The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Toll-like receptor 4 Asp299Gly/Thr399Ile polymorphisms are a risk factor for Candida bloodstream infection

Toll-like receptor (TLR)-4 is an important pattern recognition receptor for Candida albicans, playing a role in innate host defense. We investigated whether there is an association between the TLR4 Asp299Gly or TLR4 Thr399Ile polymorphism, and the occurrence of Candida bloodstream infection. We performed a case-control study, involving 43 patients with a Candida bloodstream infection and 166 healthy individuals. TLR4 Asp299Gly and Thr399Ile polymorphisms were assessed, as well as cytokine production after stimulation of peripheral blood mononuclear cells (PBMC) with Candida albicans. We observed that the prevalence of TLR4 Asp299Gly polymorphism was found to be higher in patients with Candida bloodstream infection than in controls (26% versus 10%; OR 3.0; 95%CI 1.3-6.9). All patients bearing the Asp299Gly polymorphism were also positive for the Thr399Ile allele, a linkage well described in literature. IL-10 production was higher in C. albicans-stimulated PBMC from volunteers bearing the TLR4 Asp299Gly polymorphism, and a similar tendency was observed in TLR4 Asp299Gly heterozygous patients who had recovered from candidemia. These findings show that the TLR4 Asp299Gly/Thr399Ile polymorphisms are associated with an increased susceptibility to Candida bloodstream infections, and an increased production of IL-10 is probably involved in this effect.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

The Toll-Like Receptor 4 Polymorphism Asp299Gly but Not Thr399Ile Influences TLR4 Signaling and Function

The common, co-segregating Toll-like receptor 4 (TLR4) non-synonymous single nucleotide polymorphisms (SNPs), Asp299Gly and Thr399Ile, are associated with hyporesponsiveness to inhaled lipopolysaccharide (LPS) and increased susceptibility to Gram negative pathogens in humans. The purpose of this study was to identify the relative contributions of the Asp299Gly and the Thr399Ile variants in inhibiting the function of TLR4. 293/hMD2-CD14 cell line was transfected with lentiviral constructs containing human wild type (WT) TLR4-EGFP or TLR4-EGFP with Asp299Gly, Thr399Ile or Asp299Gly/Thr399Ile complementary DNA (cDNA). Multiple stable cell lines were established for each construct: three for WT TLR4, Asp299Gly, and Thr399Ile, and only two for Asp299Gly/Thr399Ile mutants and EGFP control. We did not observe a significant effect of polymorphisms on cell surface and intracellular TLR4 expression nor were there any significant differences in TLR4 and EGFP protein levels assessed by Western blotting and confocal microscopy among the multiple cell lines of each of the constructs. All cell lines had a dose-dependent responsiveness to LPS stimulation. However, compared to the WT TLR4, cells expressing TLR4 with Asp299Gly but not Thr399Ile polymorphism produced significantly less (P<0.05) IL-8 following LPS stimulation. Similarly, cells expressing TLR4 Asp299Gly but not Thr399Ile allele had significantly lower percentage of phosphorylated and total NF-κB P65 following LPS stimulation. While we could not do statistics on the Asp299Gly/Thr399Ile group, we observed a reduced responsiveness to LPS compared to WT TLR4. Taken together, we observed that the TLR4 Asp299Gly variant, but not the Thr399Ile variant, is responsible for impaired responsiveness of TLR4 to LPS and corresponding activation of NF-κB.     

Residues within the HFRIGC sequence of HIV-1 vpr involved in growth arrest activities.

HIV-1 Vpr is a virion-associated protein that can cause growth arrest when produced inside the cell but when added externally it can cause cell death. Employing the yeast model system, the C-terminal domain, in particular the sequence HFRIGCRHSRIG (Vpr(71-82)), is essential for both the growth arrest and cytocidal activities. Conservation of this sequence in HIV-2 and SIV suggests that these residues may be functionally important. Using site-directed mutagenesis we show that the most highly conserved aa residues, His71 and Gly75, were important for the cell cycle inhibitory effects. In contrast, we show that the wild-type Vpr(71-82) peptide and three variants of this peptide with Gly75 changed to Ser, Ala, and Ile all exhibited the same cytocidal activity suggesting that the intracellular and extracellular effects are unrelated.     

Aldehyde dehydrogenase enzyme ALDH3H1 from Arabidopsis thaliana: Identification of amino acid residues critical for cofactor specificity

The cofactor-binding site of the NAD⁺-dependent Arabidopsis thaliana aldehyde dehydrogenase ALDH3H1 was analyzed to understand structural features determining cofactor-specificity. Homology modeling and mutant analysis elucidated important amino acid residues. Glu149 occupies a central position in the cofactor-binding cleft, and its carboxylate group coordinates the 2′- and 3′-hydroxyl groups of the adenosyl ribose ring of NAD⁺ and repels the 2′-phosphate moiety of NADP⁺. If Glu149 is mutated to Gln, Asp, Asn or Thr the binding of NAD⁺ is altered and rendered the enzyme capable of using NADP⁺. This change is attributed to a weaker steric hindrance and elimination of the electrostatic repulsion force of the 2′-phosphate of NADP⁺. Simultaneous mutations of Glu149 and Ile200, which is situated opposite of the cofactor binding cleft, improved the enzyme efficiency with NADP⁺. The double mutant ALDH3H1_{Glu149Thr/Ile200Val} showed a good catalysis with NADP⁺. Subsequently a triple mutation was generated by replacing Val178 by Arg in order to create a “closed” cofactor binding site. The cofactor specificity was shifted even further in favor of NADP⁺, as the mutant ALDH3H1_{E149T/V178R/I200V} uses NADP⁺ with almost 7-fold higher catalytic efficiency compared to NAD⁺. Our experiments suggest that residues occupying positions equivalent to 149, 178 and 200 constitute a group of amino acids in the ALDH3H1 protein determining cofactor affinity.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

C-Terminal Turn Stability Determines Assembly Differences between Aβ40 and Aβ42

Oligomerization of the amyloid β-protein (Aβ) is a seminal event in Alzheimer's disease. Aβ42, which is only two amino acids longer than Aβ40, is particularly pathogenic. Why this is so has not been elucidated fully. We report here results of computational and experimental studies revealing a C-terminal turn at Val36–Gly37 in Aβ42 that is not present in Aβ40. The dihedral angles of residues 36 and 37 in an Ile31–Ala42 peptide were consistent with β-turns, and a β-hairpin-like structure was indeed observed that was stabilized by hydrogen bonds and by hydrophobic interactions between residues 31–35 and residues 38–42. In contrast, Aβ(31–40) mainly existed as a statistical coil. To study the system experimentally, we chemically synthesized Aβ peptides containing amino acid substitutions designed to stabilize or destabilize the hairpin. The triple substitution Gly33Val–Val36Pro–Gly38Val (“VPV”) facilitated Aβ42 hexamer and nonamer formation, while inhibiting formation of classical amyloid-type fibrils. These assemblies were as toxic as were assemblies from wild-type Aβ42. When substituted into Aβ40, the VPV substitution caused the peptide to oligomerize similarly to Aβ42. The modified Aβ40 was significantly more toxic than Aβ40. The double substitution d-Pro36–l-Pro37 abolished hexamer and dodecamer formation by Aβ42 and produced an oligomer size distribution similar to that of Aβ40. Our data suggest that the Val36–Gly37 turn could be the sine qua non of Aβ42. If true, this structure would be an exceptionally important therapeutic target.     

Occurrence of a new corticotropin-like intermediate lobe peptide in salmon pituitary glands

A new corticotropin-like intermediate lobe peptide (CLIP) has been identified in the pituitary of chum salmon, Oncorhynchus keta. The newly isolated peptide is a tetracosa peptide, which is two residues longer than the predominant form, CLIP I, with the following amino acid sequence, H-ArgProIleLysValTyrAlaSerSerLeuGlu GlyGlyAspSerSerGluGlyThrPheProLeuGlnAlaOH. This peptide, named CLIP II is the fourth line of evidence in the teleost that the pituitary gland secretes two different forms of processed hormones, for which precursor molecules are coded on two separate genes. Together with the structures of α-melanotropin I and II, two putative ACTH molecules are proposed.     

The formation of cysteine-linked dimers of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for sensitivity to Vpu

Background

The integrase (IN) of human immunodeficiency virus type 1 (HIV-1) has been implicated in different steps during viral replication, including nuclear import of the viral pre-integration complex. The exact mechanisms underlying the nuclear import of IN and especially the question of whether it bears a functional nuclear localization signal (NLS) remain controversial.

Results

Here, we studied the nuclear import pathway of IN by using multiple in vivo and in vitro systems. Nuclear import was not observed in an importin α temperature-sensitive yeast mutant, indicating an importin α-mediated process. Direct interaction between the full-length IN and importin α was demonstrated in vivo using bimolecular fluorescence complementation assay (BiFC). Nuclear import studies in yeast cells, with permeabilized mammalian cells, or microinjected cultured mammalian cells strongly suggest that the IN bears a NLS domain located between residues 161 and 173. A peptide bearing this sequence -NLS-IN peptide- inhibited nuclear accumulation of IN in transfected cell-cycle arrested cells. Integration of viral cDNA as well as HIV-1 replication in viral cell-cycle arrested infected cells were blocked by the NLS-IN peptide.

Conclusion

Our present findings support the view that nuclear import of IN occurs via the importin α pathway and is promoted by a specific NLS domain. This import could be blocked by NLS-IN peptide, resulting in inhibition of viral infection, confirming the view that nuclear import of the viral pre-integration complex is mediated by viral IN.     

用合成多肽免疫制备尿激酶原单克隆抗体

根据尿激酶原与尿激酶一级结构的区别并结合计算机分子模拟,设计合成了包括尿激酶原Thr152-Glu163肽段的13肽,然后与载体蛋白KLH偶联作为免疫原,用BI林巴细胞融合技术获得了3种尿激酶原特异性单克隆抗体,这3种抗体仅与尿激酶原和合成多肽反应 ,而不与尿激酶及其结构类似物组织型纤溶酶原激活剂,凝血酶,纤维蛋白原反应,琼脂双向免疫扩散实验及酶活性抑制实验表明,3种抗体均为IgG类的IgG1亚类,所有3种抗体均不抑制酶活力,探讨了这组抗体用于尿激酶原结构与功能及其定量,定性分析研究方面的可能性。     

1H NMR study on amide proton exchange of calmodulin-mastoparan complex

Amide proton exchange rates of Ca2(+)-saturated calmodulin and Ca2(+)-saturated calmodulin-mastoparan complex were studied by 1H NMR spectroscopy. Exchange rates of Gly25, Gly61, Gly98, Gly134, Ile27, Ile100, and Asn137 were determined for Ca2(+)-saturated calmodulin and for Ca2(+)-saturated calmodulin-mastoparan complex, and were found to be less than 10(-4)s-1. All these residues of which the amide proton resonances appear at lower fields were considered to form hydrogen bonds, based on the results of X-ray analysis. Exchange rates of Ile27 and Asn137 became an order of magnitude smaller when mastoparan bound to Ca2(+)-saturated calmodulin, while those of the four glycines and Ile100 did not change appreciably. The reduction in accessibility of Asn137 to water cased by mastoparan binding suggests that a part of the mastoparan binding site is probably located in or near the hydrophobic cluster of the C-terminal-half domain. The reduction in accessibility of Ile27 also suggests that another part of the mastoparan binding site is located in or near the hydrophobic cleft of the N-terminal-half domain.     

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs.

Ascidiacyclamide (ASC), cyclo(-Ile1-Oxz2-d-Val3-Thz4-)2 (Oxz=oxazoline and Thz=thiazole) has a C2-symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers49, 459-469]. In this study, the modifications were extended. The Ile1 residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d-Ile, and the d-Val3 residue was replaced by Val. The structures of these analogs were analyzed by X-ray diffraction, 1H NMR and CD techniques. X-Ray diffraction analyses revealed that the [Ala1], [Aib1] and [Phe1]ASC analogs are folded, whereas [Val1]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly1] and [Phe1]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib1], [d-Ile1] and [Val3]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d-Ile1] and [Val3]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly1]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val3]ASC. In the cytotoxicity test using P388 cells, [Val1]ASC exhibited the strongest activity, whereas the epimers of ASC ([d-Ile1] and [Val3]ASCs), showed fairly moderate activities.     

Mechanism of action of the HIV-1 integrase inhibitory peptide LEDGF 361-370

The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.     

Residues in Human Arsenic (+3 Oxidation State) Methyltransferase Forming Potential Hydrogen Bond Network around S-adenosylmethionine

Residues Tyr59, Gly78, Ser79, Met103, Gln107, Ile136 and Glu137 in human arsenic (+3 oxidation state) methyltransferase (hAS3MT) were deduced to form a potential hydrogen bond network around S-adenosylmethionine (SAM) from the sequence alignment between Cyanidioschyzon merolae arsenite S-adenosylmethyltransferase (CmArsM) and hAS3MT. Herein, seven mutants Y59A, G78A, S79A, M103A, Q107A, I136A and E137A were obtained. Their catalytic activities and conformations were characterized and models were built. Y59A and G78A were completely inactive. Only 7.0%, 10.6% and 13.8% inorganic arsenic (iAs) was transformed to monomethylated arsenicals (MMA) when M103A, Q107A and I136A were used as the enzyme. The V_max (the maximal velocity of the reaction) values of M103A, Q107A, I136A and E137A were decreased to 8%, 22%, 15% and 50% of that of WT-hAS3MT, respectively. The K_M(SAM) (the Michaelis constant for SAM) values of mutants M103A, I136A and E137A were 15.7, 8.9 and 5.1 fold higher than that of WT-hAS3MT, respectively, indicating that their affinities for SAM were weakened. The altered microenvironment of SAM and the reduced capacity of binding arsenic deduced from K_M(As) (the Michaelis constant for iAs) value probably synergetically reduced the catalytic activity of Q107A. The catalytic activity of S79A was higher than that of WT despite of the higher K_M(SAM), suggesting that Ser79 did not impact the catalytic activity of hAS3MT. In short, residues Tyr59 and Gly78 significantly influenced the catalytic activity of hAS3MT as well as Met103, Ile136 and Glu137 because they were closely associated with SAM-binding, while residue Gln107 did not affect SAM-binding regardless of affecting the catalytic activity of hAS3MT. Modeling and our experimental results suggest that the adenine ring of SAM is sandwiched between Ile136 and Met103, the amide group of SAM is hydrogen bonded to Gly78 in hAS3MT and SAM is bonded to Tyr59 with van der Waals, cation-π and hydrogen bonding contacts.     

Identification and bioactivity evaluation of a novel bradykinin inhibitory peptide from the skin secretion of Chinese large odorous frog,Odorrana livida

A novel peptide was isolated from the skin secretion of Chinese large odorous frog, Odorrana livida, and was named as Rana‐BI. The cDNA sequencing was obtained by ‘shotgun’ cloning. The amino acid sequence of the mature peptide was identified as Gly‐Leu‐Leu‐Ser‐Gly‐Lys‐Ser‐Val‐Lys‐Gly‐Ser‐Ile‐OH by automated Edman degradation, and the molecular weight of the peptide was confirmed to be 1144.68 Da by MALDI‐TOF and liquid chromatography/MS. Subsequently, the bioactivity of synthetic peptide was evaluated by smooth muscle assay using isolated rat bladder preparation. It was demonstrated that Rana‐BI inhibited the contraction of rat bladder induced by bradykinin. Comparing with other peptides by searching from database, the primary structure of Rana‐BI showed high similarity with that of an antimicrobial peptide of Rana family (12/12 residues). These data revealed a novel biological function of this peptide. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Simulation, construction and characterization of a multi-functional thrombolytic agent with anti-thrombosis activity

Prourokinase (scu-PA), a thrombolytic agent, was inserted between Gly118 and Ile119 with foreign antithrombosis functional motif (Lys-Gly-Asp-Trp-motif) to construct a multi-functional chimeric molecule. The molecular model of a chimera was simulated and predicted. The recombinant chimeric protein was expressed by the baculovirus-insect cell expression system and purified by affinity chromatography. The physico-chemical characteristics of the chimeric molecule were assayed. The thrombolytic activity was determined to be 90000 IU/mg of fibrinolytic special activity by the fibrin-plate method. The anti-thrombosis activities were also assayed with IC₅₀ of 9.6 μM by an inhibition test of ADP-induced platelet aggregation.