共查询到20条相似文献,搜索用时 73 毫秒
1.
Kohji Yamamoto Akifumi Higashiura Mamoru Suzuki Kosuke Aritake Yoshihiro Urade Nobuko Uodome Atsushi Nakagawa 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Glutathione transferases (GSTs) are members of a major family of detoxification enzymes. Here, we report the crystal structure of a sigma-class GST of Bombyx mori, bmGSTS1, to gain insight into the mechanism catalysis.Methods
The structure of bmGSTS1 and its complex with glutathione were determined at resolutions of 1.9 Å and 1.7 Å by synchrotron radiation and the molecular replacement method.Results
The three-dimensional structure of bmGSTS1 shows that it exists as a dimer and is similar in structure to other GSTs with respect to its secondary and tertiary structures. Although striking similarities to the structure of prostaglandin D synthase were also detected, we were surprised to find that bmGSTS1 can convert prostaglandin H2 into its E2 form. Comparison of bmGSTS1 with its glutathione complex showed that bound glutathione was localized to the glutathione-binding site (G-site). Site-directed mutagenesis of bmGSTS1 mutants indicated that amino acid residues Tyr8, Leu14, Trp39, Lys43, Gln50, Met51, Gln63, and Ser64 in the G-site contribute to catalytic activity.Conclusion
We determined the tertiary structure of bmGSTS1 exhibiting prostaglandin E synthase activity.General significance
These results are, to our knowledge, the first report of a prostaglandin synthase activity in insects. 相似文献2.
Background
The Theta class glutathione transferase GST T1-1 is a ubiquitously occurring detoxication enzyme. The rat and mouse enzymes have high catalytic activities with numerous electrophilic compounds, but the homologous human GST T1-1 has comparatively low activity with the same substrates. A major structural determinant of substrate recognition is the H-site, which binds the electrophile in proximity to the nucleophilic sulfur of the second substrate glutathione. The H-site is formed by several segments of amino acid residues located in separate regions of the primary structure. The C-terminal helix of the protein serves as a lid over the active site, and contributes several residues to the H-site.Methods
Site-directed mutagenesis of the H-site in GST T1-1 was used to create the mouse Arg234Trp for comparison with the human Trp234Arg mutant and the wild-type rat, mouse, and human enzymes. The kinetic properties were investigated with an array of alternative electrophilic substrates to establish substrate selectivity profiles for the different GST T1-1 variants.Results
The characteristic activity profile of the rat and mouse enzymes is dependent on Arg in position 234, whereas the human enzyme features Trp. Reciprocal mutations of residue 234 between the rodent and human enzymes transform the substrate-selectivity profiles from one to the other.Conclusions
H-site residue 234 has a key role in governing the activity and substrate selectivity profile of GST T1-1.General significance
The functional divergence between human and rodent Theta class GST demonstrates that a single point mutation can enable or suppress enzyme activities with different substrates. 相似文献3.
Julia Knöckel Rositsa Jordanova Ingrid B. Müller Carsten Wrenger Matthew R. Groves 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
Vitamin B6 synthesis requires a functional Pdx1 assembly that is dodecameric in vivo. We have previously shown that mutation of a catalytic lysine in the plasmodial Pdx1 protein results in a protein that is both inactive and hexameric in vitro.Methods
Static and dynamic light scattering, circular dichroism, co-purification and enzyme assays are used to investigate the role of a glycine conserved in all Pdx1 family members.Results
Static light scattering indicates that a glycine to alanine mutant is present as a hexamer in vitro. Subsequent circular dichroism experiments demonstrate that a significant change in secondary structure content is induced by this mutation. However, this mutant is still competent to bind and support Pdx2 activity.Conclusions
As the mutated glycine occupies an unrestricted region of the Ramachandran plot the additional stereo-chemical restrictions imposed on alanine residues strongly support our hypothesis that significant structural rearrangement of Pdx1 is required during the transition from hexamer to dodecamer.General significance
The presented results demonstrate that reduction in the mobility of this region in Pdx1 proteins is required for formation of the in vivo dodecamer, negatively affecting the activity of Pdx1, opening the possibility of allosteric Pdx1 inhibitors. 相似文献4.
Carine F. Djuika Sabine Fiedler Martina Schnölzer Cecilia Sanchez Michael Lanzer Marcel Deponte 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Peroxiredoxins are important heterogeneous thiol-dependent hydroperoxidases with a variety of isoforms and enzymatic mechanisms. A special subclass of glutaredoxin/glutathione-dependent peroxiredoxins has been discovered in bacteria and eukaryotes during the last decade, but the exact enzymatic mechanisms of these enzymes remain to be unraveled.Methods
We performed a comprehensive analysis of the enzyme kinetics and redox states of one of these glutaredoxin/glutathione-dependent peroxiredoxins, the antioxidant protein from the malaria parasite Plasmodium falciparum, using steady-state kinetic measurements, site-directed mutagenesis, redox mobility shift assays, gel filtration, and mass spectrometry.Results
P. falciparum antioxidant protein requires not only glutaredoxin but also glutathione as a true substrate for the reduction of hydroperoxides. One peroxiredoxin cysteine residue and one glutaredoxin cysteine residue are sufficient for catalysis, however, additional cysteine residues of both proteins result in alternative redox states and conformations in vitro with implications for redox regulation. Our data furthermore point to a glutathione-dependent peroxiredoxin activation and a negative subunit cooperativity.Conclusions
The investigated glutaredoxin/glutathione/peroxiredoxin system provides numerous new insights into the mechanism and redox regulation of peroxiredoxins.General significance
As a member of the special subclass of glutaredoxin/glutathione-dependent peroxiredoxins, the P. falciparum antioxidant protein could become a reference protein for peroxiredoxin catalysis and regulation. 相似文献5.
Iveta Uhliariková Mária Vršanská Barry V. McCleary Peter Biely 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Microbial degradation of acetylated plant hemicelluloses involves besides enzymes cleaving the glycosidic linkages also deacetylating enzymes. A detailed knowledge of the mode of action of these enzymes is important in view of the development of efficient bioconversion of plant materials that did not undergo alkaline pretreatment leading to hydrolysis of ester linkages.Methods
In this work deacetylation of hardwood acetylglucuronoxylan by acetylxylan esterases from Streptomyces lividans (carbohydrate esterase family 4) and Orpinomyces sp. (carbohydrate esterase family 6) was monitored by 1H-NMR spectroscopy.Results
The 1H-NMR resonances of all acetyl groups in the polysaccharide were fully assigned. The targets of both enzymes are 2- and 3-monoacetylated xylopyranosyl residues and, in the case of the Orpinomyces sp. enzyme, also the 2,3-di-O-acetylated xylopyranosyl residues. Both enzymes do not recognize as a substrate the 3-O-acetyl group on xylopyranosyl residues α-1,2-substituted with 4-O-methyl-d-glucuronic acid.Conclusions
The 1H-NMR spectroscopy approach to study positional and substrate specificity of AcXEs outlined in this work appears to be a simple way to characterize catalytic properties of enzymes belonging to various CE families.Significance
The results contribute to development of efficient and environmentally friendly procedures for enzymatic degradation of plant biomass. 相似文献6.
Joel L. Asenjo Heide C. Ludwig Cristian A. Droppelmann Juan G. Cárcamo Ilona I. Concha Alejandro J. Yáñez María L. Cárdenas Athel Cornish-Bowden Juan C. Slebe 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P2 and by high concentrations of its substrate Fru-1,6-P2. The mechanism that produces substrate inhibition continues to be obscure.Methods
Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P2 and Fru-1,6-P2 on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244.Results
The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P2 induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P2, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a “stapler” that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition.Conclusions
Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits.General significance
Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia. 相似文献7.
Hazuki E. Miwa Thomas A. Gerken Tru D. Huynh Lori R. Duesler Meghan Cotter Thomas M. Hering 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
Cleavage of aggrecan by ADAMTS proteinases at specific sites within highly conserved regions may be important to normal physiological enzyme functions, as well as pathological degradation.Methods
To examine ADAMTS selectivity, we assayed ADAMTS-4 and -5 cleavage of recombinant bovine aggrecan mutated at amino acids N-terminal or C-terminal to the interglobular domain cleavage site.Results
Mutations of conserved amino acids from P18 to P12 to increase hydrophilicity resulted in ADAMTS-4 cleavage inhibition. Mutation of Thr, but not Asn within the conserved N-glycosylation motif Asn-Ile-Thr from P6 to P4 enhanced cleavage. Mutation of conserved Thr residues from P22 to P17 to increase hydrophobicity enhanced ADAMTS-4 cleavage. A P4′ Ser377Gln mutant inhibited cleavage by ADAMTS-4 and -5, while a neutral Ser377Ala mutant and species mimicking mutants Ser377Thr, Ser377Asn, and Arg375Leu were cleaved normally by ADAMTS-4. The Ser377Thr mutant, however, was resistant to cleavage by ADAMTS-5.Conclusion
We have identified multiple conserved amino acids within regions N- and C-terminal to the site of scission that may influence enzyme–substrate recognition, and may interact with exosites on ADAMTS-4 and ADAMTS-5.General significance
Inhibition of the binding of ADAMTS-4 and ADAMTS-5 exosites to aggrecan should be explored as a therapeutic intervention for osteoarthritis. 相似文献8.
Demei Meng Lin Shen Rui Yang Xinhua Zhang Jiping Sheng 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
1-Aminocyclopropane-1-carboxylate oxidase (ACO) is a key enzyme that catalyses the final step in the biosynthesis of the plant hormone ethylene. Recently, the first ACO homologue gene was isolated in Agaricus bisporus, whereas information concerning the nature of the ethylene-forming activity of this mushroom ACO is currently lacking.Methods
Recombinant ACO from A. bisporus (Ab-ACO) was purified and characterised for the first time. Molecular modelling combined with site-directed mutagenesis and kinetic and spectral analysis were used to investigate the property of Ab-ACO.Results
Ab-ACO has eight amino acid residues that are conserved in the Fe (II) ascorbate family of dioxygenases, including four catalytic residues in the active site, but Ab-ACO lacks a key residue, S289. In comparison to plant ACOs, Ab-ACO requires ACC and Fe (II) but does not require ascorbate. In addition, Ab-ACO had relatively low activity and was completely dependent on bicarbonate, which could be ascribed to the replacement of S289 by G289. Moreover, the ferrous ion could induce a change in the tertiary, but not the secondary, structure of Ab-ACO.Conclusions
These results provide crucial experimental support for the ability of Ab-ACO to catalyse ethylene formation in a similar manner to that of plant ACOs, but there are differences between the biochemical and catalytic characteristics of Ab-ACO and plant ACOs.General significance
This work enhances the understanding of the ethylene biosynthesis pathways in fungi and could promote profound physiological research of the role of ethylene in the regulation of mushroom growth and development. 相似文献9.
Aim
The tumor suppressor gene Ras association domain family 1 isoform A (RASSF1A) regulates cell cycle regulation, apoptosis and microtubule stability and is inactivated by promoter hypermethylation at a high frequency in hepatocellular carcinoma (HCC). A guanine (G)/thymine (T) common single nucleotide polymorphism (SNP) at first position of codon 133 in RASSF1A gene determines an alanine (Ala) to serine (Ser) (Ala133Ser) amino acidic substitution which may alter cancer risk by influencing the function of RASSF1A protein.Methods
To determine the association of the RASSF1A Ala133Ser polymorphism with the risk of HCC development in a Turkish population, a hospital-based case–control study was designed consisting of 236 subjects with HCC and 236 cancer-free control subjects matched for age, gender, smoking and alcohol status. The genotype frequency of the RASSF1A Ala133Ser polymorphism was determined by using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay.Results
Allele and genotype associations of RASSF1A Ala133Ser polymorphism with HCC susceptibility were observed in comparisons between the patient and control samples (P < 0.001). Risk of HCC development in this Turkish population was significantly increased in carriers of the Ser133 variant allele of Ala133Ser polymorphism (Ala/Ser and Ser/Ser genotypes) when compared with homozygote Ala/Ala genotype (OR = 5.47, 95% CI = 3.63–8.25, P = 0.001).Conclusion
Because our results suggest for the first time that the Ser133 allele of RASSF1A Ala133Ser polymorphism may be a genetic susceptibility factor for HCC in the Turkish population, further independent studies are required to validate our findings in a larger series, as well as in patients of different ethnic origins. 相似文献10.
Background and objective
The genetic variants of xenobiotic-metabolizing enzymes, such as those encoded by glutathione-S-transferase (GST) genes, may be associated with the risk of coronary artery disease (CAD). To investigate the genetic factors for CAD, we examined the GSTM1, GSTT1, GSTP1, and GSTA1 genotypes in a CAD cohort in Taiwan.Methods
Our study included 458 CAD participants and 209 control participants who received coronary angiography to assess CAD. The severity of CAD was defined as the number of coronary vessels with 50% or greater stenosis. Sequence variation of the GSTM1 and GSTT1 genes was determined using a polymerase chain reaction (PCR). The GSTP1 (Ile105Val), and GSTA1 (-69C > T) genetic variants were identified using a combination of PCR and restriction fragment length polymorphism analysis. Logistic regression analysis was used to calculate the odds ratios (ORs) and 95% confidence intervals.Results
Among the GST genetic variants examined, the GSTT1 null genotype was more prevalent in CAD participants with 3 stenosed vessels than in control participants (OR = 1.64, P = .02). This association was no longer observed after adjusting for age, sex, smoking, alcohol use, diabetes mellitus, and serum levels of total cholesterol and high-density lipoprotein cholesterol (OR = 1.28, P = .40). Both univariate and multivariate logistic regression analyses found no significant associations between CAD and the other genetic variants, either separately or in combination. In addition, no effects of interactions between the genotypes and environmental factors, such as cigarette smoking, were significantly associated with the risk of CAD.Conclusion
The GST genetic variants examined were not associated with susceptibility to CAD in our Taiwanese cohort. This null association requires further confirmation with larger samples. 相似文献11.
Background
Asthma is a complex multifactorial disease with an obvious genetic predisposition. Polymorphisms of the glutathione-S-transferase (GST) genes are known risk factors for some environmentally-related diseases. The aim of the present study was to investigate the role of polymorphisms in the GSTT1, GSTM1 and GSTP1 genes and asthma susceptibility in Egyptian children, and to analyze their effect on GST activity and lung function.Methods
GSTT1 and GSTM1 gene polymorphism was genotyped using the multiplex polymerase chain reaction (PCR) and GSTP1 ILe105Val polymorphism was determined using PCR-restriction fragment length polymorphism (PCR-RFLP) in 168 healthy and 126 asthmatic children (82 atopic and 44 nonatopic). Also GST enzyme activity and lung function were evaluated.Results
Asthmatic children had a significant higher prevalence of the GSTM1 null (P = 0.003) and significant lower prevalence of GSTP1 Val/Val genotypes (P = 0.02) than control group. Lung function was significantly decreased in GSTM1 null genotype and GSTP1 Ile/Ile genotype. GSTP1 Val/Val genotypes and GSTM1 null genotype had a significant decrease in plasma GST activity.Conclusions
GST genes polymorphisms may play an important role in pathogenesis and susceptibility to asthma in children. 相似文献12.
Babu Sudhamalla Mahesh Kumar Karnati R. Roy R. Sunil Kumar Abani K. Bhuyan 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
It is known that tandem domains of enzymes can carry out catalysis independently or by collaboration. In the case of cysteine proteases, domain sequestration abolishes catalysis because the active site residues are distributed in both domains. The validity of this argument is tested here by using isolated human ribosomal protein S4, which has been recently identified as an unorthodox cysteine protease.Methods
Cleavage of the peptide substrate Z-FR↓-AMC catalyzed by recombinant C-terminal domain of human S4 (CHS4) is studied by fluorescence-monitored steady-state and stopped-flow kinetic methods. Proteolysis and autoproteolysis were analyzed by electrophoresis.Results
The CHS4 domain comprised of sequence residues 116–263 has been cloned and ovreexpressed in Escherichia coli. The purified domain is enzymatically active. Barring minor differences, steady-state kinetic parameters for catalysis by CHS4 are very similar to those for full-length human S4. Further, stopped-flow transient kinetics of pre-steady-state substrate binding shows that the catalytic mechanism for both full-length S4 and CHS4 obeys the Michaelis–Menten model adequately. Consideration of the evolutionary domain organization of the S4e family of ribosomal proteins indicates that the central domain (residues 94–170) within CHS4 is indispensable.Conclusion
The C-terminal domain can carry out catalysis independently and as efficiently as the full-length human S4 does.Significance
Localization of the enzyme function in the C-terminal domain of human S4 provides the only example of a cysteine endoprotease where substrate-mediated intramolecular domain interaction is irrelevant for catalytic activity. 相似文献13.
Background
Chinese Tibetans have a series of distinctive physiological traits which enable them to tolerate the extreme environment of the Tibetan plateau. P-selectin gene has been proved to be highly polymorphic in Europeans and Americans. Nevertheless, studies on either the frequency distributions of single nucleotide polymorphisms (SNPs) or haplotype diversity and linkage disequilibrium of P-selectin gene in Chinese Tibetan population are still unavailable.Methods
The frequency distributions of 3 SNPs in P-selectin gene promoter (− 2123C/G, − 1969A/G, − 1817T/C) and 3 SNPs in exon region (Ser290Asn, Val599Leu, Thr715Pro) were investigated by real-time PCR and high-resolution melting method among 314 Chinese Tibetans and 328 age- and sex-matched Han people.Results
The frequencies of the − 2123G and − 1817T alleles among the Tibetan population had no significant differences from those of the Han population. Among the Tibetan population, the G allele frequency of − 1969A/G and Ser290Asn were both higher than those of the Han population. Val599Leu and Thr715Pro did not show any polymorphism in the two populations. In the Tibetan population, − 2123C/G, − 1969A/G, − 1817T/C and Ser290Asn were in tight linkage disequilibrium with each other.Conclusions
The frequency distributions of − 1969A/G and Ser290Asn polymorphisms in the Tibetan population were different from those in the Han population. 相似文献14.
Yuki Masahara-Negishi Akira Hosomi Massimiliano Della Mea Donatella Serafini-Fracassini Tadashi Suzuki 《Biochimica et Biophysica Acta (BBA)/General Subjects》2012
Background
The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme involved in the ER-associated degradation (ERAD) process, while ERAD-independent activities are also reported. Previous biochemical analyses indicated that the cytoplasmic PNGase orthologue in Arabidopsis thaliana (AtPNG1) can function as not only PNGase but also transglutaminase, while its in vivo function remained unclarified.Methods
AtPNG1 was expressed in Saccharomyces cerevisiae and its in vivo role on PNGase-dependent ERAD pathway was examined.Results
AtPNG1 could facilitate the ERAD through its deglycosylation activity. Moreover, a catalytic mutant of AtPNG1 (AtPNG1(C251A)) was found to significantly impair the ERAD process. This result was found to be N-glycan-dependent, as the AtPNG(C251A) did not affect the stability of the non-glycosylated RTA? (ricin A chain non-toxic mutant). Tight interaction between AtPNG1(C251A) and the RTA? was confirmed by co-immunoprecipitation analysis.Conclusion
The plant PNGase facilitates ERAD through its deglycosylation activity, while the catalytic mutant of AtPNG1 impair glycoprotein ERAD by binding to N-glycans on the ERAD substrates.General significance
Our studies underscore the functional importance of a plant PNGase orthologue as a deglycosylating enzyme involved in the ERAD. 相似文献15.
Besma Lakhal Sonia Ben-Hadj-Khalifa Nouha Bouali Pascal Philipert Françoise Audran Rim Braham Elghezal Hatem Charles Sultan Ali Saad 《Gene》2012
Background
WNT4 and SF1 genes play an important role in ovarian development. They constitute coherent candidate genes associated with premature ovarian failure (POF) pathogenesis.Methods
We sequenced the coding region of WNT4 and SF1 in 55 Tunisian women with POF and 100 healthy controls.Results
We identified a synonymous variation in WNT4 (c.99G>A, p.Ser33Ser) and a substitution (c.G437C) in SF1 gene inducing G146 to Ala (GGG–GCG) missense mutation. WNT4 (c.99G>A, p.Ser33Ser) was not associated with POF pathology. However, a positive association of SF1 Gly146Ala polymorphism was noted. Gly146Ala minor allele frequency was significantly higher (p = 0.029) in POF patients versus controls and Ala allele containing genotypes (p = 0.005) were positively associated with POF pathology. The carriage of 146Ala allele was also associated with a significant reduction in estradiol plasma levels.Conclusions
SF1 Gly146Ala polymorphism seems to be associated with POF pathology in the Tunisian population likely by reducing estradiol levels. 相似文献16.
Chen BY Ma XX Guo PC Tan X Li WF Yang JP Zhang NN Chen Y Xia Q Zhou CZ 《Journal of molecular biology》2011,412(2):204-211
Glutathione transferases (GSTs) are ubiquitous detoxification enzymes that conjugate hydrophobic xenobiotics with reduced glutathione. The silkworm Bombyx mori encodes four isoforms of GST Omega (GSTO), featured with a catalytic cysteine, except that bmGSTO3-3 has an asparagine substitution of this catalytic residue. Here, we determined the 2.20-Å crystal structure of bmGSTO3-3, which shares a typical GST overall structure. However, the extended C-terminal segment that exists in all the four bmGSTOs occupies the G-site of bmGSTO3-3 and makes it unworkable, as shown by the activity assays. Upon mutation of Asn29 to Cys and truncation of the C-terminal segment, the in vitro GST activity of bmGSTO3-3 could be restored. These findings provided structural insights into the activity regulation of GSTOs. 相似文献
17.
Keisuke Kaneko Yasunori Sugiyama Yusuke Yamada Noriyuki Sueyoshi Akira Watanabe Yasuhiko Asada Atsuhiko Ishida Isamu Kameshita 《Biochimica et Biophysica Acta (BBA)/General Subjects》2011
Background
In a previous study, we conducted an expression cloning screen of a cDNA library prepared from Coprinopsis cinerea mycelia using Multi-PK antibodies and detected a wide variety of Ser/Thr protein kinases. One of the isolated clones, CMZ032, was found to encode a putative Ser/Thr protein kinase designated CoPK32. In the present study, we investigated the biochemical properties and physiological significance of CoPK32.Methods
CoPK32 was expressed in Escherichia coli, and its biochemical properties were examined. The effects of high osmotic stresses on the growth of C. cinerea and on the endogenous CoPK32 activity in mycelia were also examined.Results
CoPK32 showed autophosphorylation activity and effectively phosphorylated exogenous protein substrates. CoPK32S, a splice variant that was 18 amino acids shorter than CoPK32, showed much lower protein kinase activity than CoPK32. The catalytic properties of CoPK32 deletion mutants suggested that the C-terminal region of CoPK32 was important for the kinase activity and recognition of substrates. CoPK32 was highly expressed in the actively growing region of the mycelial colony. When mycelia were stimulated by high osmotic stresses, endogenous CoPK32 was markedly activated and the mycelial growth was severely inhibited. The activation of CoPK32 activity by high osmotic stresses was abrogated by SB202190 or SB239063 as well-known inhibitors of p38 mitogen-activated protein kinase.Conclusions
CoPK32 is involved in the stress response pathway in mycelia of C. cinerea in response to environmental stresses.General significance
In C. cinerea, protein kinases such as CoPK32 play important roles in signal transduction pathways involved in stress responses. 相似文献18.
Fabian Milz Alexander Harder Phillipp Neuhaus Olga Breitkreuz-Korff Volker Walhorn Torben Lübke Dario Anselmetti Thomas Dierks 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Sulf1 is a cell-surface sulfatase removing internal 6-O-sulfate groups from heparan sulfate (HS) chains. Thereby it modulates the activity of HS-dependent growth factors. For HS interaction Sulf1 employs a unique hydrophilic domain (HD).Methods
Affinity-chromatography, AFM-single-molecule force spectroscopy (SMFS) and immunofluorescence on living cells were used to analyze specificity, kinetics and structural basis of this interaction.Results
Full-length Sulf1 interacts broadly with sulfated glycosaminoglycans (GAGs) showing, however, higher affinity toward HS and heparin than toward chondroitin sulfate or dermatan sulfate. Strong interaction depends on the presence of Sulf1-substrate groups, as Sulf1 bound significantly weaker to HS after enzymatic 6-O-desulfation by Sulf1 pretreatment, hence suggesting autoregulation of Sulf1/substrate association. In contrast, HD alone exhibited outstanding specificity toward HS and did not interact with chondroitin sulfate, dermatan sulfate or 6-O-desulfated HS. Dynamic SMFS revealed an off-rate of 0.04/s, i.e., ~ 500-fold higher than determined by surface plasmon resonance. SMFS allowed resolving the dynamics of single dissociation events in each force–distance curve. HD subdomain constructs revealed heparin interaction sites in the inner and C-terminal regions of HD.Conclusions
Specific substrate binding of Sulf1 is mediated by HD and involves at least two separate HS-binding sites. Surface plasmon resonance KD-values reflect a high avidity resulting from multivalent HD/heparin interaction. While this ensures stable cell–surface HS association, the dynamic cooperation of binding sites at HD and also the catalytic domain enables processive action of Sulf1 along or across HS chains.General significance
HD confers a novel and highly dynamic mode of protein interaction with HS. 相似文献19.
Li Xing Meijuan Niu Xia ZhaoLawrence Kleiman 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.Methods
The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A–RNA interaction and RNA helicase A-stimulated viral RNA processes.Results
Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNALys3 annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A–HIV-1 RNA interaction in the cells.Conclusions
The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.General significance
The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro. 相似文献20.
Preeyanuch Thongpoo Lauren S. McKee Ana Catarina Araújo Prachumporn T. Kongsaeree Harry Brumer 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013