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81.
Design of inhibitors of Ras--Raf interaction using a computational combinatorial algorithm 总被引:1,自引:0,他引:1
Zeng J Nheu T Zorzet A Catimel B Nice E Maruta H Burgess AW Treutlein HR 《Protein engineering》2001,14(1):39-45
Drugs that inhibit important protein-protein interactions are hard to find either by screening or rational design, at least so far. Most drugs on the market that target proteins today are therefore aimed at well-defined binding pockets in proteins. While computer-aided design is widely used to facilitate the drug discovery process for binding pockets, its application to the design of inhibitors that target the protein surface initially seems to be limited because of the increased complexity of the task. Previously, we had started to develop a computational combinatorial design approach based on the well-known 'multiple copy simultaneous search' (MCSS) procedure to tackle this problem. In order to identify sequence patterns of potential inhibitor peptides, a three-step procedure is employed: first, using MCSS, the locations of specific functional groups on the protein surface are identified; second, after constructing the peptide main chain based on the location of favorite locations of N-methylacetamide groups, functional groups corresponding to amino acid side chains are selected and connected to the main chain C(alpha) atoms; finally, the peptides generated in the second step are aligned and probabilities of amino acids at each position are calculated from the alignment scheme. Sequence patterns of potential inhibitors are determined based on the propensities of amino acids at each C(alpha) position. Here we report the optimization of inhibitor peptides using the sequence patterns determined by our method. Several short peptides derived from our prediction inhibit the Ras--Raf association in vitro in ELISA competition assays, radioassays and biosensor-based assays, demonstrating the feasibility of our approach. Consequently, our method provides an important step towards the development of novel anti-Ras agents and the structure-based design of inhibitors of protein--protein interactions. 相似文献
82.
Maruta K Yoshiga T Katagiri C Ochiai M Tojo S 《Archives of insect biochemistry and physiology》2002,50(2):97-106
Biliverdin-binding vitellogenin (Vg) was purified from adult female hemolymph of the common cutworm, Spodoptera litura, by using gel filtration and ion exchange chromatographies. The molecular mass of the protein was 490 kDa and it was composed of two 188-kDa subunits. Three internal amino acid sequences obtained by digestion of the protein with lysylendopeptidase showed high similarity to those of Bombyx mori Vg, supporting the purified blue protein to be vitellogenin. latroscan analyses demonstrated the presence of biliverdin in Vg that occupied 2.4% of total lipid components. Among the lipids of Vg (9.5 micrograms total lipids per 100 micrograms protein), diacylglycerol was the most predominant, followed by phospholipid, hydrocarbons, and then triacylglycerol, while in biliverdin-binding proteins (BPs) purified from larval hemolymph (3.1 micrograms total lipids per 100 micrograms protein), phospholipid was the most abundant lipid followed by diacylglycerol; hydrocarbons and triacylglycerol were minor components. Vg was first detected in the hemolymph of female pupae one day before eclosion, but injection of 5 micrograms of methoprene into a 3-day-old pupa induced Vg in the hemolymph 4 days earlier than in the control. Methoprene also induced a faster decline in BP-A and BP-B titers in the hemolymph with a corresponding increase of the Vg titer. These results suggest that juvenile hormone (JH) induces not only vitellogenesis but also the uptake of these proteins by stimulating the metamorphosis of fat body during the pupal stage. 相似文献
83.
84.
p190-A, a human tumor suppressor gene, maps to the chromosomal region 19q13.3 that is reportedly deleted in some gliomas 总被引:3,自引:0,他引:3
To date, two distinct genes coding for Ras GAP-binding phosphoproteins of 190kDa, p190-A and p190-B, have been cloned from mammalian cells. Rat p190-A of 1513 amino acids shares 50% sequence identity with human p190-B of 1499 amino acids. We have previously demonstrated, using rat p190-A cDNA, that full-length p190-A is a tumor suppressor, reversing v-Ha-Ras-induced malignancy of NIH 3T3 cells through both the N-terminal GTPase (residues 1-251) and the C-terminal Rho GAP (residues 1168-1441) domains. Here we report the cloning of the full-length human p190-A cDNA and its first exon covering more than 80% of this protein, as well as its chromosomal mapping. Human p190-A encodes a protein of 1514 amino acids, and shares overall 97% sequence identity with rat p190-A. Like the p190-B exon, the first exon of p190-A is extremely large (3.7 kb in length), encoding both the GTPase and middle domains (residues 1-1228), but not the remaining GAP domain, suggesting a high conservation of genomic structure between two p190 genes. Using a well characterized monochromosome somatic cell hybrid panel, fluorescent in situ hybridization (FISH) and other complementary approaches, we have mapped the p190-A gene between the markers D19S241E and STD (500 kb region) of human chromosome 19q13.3. Interestingly, this chromosomal region is known to be rearranged in a variety of human solid tumors including pancreatic carcinomas and gliomas. Moreover, at least 40% glioblastoma/astrocytoma cases with breakpoints in this region were previously reported to show loss of the chromosomal region encompassing p190-A, suggesting the possibility that loss or mutations of this gene might be in part responsible for the development of these tumors. 相似文献
85.
Maruta K Mukai K Yamashita H Kubota M Chaen H Fukuda S Kurimoto M 《Bioscience, biotechnology, and biochemistry》2002,66(9):1976-1980
A gene encoding a trehalose phosphorylase was cloned from Thermoanaerobacter brockii ATCC 35047. The gene encodes a polypeptide of 774 amino acid residues. The deduced amino acid sequence was homologous to bacterial maltose phosphorylases and a trehalose 6-phosphate phosphorylase catalyzing anomer-inverting reactions. On the other hand, no homology was found between the T. brockii enzyme and an anomer-retaining trehalose phosphorylase from Grifola frondosa. 相似文献
86.
Tsubasa Sodeyama Hitoshi Nishikawa Kenji Harai Daiki Takeshima Yoshihiro Sawa Takanori Maruta Takahiro Ishikawa 《The Plant journal : for cell and molecular biology》2021,107(6):1724-1738
Ascorbate is an abundant and indispensable redox compound in plants. Genetic and biochemical studies have established the d -mannose/l -galactose (d -Man/l -Gal) pathway as the predominant ascorbate biosynthetic pathway in streptophytes, while the d -galacturonate (d -GalUA) pathway is found in prasinophytes and euglenoids. Based on the presence of the complete set of genes encoding enzymes involved in the d -Man/l -Gal pathway and an orthologous gene encoding aldonolactonase (ALase) – a key enzyme for the d -GalUA pathway – Physcomitrium patens may possess both pathways. Here, we have characterized the moss ALase as a functional lactonase and evaluated the ascorbate biosynthesis capability of the two pathways using knockout mutants. Physcomitrium patens expresses two ALase paralogs, namely PpALase1 and PpALase2. Kinetic analyses with recombinant enzymes indicated that PpALase1 is a functional enzyme catalyzing the conversion of l -galactonic acid to the final precursor l -galactono-1,4-lactone and that it also reacts with dehydroascorbate as a substrate. Interestingly, mutants lacking PpALase1 (Δal1) showed 1.2-fold higher total ascorbate content than the wild type, and their dehydroascorbate content was increased by 50% compared with that of the wild type. In contrast, the total ascorbate content of mutants lacking PpVTC2-1 (Δvtc2-1) or PpVTC2-2 (Δvtc2-2), which encode the rate-limiting enzyme GDP-l -Gal phosphorylase in the d -Man/l -Gal pathway, was markedly decreased to 46 and 17%, respectively, compared with that of the wild type. Taken together, the dominant ascorbate biosynthetic pathway in P. patens is the d -Man/l -Gal pathway, not the d -GalUA pathway, and PpALase1 may play a significant role in ascorbate metabolism by facilitating dehydroascorbate degradation rather than ascorbate biosynthesis. 相似文献
87.
Takanori Maruta Takahiro Inoue Masahiro Noshi Masahiro Tamoi Yukinori Yabuta Kazuya Yoshimura Takahiro Ishikawa Shigeru Shigeoka 《Biochimica et Biophysica Acta (BBA)/General Subjects》2012
Background
Reactive oxygen species (ROS) are not only cytotoxic compounds leading to oxidative damage, but also signaling molecules for regulating plant responses to stress and hormones. Arabidopsis cytosolic ascorbate peroxidase 1 (APX1) is thought to be a central regulator for cellular ROS levels. However, it remains unclear whether APX1 is involved in plant tolerance to wounding and methyl jasmonate (MeJA) treatment, which are known to enhance ROS production.Methods
We studied the effect of wounding and MeJA treatment on the levels of H2O2 and oxidative damage in the Arabidopsis wild-type plants and knockout mutants lacking APX1 (KO-APX1).Results
The KO-APX1 plants showed high sensitivity to wounding and MeJA treatment. In the leaves of wild-type plants, H2O2 accumulated only in the vicinity of the wound, while in the leaves of the KO-APX1 plants it accumulated extensively from damaged to undamaged regions. During MeJA treatment, the levels of H2O2 were much higher in the leaves of KO-APX1 plants. Oxidative damage in the chloroplasts and nucleus was also enhanced in the leaves of KO-APX1 plants. These findings suggest that APX1 protects organelles against oxidative stress by wounding and MeJA treatment.General significance
This is the first report demonstrating that H2O2-scavenging in the cytosol is essential for plant tolerance to wounding and MeJA treatment. 相似文献88.
89.
Yoshimura K Mori T Yokoyama K Koike Y Tanabe N Sato N Takahashi H Maruta T Shigeoka S 《Plant & cell physiology》2011,52(10):1786-1805
90.
Satoshi Yasuda Takanori Yanagi Masafumi D. Yamada Shoji Ueki Shinsaku Maruta Akio Inoue Toshiaki Arata 《Biochemical and biophysical research communications》2014
In kinesin X-ray crystal structures, the N-terminal region of the α-1 helix is adjacent to the adenine ring of the bound nucleotide, while the C-terminal region of the helix is near the neck-linker (NL). Here, we monitor the displacement of the α-1 helix within a kinesin monomer bound to microtubules (MTs) in the presence or absence of nucleotides using site-directed spin labeling EPR. Kinesin was doubly spin-labeled at the α-1 and α-2 helices, and the resulting EPR spectrum showed dipolar broadening. The inter-helix distance distribution showed that 20% of the spins have a peak characteristic of 1.4–1.7 nm separation, which is similar to what is predicted from the X-ray crystal structure, albeit 80% were beyond the sensitivity limit (>2.5 nm) of the method. Upon MT binding, the fraction of kinesin exhibiting an inter-helix distance of 1.4–1.7 nm in the presence of AMPPNP (a non-hydrolysable ATP analog) and ADP was 20% and 25%, respectively. In the absence of nucleotide, this fraction increased to 40–50%. These nucleotide-induced changes in the fraction of kinesin undergoing displacement of the α-1 helix were found to be related to the fraction in which the NL undocked from the motor core. It is therefore suggested that a shift in the α-1 helix conformational equilibrium occurs upon nucleotide binding and release, and this shift controls NL docking onto the motor core. 相似文献