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1.
Preeti Subramanian Silvia Locatelli-Hoops Jason Kenealey Jacqueline DesJardin Luigi Notari S. Patricia Becerra 《The Journal of biological chemistry》2013,288(33):23928-23942
The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell surfaces. We have previously reported that PEDF binds and stimulates PEDF receptor (PEDF-R), a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (Leu159–Met325) with affinity similar to the full-length PEDF-R (Met1–Leu504). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile193–Leu232) and P1 (Thr210–Leu249) peptides. Recombinant C-terminal truncated PEDF-R4 (Met1–Leu232) and internally truncated PEDF-R and PEDF-R4 (ΔHis203–Leu232) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His203–Leu232 region lost the PEDF affinity that stimulated their enzymatic activity. Cell surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knock down PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF·PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation. 相似文献
2.
Alexandre Beautrait Kevin R. Michalski Thomas S. Lopez Katelynn M. Mannix Devin J. McDonald Amber R. Cutter Christopher B. Medina Aaron M. Hebert Charnelle J. Francis Michel Bouvier John J. G. Tesmer Rachel Sterne-Marr 《The Journal of biological chemistry》2014,289(36):25262-25275
G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α2A-adrenergic receptors (α2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α2AAR with EC50 values of 15 nm and 8 μm, respectively. We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu4, Val7, Leu8, Val11, and Ser12, directly interacts with receptors, whereas residues such as Asp10, Tyr13, Ala16, Met17, Gly475, Val477, and Ile485 are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity. 相似文献
3.
Context
Recurrent miscarriage (RM), defined as three or more consecutive losses before the 20th week of gestation, affects 0.5–2% of pregnant women. In over 80% of cases, RM remains unexplained after investigations, suggesting the involvement of genetic factors.Objectives
The present study investigates the common polymorphisms of chemokine receptors CCR5 (NG_012637.1:g.5303A>G) and CX3CR1 (NG_016362.1:g.21065C>T, Thr280Met and NG_016362.1:g.20971G>A, Val249Ile) and their association with recurrent miscarriages (RM) among north Indian women.Participants and Methods
In a retrospective case-control study 200 well characterized patients with unexplained RM and 300 controls were genotyped for three polymorphic markers of CCR5 and CX3CR1 by restriction digestion of PCR amplified fragments.Results
Alleles and genotypes of CX3CR1 Val249Ile revealed statistically significant associations with RM cases when compared with the controls. The homozygous variant genotype Ile/Ile was found to be significantly higher among patients (p = 0.0002) when compared with the homozygous wild type Val/Val genotype. The haplotype of CX3CR1 that carried major alleles of Thr280Met and Val249Ile (T-V) showed statistically significant protective association (p < 0.0001, OR = 0.41, 95% CI = 0.31–0.54). The haplotype A-T-V (all wild type alleles) revealed a statistically significant protective association (p < 0.0001, OR = 0.41, 95% CI = 0.34–0.62), whereas the haplotypes G-T-I, A-T-I and A-M-V modified the risk of RM 1.9-fold, 5.5-fold and 5.1-fold respectively.Conclusions
A common polymorphism of CX3CR1 gene, Val240Ile is associated with the risk of RM in north Indian women. Risk of RM may also be modified by the presence of haplotypes T-I, M-V, G-T-I, A-T-I and A-M-V. 相似文献4.
Ranjana Sarma Stephen Keable Lance C. Seefeldt 《Journal of inorganic biochemistry》2010,104(4):385-1555
The X-ray crystal structure is presented for a nitrogenase MoFe protein where the alpha subunit residue at position 70 (α-70Val) has been substituted by the amino acid isoleucine (α-70Ile). Substitution of α-70Val by α-70Ile results in a MoFe protein that is hampered in its ability to reduce a range of substrates including acetylene and N2, yet retains normal proton reduction activity. The 2.3 Å structure of the α-70Ile MoFe protein is compared to the α-70Val wild-type MoFe protein, revealing that the δ methyl group of α-70Val is positioned over Fe6 within the active site FeMo-cofactor. This work provides strong crystallographic support for the previously proposed model that substrates bind and are reduced at a single 4Fe-4S face of the FeMo-cofactor and that when α-70Val is substituted by α-70Ile access of substrates to Fe6 of this face is effectively blocked. Furthermore the detailed examination of the structure provides the basis for understanding the ability to trap and characterize hydrides in the variant, contributing significantly to our understanding of substrate access and substrate reduction at the FeMo-cofactor active site of nitrogenase. 相似文献
5.
Masataka Nakagawa Megumi Ueyama Hiroki Tsuruta Tomohide Uno Kengo Kanamaru Bunzo Mikami Hiroshi Yamagata 《The Journal of biological chemistry》2010,285(39):29797-29807
Cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (Cucumis melo L.). It is synthesized as a preproprotein consisting of a signal peptide, NH2-terminal propeptide, and 67-kDa protease domain. We investigated the role of this propeptide (88 residues) in the cucumisin precursor. Complementary DNAs encoding the propeptides of cucumisin, two other plant subtilases (Arabidopsis ARA12 and rice RSP1), and bacterial subtilisin E were expressed in Escherichia coli independently of their mature enzymes. The cucumisin propeptide strongly inhibited cucumisin in a competitive manner with a Ki value of 6.2 ± 0.55 nm. Interestingly, cucumisin was also strongly inhibited by ARA12 and RSP1 propeptides but not by the subtilisin E propeptide. In contrast, the propeptides of cucumisin, ARA12, and RSP1 did not inhibit subtilisin. Deletion analysis clearly showed that two hydrophobic regions, Asn32–Met38 and Gly97–Leu103, in the cucumisin propeptide were important for its inhibitory activity. Site-directed mutagenesis also confirmed the role of a Val36-centerd hydrophobic cluster within the Asn32–Met38 region in cucumisin inhibition. Circular dichroism spectroscopy revealed that the cucumisin propeptide had a secondary structure without a cognate protease domain and that the thermal unfolding of the propeptide at 90 °C was only partial and reversible. A tripeptide, Ile35-Val36-Tyr37, in the Asn32–Met38 region was thought to contribute toward the formation of a proper secondary structure necessary for cucumisin inhibition. This is the first report on the function and structural information of the propeptide of a plant serine protease. 相似文献
6.
The wild-type (wt) N-terminal 23-residue fusion peptide (FP) of the human immunodeficiency virus (HIV) fusion protein gp41 and its V2E mutant have been studied by nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles as membrane mimics. A number of NMR techniques have been used. Pulsed field-gradient diffusion measurements in DPC and in 4:1 DPC/sodium dodecylsulfate mixed micelles showed that there is no major difference between the partition coefficients of the fusogenic wt peptide and the V2E mutant in these micelles, indicating that there is no correlation between the activity of the fusion peptides and their membrane affinities. The nuclear Overhauser enhancement (NOE) patterns and the chemical shift index for these two peptides indicated that both FP are in an α helical conformation between the Ile4 to Leu12 or to Ala15 region. Simulated annealing showed that the helical region extends from Ile4 to Met19. The two FPs share similar conformational characteristics, indicating that the conformation of the FP is not an important factor determining its activity. The spin-label studies, utilizing spin labels 5- and 16-doxystearic acids in the DPC micelles, provided clear indication that the wt FP inserts its N-terminus into the micelles while the V2E mutant does not insert into the micelles. The conclusion from the spin-label results is corroborated by deuterium amide proton exchange experiments. The correlation between the oblique insertion of the FP and its fusogenic activity is in excellent agreement with results from our molecular dynamics simulation and from other previous studies. 相似文献
7.
Shailesh K. Shahi Vinay K. Singh Ashok Kumar Sanjeev K. Gupta Surya K. Singh 《Journal of molecular modeling》2013,19(3):973-983
Klebsiella pneumoniae strain DF12SA (HQ114261) was isolated from diabetic foot wounds. The strain showed resistance against ampicillin, kanamycin, gentamicin, streptomycin, spectinomycin, trimethoprim, tetracycline, meropenem, amikacin, piperacillin/tazobactam, augmentin, co-trimoxazole, carbapenems, penicillins and cefoperazone, and was sensitive to clindamycin. Molecular characterization of the multidrug-resistance phenotype revealed the presence of a class 1 integron containing two genes, a dihydrofolate reductase (DHFR) (PF00186), which confers resistance to trimethoprim; and aminoglycoside adenyltransferase (AadA) (PF01909), which confers resistance to streptomycin and spectinomycin. A class 1 integron in K. pneumoniae containing these two genes was present in eight (18.18 %) out of 44 different diabetic foot ulcer (DFU) patients. Hence, there is a need to develop therapeutics that inhibit growth of multidrug resistant K. pneumoniae in DFU patients and still achieve amputation control. Am attempt was made to create a 3D model and find a suitable inhibitor using an in silico study. Rational drug design/testing requires crystal structures for DHFR and AadA. However, the structures of DHFR and AadA from K. pneumoniae are not available. Modelling was performed using Swiss Model Server and Discovery Studio 3.1. The PDBSum server was used to check stereo chemical properties using Ramachandran plot analysis of modeled structures. Clindamycin was found to be suitable inhibitor of DHFR and AadA. A DockingServer based on Autodock & Mopac was used for docking calculations. The amino acid residues Ser32, Ile46, Glu53, Gln54, Phe57, Thr72, Met76, Val78, Leu79, Ser122, Tyr128, Ile151 in case of DHFR and Phe34, Asp60, Arg63, Gln64, Leu68, Glu87, Thr89, Val90 for AadA were found to be responsible for positioning clindamycin into the active site. The study identifies amino acid residues crucial to ‘DHFR and AadA -drug’ and ‘DHFR and AadA -inhibitor’ interactions that might be useful in the ongoing search for a versatile DHFR and AadA -inhibitor. 相似文献
8.
Activation of several tRNAs of Escherichia coli by the phenoxyacetyl derivative of N-hydroxysuccinimide 总被引:3,自引:0,他引:3
Escherichia coli 15T? treated with chloramphenicol produces tRNAphe which is deficient in minor nucleosides. Undermodified tRNAphe chromatographs as two new peaks from a benzoylated diethylaminoethyl-cellulose column. Chloramphenicol tRNAphe was purified by phenoxyacetylation of phenylalanyl-tRNA and subsequent chromatography on benzoylated diethylaminoethyl-cellulose. Purified tRNAphe had an altered Chromatographie profile as a result of the purification procedure. Phenoxyacetylation of an unpurified tRNA preparation, which was either charged with phenylalanine or kept discharged, resulted in a permanent alteration of tRNAphe which was similar to the alteration of the purified tRNAphe. The altered tRNAs eluted with higher salt or ethanol concentrations from benzoylated diethylaminoethyl-cellulose. The alteration was also shown for tRNAphe of phenoxyacetylated tRNA from late log phase E. coli 15T?. tRNAglu and tRNALeu were not changed, but both tRNAArg and tRNAIle were altered. tRNA2Val and tRNAMet shifted in the elution profile; tRNA1Val and tRNAfMet were not affected.Comparison of the primary structures of the alterable and nonalterable tRNA's revealed that all alterable tRNA's have the undefined nucleoside X in the extra loop. Phenoxyacetylation of nucleoside X probably was the cause of the altered profiles.tRNAphe from E. coli 15T? treated with chloramphenicol was less reactive towards phenoxyacetylation than normal tRNA, possibly because of a different conformation of the modification-deficient molecule relative to the normal tRNAphe. tRNAphe from E. coli 15T?, starved for cysteine and methionine and treated with chloram-phenicol, is more deficient in minor nucleosides and showed even less reactivity.Acceptor capacities of the altered tRNA species were not changed significantly; only the acceptor capacity for tRNAIle decreased approximately 25%. The recognition site for the aminoacyl-tRNA synthetases probably is not affected. 相似文献
9.
David T.W. Fei Bruce A. Scoggins Geoffrey W. Tregear John P. Coghlan 《Life sciences》1981,29(2):157-163
The present study examines the effect of [Sar1, Ile8] angiotensin II ([Sar1, Ile8] ANG II) on the blood clearance rate of [Val5] angiotensin II ([Val5] ANG II) in conscious, sodium-replete sheep. Animals were infused simultaneously with [Val5] ANG II and [Sar1, Ile8] ANG II at a rate of 42 nmol/h and 6 μmol/h respectively. Blood [Val5] ANG II was quantitatively determined with care taken in separating [Val5] ANG II from [Sar1, Ile8] ANG II prior to radioimmunoassay. The blood clearance rate of [Val5] ANG II calculated from infusion rate/blood concentration was significantly different before and during [Sar1, Ile8] ANG II infusion, being 141 ± 13 L/h (n = 12) and 95 ± 10 L/h (n = 12) respectively. Plasma renin concentration remained suppressed after the commencement of [Sar1, Ile8] ANG II infusion. studies showed no significant decrease in the rate of degradation of [Val5] ANG II in blood in the presence of [Sar1, Ile8] ANG II. Possible interpretation of this reduction of blood clearance rate of [Val5] ANG II by 45 ± 15 L/h (n = 6) was discussed. 相似文献
10.
Hiroshi Sekine 《Bioscience, biotechnology, and biochemistry》2013,77(8):1945-1952
Some physicochemical properties of neutral proteinases I and II, zinc-containing metalloenzymes, from Aspergillus sojae were investigated.Neutral proteinase I: The enzyme protein had a sedimentation coefficient of 3.90S, an intrinsic viscosity of 0.0315 dl/g, and a partial specific volume, calculated from the amino acid and carbonhydrate composition, of 0.715 cm3/g. The molecular weight was 42,200 from the Yphantis’ procedure, and was 42,500 from the calculation according to the Scheraga-Mandel-kern’s formula. The integral numbers of amino acid residues per molecule calculated on the basis of 42,200 as molecular weight were as follows; Lys16, His6, Arg13, Trp8, Asp56, Thr25, Ser23, Glu31, Pro18, Gly40, Ala33, l/2Cys4, Val11, Met6, Ile15, Leu25, Tyr20, Р?е10, (amide-ammonia)29, in addition to mannose6, galactose1, hexosamine3.Neutral proteinase II: The enzyme protein had a sedimentation coefficient of 2.32S, an intrinsic viscosity of 0.0270 dl/g, and a calculated partial specific volume of 0.714 cm3/g. The molecular weight was 16,800 from the Yphantis’ procedure, and was 18,000 from the sedimentation and intrinsic viscosity. The following amino acid compositions was calculated on the basis of 16,800 as molecular weight; Lys8, His3, Arg3, Asp19, Thr17, Ser11, GIu23, Pro5, Gly9, Ala24, l/2Cys4, Val5, Ile3, Leu13, Tyr10, Phe3, (amide-ammonia)15. In the enzyme preparation, neither methionine nor tryptophan was detected and carbohydrate was also absent.In both neutral proteinases I and II, no free SH group was detected by the PCMB-titration in the presence of 8 M urea. 相似文献
11.
The molecular weight of γ3 globulin was determined to be 120,000 daltons by means of both sedimentation equilibrium and gel filtration methods. The protein was composed of 3 identical major and 1 minor subunits, and the molecular weights of them were found to be 35,000 and 13,000 daltons, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major subunit has an arginyl residue as the amino terminal amino acid. The amino acid and carbohydrate composition of γ3 globulin was determined as follows: Lys37His37Arg92Asp60Glu139Gly120Ala83Val76Leu70Ile31Pro54Ser77Thr33Cys11Met9Phe51Tyr26Trp8 (Amide NH3)69Hexose15Pentose4Hexosamine4. The structure of γ3 globulin was discussed with comparing that of γ1 globulin. 相似文献
12.
Summary Familial amyloid cardiomyopathy in a Danish kindred is associated with a specific mutation (Met for Leu111) in the transthyretin (TTR) gene, causing the loss of a recognition site for the restriction enzyme DdeI in the gene. We describe a diagnostic test for the molecular detection of this mutation. A sequence of the TTR gene containing the mutation was amplified by the polymerase chain reaction from isolated genomic DNA of two affected patients and several controls. DdeI digestion of the amplified DNA from the patients revealed 3 bands by gel-electrophoresis, whereas amplified DNA of the controls showed only 2 bands, consistent with complete digestion. Thus, the assumed heterozygous TTR Met111 mutation was confirmed in the affected patients. 相似文献
13.
Hyun Young Yu Farooqahmed S. Kittur David R. Bevan Asim Esen 《Phytochemistry》2009,70(11-12):1355-1365
β-Glucosidases (Glu1 and Glu2) in maize specifically interact with a lectin called β-glucosidase aggregating factor (BGAF). We have shown that the N-terminal (Glu50–Val145) and the C-terminal (Phe466–Ala512) regions of maize Glu1 are involved in binding to BGAF. Sequence comparison between sorghum β-glucosidases (dhurrinases, which do not bind to BGAF) and maize β-glucosidases, and the 3D-structure of Glu1 suggested that the BGAF-binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF-binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile72–Thr82) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser1–Thr29, together with C-terminal region Phe466–Ala512, affects the size of Glu1–BGAF complexes. The dissociation constants (Kd) of chimeric β-glucosidase–BGAF interactions also demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile72–Thr82 on Glu1 for BGAF binding, we constructed a chimeric sorghum β-glucosidase, Dhr2 (C-11, Dhr2 whose Val72–Glu82 region was replaced with the Ile72–Thr82 region of Glu1). C-11 binds to BGAF, indicating that the Ile72–Thr82 region is indeed a major interaction site on Glu1 involved in BGAF binding. 相似文献
14.
High‐performance liquid chromatographic enantioseparation of N‐aryl‐thioureidoalkylphosphonates and thiourylenedi(alkylphosphonates) on polysaccharide‐based chiral stationary phases
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Józef Drabowicz Marcin H. Kudzin Zbigniew H. Kudzin Patrycja Pokora‐Sobczak 《Chirality》2018,30(2):131-140
The first successful enantioseparation of representative O,O‐diphenyl‐N‐arylthioureidoalkylphosphonates, (±)‐Ptc‐ValP(OPh)2 & (±)‐Ptc‐LeuP(OPh)2 and thiourylenedi(isobutyl phosphonate), Tcm[ValP(OPh)2]2 on analytical and semipreparative scale was achieved by high‐performance liquid chromatography using polysaccharide‐based chiral stationary phases (CPs). Atc‐AAP(OPh)2 was obtained using modified tricomponent condensations of the corresponding aldehydes, N‐arylthiourea and triphenyl phosphite whereas Tcm[ValP(OPh)2]2 by the condensations of aldehydes, thiourea, and triphenyl phosphite. The prepared, racemic (±)‐Atc‐AAP(OPh)2 [(±)‐Ptc‐ValP(OPh)2, (±)‐Ptc‐LeuP(OPh)2, (±)‐Ptc‐PglyP(OPh)2 and (±)‐Ntc‐PglyP(OPh)2] and racemic (±)‐Tcm[AAP(OPh)2]2 [(±)‐Tcm[NvaP(OPh)2]2 & (±)‐Tcm[ValP(OPh)2]2] were adequately characterized and used for chromatographic separations on high‐performance liquid chromatography–chiral stationary phases. The best results were obtained for (±)‐Ptc‐ValP(OPh)2, (±)‐Ptc‐LeuP(OPh)2 and (±)‐Tcm[ValP(OPh)2]2. 相似文献
15.
An alkaline proteinase of Aspergillus Candidus was purified from wheat bran solid culture by batchwise treatment with Amberlite IRC–50 and sequential chromatography on DEAE-cellulose, hydroxylapatite and Sephadex G–100 gel. This purification results in a 18-fold increase of proteolytic activity and the enzyme preparation was homogeneous in sedimentation analysis of the ultracentrifuge and polyacrylamide gel disc electrophoresis. The molecular weight was estimated to be about 23,000 by gel glltration and 22,000 by calculation from the amino acid composition. The enzyme consisted of Lys14, His4, Arg3, Asp25, Thr15, Ser23, Glu15, Pro7, Gly22, Ala24, Met2, Val16, Ile11, Leu10, Tyr6, Phe7, Trp2 and amide ammonia14 and did not contain cysteine or cystine. 相似文献
16.
Comparison of amide proton exchange in reduced and oxidizedRhodobacter capsulatus cytochrome c2: A1H−15N NMR study 总被引:6,自引:0,他引:6
Summary The hydrogen-deuterium exchange rates of the reduced and oxidized forms ofRhodobacter' capsulatus cytochrome c2 were studied by1H–15N homonuclear multiple quantum correlation spectroscopy. Minimal differences were observed for the N- and C-terminal helices on changing redox state suggesting that although these helices are structurally important they do not affect the relative stability of the two redox states and hence may not be important in determining the redox potential differences observed amongst the class I c-type cytochromes. However, significant differences were observed for other regions of the protein. For example, all slow exchanging protons of the helix spanning Phe82 to Asp87 are similarly affected on reduction indicating that the unfolding equilibrium of this helix is altered between the two redox states. Other regions are not as simple to interpret; however, the difference in NH exchange rates between the redox states for a number of residues including His17, Leu37, Arg43, Ala45, Gly46, Ile57, Val58, Leu60, Gly61 and Leu100 suggest that interactions affecting the causes of these differences may be important factors in determining redox potential.Abbreviations NMR
nuclear magnetic resonance
- HMQC
homonuclear multiple quantum correlation
- NOESY
nuclear Overhauser effect spectroscopy 相似文献
17.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(2):238-244
A group of 2-alkoxy-5-methoxyallylbenzene were designed, synthesised and evaluated as potential inhibitors of the soybean 15-lipoxygenase (SLO) on the basis of the eugenol and esteragol structures. Compound 4d showed the best half maximal inhibitory concentration (IC50) for SLO inhibition (IC50?=?5.9?±?0.6 µM). All the compounds were docked in the SLO active site retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB entry: 1IK3) and showed that the allyl group of the synthetic compounds similar to the linoleic acid double bond, were oriented toward the Fe3+-OH moiety in the active site of the enzyme and this conformation was especially fixed by the hydrophobic interaction of the 2-alkoxy group with Leu515, Trp519, Val566 and Ile572. It was concluded that the molecular volume and shape of the alkoxy moiety was a major factor in the inhibitory potency variation of the synthetic compounds. 相似文献
18.
Hairat Sabit Sairam S. Mallajosyula Alexander D. MacKerell Jr. Peter W. Swaan 《The Journal of biological chemistry》2013,288(45):32394-32404
Human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is responsible for intestinal reabsorption of bile acids and plays a key role in cholesterol homeostasis. We used a targeted and systematic approach to delineate the role of highly conserved transmembrane helix 2 on the expression and function of hASBT. Cysteine mutation significantly depressed transport activity for >60% of mutants without affecting cell surface localization of the transporter. All mutants were inaccessible toward chemical modification by membrane-impermeant MTSET reagent, strongly suggesting that transmembrane 2 (TM2) plays an indirect role in bile acid substrate translocation. Both bile acid uptake and sodium dependence of TM2 mutants revealed a distinct α-helical periodicity. Kinetic studies with conservative and non-conservative mutants of sodium sensitive residues further underscored the importance of Gln75, Phe76, Met79, Gly83, Leu86, Phe90, and Asp91 in hASBT function. Computational analysis indicated that Asp91 may coordinate with sodium during the transport cycle. Combined, our data propose that a consortium of sodium-sensitive residues along with previously reported residues (Thr134, Leu138, and Thr149) from TM3 may form the sodium binding and translocation pathway. Notably, residues Gln75, Met79, Thr82, and Leu86 from TM2 are highly conserved in TM3 of a putative remote bacterial homologue (ASBTNM), suggesting a universal mechanism for the SLC10A transporter family. 相似文献
19.
Nathan R. West Benjamin Farnell Jill I. Murray Fraser Hof Peter H. Watson Martin J. Boulanger 《Protein science : a publication of the Protein Society》2009,18(12):2615-2623
S100A7 (psoriasin) is a calcium‐ and zinc‐binding protein implicated in breast cancer. We have shown previously that S100A7 enhances survival mechanisms in breast cells through an interaction with c‐jun activation domain binding protein 1 (Jab1), and an engineered S100A7 triple mutant (Asp56Gly, Leu78Met, and Gln88Lys—S100A73) ablates Jab1 binding. We extend these results to include defined breast cancer cell lines and demonstrate a disrupted S100A73/Jab1 phenotype is maintained. To establish the basis for the abrogated Jab1 binding, we have recombinantly produced S100A73, demonstrated that it retains the ability to form an exceptionally thermostable dimer, and solved the three dimensional crystal structure to 1.6 Å. Despite being positioned at the dimer interface, the Leu78Met mutation is easily accommodated and contributes to a methionine‐rich pocket formed by Met12, Met15, and Met34. In addition to altering the surface charge, the Gln88Lys mutation results in a nearby rotameric shift in Tyr85, leading to a substantially reorganized surface cavity and may influence zinc binding. The final mutation of Asp56 to Gly results in the largest structural perturbation shortening helix IV by one full turn. It is noteworthy that position 56 lies in one of two divergent clusters between S100A7 and the functionally distinct yet highly homologous S100A15. The structure of S100A73 provides a unique perspective from which to characterize the S100A7‐Jab1 interaction and better understand the distinct functions between S100A7, and it is closely related paralog S100A15. 相似文献
20.
A live,attenuated dengue virus type 1 vaccine candidate with a 30-nucleotide deletion in the 3' untranslated region is highly attenuated and immunogenic in monkeys 总被引:1,自引:0,他引:1
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Whitehead SS Falgout B Hanley KA Blaney Jr JE Markoff L Murphy BR 《Journal of virology》2003,77(2):1653-1657
The Delta30 deletion mutation, which was originally created in dengue virus type 4 (DEN4) by the removal of nucleotides 172 to 143 from the 3' untranslated region (3' UTR), was introduced into a homologous region of wild-type (wt) dengue virus type 1 (DEN1). The resulting virus, rDEN1Delta30, was attenuated in rhesus monkeys to a level similar to that of the rDEN4Delta30 vaccine candidate. rDEN1Delta30 was more attenuated in rhesus monkeys than the previously described vaccine candidate, rDEN1mutF, which also contains mutations in the 3' UTR, and both vaccines were highly protective against challenge with wt DEN1. Both rDEN1Delta30 and rDEN1mutF were also attenuated in HuH-7-SCID mice. However, neither rDEN1Delta30 nor rDEN1mutF showed restricted replication following intrathoracic inoculation in the mosquito Toxorhynchites splendens. The ability of the Delta30 mutation to attenuate both DEN1 and DEN4 viruses suggests that a tetravalent DEN vaccine could be generated by introduction of the Delta30 mutation into wt DEN viruses belonging to each of the four serotypes. 相似文献