Replacement Of Lysine Residue Research Articles

An evolutionary analysis identifies a conserved pentapeptide stretch containing the two essential lysine residues for rice L-myo-inositol 1-phosphate synthase catalytic activity.

A molecular evolutionary analysis of a well conserved protein helps to determine the essential amino acids in the core catalytic region. Based on the chemical properties of amino acid residues, phylogenetic analysis of a total of 172 homologous sequences of a highly conserved enzyme, L-myo-inositol 1-phosphate synthase or MIPS from evolutionarily diverse organisms was performed. This study revealed the presence of six phylogenetically conserved blocks, out of which four embrace the catalytic core of the functional protein. Further, specific amino acid modifications targeting the lysine residues, known to be important for MIPS catalysis, were performed at the catalytic site of a MIPS from monocotyledonous model plant, Oryza sativa (OsMIPS1). Following this study, OsMIPS mutants with deletion or replacement of lysine residues in the conserved blocks were made. Based on the enzyme kinetics performed on the deletion/replacement mutants, phylogenetic and structural comparison with the already established crystal structures from non-plant sources, an evolutionarily conserved peptide stretch was identified at the active pocket which contains the two most important lysine residues essential for catalytic activity.

Abstract 2167: Differential roles of palmitoylation on oncogenic potential of NRAS and KRAS4A

Abstract The RAS family includes three RAS genes, which encode four highly homologous proteins: H-, N-, and KRAS4A and 4B, the latter two being alternatively spliced forms differing only at the carboxyl terminus. Hyperactivation of RAS is common in human cancer, including hematological malignancies. Since RAS proteins are difficult to target, identification of alternative means to block RAS oncogenic signaling is critical for developing therapies against RAS-driven cancers. The biological activity of RAS proteins relies upon post-translational modifications (PTMs) that anchor RAS to cellular membranes. Among RAS PTMs, palmitoylation is required for the high affinity plasma membrane binding of NRAS, HRAS and KRAS4A. We have previously shown that palmitoylation is essential for NRAS leukemogenesis, suggesting that targeting RAS palmitoylation may be an effective therapy for NRAS related cancers. In addition, it has been shown that KRAS4A plays an essential role in the development of KRAS-driven lung cancer, suggesting that targeting palmitoylation may impact on cancers with KRAS mutations as well. However, the exact role of palmitoylation in KRAS4A oncogenic activity is not known. In this study we compared the effect of palmitoylation on leukemogenic potential of oncogenic NRAS and KRAS4A whose high expression level is detected in some KRAS mutated leukemia cell lines. We found that palmitoylation of oncogenic NRAS and KRAS4A is both critical for their plasma membrane attachment and their activity to activate the MEK-ERK signaling pathway. Mutation at the palmitoylation site of the oncogenic KRAS4A significantly abrogates its leukemogenic potential. However, unlike NRAS, palmitoylation defective KRAS4A still induces leukemia in mice, albeit with a long latency. Using NRAS/KRAS4A chimeric constructs, we found that the KIKK (Lys-Ile-Lys-Lys) motif between amino acids 181 and 189 in the hypervariable region of KRAS4A effects on the oncogenic activity of KRAS4A, though its contribution is less than that of palmitoylation. After replacing the three lysine residues in the KIKK motif with glutamine residues, the leukemogenic potential of oncogenic KRAS4A is partially inhibited, while inactivating mutations of both palmitoylation site and KIKK motif completely suppress the ability of oncogenic KRAS4A to induce leukemia in mice. On the other hand, replacement of lysine residues in KIKK motif with arginine residues does not change the oncogenic activity of KRAS4A, indicating that the KIKK motif functions by providing positive charged residues. These studies demonstrate that KRAS4A contains an additional membrane association motif comparing to NRAS and suggest that though therapies targeting RAS palmitoylation would inhibit KRAS4A tumorigenesis, interfering the KIKK motif of KRAS4A would enhance the therapeutic effectiveness. Citation Format: Huanbin Zhao, Ruibao Ren. Differential roles of palmitoylation on oncogenic potential of NRAS and KRAS4A. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2167. doi:10.1158/1538-7445.AM2015-2167

Mutants of immunotoxin anti-Tac(dsFv)-PE38 with variable number of lysine residues as candidates for site-specific chemical modification. 1. Properties of mutant molecules.

Chemical modification of proteins with substances such as poly(ethylene glycol) can add useful properties to proteins. Currently PEGylation is done in a random manner utilizing amino residues dispersed throughout a protein. For proteins such as immunotoxins, which have several different functional domains, random modification leads to inactivation. To determine if we could produce an immunotoxin with a diminished number of lysine residues so that chemical modification could be restricted to certain regions of the protein, we chose the recombinant immunotoxin anti-Tac(dsFv)-PE38 that has 13 lysine residues in the Fv portion and 3 in the toxin. We prepared a series of mutants with 0-12 lysines in the Fv and 0 or 3 in the toxin. Almost all of these molecules retain full biological activity. Our data indicate that replacement of lysine residues can be achieve without loss of biological potency. These molecules are a useful starting point to carry out site-specific PEGylation experiments.

Endocytosis of the AT 1A angiotensin receptor is independent of ubiquitylation of its cytoplasmic serine/threonine-rich region

Agonist-induced internalisation of the rat type 1A (AT 1A) angiotensin II receptor is associated with phosphorylation of a serine/threonine-rich region in its cytoplasmic tail. In yeast, hyperphosphorylation of the α-factor pheromone receptor regulates endocytosis of the receptor by facilitating the monoubiquitylation of its cytoplasmic tail on lysine residues. The role of receptor ubiquitylation in AT 1A receptor internalisation was evaluated by deletion or replacement of lysine residues in its agonist-sensitive serine/threonine-rich region. Expression of such receptor mutants in CHO cells showed that these modifications had no detectable effect on the angiotensin II-induced endocytosis of the AT 1A receptor. Furthermore, fusion of ubiquitin in-frame to an internalisation-deficient AT 1A receptor mutant with a truncated carboxyl-terminal tail did not restore the endocytosis of the resulting chimeric receptor. No impairment of receptor internalisation was observed after substitution of all lysine residues in the serine/threonine-rich region at saturating angiotensin II concentrations, where endocytosis occurs by a β-arrestin and dynamin independent mechanism. Taken together, these data demonstrate that ubiquitylation of the cytoplasmic serine/threonine-rich region of the AT 1A receptor on lysine residues is not required for its agonist-induced internalisation, and suggest that endocytosis of mammalian G protein-coupled receptors (GPCRs) occurs by a different mechanism than that of yeast GPCRs.

Beta-endorphin: synthesis and properties of analogs with replacement of lysine residues by arginine.

Human β‐endorphin analogs, [Arg9,19,24,28,29]‐β‐endorphin (I) and [Arg24,28,29]‐β‐endorphin (II), have been synthesized by the solid‐phase method. Peptide II had 86% of the analgesic potency and 216% of the receptor binding activity of the parent molecule. Peptide I had only 18% analgesic potency but its binding activity was more than three time greater than that of human β‐endorphin.