this tumor type. This finding might have represented an important difference, or solely a chance numerical variation. To address this question, we studied an independent panel of 20 breast cancer cell lines and xenografts for MAP2K4 alterations. We found a splice acceptor mutation accompanied by loss of the other allele in the cell line MPE600. This was the sole alteration in this panel (5% of tumors). These data seem to re-establish a rather consistent rate of genetic inactivation of MAP2K4 among most tumor types, including breast cancer. The genetic evaluation of other mediators of the SAPK pathways might offer insight into a promising, but as yet poorly defined, tumor-suppressive system. © 2001 Wiley-Liss, Inc. [source] Bombyx mori Ras proteins BmRas1, BmRas2 and BmRas3 are neither farnesylated nor palmitoylated but are geranylgeranylatedINSECT MOLECULAR BIOLOGY, Issue 3 2010K. Moriya Abstract The lipid modifications which occur on Bombyx mori Ras proteins BmRas1, BmRas2 and BmRas3 were studied by metabolic labelling in an insect cell-free protein synthesis system and in a baculovirus expression system, using specific inhibitors of protein prenylation and protein palmitoylation. In addition, the subcellular localization of BmRas proteins was examined using EGFP fusion proteins of constitutively active forms of BmRas proteins transiently expressed in Sf9 cells. As a result, it was revealed that the three B. mori Ras proteins BmRas1, BmRas2 and BmRas3 are neither farnesylated nor palmitoylated but are geranylgeranylated for localization to the plasma membrane of insect cells. Thus, the mechanism of membrane binding of insect Ras proteins is quite different from that reported for mammalian Ras proteins. [source] Cytotoxic effects of ,, T cells expanded ex vivo by a third generation bisphosphonate for cancer immunotherapyINTERNATIONAL JOURNAL OF CANCER, Issue 1 2005Kiyoshi Sato Abstract Nitrogen containing-bisphosphonates (N-BPs), widely used to treat bone diseases, have direct antitumor effects via the inactivation of Ras proteins. In addition to the direct antitumor activities, N-BPs expand gd,,T cells, which exhibit major histocompatibility complex-unrestricted lytic activity. BPs accumulate intermediate metabolites which may be tumor antigens in target cells. The purpose of our study was to clarify the cytotoxicity of gd,, T cells expanded ex vivo by the most potent N-BP, zoledronate (ZOL). Especially, we focused on the importance of pretreatment against target cells also with ZOL; 1 m,M ZOL plus IL-2 increased the absolute number of gd,,T cells 298,768 fold for 14 days incubation. The small cell lung cancer and fibrosarcoma cell lines pretreated with 5 m,M ZOL showed a marked increase in sensitivity to lysis by gd,,T cells. While, untreated cell lines were much less sensitive to lysis by gdT cells. Video microscopy clearly demonstrated that gd,,T cells killed target cells pre-treated with ZOL within 3 hr. Pretreatment with 80 m,g/kg ZOL also significantly enhanced the antitumor activity of gd,,T cells in mice xenografted with SBC-5 cells. These findings show that ZOL significantly stimulated the proliferation of gd,,T cells and that gd,,T cells required pre-treatment with ZOL for cytotoxic activity against target cells. © 2005 Wiley-Liss, Inc. [source] Ras family genes: An interesting link between cell cycle and cancerJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2002M. Macaluso Ras genes are evolutionary conserved and codify for a monomeric G protein binding GTP (active form) or GDP (inactive form). The ras genes are ubiquitously expressed although mRNA analysis suggests different level expression in tissue. Mutations in each ras gene frequently were found in different tumors, suggesting their involvement in the development of specific neoplasia. These mutations lead to a constitutive active and potentially oncogenic protein that could cause a deregulation of cell cycle. Ras protein moderates cellular responses at several mitogens and/or differentiation factors and at external stimuli. These stimuli activate a series of signal transduction pathways that either can be independent or interconnected at different points. Recent observations begin to clarify the complex relationship between Ras activation, apoptosis, and cellular proliferation. A greater understanding of these processes would help to identify the factors directly responsible for cell cycle deregulation in several tumors, moreover it would help the design of specific therapeutic strategies, for the control on the proliferation of neoplastic cells. We summarize here current knowledge of ras genes family: structural and functional characteristics of Ras proteins and their links with cell cycle and cancer. © 2002 Wiley-Liss, Inc. [source] Purification, crystallization and preliminary X-ray analysis of the GTP-binding protein Rab9 implicated in endosome-to-TGN vesicle traffickingACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2004Julia G. Wittmann Rab GTP-binding proteins are involved in the regulation of distinct vesicular-transport events involving membrane targeting and fusion. They differ from other small GTPases by the presence of specific loop regions that serve as effector-binding sites in addition to the classical switch I and switch II regions. While the structures of many small GTP-binding proteins of the Ras superfamily are available in both GDP- and GTP-bound forms, Rab proteins are less well characterized than Ras proteins at the structural level. The crystallization of Rab9, a key regulatory component in the recycling of mannose-6-phosphate receptors from endosomes to the trans-Golgi network, is described here. [source] A rapid and simple HPLC-UV method for the determination of inhibition characteristics of farnesyl transferase inhibitorsBIOMEDICAL CHROMATOGRAPHY, Issue 2 2006Natalie M. G. M. Appels Abstract Ras proteins play an important role in the development of cancer. Farnesyl transferase inhibitors (FTIs) block the first obligatory post-translational step for activation, prenylation, of Ras proteins. To find new potent FTIs, rapid enzyme activity assays are required to reduce FTI development time. Most assays to date are based on radioactive labelled substrates. We developed a new, in vitro, farnesyl transferase assay based on gradient chromatography coupled to UV detection. Unfarnesylated and farnesylated H-Ras proteins were resolved on a C18 wide-pore HPLC column and their concentrations were determined with use of a calibration curve of unfarnesylated H-Ras. The assay was used to investigate inhibition characteristics of FTIs. The IC50 values of the FTIs L778,123 and SCH66336 were 4.2 nm and 78 µm, respectively. This assay could support the screening and development of FTIs to obtain rapid insights into their inhibitory properties. Copyright © 2005 John Wiley & Sons, Ltd. [source] Site-Specific Functionalization of Proteins by Organopalladium Reactions,CHEMBIOCHEM, Issue 2 2007Koichiro Kodama Abstract A new carbon,carbon bond has been regioselectively introduced into a target position (position 32 or 174) of the Ras protein by two types of organopalladium reactions (Mizoroki,Heck and Sonogashira reactions). Reaction conditions were screened by using a model peptide, and the stability of the Ras protein under the reaction conditions was examined by using the wild-type Ras protein. Finally, the iF,Ras proteins containing a 4-iodo- L -phenylalanine residue were subjected to organopalladium reactions with vinylated or propargylated biotin. Site-specific biotinylations of the Ras protein were confirmed by Western blot and LC-MS/MS. [source] |