Dissertations / Theses: 'Ras proteins'

1

Clyde-Smith, Jodi. "Characterization of ras isoform activation by ras guanine nucleotide exchange factors /." St. Lucia, Qld, 2002. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16393.pdf.

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2

Iritani, Brian Masao. "Control of B lymphocyte development by Ras and Raf /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/8322.

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3

Chan, Yuk-shing, and 陳旭勝. "Expression of RAs-related Nuclear (RAN) protein in breast cancer." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44671003.

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4

Castillo, Chabeco Boris. "Redox Regulation of Ras Proteins in Dictyostelium discoideum." FIU Digital Commons, 2015. http://digitalcommons.fiu.edu/etd/1864.

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Reactive oxygen species are a normal consequence of life in an aerobic environment. However when they deviate from the narrow permissible range in cells, oxidative damage can occur. Dictyostelium discoideum is a model organism ideal for the study of cell signaling events such as those affected by oxidative stress. It was previously shown that Ras signaling in Dictyostelium is affected by genetic inactivation of the antioxidant enzyme Superoxide dismutase C (SodC) and in vitro data suggests that the NKCD motif of Ras is the redox target of superoxide. The main objective of this project was to determine the mechanism of superoxide mediated Ras regulation in vivo. To accomplish the main objective, we cloned, and in some cases, mutated different Ras proteins and later determined their activity in wild type and sodC- cells. RasC and RasD showed normal activation in sodC- cells, however RasG and RasS displayed high Ras activity. These last two Ras proteins contain the NKC118D motif inside the nucleotide binding region. A mutation of cysteine118 to alanine in RasG rendered the protein less active in sodC- than the wild type RasG protein and a mutation alanine118 to cysteine in RasD conferred redox sensitivity to this small GTPase. Additionally, the propensity of RasG to be targeted by superoxide was evident when the environment of wild type cells was manipulated to induce the internal generation of superoxide through changes in the extracellular ion levels mainly magnesium. Lack of magnesium ions increased the intracellular level of superoxide and severely hampered directional cell migration. Chemotaxis of cells expressing RasG was negatively impacted by the absence of magnesium ions; however rasG- cells did not seem to be affected in their ability to perform chemotaxis. The last experiment implies that RasG is an important mediator of cell signaling during oxidative stress, responsible for preventing cells from continuing their developmental program. Our study suggests that the cysteine residue in the NKCD motif is essential for mediating the redox sensitivity of Ras proteins in Dictyostelium and that RasG is an essential mediator of the response to oxidative stress in this organism.

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5

Iuga, Adriana. "Solid-state 31P NMR of nucleotide binding proteins." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=973225238.

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6

McGee, John Hanney. "Evolving a Direct Inhibitor of the Ras Proteins." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10915.

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In recent years, great advances have been made in understanding the molecular causes of human disease, but our ability to exploit these discoveries for therapeutic benefit is frequently limited by the inability to make drugs that target the processes responsible. Many diseases can be linked to the aberrant activity of proteins, and while the development of inhibitors for enzymes and extracellular targets is often feasible, these proteins account for only a small fraction of all the proteins in cells. The remaining proteins are, in most cases, considered therapeutically intractable and are sometimes referred to as "undruggable." Many proteins, particularly in higher organisms, carry out their activity in part through interactions with other proteins and biomolecules. The ability to specifically disrupt these interactions could have great therapeutic benefit, as it may provide a means of targeting otherwise intractable processes. The focus of this dissertation is on the development and characterization of molecules that inhibit the interactions of an “undruggable” protein target, Ras, which is linked to both the initiation and progression of a wide array of human cancers. Our approach has been to use high-throughput screening, coupled with directed evolution, to identify and improve small proteins (peptides) that bind Ras and block its ability to engage the effector proteins necessary for its oncogenic activity. We report these efforts, along with a series of biochemical experiments aimed at characterizing the properties and binding mechanism of the peptides discovered in the screen. These peptides bind the three human Ras proteins with mid-to-low nanomolar affinity, and with high specificity for Ras proteins over their close family members. The peptides directly engage the Ras effector domain, and can block Ras from binding a canonical effector protein in the context of cancer cell lysates. Based on a series of observations, we hypothesize that the peptides bind Ras as head-to-tail homodimers, and report preliminary attempts to exploit this observation and identify peptides with improved affinity to Ras. Finally, we discuss the preliminary results from a conceptually related effort to identify peptide inhibitors of the Myc transcription factor, which is another protein heavily implicated in human cancer.

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7

Worth, Graham Alan. "The energetics of nucleotide binding to RAS proteins." Thesis, University of Oxford, 1992. http://ora.ox.ac.uk/objects/uuid:44524415-2f2b-4601-998c-56110f332153.

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Ras proteins are a special class of proteins that mediate cell growth signals. Their importance lies in the fact that they are products of a proto-oncogene. This means that under certain conditions the gene that determines its structure is altered and a mutant protein results that is involved in the transformation of normal cells to cancer cells. The actual function by which the protein acts in the signal pathway is not known. However it is known that they act as a switch, undergoing a cycle involving the exchange of guaninosine nucleotides in the binding site. This thesis uses computer simulations to study the energetics of this binding, with the long term aim of developing a drug to inhibit the transforming activity of the oncogenic protein. To begin with, a model of the protein based on a crystal structure is built. Using Molecular dynamics the motion of this model is studied. A possible mechanism by which one half of the nucleotide cycle could be induced is investigated, with the result that phosphorylation of the protein may be involved. The main part of the thesis is then devoted to using the free energy perturbation (FEP) method to calculate the difference in Gibbs binding free energy between the nucleotides in the protein. Using histamine as a model, a method of dealing with charged, flexible molecules is developed; namely the inclusion of a reaction field and comprehensive conformational analysis. The results from the associated calculations are seen to be very close to experimental data. The same procedures are then applied to the much more complex ras: nucleotide system with less successful results, the reason for which is mostly due to the restriction of limited computer resources to tackle such a problem. The conclusion is that given the resources and by using the techniques developed in this thesis, this type of calculation is a feasible way to study such systems.

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8

Wilkins, Andrew. "The function of Ras proteins in Dictyostelium discoideum." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313544.

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9

Naim, Adnan. "The Role of G3BPs in the Stress Response Pathway." Thesis, Griffith University, 2016. http://hdl.handle.net/10072/367499.

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The ras-GTPase SH3-domain Binding Proteins (G3BP) are a family of RNA-binding proteins that have been implicated in multiple cellular activities ranging from signal transduction to regulation of messenger RNA (mRNA). G3BPs were named after their interaction with the SH3 domain of Ras-GTPase-activating protein; however recent research did not find this interaction. All three members of the G3BPs family, G3BP1, G3BP2a and G3BP2b, share structural similarities with each other by having four distinct regions (1) the Nuclear Transporting Factor 2, (NTF2) domain at the N-terminal, (2) the acidic and proline-rich domain in the centre, (3) the RNA recognition motif (RRM) and (4) the arginine glycine (RGG)-rich region rich at the C-terminal. The presence of the NTF2 domain in its structure suggests G3BP might play a role in nucleocytoplasmic transportation, which was observed after serum stimulation where G3BP1 was translocated to the nucleus from the cytoplasm. The RNA recognition motif (RRM) region plays a vital role in its interaction with the target RNA. The RGG-rich box is a region rich in arginine and glycine residues, which plays a role assisting RRM in interactions with protein or RNA. G3BP1 is found to be overexpressed in many cancers, including breast cancer, and head and neck tumours, as well as cell lines derived from human lung, prostrate, colon, thyroid and breast cancer. G3BPs have also been implicated in translational control within differentiating neurons, suggesting that G3BP may play several roles in controlling the translational fate of its cargo and that its role may be cell-specific. G3BP1 has also been found in β-integrin- induced adhesion complexes. This information highlights G3BPs as a dynamic protein that is involved in several biological functions.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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10

De, Cristofano Sabrina. "The role of Ras and Kinase Suppressor of Ras 1 (KSR-1) in breast cancer in progression and metastasis /." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112613.

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The Ras signaling cascade is a vital component in the processes that mediate cell survival, growth, differentiation and transformation through activation of MAP kinase (mitogen-activated protein kinase). The recent discovery of a new scaffold of the Ras signaling pathway, Kinase Suppressor of Ras (KSR), is found to be a positive effector of Ras signaling which further contributes to proliferation and transformation in the ERK/MAPK pathway. This thesis describes the roles of Ras and Kinase Suppressor of Ras 1 (KSR-1) in regulating the expression of tumor promoting genes such as urokinase plasminogen activator (uPA) in the development and progression of breast cancer in vitro and in vivo. Ras and KSR increase the proliferative capacity and migration of MDAMB-231 human breast cancer cells in vitro. In contrast, Ras and KSR decrease the invasiveness of MDA-MB-231 human breast cancer cells in vitro. Furthermore, uPA gene expression levels do not correlate with uPA protein expression levels suggesting a possible mutation induced by KSR and/or Ras. In vivo studies reveal that Ras and KSR increase tumor volume in mice, as well as more advanced osteolytic bone metastases. Collectively, these results indicate that Ras and KSR play significant roles in breast cancer development and metastasis.

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11

Bramble, Sharyl Elizabeth. "Guanine nucleotide binding properties and attempted immunopurification of ras protein from dictyostelium discoideum." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26172.

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One purpose of this study was to determine whether the ras protein from Dictyostelium discoideum (p23) binds guanine nucleotides like the ras proteins from mammals (p21) and yeast. The other purpose of this investigation was to purify or enrich for p23ras from D. discoideum by immunoaffinity chromatography. A number of different approaches were used to determine guanine nucleotide binding by p23RAS . A simple filter binding assay, binding to Western blots, and photoaffinity labeling all failed to demonstrate specific binding with lysates of D. discoideum cells. In contrast p21RAS from transformed NIH-3T3 cell lysate was successfully photoaffinity labeled in the presence of ³²P-α-guanosine 5¹-triphosphate (GTP) suggesting that the technique had been performed correctly. It was concluded that either p23RAS has a very low affinity for guanine nucleotides such that GTP binding was not detectable in these experiments or that the ras protein from D. discoideum simply does not bind guanine nucleotides. The purification of p23RAS from D. discoideum cells was attempted in order to provide a purified protein preparation for guanine nucleotide binding and for reconstitution studies. An anti-ras monoclonal antibody (Y13-259) was used as the ligand for the immunoaffinity chromatography. This approach was not successful in that the ras protein could not be enriched relative to other proteins because the immunoaffinity columns did not bind p23RAS.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

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12

Schroder, Wayne Ashley. "Cloning and Characterisation of the Human SinRIP Proteins." Thesis, Griffith University, 2003. http://hdl.handle.net/10072/366190.

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This thesis describes the cloning and characterisation of a novel human gene and its protein products, which have been designated SAPK- and Ras-interacting protein (SinRIP). SinRIP shares identity with JC310, a partial human cDNA that was previously identified a candidate Ras-inhibitor (Colicelli et al., 1991, Proc Natl Acad Sci USA 88, p. 2913). In this study, it was shown that SinRIP is a member of an orthologous family of proteins that is conserved from yeast to mammals and contains proteins involved in Ras- and SAPK-mediated signalling pathways. Comparison of this family of proteins showed that human SinRIP contains a potential Ras-binding domain (RBD; residues 279-354), a PH-like domain (PHL; 376-487), and a highly conserved novel region designated the CRIM (134-265). Several other potential targeting sites, such as nuclear localisation signals and target sites for kinases, were identified within the SinRIP sequence. The human SinRIP gene is unusually large (>280 kbp) and is located on chromosome 9 at 9q34. SinRIP mRNA was detected in a wide variety of tissue-types and cell lines by RT-PCR, and the SinRIP sequences in the EST database were derived from an diverse array of tissues, suggesting a widespread or ubiquitous expression. Northern blot analysis revealed the highest levels in skeletal muscle and heart tissue. However, the steady-state levels of SinRIP mRNA vary greatly from cell to cell, and SinRIP expression is likely to be regulated at multiple post-transcriptional levels. It was shown that SinRIP mRNA is likely to be translated inefficiently by the normal cap-scanning mechanism, due to the presence of a GC-rich and structured 5’-UTR, which also contains upstream ORFs. Alternative polyadenylation signals in the SinRIP 3’-UTR can be used, resulting in the expression of short and long SinRIP mRNA isoforms. Several potential A/T-rich regulatory elements were also identified in SinRIP mRNA, which may target specific SinRIP mRNA isoforms for rapid degradation. Importantly, it was shown that SinRIP mRNA is alternatively spliced, resulting in the production of distinct SinRIP protein isoforms. Three isoforms, SinRIP2-4, were definitively identified by RT-PCR and full-length cloning. The SinRIP isoforms contain deletions in conserved regions, and are likely to have biochemical characteristics that are different to full-length SinRIP1. SinRIP2 is C-terminally truncated and lacks the PHL domain and part of the RBD, and relatively high levels of SinRIP2 expression arelikely to occur in kidneys. The RBD is disrupted in SinRIP3, but all other domains are intact, and RT-PCR analyses suggest that SinRIP3 is present in some cells at levels comparable to SinRIP1. A rabbit polyclonal antiserum against SinRIP was generated and detected endogenous SinRIP proteins. Using the anti-SinRIP antibody in immunoblots, multiple SinRIP isoforms were observed in most cell types. SinRIP1 and another endogenous SinRIP protein, likely to be SinRIP3, were detected in most cell lines, and appear to be are the major SinRIP proteins expressed in most cells. The subcellular localisation of both recombinant and endogenous SinRIP proteins was investigated by immunofluorescence assays and biochemical fractionation. Recombinant SinRIP1 protein was found in the cytoplasm and associated with the plasma membrane. In contrast, the SinRIP2 protein was predominantly nuclear, with only low-level cytoplasmic staining observed. The endogenous SinRIP proteins, likely to comprise these and other SinRIP isoforms, were found in both the nucleus and cytoplasm. SinRIP1 interacted with GTP-bound (active) Ras, but not GDP-bound (inactive) Ras, in an in vitro assay, and also co-localised with activated H- and K-Ras in cells. The binding profile observed is typical of Ras-effectors, and SinRIP did not inhibit signalling by the Ras proteins, suggesting that it is not likely to be a Ras-inhibitor. It was also shown that SinRIP1 and SinRIP2 both interact and colocalise with c-Jun NH2- terminal kinase (JNK). Both SinRIP proteins were able to recruit JNK to their respective sub-cellular compartments. These interactions suggest an adaptor role for SinRIP in the Ras and/or JNK pathways. In addition, Sam68 was isolated as a SinRIP-binding protein in a yeast two-hybrid screen. Sam68 was shown to colocalise with SinRIP2 and endogenous SinRIP proteins, but not SinRIP1. Further colocalisation studies showed that endogenous SinRIP proteins localise in nuclear structures that may be associated with pre-mRNA splicing. Likely functions for SinRIP, as indicated by experimental results and studies of the orthologues of SinRIP in other species, are discussed.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Faculty of Science
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13

Martins, Carla Pedro. "Cip/Kip proteins in the suppression of murine lymphomagenesis." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2003. http://dare.uva.nl/document/67628.

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14

Liu, Li. "Purification and characterization of a protein palmitoyltransferase that acts on H-Ras protein and on a C-terminal N-Ras peptide /." Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/8664.

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15

Roy, Sandrine. "An Investigation of the interaction of Ras with Cell membranes /." St. Lucia, Qld, 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16356.pdf.

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16

Gibson, Janet Rae. "A study of RAS p21 and related GTP-binding proteins." Thesis, University of East Anglia, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293243.

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17

Schroder, Wayne Ashley, and n/a. "Cloning and Characterisation of the Human SinRIP Proteins." Griffith University. School of Biomolecular and Biomedical Science, 2003. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20030829.140754.

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This thesis describes the cloning and characterisation of a novel human gene and its protein products, which have been designated SAPK- and Ras-interacting protein (SinRIP). SinRIP shares identity with JC310, a partial human cDNA that was previously identified a candidate Ras-inhibitor (Colicelli et al., 1991, Proc Natl Acad Sci USA 88, p. 2913). In this study, it was shown that SinRIP is a member of an orthologous family of proteins that is conserved from yeast to mammals and contains proteins involved in Ras- and SAPK-mediated signalling pathways. Comparison of this family of proteins showed that human SinRIP contains a potential Ras-binding domain (RBD; residues 279-354), a PH-like domain (PHL; 376-487), and a highly conserved novel region designated the CRIM (134-265). Several other potential targeting sites, such as nuclear localisation signals and target sites for kinases, were identified within the SinRIP sequence. The human SinRIP gene is unusually large (>280 kbp) and is located on chromosome 9 at 9q34. SinRIP mRNA was detected in a wide variety of tissue-types and cell lines by RT-PCR, and the SinRIP sequences in the EST database were derived from an diverse array of tissues, suggesting a widespread or ubiquitous expression. Northern blot analysis revealed the highest levels in skeletal muscle and heart tissue. However, the steady-state levels of SinRIP mRNA vary greatly from cell to cell, and SinRIP expression is likely to be regulated at multiple post-transcriptional levels. It was shown that SinRIP mRNA is likely to be translated inefficiently by the normal cap-scanning mechanism, due to the presence of a GC-rich and structured 5-UTR, which also contains upstream ORFs. Alternative polyadenylation signals in the SinRIP 3-UTR can be used, resulting in the expression of short and long SinRIP mRNA isoforms. Several potential A/T-rich regulatory elements were also identified in SinRIP mRNA, which may target specific SinRIP mRNA isoforms for rapid degradation. Importantly, it was shown that SinRIP mRNA is alternatively spliced, resulting in the production of distinct SinRIP protein isoforms. Three isoforms, SinRIP2-4, were definitively identified by RT-PCR and full-length cloning. The SinRIP isoforms contain deletions in conserved regions, and are likely to have biochemical characteristics that are different to full-length SinRIP1. SinRIP2 is C-terminally truncated and lacks the PHL domain and part of the RBD, and relatively high levels of SinRIP2 expression arelikely to occur in kidneys. The RBD is disrupted in SinRIP3, but all other domains are intact, and RT-PCR analyses suggest that SinRIP3 is present in some cells at levels comparable to SinRIP1. A rabbit polyclonal antiserum against SinRIP was generated and detected endogenous SinRIP proteins. Using the anti-SinRIP antibody in immunoblots, multiple SinRIP isoforms were observed in most cell types. SinRIP1 and another endogenous SinRIP protein, likely to be SinRIP3, were detected in most cell lines, and appear to be are the major SinRIP proteins expressed in most cells. The subcellular localisation of both recombinant and endogenous SinRIP proteins was investigated by immunofluorescence assays and biochemical fractionation. Recombinant SinRIP1 protein was found in the cytoplasm and associated with the plasma membrane. In contrast, the SinRIP2 protein was predominantly nuclear, with only low-level cytoplasmic staining observed. The endogenous SinRIP proteins, likely to comprise these and other SinRIP isoforms, were found in both the nucleus and cytoplasm. SinRIP1 interacted with GTP-bound (active) Ras, but not GDP-bound (inactive) Ras, in an in vitro assay, and also co-localised with activated H- and K-Ras in cells. The binding profile observed is typical of Ras-effectors, and SinRIP did not inhibit signalling by the Ras proteins, suggesting that it is not likely to be a Ras-inhibitor. It was also shown that SinRIP1 and SinRIP2 both interact and colocalise with c-Jun NH2- terminal kinase (JNK). Both SinRIP proteins were able to recruit JNK to their respective sub-cellular compartments. These interactions suggest an adaptor role for SinRIP in the Ras and/or JNK pathways. In addition, Sam68 was isolated as a SinRIP-binding protein in a yeast two-hybrid screen. Sam68 was shown to colocalise with SinRIP2 and endogenous SinRIP proteins, but not SinRIP1. Further colocalisation studies showed that endogenous SinRIP proteins localise in nuclear structures that may be associated with pre-mRNA splicing. Likely functions for SinRIP, as indicated by experimental results and studies of the orthologues of SinRIP in other species, are discussed.

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18

Seifert, Jason Paul Harden T. Kendall. "Regulation of phospholipase C-epsilon by Rho and Ras family proteins." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1008.

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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Dec. 18, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology." Discipline: Pharmacology; Department/School: Medicine.

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19

Tuxworth, Richard Ian. "The control of cell motility and differentiation by Ras pathways." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314227.

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20

Wang, Kai Ling. "Identification and characterisation of a Ras-related GTP-binding protein, Tal-A, purified from human erythrocyte membranes." Thesis, The University of Sydney, 1997. https://hdl.handle.net/2123/29249.

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This thesis investigates the identification and characterisation of a protein with monomeric molecular mass of 28 kDa (referred as to p28) purified from human erythrocyte plasma membrane. Chapter Two describes the purification of p28 from Triton X-100 extracts of human erythrocyte membrane by calmodulin affinity chromatography. Based on internal peptide sequencing and its protein amino acid composition, this protein has been shown to be highly related, if not identical to Ral-A, a Ras-related GTP-binding protein. Results presented in Chapter Three characterises this protein as a low-molecular weight GTP-binding (LMWG) protein and further confirmed the protein assignment by showing that it binds [32P]GTP specifically and has low intrinsic GTPase activity. A novel finding described in Chapter Four shows that Ral-A is a calmodulin-binding protein and its calmodulin binding domain has been identified and characterised. The interaction of Ral-A with calmodulin was first detected on the basis of its ability to bind to a calmodulin affinity column in a Ca2+ -dependent manner and to be released upon elution by chelation of Ca2+ with EDTA. Biotinylated calmodulin overlay experiments have confirmed that biotinylated calmodulin binds to renatured Ral-A in a Ca2+-dependent manner. Based on inspection of Ral-A amino acid sequence for the presence of a basic/hydrophobic composition with the propensity to form an amphiphilic helix, a putative calmodulin-binding domain with the sequence 183 SKEKNGKKKRKSLAKRJ200R was identified within the C-terminal region ofRalA. This 18 amino acid peptide has been synthesised and its interaction with calmodulin has been characterised. Phosphorylation of Ral-A by protein kinases has been investigated. Chapter Five shows that Ral-A was phosphorylated in vitro by PKA, PKG, and PKC and thephosphorylation was regulated by calmodulin, further supporting the finding that RalA is a calmodulin-binding protein. In agreement with other literature findings that phosphorylation of Ras-related proteins does not regulate their GTP-binding and GTPase activities, Ral phosphorylation by PKC and PKG did not affect on its ability to bind to GTP. However, Ral-A phosphorylation by PKC and PKG, but not PKA, was enhanced in the presence of GTP. The identification of Ral-A putative phosphorylation sites near its isoprenylated C-terminus leads to the speculation that the possible physiological consequence of Ral-A phosphorylation may facilitate its transmembrane localisation. The unusual Ca2+ inhibition of Ral-A phosphorylation by CaM kinase II may reflect the potential significance of calmodulin binding by RalA in Ca2+/calmodulin-regulated processes. The consequences of Ral-A dephosphorylation were further examined. Ral proteins represent a distinct family of Ras-related GTP-binding proteins. The recent finding that RalGDS interacts with Ras and functions as a putative effector protein in Ras signalling pathways indicates that Ral proteins and RalGDS constitute a distinct downstream pathway from Ras in parallel with activation of the Raf/mitogen-activated protein kinase cascade. Studies presented in this thesis constitute a previously undescribed calmodulin-binding characteristic of Ral-A protein. This finding has provided an exciting new perspective that the function of Ral proteins may be modulated by Ca2+ /calmodulin and may have a potential significant function in Ca2+ signalling pathways. It also raises the possibility of cross-talk between signal transduction pathways mediated by Ca2+/calmodulin and Ras proteins.

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21

Bruce, Emily Adaline. "Role of the Rab11 pathway in influenza virus assembly and budding." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610519.

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22

Trowbridge, Amanda J. "Expression of SNAP23 and Rab3A in mouse oocytes and fertilized eggs and their role in cortical granules exocytosis." Virtual Press, 2004. http://liblink.bsu.edu/uhtbin/catkey/1307377.

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The proteins and molecular machinery mediating the release of cortical granule (CG) contents from fertilized embryos is not completely understood. The process of vesicle fusion involves linking chaperones prior to vesicle to membrane contact. Rab3A, a member of a low-molecular weight GTP-binding protein superfamily has been detected in mouse embryos from the unfertilized meiotic II stage to the 2-cell. It is believed to positively regulate the final step of CG exocytosis by binding to Rabphillin, calcium ions (Ca2+), and phospholipids. SNAP23 a member of soluble NSF [N-ethylmaleimidesensitive factor] attachment protein receptors (SNAREs) binds together with parts of the Rab3A-rabphilin3A complex and is believed to be involved in the Ca2+-dependent exocytosis of non-neuronal systems. In this study we observed the mRNA expression for SNAP23 and Rab3A in pre-Meiotic I, post-Meiotic I unfertilized eggs (pre-MI UFE and post-MI UFE), and fertilized eggs (FE) utilizing RT-PCR. The products were analyzed in 2% agarose gel stained with ethidium bromide. Density analysis using a globin external standard showed that the levels of mRNA transcripts declined from the UFE to the FE in both genes, SNAP23 and Rab3A. Immunofluorescence was used for the detection and localization of Rab3A protein within the pre-MI and post-MI UFE and FE mouse egg. Eggs were stained with anti-Rab3A primary antibody and lens culinaris agglutinin (LCA) conjugated to FITC. Rab3A showed punctate staining in pre- and post-MI UFEs on small vesicles assumed to be CGs and in FEs on vesicles of a larger size. Uniform cytoplasmic expression was also seen, throughout the cells cortical and subcortical regions in each stage (pre- and post-MI UFEs and FEs), but with decreasing intensity as the eggs matured. This cytoplasmic stain may represent inactive Rab3A in the cytosol. The LCA stain showed punctate expression of cortical granules with localization within the cortical region and the plasma membrane. The addition of information on SNAP23 and Rab3A will aid in the process of studying CG exocytosis as well as in understanding the temporal and spatial development pathways involved in stimulating the cortical reaction.
Department of Biology

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23

McCollam-Guilani, Linda Sue. "Analysis of the Mechanism of Ras Activation: Mapping of Important Functional Domains of the Son of Sevenless Protein." eScholarship@UMMS, 1998. https://escholarship.umassmed.edu/gsbs_diss/90.

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The questions outlined in this thesis dissertation were proposed in order to provide insight regarding the mechanism by which the Drosophila Son of sevenless (dSOS) protein activates Ras. Ras proteins are GTP-binding proteins which bind guanine nucleotides very tightly and cycle between the inactive GDP-bound state and the active GTP-bound state. To address the mechanism by which the dSOS proteins activates Ras, a structure-function analysis of the dSOS protein was performed using truncation and deletion mutants of dSOS. In vivo Ras activation experiments using transiently transfected cells revealed that the NH2-terminal domain of dSOS is required in order for the catalytic domain of dSOS to exhibit exchange activity in cultured mammalian cells. The COOH-terminal GRB2 (Growth Factor Receptor Binding Protein) binding domain on the otherhand was insufficient to confer Ras exchange activity to the dSOS catalytic domain. Further analysis of the NH2-terminal domain of the dSOS protein demonstrated that the function of promoting catalytic domain activity could be localized by mutational analysis to the pleckstrin (PH) and DBL (Diffuse B-cell Lymphoma) homology sequences. Fractionation studies of cells transiently transfected with various dSOS mutant proteins demonstrated that the NH2-terminus of dSOS is also necessary for membrane association. These findings suggested that the model proposing that the recruitment of SOS via the adaptor protein GRB2 to the membrane is the main mechanism by which SOS activates Ras is unlikely to be the only mechanism by which SOS can activate Ras. From our data, a model can be proposed which postulates that SOS can activate Ras as a consequence of at least two steps. One step involves the SOS/GRB2 interaction and the second step involves the NH2-terminal domain of SOS associating with unidentified cellular elements.

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24

Prober, David Aaron. "Regulation of cell growth and cell identity by Ras 1 in the developing Drosophila melanogaster wing /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/4988.

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25

BROGGI, SERENA. "Studies on active RAS proteins localization and evidences for nuclear active RAS2 involvement in invasive growth in saccharomyces cerevisiae." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2013. http://hdl.handle.net/10281/41878.

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In the yeast Saccharomyces cerevisiae, the Ras proteins are part of the cAMP/PKA signalling pathway, which plays a fundamental role in the control of many cellular processes including cells proliferation, stress resistance, metabolism, and growth. They belong to the super-family of the small GTPases that act as molecular switches by cycling between an inactive GDP-bound form and an active GTP-bound form. This process is controlled by two classes of regulatory proteins: the GEFs promote the activation of Ras by catalyzing the GDP-GTP exchange, whereas the GAPs turn off the Ras proteins by stimulating the hydrolysis of GTP to GDP. In the first section of this thesis, we investigated the localization of active Ras proteins in wild type cells and in mutants in several components of the cAMP/PKA pathway to understand how the proteins involved in this pathway influence the localization of active Ras. To this aim we used a probe in which the eGFP (enhanced green fluorescent protein) is fused to a trimeric Ras binding domain (RBD3) of the human Ras effector, c-Raf1. This RBD directly binds to the active Ras with a much higher affinity than the inactive Ras. We also investigated the influence of PKA activity on active Ras localization analyzing different mutants with either high or low/absent PKA activity. The cells of the different strains expressing the eGFP-RBD3 probe growing on glucose medium were observed under the microscope. In wild type cells, Ras-GTP was mainly localized at the plasma membrane and surprisingly in the nucleus. In cyr1∆ and gpr1∆ cells, the probe showed a similar localization as in wild type cells. In gpa2∆, hxk2∆ and hxk1∆hxk2∆ cells, the fluorescence accumulated in internal membranes and mitochondria. However, in the hxk1∆hxk2∆ cells transformed with the centromeric plasmid YCpHXK2 expressing Hxk2, the eGFP-RBD3 probe was mainly localized at the plasma membrane and in the nucleus. These results suggest that Gpa2 and Hxk2 play a role in the localization of active Ras. We also observed that the localization of active Ras is dependent on PKA activity. Indeed, in the bcy1∆ mutant, showing high PKA activity, there was a clear relocalization of active Ras to the cytoplasm and to the nucleus, while no active Ras was localized at the plasma membrane anymore. In a strain with either reduced PKA activity, the tpk1w1 tpk2∆ tpk3∆ strain or absent PKA activity, the tpk1∆ tpk2∆ tpk3∆ yak1∆ strain, active Ras was mainly localized at the plasma membrane. In the second section of this thesis, we investigated the role played by active Ras in the nucleus. To this aim, a fusion was made between the Ras2 protein and the Nuclear Export Signal (NES) from the HIV virus (HIV virus Rev protein NES) (Henderson et al., 2000), generating the NES-RAS2 strain. Our results showed that the exclusion of Ras2 protein from the nucleus did not cause a growth defect neither on fermentable nor non fermentable carbon sources and did not influence the PKA related phenotypes analyzed in our work. Cells expressing the fusion protein were only defective for the invasive growth, suggesting that nuclear active Ras2 is involved in this cellular process. These results were confirmed using also the Tlys86 strain, that is commonly used to test this phenotype. We also demonstrated that the nuclear localization of Cdc25, the main GEF of Ras proteins, is required for invasive growth and that PKA activity controls invasive growth influencing the localization of active Ras. Data in literature (Cazzaniga et al., 2008; Pescini et al., 2012) show the presence in silico of cAMP levels oscillations. In the last section of this thesis, we tested two different FRET sensors, previously used in mammalian cells, to monitor the cAMP levels (CFP-Epac1-YFP probe) and PKA activity in single cells in vivo (AKAR3 probe). We inserted the sequences coding for the CFP-Epac1-YFP sensor and for the AKAR3 sensor in a multicopy yeast expression vector and the sensors were expressed under the control of the TPI promoter in several yeast strains. We used a two-photon confocal microscope system to measure the FRET efficiency. Our preliminary results showed that in a wild type strain expressing either the Epac sensor or the AKAR3 sensor there was respectively an increase of cAMP level and PKA activity in a single yeast cell after glucose addition to glucose-starved cells.

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D'Silva, Nisha Jacinta. "Rap1, a small GTP-binding protein in the rat parotid gland : identification, investigation of function and regulation /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/6388.

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Wahlström, Annika. "Defining RCE1 and ICMT as therapeutic targets in K-RAS-induced cancer /." Göteborg : The Wallenberg Laboratory, Dept.of Molecular and Clinical Medicine, Institute of Medicine at Sahlgrenska Academy, University of Gothenburg, 2009. http://hdl.handle.net/2077/19643.

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28

Katsoulotos, Gregory Peter St George Clinical School UNSW. "The function of the signaling protein Ras guanine releasing protein 4 (RasGRP4) in human mast cells." Awarded by:University of New South Wales. St George Clinical School, 2006. http://handle.unsw.edu.au/1959.4/27341.

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Mast cells have been implicated in the pathogenesis of both atopic and non-atopic asthma. Ras guanine nucleotide-releasing protein 4 (RasGRP4) is a mast cell-restricted guanine nucleotide exchange factor and diacylglycerol (DAG)/ phorbol ester receptor whose function has not been deduced. RT-PCR analysis of 40 asthmatic patients and 40 non-asthmatic controls demonstrated a higher hRasGRP4 mRNA expression in a subgroup of the asthmatics. A RasGRP4-defective variant of the human mast cell line HMC-1 was used to create stable clones expressing green fluorescent protein-labeled human RasGRP4 for monitoring the movement of this signaling protein inside mast cells before and after exposure to phorbol-12-myristate 13-acetate (PMA) and for evaluating the protein???s ability to control the development, phenotype, and function of mast cells. Transcript-profiling approaches revealed hRasGRP4 constitutively regulates the expression of numerous genes in the HMC-1 cell line. For example, expression of hRasGRP4 in HMC-1 cells substantially decreased GATA-1 levels without altering GATA-2 levels, suggesting that hRasGRP4 regulates mast cell commitment of multipotential progenitors in part by controlling the intracellular levels of at least one lineage-dependent transcription factor for hematopoietic cells. hRasGRP4 resided primarily in the cytosol before HMC-1 cells were stimulated with PMA. After exposure to PMA, hRasGRP4 translocated to the inner leaflet of the cell???s plasma membrane and then to perinuclear and Golgi compartments. Extracellular signal-regulated kinases 1 and 2 were activated during this translocation process, and the PMA-treated cells transiently increased their expression of the transcripts encoding the interleukin 13 receptor IL-13R??2 and numerous other proteins. The accumulated data in our mast cell model suggest hRasGRP4 translocates to various intracellular compartments via its DAG/PMA-binding domain to regulate those signaling pathways that allow mast cells to respond quickly to changes in their tissue microenvironments.

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Self, Annette Jane. "Structural and functional analysis of Ras and Ruo-related small GTP-binding proteins." Thesis, Institute of Cancer Research (University Of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266353.

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30

Lemaire, Mathieu. "Intracellular signals underlying the inductive effects of agrin during neuromuscular junction formation : study on the roles of ras and Shc." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0030/MQ64388.pdf.

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31

Bolourani, Parvin. "Partitioning of the response to cAMP via two specific Ras proteins during Dictyostelium discoideum development." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/1719.

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Following starvation, Dictyostelium discoideum cells aggregate, a response that requires chemotaxis to cyclic AMP (cAMP) and the relay of the cAMP signal by the activation of adenylyl cyclase (ACA). Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG does have a role in early development. When the responses of rasG⁻ cells to cAMP were compared with the responses of rasC⁻ strain, these studies revealed that signal transduction through RasG is more important in chemotaxis and early gene expression, but that signal transduction through RasC is more important in ACA activation. Characterization of a rasC⁻/rasG⁻ mutant revealed that both cAMP chemotaxis and adenylyl cyclase (ACA) activation were negligible in this strain. The ectopic expression of carA from the actin 15 promoter restored early developmental gene expression to the rasC⁻/rasG⁻ strain, rendering it suitable for an analysis of cAMP signal transduction. Since there was negligible signaling through either the cAMP chemotactic pathway or the adenylyl cyclase activation pathway in this strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The mutational analysis of Switch I and Switch II regions also defined the key residues that generate functional differences between RasC and RasG. Rap1 is also activated in response to cAMP but its position in the signal transduction cascade was clarified by the finding that its activation was totally abolished in rasC⁻/rasG⁻/[act15]:carA and in rasG⁻ cells, but only slightly reduced in rasC⁻ cells. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC/rasG⁻4act15]:carA strain identifies RasG⁻/RasC⁻ as the presumptive monomeric GTPases required for this activation. The phenotypes of the vegetative ras null mutants were also examined. The results indicate that RasG plays an important role in cytokinesis. The partial absence of chemotaxis to folate in rase cells compared to the total absence of chemotaxis to folate in rasC⁻/rasG⁻, and rasC⁻/rasG⁻/[act15]:carA cells suggests a compensatory role of RasC for RasG during this process, a similar phenomenon to that observed for cAMP chemotaxis by aggregating cells.

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32

Jayakanth, Kankanala. "Design, synthesis and biological evaluation of inhibitors of FGFR, VEGFR-2 and Ras proteins." Thesis, University of Leeds, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.550818.

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Deregulation of kinase activity has emerged as a major mechanism by which cancer cells evade normal physiological functions such as growth and survival. The identification of mutations in FGFR-3 within non invasive tumours of bladder cancer and over expression of this receptor in invasive tumours and superficial tumours makes FGFR-3 a promising target in developing a therapy for the treatment of urothelial bladder cancer. It was found that inhibiting VEGFR-2 together with FGFR provides an attractive strategy for the development of new anti-angiogenic agents as a potential anti -cancer therapy. In this project, the three dimensional structure of both the FGFR and VEGFR-2 was used in conjunction with the de novo methods such as SPROUT to generate oxindole- based novel inhibitors of FGFR and VEGFR-2 with ICso values in the range of 1-10 flM (Compound 4.7 inhibits FGFR and VEGFR-2 with ICso values of 3.9 uM and 1.6 flM for respectively (Chapter 4). These compounds show encouraging anti-angiogenic activity in the lower u M concentrations. In Chapter 5, intramolecular H-bonding concept was used to design pyrazole-based inhibitors of FGFR and VEGFR-2 with 29% and 63% inhibition at 10 uM respectively. Benzo-fused designs based on pyrazole scaffolds yielded indazole based compounds with inhibitory effects of 45%, 41 % and 74% for FGFR-1, FGFR-3 and VEGFR-2 respectively. Shape similarity was used to find novel hinge binders replacing the indazole core with hydroxy-quinolinone moiety which showed an encouraging level of inhibition with 30% and 64% for FGFR and VEGFR-2 respectively. Purine based inhibitors were designed to exploit the smaller gatekeeper residue in both the FGFR and VEGFR-2, but all the compounds show weaker or no inhibition at 10 u M (Chapter 5). In Chapter 6, structure guided method were used to design oxadiazole-based novel VEGFR-2 inhibitor with an ICso value of 1.6 j.1M. All these approaches are novel used in complementary to HTS. In Chapter 3, SPROUT was used to design novel inhibitors of Ras protein and the preliminary analysis of these inhibitors show preferential binding of these inhibitors to the active form compared to the inactive form. These molecules were first amongst the de novo/designed inhibitors of Ras which could serve as a starting point f-or the future potential inhibitors of protein-protein interaction.

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33

Kapoor, Shobhna [Verfasser], Roland [Akademischer Betreuer] Winter, and Martin [Akademischer Betreuer] Engelhard. "Biophysical insights into the Ras-membrane ballet: orientational flexibility, conformational substates and mechanosensitivity of Ras proteins / Shobhna Kapoor. Betreuer: Roland Winter. Gutachter: Martin Engelhard." Dortmund : Universitätsbibliothek Dortmund, 2013. http://d-nb.info/1099297656/34.

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34

Ho, Peter D. "Regulation of morphology and intracellular calcium by Ras in rat neonatal cardiac myocytes /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2000. http://wwwlib.umi.com/cr/ucsd/fullcit?p9984293.

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35

Yasmin, Lubna. "Exoenzyme S of Pseudomonas aeruginosa : cellular targets and interaction with 14-3-3." Doctoral thesis, Umeå : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1411.

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36

Bradbury, Andrew W. "Cyclic AMP binding proteins and ras p21 oncogene expression in human colorectal cancer and mucosa." Thesis, University of Edinburgh, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.531024.

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37

Falsetti, Samuel C. "The Role of RalA and RalB in Cancer." [Tampa, Fla] : University of South Florida, 2008. http://purl.fcla.edu/usf/dc/et/SFE0002307.

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38

Omholt, Katarina. "Activating proto-oncogene mutations in human cutaneous melanoma /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-191-1/.

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39

Hoenerhoff, Mark James. "Bmi-1 collaborates with H-ras to promote mammary epithelial cell transformation, tumorigenesis, and metastasis." Diss., Connect to online resource - MSU authorized users, 2008.

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Thesis (Ph.D.)--Michigan State University. Dept. of Pathobiology and Diagnostic Investigation, 2008.
Title from PDF t.p. (viewed on July 10, 2009) Includes bibliographical references (p. 157-174). Also issued in print.

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40

Khan, Abdul Kareem. "Electrostaticanalisys the Ras active site." Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7161.

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La preorganització electrostàtica del centre actiu s'ha postulat com el mecanisme genèric de l'acció dels enzims. Així, alguns residus "estratègics" es disposarien per catalitzar reaccions interaccionant en una forma més forta amb l'estat de transició, baixant d'aquesta manera el valor de l'energia dactivació g cat. S'ha proposat que aquesta preorientació electrostática s'hauria de poder mostrar analitzant l'estabilitat electrostàtica de residus individuals en el centre actiu.
Ras es una proteïna essencial de senyalització i actúa com un interruptor cel.lular. Les característiques estructurals de Ras en el seu estat actiu (ON) són diferents de les que té a l'estat inactiu (OFF). En aquesta tesi es duu a terme una anàlisi exhaustiva de l'estabilitat dels residus del centre actiu deRas en l'estat actiu i inactiu.
The electrostatic preorganization of the active site has been put forward as the general framework of action of enzymes. Thus, enzymes would position "strategic" residues in such a way to be prepared to catalyze reactions by
interacting in a stronger way with the transition state, in this way decreasing the activation energy g cat for the catalytic process. It has been proposed that
such electrostatic preorientation should be shown by analyzing the electrostatic stability of individual residues in the active site.
Ras protein is an essential signaling molecule and functions as a switch in the
cell. The structural features of the Ras protein in its active state (ON state) are different than those in its inactive state (OFF state). In this thesis, an exhaustive analysis of the stability of residues in the active and inactive Ras active site is performed.

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41

Phan, Vernon Truong. "The Ras-GAP proteins Ira2 and neurofibromin are negatively regulated by ubiquitin-associated proteins Gpb1 in yeast and ETEA/UBXD8 in human cells." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3297797.

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42

Huynh, Carl. "The cytoprotective role of Ras signaling in glomerular epithelial cell injury /." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112639.

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In experimental membranous nephropathy, complement C5b-9-induced glomerular epithelial cell (GEC) injury leads to breakdown of glomerular peimselectivity and proteinuria. This study addresses mechanisms that limit complement-mediated injury, focusing on Ras. Complement-mediated injury was attenuated in cultured GEC expressing a constitutively active form of Ras (V12Ras), compared with Neo (control) GEC. V12Ras GEC showed constitutive activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways, but inhibition of these pathways did not reverse the protective effect of Ras. V12Ras GEC showed smaller and rounder morphology, decreased F- to G-actin ratio, decreased activity of the Rho GTPase, Rac, and decreased Src activity. In V12Ras GEC, disruption or stabilization of the F-actin cytoskeleton reversed the protective effect of V12Ras on complement-mediated injury. Thus, the protective effect of V12Ras may be dependent on remodeling of the actin cytoskeleton. Furthermore, the reduction of Src activity due to Ras activation may alter the equilibrium in activities of Rho GTPases, a family of proteins known regulate the actin cytoskeleton. Activation of Ras signaling is a novel pathway to consider in developing strategies for cytoprotection in complement-mediated injury.

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43

Winge, Per. "The evolution of small GTP binding proteins in cellular organisms. Studies of RAS GTPases in arabidopsis thaliana and the Ral GTPase from Drosophila melanogaster." Doctoral thesis, Norwegian University of Science and Technology, Faculty of Natural Sciences and Technology, 2002. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-169.

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Small GTP binding proteins function as molecular switches which cycles between GTP-bound ON and GDP-bound OFF states, and regulate a wide variety of cellular processes as biological timers. The first characterized member of the small GTPase family, the mutated oncogene p21 src, later known as Harvey-Ras, was identified in the early 1980s (Shih, T. Y. et al. 1980). In the following years small Ras-lik GTPases were found in several organisms and it was soon discovered that they took part in processes, such as signal transduction, gene expression, cytoskeleton reorganisation, microtubule organisation, and vesicular and nuclear transport. The first Rho (Ras homology) gene was cloned in 1985 from the sea slug Aplysia (Madaule, P. et al. 1985) and because of their homology to Ras it was first suspected that they could act as oncogenes. Later studies have shown that even though they participate in processes such as cell migration and motility they are not mutated in cancers.

The first indications that Rho was a signaling protein regulating the actin cytoskeleton, came from experiments where activated forms of human RhoA was microinjected into 3T3 cells (Paterson, H. F. et al. 1990). Another Rho-like GTPase Rac1 (named after Ras-related C3 botulinum toxin substrate) was later shown to regulate actin cytoskeletal dynamics as well, suggesting that Rho-family members cooperate in controlling these processes (Ridley, A. J. et al. 1992). The Rac GTPase was also implicated in regulating the phagocytic NADPH oxidase, which produce superoxide for killing phagocytized microorganisms (Abo, A. et al. 1991). Thus, it soon became clear that Rac/Rho and the related GTPase Cdc42 (cell division cycle 42) had central functions in many important cellular processes.

There are at least three types of regulators for Rho-like proteins. The GDP/GTP exchange factors (GEFs) which stimulates conversion from the GDPbound form to the GTP-bound form. GDP dissociation inhibitors (GDIs) decrease the nucleotide dissociation from the GTPase and retrieve them from membranes to the cytosol. GTPase activating proteins (GAPs) stimulates the intrinsic GTPase activity and GTP hydrolysis. In addition there are probably regulators that dissociate GDI from the GTPase leaving it open for activation by the RhoGEFs.

Ras and Rho-family proteins participate in a coordinated regulation of cellular processes such as cell motility, cell growth and division. The Ral GTPase is closely related to Ras and recent studies have shown that this GTPase is involved in crosstalk between both Ras and Rho proteins (Feig, L. A. et al. 1996; Oshiro, T. et al. 2002). Ral proteins are not found in plants and they appear to be restricted to animalia and probably yeast. During a screen for small GTPases in Drosophila melanogaster I discovered in 1993 several new members of the Ras-family, such as Drosophila Ral (DRal), Ric1 and Rap2. The functions of Ral GTPases in Drosophila have until recently been poorly known, but in paper 2 we present some of the new findings.

Rho-like GTPases have been identified in several eukaryotic organisms such as, yeast (Bender, A. et al. 1989), Dictyostelium discoideum (Bush, J. et al. 1993), plants (Yang, Z. et al. 1993), Entamoeba histolytica (Lohia, A. et al. 1993) and Trypanosoma cruzi (Nepomuceno-Silva, J. L. et al. 2001). In our first publication, (Winge, P. et al. 1997), we describe the cloning of cDNAs from RAC-like GTPases in Arabidopsis thaliana and show mRNA expressions pattern for five of the genes. The five genes analyzed were expressed in most plant tissues with the exception of AtRAC2 (named Arac2 in the paper), which has an expression restricted to vascular tissues. We also discuss the evolution and development of RAC genes in plants. The third publication, (Winge, P. et al. 2000), describe the genetic structure and the genomic sequence of 11 RAC genes from Arabidopsis thaliana. As most genomic sequences of the AtRACs we analyzed came from the Landsberg erecta ecotype and the Arabidopsis thaliana genome was sequenced from the Columbia ecotype, it was possible to compare the sequences and identify new polymorphisms. The genomic location of the AtRAC genes plus the revelation of large genomic duplications provided additional information regarding the evolution of the gene family in plants. A summary and discussion of these new findings are presented together with a general study of small Ras-like GTPases and their evolution in cellular organisms. This study suggests that the small GTPases in eukaryots evolved from two bacterial ancestors, a Rab-like and a MglA/Arp-like (Arf-like) protein. The MglA proteins (after the mgl locus in Myxococcus xanthus) are required for gliding motility, which is a type of movement that take place without help of flagella.

The second publication describes experiments done with the Drosophila melanogaster DRal gene and its effects on cell shape and development. Ectopic expression of dominant negative forms of DRal reveals developmental defects in eye facets and hairs, while constitutive activated forms affects dorsal closure, leaving embryos with an open dorsal phenotype. Results presented in this publication suggest that DRal act through the Jun N-terminal kinase (JNK) pathway to regulate dorsal closure, but recent findings may point to additional explanations as well. The results also indicate a close association between processes regulated by Rac/Rho and Ral proteins in Drosophila.

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44

Appleman, Victoria A. "Mechanisms of KRAS-Mediated Pancreatic Tumor Formation and Progression: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/600.

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Pancreatic cancer is the 4th leading cause of cancer related death in the United States with a median survival time of less than 6 months. Pancreatic ductal adenocarcinoma (PDAC) accounts for greater than 85% of all pancreatic cancers, and is marked by early and frequent mutation of the KRAS oncogene, with activating KRAS mutations present in over 90% of PDAC. To date, though, targeting activated KRAS for cancer treatment has been very difficult, and targeted therapies are currently being sought for the downstream effectors of activated KRAS. Activation of KRAS stimulates multiple signaling pathways, including the MEK-ERK and PI3K-AKT signaling cascades, but the role of downstream effectors in pancreatic tumor initiation and progression remains unclear. I therefore used primary pancreatic ductal epithelial cells (PDECs), the putative cell of origin for PDAC, to determine the role of specific downstream signaling pathways in KRAS activated pancreatic tumor initiation. As one third of KRAS wild type PDACs harbor activating mutations in BRAF , and KRAS and BRAF mutations appear to be mutually exclusive, I also sought to determine the effect of activated BRAF (BRAF V600E ) expression on PDECs and the signaling requirements downstream of BRAF. I found that both KRAS G12D and BRAF V600E expressing PDECs displayed increased proliferation relative to GFP expressing controls, as well as increased PDEC survival after challenge with apoptotic stimuli. This survival was found to depend on both the MEK-ERK and PI3K-AKT signaling cascades. Surprisingly, I found that this survival is also dependent on the IGF1R, and that activation of PI3K/AKT signaling occurs downstream of MEK/ERK activation, and is dependent on signaling through the IGF1R. Consistent with this, I find increased IGF2 expression in KRAS G12D and BRAF V600E expressing PDECs, and show that ectopic expression of IGF2 rescues survival in PDECs with inhibited MEK, but not PI3K. Finally, I showed that the expression of KRAS G12D or BRAF V600E in PDECs lacking both the Ink4a/Arf and Trp53 tumor suppressors is sufficient for tumor formation following orthotopic transplant of PDECs, and that IGF1R knockdown impairs KRAS and BRAF-induced tumor formation in this model. In addition to these findings within PDECs, I demonstrate that KRAS G12D or BRAF V600E expressing tumor cell lines differ in MEK-ERK and PI3K-AKT signaling from PDECs. In contrast to KRAS G12D or BRAF V600E expressing PDECs, activation of AKT at serine 473 in the KRAS G12D or BRAF V600E expressing tumor cell lines does not lie downstream of MEK, and only the inhibition of PI3K alone or both MEK and the IGF1R simultaneously results in loss of tumor cell line survival. However, inhibition of MEK, PI3K, or the IGF1R in KRAS G12D or BRAF V600E expressing tumor cell lines also resulted in decreased proliferation relative to DMSO treated cells, demonstrating that all three signaling cascades remain important for tumor cell growth and are therefore viable options for pancreatic cancer therapeutics.

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Adhikari, Anirban. "Regulation of guanine nucelotide exchange in inhibitory G protein alpha subunit by activator of G protein signaling 3 and novel regulatory peptides." Embargoed access until after 12/19/2006, 2005. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=114.

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Pickford, Christopher. "Mechanism of nucleotide exchange by guanine nucleotide exchange factors on the Ras superfamily of small G-proteins." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325813.

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47

Khanna, Ankita, Pouya Lotfi, Anita J. Chavan, Nieves M. Montaño, Parvin Bolourani, Gerald Weeks, Zhouxin Shen, et al. "The small GTPases Ras and Rap1 bind to and control TORC2 activity." NATURE PUBLISHING GROUP, 2016. http://hdl.handle.net/10150/614747.

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Target of Rapamycin Complex 2 (TORC2) has conserved roles in regulating cytoskeleton dynamics and cell migration and has been linked to cancer metastasis. However, little is known about the mechanisms regulating TORC2 activity and function in any system. In Dictyostelium, TORC2 functions at the front of migrating cells downstream of the Ras protein RasC, controlling F-actin dynamics and cAMP production. Here, we report the identification of the small GTPase Rap1 as a conserved binding partner of the TORC2 component RIP3/SIN1, and that Rap1 positively regulates the RasC-mediated activation of TORC2 in Dictyostelium. Moreover, we show that active RasC binds to the catalytic domain of TOR, suggesting a mechanism of TORC2 activation that is similar to Rheb activation of TOR complex 1. Dual Ras/Rap1 regulation of TORC2 may allow for integration of Ras and Rap1 signaling pathways in directed cell migration.

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Estrozi, Bruna. "Avaliação anatomoclínica e molecular do melanoma cutâneo em pacientes jovens (idade 18-30 anos)." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/5/5144/tde-01042015-144721/.

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A incidência do melanoma cutâneo em pacientes adultos jovens tem aumentado consideravelmente nos últimos anos. Há, contudo, carência de conhecimentos clinicopatológicos e moleculares sobre os melanomas que ocorrem nessa faixa etária. O presente estudo teve por objetivo avaliar 132 casos de melanoma cutâneo primário em pacientes com idade entre 18 e 30 anos, com ênfase no estudo das características clínicas, histopatológicas e avaliação molecular das mutações nos genes BRAF, NRAS e KIT. Em relação aos achados clínicos e histopatológicos, houve predomínio de indivíduos do sexo feminino (61,4%), sendo o tronco o sítio anatômico mais comumente envolvido (44,3%) e o melanoma extensivo superficial o tipo histológico predominante (79,5%). A mutação V600E no gene BRAF (BRAFV600E) foi analisada em 93 casos, utilizando-se a técnica de RT-PCR. Essa mutação foi identificada em 38,7% (36/93) e, estatisticamente, associada à fase vertical de crescimento (p = 0,01), infiltrado inflamatório discreto (p = 0,02) e presença de mitose intradérmica (p = 0,004). Houve, ainda, forte indício de associação com a presença de ulceração (p = 0,05). Todas essas variáveis apresentaram associação com pior prognóstico do melanoma cutâneo. Observou-se predomínio da mutação BRAFV600E em regiões anatômicas relacionadas à exposição solar intermitente. Nenhum caso de melanoma com fenômeno de regressão apresentou mutação BRAFV600E (p < 0,05). Não houve associação significativa entre BRAFV600E e sexo, tipo histológico, nível de Clark, índice de Breslow, elastose solar, invasão angiolinfática e perineural, satelitose, nevo melanocítico coexistente e sobrevida. A pesquisa de mutações NRAS, pela técnica de RT-PCR, detectou frequência de 3,95% (3/76). As três mutações encontradas foram do tipo 61K e ocorreram em pacientes do sexo masculino e em região de cabeça e pescoço. As mutações BRAFV600E e NRAS, quando presentes, eram mutuamente exclusivas. A frequência de mutações KIT, analisadas por sequenciamento, foi de 11,1% (3/27). As três mutações identificadas estavam localizadas no éxon 9 (G510, G498S e 489I). Houve concomitância de casos com mutação KIT tanto com NRAS, como com BRAFV600E. Devido ao pequeno número de casos com mutação em KIT e NRAS, não foi possível estabelecer correlações clínicas e histopatológicas com esses genes. Este estudo é o primeiro a descrever as mutações G510D e G498S no gene KIT em melanomas cutâneos. No presente estudo, a mutação BRAFV600E, em melanomas cutâneos de adultos jovens, correlacionou-se com características anatomoclínicas de pior prognóstico em relação aos melanomas selvagens para BRAFV600E
The incidence of cutaneous melanoma in young adults has dramatically increased in recent years. However, there is scarce data about the clinicopathological and molecular characteristics on the melanomas occurring at this age group. The present study aimed to evaluate 132 patients aged between 18 and 30 years with primary cutaneous melanoma with emphasis on the study of clinical, histopathological characteristics and molecular evaluation of mutations in BRAF, NRAS and KIT genes. Regarding the clinical and histopathological findings, the following results were found: female predominance (61.4%), trunk was the most commonly anatomical site involved (44.3%) and superficial spreading melanoma, was the most common histological type (79.5 %). The V600E mutation in BRAF (BRAFV600E) gene was analyzed in 93 cases, using RT-PCR. It was present in 38.7% (36/93) and statistically related to the vertical growth phase (p = 0.01), mild inflammatory infiltration (p = 0.02) and the presence of intradermal mitosis (p = 0.004). There was, also, strongly evidence of an association with the presence of ulceration (p = 0.05). Worse prognosis was associated with these variables. There was a predominance of BRAFV600E mutation in anatomical regions related to intermittent sun exposure. No cases of melanoma with BRAFV600E mutation showed regression phenomenon (p < 0.05). There was no significant association between BRAFV600E and gender, histological type, Clark level, Breslow thickness, solar elastosis, angiolymphatic and perineural invasion, sattelitosis, coexisting melanocytic nevus and survival. The presence of a mutation in NRAS, by RT-PCR was seen in 3.95% (3/76) of the cases. All these three mutations were of type 61K, occurred in male patients and the head and neck region. BRAFV600E and NRAS mutations, when present, were mutually exclusive. The frequency of KIT mutations, analyzed by sequencing, was 11.1% (3/27). The three mutations identified in this gene were located in exon 9 (G510, G498S and 489I). Concomitant mutations were found between KIT and NRAS and BRAFV600E. Due to the small number of KIT and NRAS mutated cases, it was not possible to establish clinical and histopathological correlations and mutation status in these genes. This study was the first to describe the G510D and G498S mutations in KIT gene in cutaneous melanomas. In the present study, BRAFV600E mutation in cutaneous melanoma of young adults correlated with anatomic and clinical features of worse prognosis compared to wild type

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Jeong, Sun Yong. "Functional investigation of arabidopsis long coiled-coil proteins and subcellular localization of plant rangap1." The Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=osu1086119855.

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Müller, Jasmin [Verfasser]. "Design of drugs for the inhibition of the cancer related proteins MIA, Rheb and K-Ras / Jasmin Müller." Wuppertal : Universitätsbibliothek Wuppertal, 2019. http://d-nb.info/1192844750/34.

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Dissertations / Theses on the topic 'Ras proteins'

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