K-ras protein

In an initial study,54 the heptapeptide KTKCVFM (Kj 4.5 pM) was employed as a mimic of the K-Ras protein. Several intraresidue TRNOEs were found in the terminal tetrapeptide sequence, including the notable NHi/NHi+1 interactions at Val-Phe and Phe-Met, as well as between the Val-Phe sidechains. After incorporation of the distance constraints, semiquantitative and qualitative manipulations provided a structure which adopts a type I P-turn about the CVFM fragment,... 

We have also demonstrated the utility of analytical DEAR coupled with ESI-MS in the determination of bound nucleotide. This technique may be useful in evaluation of K-ras protein that has been loaded with GTP analogues in order to activate it (7). 

Farnesylation of the Ras protein occurs at the C-terminal CAAX sequence (A aliphatic amino acid, X Ser or Thr). The farnesyl residue is attached, with the help of a farnesyl protein transferase and via a thioether bond, to the Cys residue of the CAAX sequence. Next, the last three amino acids are cleaved offby proteases, and the carboxyl group of the C-terminal cysteine residue undergoes a methylesterification (Fig. 9.6). In addition, the Ras proteins have a palmitinic acid anchor at different Cys residues in the vicinity of the C terminus. The membrane localization of the K-Ras protein is also supported by a polybasic sequence close to the C terminus (see Section 3.7). 

Our understanding of the mechanism of activation of Raf kinase by Ras protein is still incomplete, mainly because full-length Raf kinase cannot yet be studied biochemically and structurally. Preliminary information was obtained in experiments using a fusion protein consisting of the membrane localization signal CAAX of K-Ras protein linked to the C terminus of Raf kinase. The presence of the membrane localization sequence of the Ras protein in Raf kinase leads to its constitutive activation, and the activity of... 

K-Ras-dependent cells are cancer cell lines that require sustained K-Ras function for viability. These cells were identified from a large panel of human cancer cell lines harboring mutant K-Ras after treatment with specific shRNAs directed to K-Ras. In K-Ras-dependent cells, a decrease in K-Ras protein expression results in a marked growth suppression, whereas in K-Ras-independent cells,a decrease in protein expression is not correlated with grovrth inhibition [14]. 

The membrane-associated small G proteins H-Ras and K-Ras have been studied with respect to their association with cytoplasmic leaflets. These two proteins have nearly identical structures and functions but different membrane anchors, membrane distributions and effector responses. Application of the FRAP method to fluorescent constructs of H-Ras and K-Ras revealed that only H-Ras in its guanosine 5 diphosphate (GDP)-bound form associates with cholesterol-dependent rafts [26]. 

A first approach to study the interaction of posttranslational modified Ras proteins with membranes was the analysis of binding and exchange of isoprenyl-ated peptides with and between lipid vesicles utilizing a fluorescent bimanyl label. Studies with K-Ras peptides revealed that a single isoprenyl group is sufficient for membrane association only if supported by carboxymethylation of the C-terminal cysteine [227,228]. 

The less polar methyl ester 2 as prodrug showed better results in vivo and inhibits both farnesylation of the Ras protein and growth of Ras-transformed cells, whilst proliferation of Raf- or Mos-transformed cells was not influenced. Growth of human pancreatic adenocarcinoma cells with mutated K-Ras, c-Myc and p53 genes was inhibited by application of 2. If the compound is administered over a period of 5 days to mice with implanted Ras-dependent tumors, tumor growth can be reduced by up to 66% compared to untreated mice, whereas application of the antitumor antibiotic doxorubicin only resulted in 33% reduction under the same conditions. It is particularly noteworthy that treatment with the /1-turn mimetic - in contrast to treatment with doxorubicin - was without any visible side effects, such as weight loss. 

Lipid modified proteins are often attached to cell membranes. In many cases, they play crucial roles in the transduction of extracellular signals across the plasma membrane and into the nucleus. A particularly important example are the N-, K-, and H-Ras proteins. All Ras proteins terminate in a fame... 

Bioorganic Synthesis of Ras Proteins for the Study of Signal Transduction, B. Bader, K. Kuhn, D. J. Owen, H. Waldmann, A. Wittinghofer, J. Kuhlmann, Nature 2000, 403, 223-226. 

Proteins can undergo different rounds of palmitoylation and depalmitoylation, either constitutively or as a response to signals." " Here the Ras proteins are the most commonly discussed examples. As described above, all Ras proteins are expressed with the CAAX-box and are subject to post-translational modifications. First, they get farnesylated and after proteolysis and methylation of the C-terminus, H-/N-Ras as well as K-Ras 4A get further palmitoylated at additional cysteines present in their C-terminus. Palmitoylation occurs in the Golgi apparatus and via vesicular transport the farnesylated and palmitoylated proteins are directed to the plasma membrane (PM). The palmitoyl thioester is hydrolyzed at multiple cellular sites and the protein is transported back to the Golgi via a nonvesicular pathway (Scheme 3)." ... 

For Ras proteins, there is no chaperone protein available or known to solubilize the lipidated protein thus the refolding of, for example, lipidated N- and H-Ras is not feasible. However, lipidated K-Ras 110 can be generated via expressed protein ligation (EPL), since the polybasic C-terminal region of this protein helps to solubilize the protein even when farnesylated and facilitates the purification (Scheme 36). ... 

Scheme 36 Expressed protein ligation of the farnesylated K-Ras 4B C-terminus.

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