Amino acid sequence of an extracellular, phosphate-starvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells

The primary structure of an extracellular ribonuclease (RNase LE) from Pi-depleted media of cultured cells of Lycopersicon esculentum L. cv. Lukullus has been determined. This was carried out by analysis of peptides isolated after enzymatic and chemical cleavage of the reduced and S-ethylpyridylated protein. RNase LE consists of 205 amino acid residues and has a molecular mass of 22,666 Da and an isoelectric point of 4.24. The enzyme contains 10 half-cystines. There are no potential N-glycosylation sites in the sequence. The sequence of RNase LE is homologous with those of self-incompatibility proteins of several higher plant species and with those of a number of fungal RNases. The sequence similarity with the family of self-incompatibility proteins is greater than with the fungal RNases, suggesting that the self-incompatibility proteins arose from ancestral RNase by gene duplication after the divergence of higher plants and fungi. Two pentapeptide sequences, i.e. HGLWP and KHGTC (or KHGSC), are present at identical positions in all the aligned proteins, suggesting that they contribute to the active site.     

Amino acids and peptides. XLII. Synthesis of an octapeptide sequence (A20-A27) of rubredoxin.

A protected octapeptide corresponding to a section found in the first half of the protein chain of rubredoxin has been prepared by standard peptide methods. Alternative approaches of this fragment are discussed in some detail. This work completes the series of subunits necessary to synthesize the molecule.     

An antigenic HIV-1 peptide sequence engineered into the surface structure of transferrin does not elicit an antibody response.

One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein such as human serum transferrin (HST). However, a potential drawback is that the HST may increase the immunoreactivity of the peptide, in the same way that carrier proteins can be used to generate highly immunogenic peptide hapten conjugates. In this study we have generated a recombinant HST carrier protein that contains a peptide substrate of HIV-1 protease (VSQNYPIVL). The protein retained native HST function, and the peptide was surface exposed since it was immunoreactive in native dot blots, and was cleaved by HIV-1 protease. Immunisation of rabbits with the recombinant protein elicited only a very poor anti-peptide immune response. In contrast, strong anti-peptide immune responses were raised against both the peptide alone, and a chemical conjugate of the peptide with HST. These data demonstrate that it is possible to attenuate the immune response normally directed against an immunogenic peptide sequence by engineering into a surface exposed loop of HST. These findings may have an important impact on the future design of peptide delivery systems.     

Amino acid sequence of a ferredoxin from thermoacidophilic archaebacterium, Sulfolobus acidocaldarius. Presence of an N6-monomethyllysine and phyletic consideration of archaebacteria

The amino acid sequence of a ferredoxin from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius, was determined by a combination of various conventional methods to be as follows: Gly-Ile-Asp-Pro-Tyr-Arg-Thr-His-Lys-Pro-Val-Val-Gly-Asp-Ser-Ser-Gly-His- Lys-Ile -Tyr-Gly-Pro-Val-Glu-Ser-Pro-Lys(Me)-Val-Leu-Gly-Val-His-Gly-Thr-Ile-Val -Gly-Va l-Asp-Phe-Asp-Leu-Cys-Ile-Ala-Asp-Gly-Ser-Cys-Ile-Thr-Ala-Cys-Pro-Val-As n-Val-P he-Gln-Trp-Tyr-Glu-Thr-Pro-Gly-His-Pro-Ala-Ser-Glu-Lys-Lys-Ala-Asp-Pro-V al-Asn- Glu-Gln-Ala-Cys-Ile-Phe-Cys-Met-Ala-Cys-Val-Asn-Val-Cys-Pro-Val-Ala-Ala- Ile-Asp -Val-Lys-Pro-Pro. It was composed of 103 amino acid residues giving a molecular weight of 10,908 excluding Fe and S atoms. This ferredoxin contained an N6-monomethyllysine residue at position 29 which was determined by a comparison of the elution profile of the acid hydrolysates of the protein and peptides on an amino acid analyzer with three methyl derivatives of lysine and also by field desorption mass spectrometry of a purified peptide. The ferredoxin has only 7 cysteine residues, which probably participate in constructing the Fe-S clusters of this ferredoxin, indicating the presence of a unique chelate structure. Comparison of this ferredoxin with other archaebacterial ferredoxins indicated that the archaebacteria might have multiple origins in an evolutionary tree.     

Amino acid sequence and antimicrobial activity of chitin-binding peptides, Pp-AMP 1 and Pp-AMP 2, from Japanese bamboo shoots (Phyllostachys pubescens)

Two novel chitin-binding peptides, designated Pp-AMP 1 and Pp-AMP 2, which had antimicrobial activity against pathogenic bacteria and fungi, were purified from Japanese bamboo shoots (Phyllostachys pubescens) by a simple procedure based on chitin affinity chromatography. They had the common structural features of the plant defensin family, but they could not be grouped in any type of that family. They showed a high degree of homology to mistletoe toxins.     

Synthetic peptide analogs of DARPP-32 (Mr 32,000 dopamine- and cAMP-regulated phosphoprotein), an inhibitor of protein phosphatase-1. Phosphorylation, dephosphorylation, and inhibitory activity

Synthetic peptides based on the threonine phosphorylation site and proposed inhibitory site of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were prepared and analyzed as substrates for cAMP-dependent protein kinase and protein phosphatases-1c, -2Ac (the catalytic subunits of protein phosphatase-1 and 2A, respectively) and -2B, and as inhibitors of protein phosphatase-1c. Studies of the kinetics of phosphorylation of the peptides by cAMP-dependent protein kinase indicated an important role in facilitating phosphorylation for the region COOH-terminal to the phosphorylatable threonyl residue. Studies of the dephosphorylation of the phosphopeptides demonstrated that they were effectively dephosphorylated by protein phosphatase-2A and -2B and poorly dephosphorylated by protein phosphatase-1. The active inhibitory region of phospho-DARPP-32 was analyzed by determining the effects of synthetic phosphopeptides on the activity of protein phosphatase-1c. Phospho-D32-(8-48) and phospho-D32-(8-38) inhibited protein phosphatase-1c with IC50 values of 2 x 10(-8) and 4 x 10(-8) M, respectively, compared with an IC50 of 8 x 10(-9) M for intact phospho-DARPP-32. Phospho-D32-(9-38) was equipotent with phospho-D32-(8-38); however, further NH2-terminal deletions resulted in marked reductions in IC50 values. An analog of an active DARPP-32 phosphopeptide containing a phosphoseryl residue in place of the phosphothreonyl residue also exhibited a much reduced IC50. These data identify the essential inhibitory region of phospho-DARPP-32 as residues 9-38, which contains the phosphorylation site (Thr34). This region exhibits extensive amino acid sequence identity with phosphatase inhibitor-1, a distinct inhibitor of protein phosphatase-1. Kinetic studies of the inhibition of protein phosphatase-1c by phospho-D32-(9-38), a potent inhibitor, as well as by phospho-D32-(10-38), a weak inhibitor, indicated a mixed competitive/noncompetitive mechanism of inhibition, as has been previously found for both intact phospho-DARPP-32 and intact phospho-inhibitor-1. These findings support the hypothesis that a 30-amino acid domain in the NH2-terminal region of phospho-DARPP-32 is sufficient for the inhibition of protein phosphatase-1.     

Increased alkalotolerant and thermostable ribonuclease (RNase) production from alkaliphilic Streptomyces sp. M49-1 by optimizing the growth conditions using response surface methodology

Total of 171 alkaliphilic actinomycetes were evaluated for extracellular RNase production and Streptomyces sp. M49-1 was selected for further experiments. Fermentation optimization for RNase production was implemented in two steps using response surface methodology with central composite design. In the first step, the effect of independent fermentation variables including temperature, initial pH and process time were investigated. After identification of carbon and nitrogen sources affecting the production by one variable at a time method, concentrations of glucose and yeast extract and also inoculum size were chosen for the second central composite design. A maximum RNase activity was obtained under optimal conditions of 4.14 % glucose concentration, 4.63 % yeast extract concentration, 6.7 × 10⁶ spores as inoculum size for 50 ml medium, 42.9 °C, 91.2 h process time and medium initial pH 9.0. Optimum activity of the enzyme is achieved at pH 11 and temperature 60 °C. The enzyme is highly stable at pH range 9.0–12.0 and at 90 °C after 2 h. Statistical optimization experiments provide 2.25 fold increases in the activity of alkalotolerant and thermostable RNase and shortened the fermentation time compared to that of unoptimized condition. The members of Streptomyces can be promising qualified RNase producer for pharmaceutical industries.     

The highly conserved extracellular peptide, DSYG(893-896), is a critical structure for sodium pump function.

The peptide sequence DSYG(893-896) of the sheep sodium pump alpha 1 subunit is highly conserved among all K(+)-transporting P-type ATPases. To obtain information about its function, single mutations were introduced and the mutants were expressed in yeast and analysed for enzymatic activity, ion recognition, and alpha/beta subunit interactions. Mutants of Ser894 or Tyr895 were all active. Conservative phenylalanine and tryptophan mutants of Tyr895 displayed properties that were similar to the properties of the wild-type enzyme. Replacement of the same amino acid by cysteine, however, produced heat-sensitive enzymes, indicating that the aromatic group contributes to the stability of the enzyme. Mutants of the neighbouring Ser894 recognized K(+) with altered apparent affinities. Thus, the Ser894-->Asp mutant displayed a threefold higher apparent affinity for K(+) (EC(50) = 1.4 +/- 0.06 mm) than the wild-type enzyme (EC(50) = 3.8 +/- 0.33 mm). In contrast, the mutant Ser894-->Ile had an almost sixfold lower apparent affinity for K(+) (EC(50) = 21.95 +/- 1.41 mm). Mutation of Asp893 or Gly896 produced inactive proteins. When an anti-beta 1 subunit immunoglobulin was used to co-immunoprecipitate the alpha 1 subunit, neither the Gly896-->Arg nor the Gly896-->Ile mutant could be visualized by subsequent probing with an anti-alpha 1 subunit immunoglobulin. On the other hand, co-immunoprecipitation was obtained with the inactive Asp893-->Arg and Asp893-->Glu mutants. Thus, it might be that Asp893 is involved in enzyme conformational transitions required for ATP hydrolysis and/or ion translocation. The results obtained here demonstrate the importance of the highly conserved peptide DSYG(893-896) for the function of alpha/beta heterodimeric P-type ATPases.     

Basic peptides in bee venom, II. Synthesis of two pentapeptides from the sequence of the mast-cell-degrading peptide (author's transl)]

The synthesis of two protected pentapeptides is described. The peptides are fragments of the sequence of a mast-cell degranulating peptide from bee venom. The fragments Boc-Lys(Z)-Ile-Cys(SiPr)-Gly-Lys(Z)(I) and Boc-Pro-His(Trt)-Ile-Cys(Trt)-Arg(Tos) (II) were synthesized conventionally. The deprotection of the alpha-amino group by HCl/acetic acid of Boc-Ile-Cys(SiPr)-Gly-Lys(Z) was accompanied by a disulfide exchange at the cysteine residue. After the hydrolysis of fragment II with 6N HCl, allo-isoleucine could be detected by gas chromatography and amino-acid analysis.     

A novel 31-amino-acid-length endothelin, ET-1(1-31), can act as a biologically active peptide for vascular smooth muscle cells

Human chymase produces a novel endothelin-1 with 31 amino-acid length ?ET-1(1-31)?, which is longer than conventional ET-1, ?ET-1(1-21)?. The aim of our study was to investigate the role of ET-1(1-31) on porcine coronary vascular smooth muscle cell (VSMC). Although the increase in [Ca(2+)](i) by ET-1(1-31) was 10 times weaker than that of ET-1(1-21), ET-1(1-31) showed equivalent potency in VSMC proliferation, c-fos/c-myc mRNA expression and cell cycle analysis with ET-1(1-21). ET-1(1-31) significantly induced expression of cyclin D1 but not those of cyclin D2 or D3. These effects were specifically inhibited by BQ485, an ET(A) receptor antagonist, although that of ET-1(1-21) was not specific to BQ485, suggesting different receptor specificity from ET-1(1-21). These results indicate that ET-1(1-31) also can involve a VSMC proliferation process such as atherosclerosis, although it has weaker vasoconstricting potency and different receptor subtypes on VSMC from those of ET-1(1-21).     

Design of novel analogue peptides with potent antibiotic activity based on the antimicrobial peptide,HP (2-20), derived from N-terminus of Helicobacter pylori ribosomal protein L1

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is the antimicrobial sequence derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RPL1). In order to develop novel antibiotic peptides useful as therapeutic agents, potent antibiotic activities against bacteria, fungi and cancer cells without a cytotoxic effect are essential. To this end, several analogues with amino acid substitutions were designed to increase or decrease only the net hydrophobicity. In particular, the substitution of Trp for the hydrophobic amino acids, Gln and Asp at positions 17 and 19 of HP (2-20) (Anal 3), caused a dramatic increase in antibiotic activity without a hemolytic effect.In contrast, the decrease of hydrophobicity brought about by substituting Ser for Leu and Phe at positions 12 and 19 of HP (2-20), respectively (Anal 4, Anal 5), did not have a significant effect on the antibiotic activity. The antibiotic effects of these synthetic peptides were further investigated by treating prepared protoplasts of Candida albicans and conducting an artificial liposomal vesicle (PC/PS; 3:1, w/w) disrupting activity test. The results demonstrated that the Anal 3 prevented the regeneration of fungal cell walls and induced an enhanced release of fluorescent dye (carboxyfluorescein) trapped in the artificial membrane vesicles to a greater degree than HP (2-20).The potassium-release test conducted on C. albicans indicated that Anal 3 induced greater amounts of potassium ion to be released than the parent peptide, HP (2-20) did. These results indicated that the hydrophobic region of peptides is prerequisite for its effective antibiotic activity and may facilitate easy penetration of the lipid bilayers of the cell membrane.     

Multiple isoforms of a protein kinase C inhibitor (KCIP-1/14-3-3) from sheep brain. Amino acid sequence of phosphorylated forms.

A potent inhibitor of protein kinase C (PKC), inhibitor protein-1 (KCIP-1), isolated from sheep brain has been shown to consist of eight isoforms by reverse-phase HPLC. Direct protein sequence analysis has revealed these to be the same as those of 14-3-3 protein, described as an activator of tyrosine and tryptophan hydroxylases involved in neurotransmitter biosynthesis. The N-termini of KCIP-1 isoforms were shown to be acetylated, and secondary structure predictions revealed a high degree of alpha-helix with an amphipathic nature. KCIP-1 showed no inhibitory activity towards protein kinase M (the catalytic fragment of PKC) and had no effect on the activities of three other protein kinases, cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase II and casein kinase 2. Four forms of KCIP-1 were shown to be substrates for PKC in vitro, but none were phosphorylated by the other protein kinases mentioned above.     

Purification and amino acid sequence of vasoactive intestinal peptide, peptide histidine isoleucinamide (1-27) and secretin from the small intestine of guinea pig

The neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucinamide (1-27) (PHI) and the hormone secretin were purified from the small intestine of guinea pig, being detected by radioimmunoassay and radioreceptor assay throughout six to seven chromatographic steps. After elution on a reverse-phase C18 column, the three peptides were separated on a Fractogel column. After cation-exchange chromatography of each peptide on Mono S, the final steps were performed using a reverse-phase RP8-e column. Guinea pig PHI differed from porcine PHI in having Tyr and Arg residues instead of Phe and Lys in, respectively, position 10 and 20. We confirmed the original sequence of guinea pig VIP previously documented (with Leu5, Thr9, Met19 and Val26). We also established the similarity of the primary structure of guinea pig secretin with that of porcine and bovine.     

4-1BB (CD137), an inducible costimulatory receptor,as a specific target for cancer therapy

Although considerable progress has been made in understanding how tumors evade immune surveillance, measures to counter the same have not kept pace with the advances made in designing effective strategies. 4-1BB (CD137; TNFRS9), an activation-induced costimulatory molecule, is an important regulator of immune responses. Targeting 4-1BB or its natural ligand 4-1BB ligand (4-1BBL) has important implications in many clinical conditions, including cancer. In-depth analysis revealed that 4-1BB-mediated anti-cancer effects are based on its ability to induce activation of cytotoxic T lymphocytes (CTL), and among others, high amounts of IFN-γ. In this review, we will discuss the various aspects of 4-1BB-mediated anti-tumor responses, the basis of such responses, and future directions. [BMB Reports 2014; 47(3): 122-129]