Structure‐dependent nonenzymatic deamidation of glutaminyl and asparaginyl pentapeptides

Abstract: Nonenzymatic deamidation rates for 52 glutaminyl and 52 asparaginyl pentapeptides in pH 7.4, 37.0 °C. 0.15 m Tris‐HCl buffer have been determined by direct injection mass spectrometry. These and the previously reported 306 asparginyl rates have been combined in a self‐consistent model for peptide deamidation. This model depends quantitatively upon peptide structure and involves succinimide, glutarimide and hydrolysis mechanisms. The experimental values and suitable interpolated values have been combined to provide deamidation rate values in pH 7.4, 37.0 °C. 0.15 m Tris‐HCl buffer for the entire set of 648 single‐amide permutations of ordinary amino acid residues in GlyXxxAsnYyyGly and GlyXxxGlnYyyGly. Thus, knowledge about sequence‐dependent deamidation in peptides is extended to include very long deamidation half‐times in the range of 2–50 years.     

Mass spectrometric evaluation of synthetic peptides as primary structure models for peptide and protein deamidation

Abstract: Solid‐phase peptide synthesis and deamidation measurements using a novel mass spectrometric technique were carried out for 94 model asparaginyl peptides from 3 to 13 residues in length. Deamidation rates of these peptides in pH 7.4, 37.0°C, 0.15 m Tris?HCl buffer were measured and evaluated. It was found that they validate the use of pentapeptide models as surrogates for the primary sequence dependence of peptide and protein deamidation rates and the discovery by difference of secondary, tertiary and quaternary structure effects. Deamidation of the pentapeptide models, compared with that of longer peptides of more intricate structure, is discussed, and the application of this technique to deamidation measurement of intact proteins is demonstrated.     

Efficiency of cell-penetrating peptides on the nasal and intestinal absorption of therapeutic peptides and proteins

The purpose of our study was to investigate the potential of cell-penetrating peptides; penetratin as novel delivery vector, on the systemic absorption of therapeutic peptides and proteins across different mucosal administration sites. The absorption-enhancing feasibility of l- and d-penetratin (0.5 mM) was used for glucagon-like peptide-1 (GLP-1), and exendin-4 as novel antidiabetic therapy, in addition to interferon-β (IFN-β) as protein biotherapeutic model from nasal and intestinal route of administration was evaluated as first time in rats. Nasal route is the most feasible for the delivery of therapeutic peptides coadministered with penetratin whereas the intestinal route appears to be more restricted. The absolute bioavailability (BA (%)) values depend on the physichochemical characters of drugs, stereoisomer character of penetratin, and site of administration. Penetratin significantly increased the nasal more than intestinal absorption of GLP-1 and exendin-4, as the BA for nasal and intestinal administration of GLP-1 was 15.9% and 5%, and for exendin-4 were 7.7% and 1.8%, respectively. Moreover, the BA of IFN-β coadministered with penetratin was 11.1% and 0.17% for nasal and intestinal administration, respectively. From these findings, penetratin is a promising carrier for transmucosal delivery of therapeutic peptides and macromolecules as an alternative to conventional parenteral routes.     

Effect of adjacent histidine and cysteine residues on the spontaneous degradation of asparaginyl- and aspartyl-containing peptides

Aspartate and asparagine residues in polypeptides are subject to nonenzymatic reactions that lead to deamidation, isomerization, peptide bond cleavage and racemization. Much of this reactivity is due to the propensity for the initial formation of a cyclic succinimide intermediate. We have been interested in determining the effect of the side chains of neighboring histidine and cysteine residues in facilitating these reactions, particularly in the possibility that they can act as general acids and bases. In this study, we found little or no effect of histidine residues preceding an asparagine residue in hexapeptides derived from the sequence of adrenocorticotropic hormone, while a histidine residue preceding an aspartic acid residue was found to increase the rate of succinimide formation 8- to 11-fold. The presence of a histidine residue following either an asparagine or aspartic acid residue did not effect the rate of succinimide formation by peptide-bond nitrogen attack, but did increase the rate of the competing side-chain nitrogen attack leading to cleavage in the asparaginyl-containing peptide. We found that the effect of a cysteine residue following an asparagine or aspartic acid residue was in general similar to that of a serine residue, although the cleavage reaction appeared to be enhanced. These results suggest that His-Asp sequences may be particularly labile to spontaneous degradation in proteins and peptides, possibly owing to the ability of the histidine residue to facilitate succinimide formation by protonating the OH^? leaving group on the side chain carboxylic acid of the aspartic acid residue. Finally, we have also utilized these results, along with previously accumulated data on succinimide formation in related peptides, to correlate the rate of succinimide formation with the predicted acidity of the peptide bond nitrogen atom that is involved in the initial nucleophilic attack. © Munksgaard 1995.     

Efficacy of the phosphorylation of synthetic peptides by purified catalytic subunit of PKA (PKAcat) from bovine lens depends on the amino acid sequence of the peptides

Abstract: Protein kinase (PK) A catalytic (PKAcat) subunit was purified to homogeneity from bovine lens using a 100‐kDa cut‐off membrane filtration followed by different chromatographic procedures. The molecular weight of PKAcat was found to be 41 kDa. The kinase phosphorylates histone IIIs and other synthetic modified peptides of VRKRTLRRL with different amino acid environment. The extent of phosphorylation depends not only on the presence of Ser or Thr (phosphorylating residues) but also on other surrounding amino acid residues. Although some peptides compete in phosphorylating histone, they are not very significant. The result suggests that the extent of phosphorylation depends on the amino acid residue(s) surrounding phosphorylable residue(s) on the peptide.     

The application of polysaccharide-based nanogels in peptides/proteins and anticancer drugs delivery

Finding adequate carriers for proteins/peptides and anticancer drugs delivery has become an urgent need, owing to the growing number of therapeutic macromolecules and the increasing amount of cancer incidence. Polysaccharide-based nanogels have attracted interest as carriers for proteins/peptides and anticancer drugs because of their characteristic properties like biodegradability, biocompatibility, stimuli-responsive behaviour, softness and swelling to help achieve a controlled, triggered response at the target site. In addition, the groups of the polysaccharide backbone are able to be modified to develop functional nanogels. Some polysaccharides have the intrinsic ability to recognise specific cell types, allowing the design of targeted drug delivery systems through receptor-mediated endocytosis. This review is aimed at describing and exploring the potential of polysaccharides that are used in nanogels which can help to deliver proteins/peptides and anticancer drugs.     

Synthesis of bis‐ and tris‐branched COOH‐terminal pegylating reagents: conjugation to NH2‐terminal peptides

Abstract: In previous studies we reported an orthogonal protection scheme that was developed for the solution‐phase synthesis of a family of bis‐ and tris‐pegylating reagents which contain a free NH₂‐terminus. These pegylating reagents were coupled to the COOH‐terminus of a model peptide. In the present study we report on the solution synthesis of a novel family of bis‐ and tris‐pegylating reagents which contain a free COOH‐terminus. To illustrate their general utility, conditions were developed for the coupling of these novel pegylating reagents to the NH₂‐function of a model pentapeptide. Taken together, our studies demonstrate that these pegylating reagents are well suited for conjugation to peptides and proteins that contain either free COOH‐ or NH₂‐functions. These reagents may have general utility in therapeutic development as branched pegylation has been shown to provide more effective protection of proteins from proteolysis by shielding the protein surface from approaching macromolecules.     

Isolation and identification of C-terminal peptides of proteins

A method for the isolation and identification of C-terminal peptides of proteins has been developed. The procedure entails the racemization of the C-terminal amino acid by reaction of the N-trifluoroacetylated protein with acetic anhydride and pyridine. After deprotection, the protein is fragmented to yield a mixture of peptides, which in turn are digested with carboxypeptidases. All peptides are hydrolyzed to L-amino acids except the C-terminal peptide with the terminal D-amino-acid residue. It is resistant to the action of carboxypeptidases and is readily identified by peptide mapping. © Munksgaard 1995.     

应用A549细胞单层模型研究蛋白多肽类药物肺部吸收的特性应用A549细胞单层模型研究蛋白多肽类药物肺部吸收的特性

药物通过呼吸进行吸收的路径依次为气管→支气管→肺泡,其中通过肺泡上皮细胞吸收占90％以上。由于肺泡上皮细胞使大分子的蛋白多肽类药物不易通过,是它们渗透的主要屏障,因此对肺泡膜屏障特性、药物透过过程及渗透特性的研究有助于更     

A review of approaches to 18F radiolabelling affinity peptides and proteins

Affinity peptide and protein‐ (APP) based radiotracers are an increasingly popular class of radiotracer in positron emission tomography (PET), which was once dominated by the use of small molecule radiotracers. Radiolabelled monoclonal antibodies (mAbs) are important examples of APPs, yet a preference for smaller APPs, which exhibit fast pharmacokinetics and permit rapid PET aided diagnosis, has become apparent. ¹⁸F exhibits favourable physical characteristics for APP radiolabelling and has been described as an ideal PET radionuclide. Notwithstanding, ¹⁸F radiolabelling of APP is challenging, and this is echoed in the literature where a number of diverse approaches have been adopted. This review seeks to assess and compare the approaches taken to ¹⁸F APP radiolabelling with the intention of highlighting trends within this expanding field. Generic themes have emerged in the literature, namely the use of mild radiolabelling conditions, a preference of site‐specific methodologies with an impetus for short, automated procedures which produce high‐yielding [¹⁸F]APPs.     

Evidence for the steric proximity of Tyr 174 and Lys 111 in bovine growth hormone

Bovine growth hormone was modified by reaction with 1,5-difluoro-2,4-dinitro-benzene under conditions favouring production of intramolecularly crosslinked derivatives from monomeric molecules. The monomeric fraction, isolated by chromatography on Sephadex G-100, was oxidized or reduced and carbamido-methylated and trypsin digested. The resulting peptides were fractionated on SP-Sephadex and further purified by peptide mapping or HPLC. Two modified peptides containing sequences 108–112 or 108–113 and 171–176 of bGH were obtained, including a dinitrophenylene bridge between lysine 111 and tyrosine 174, thus suggesting the stereochemical proximity of these residues.     

Delivery of peptides and proteins through the blood-brain barrier

Peptide and protein therapeutics are generally excluded from transport from blood to brain, owing to the negligible permeability to these drugs of the brain capillary endothelial wall, which makes up the blood—brain barrier (BBB) in vivo. However, peptides or protein therapeutics may be delivered to brain with the use of the chimeric peptide strategy for peptide drug delivery. Chimeric peptides are formed when a non-transportable peptide therapeutic is coupled to a BBB drug transport vector. Transport vectors are proteins such as cationized albumin, or the OX26 monoclonal antibody to the transferrin receptor; these proteins undergo absorptive-mediated and receptor-mediated transcytosis through the BBB, respectively. In addition to vector development, another important element of the chimeric peptide strategy is the design of strategies for coupling drugs to the vector that give high efficiency coupling and result in the liberation of biologically active peptide following cleavage of the bond linking the therapeutic and the transport vector. The avidin/biotin system has been recently shown to be advantageous in fulfilling these criteria for successful linker strategies. The use of the OX26 monoclonal antibody, the use of the avidin/biotin system as a linker strategy, and the design of a vasoactive intestinal peptide (VIP) analogue that is suitable for monobiotinylation and retention of biologic activity following cleavage, allowed for the recent demonstration of in vivo pharmacologic effects in brain following the systemic administration of relatively low doses (12 μg/kg) of neuropeptide.     

Synthesis and reactivity of 6,7-dihydrogeranylazides: reagents for primary azide incorporation into peptides and subsequent staudinger ligation

Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranylazide moiety to a peptide substrate, N-dansyl-GCVIA. Because geranylazide is actually a mixture of isomeric, interconverting primary and secondary azides, incorporation of this isoprenoid into peptides can potentially result in a corresponding mixture of prenylated peptides. Here, we first examined the reactivity of geranyl azide in a model Staudinger reaction and determined that a mixture of products is formed. We then describe the synthesis of 6,7-dihydrogeranylazide diphosphate and demonstrate that this compound allows exclusive incorporation of a primary azide into a peptide. The resulting azide-containing peptide was derivatized with a triphenylphosphine-based reagent to generate an O-alkyl imidate-linked product. Finally, we show, using a series of model reactions, that the Staudinger ligation frequently produces small amounts of O-alkyl imidate products in addition to the major amide-linked products. Thus, the alkoxyimidates we have observed as the exclusive products in the reactions of peptides containing prenylated azides also appear to be a common type of product formed using other azide-containing reactants, although at greatly reduced levels. This method for chemical modification of the C-terminus of a protein should be useful for a variety of applications in protein chemistry.     

Switching cell penetrating and CXCR4-binding activities of nanoscale-organized arginine-rich peptides

Arginine-rich protein motifs have been described as potent cell-penetrating peptides (CPPs) but also as rather specific ligands of the cell surface chemokine receptor CXCR4, involved in the infection by the human immunodeficiency virus (HIV). Polyarginines are commonly used to functionalize nanoscale vehicles for gene therapy and drug delivery, aimed to enhance cell penetrability of the therapeutic cargo. However, under which conditions these peptides do act as either unspecific or specific ligands is unknown. We have here explored the cell penetrability of differently charged polyarginines in two alternative presentations, namely as unassembled fusion proteins or assembled in multimeric protein nanoparticles. By this, we have observed that arginine-rich peptides switch between receptor-mediated and receptor-independent mechanisms of cell penetration. The relative weight of these activities is determined by the electrostatic charge of the construct and the oligomerization status of the nanoscale material, both regulatable by conventional protein engineering approaches.     

梭曼单链抗体酶EP_6一级结构的确定和三维结构的模建

以梭曼水解反应过渡态类似物 O1-甲基 - O2 -(1 ,2 ,2 -三甲基丙基 ) - 2 -羟基 - 5-硝基苯基甲基膦酸为半抗原得到了梭曼单链抗体酶 EP6.对这一株抗体进行了核苷酸序列的测定 ,测序结果表明这一单链抗体基因全长 71 4bp,其中重链长 351 bp,编码1 1 7个氨基酸 ,轻链长 31 8bp,编码 1 0 6个氨基酸 ,二者由编码 (Gly4 Ser) 3的 45bp的连接肽基因相连 .重 ,轻链均为开放读框 ,有明确的四个框架区和三个互补决定区 (CDR)以及抗体特征性的两个半胱氨酸残基 ,表明重 ,轻链的氨基酸序列符合鼠抗体可变区特征 .在 SGI工作站上对 EP6的三维结构进行了同源模建 .计算机模型显示 EP6表面包含一个由重轻链 CDRs围成的 ,可能用于半抗原结合和催化活性的长裂缝 ,其中轻链的 CDR3(L- CDR3)和重链三个 CDRs(H- CDRs)都可能参与抗原或半抗原的接触 ,L- CDR3和 H- CDR2的贡献较大 ,对抗体酶功能可能起重要作用     

Conformational analysis and aqueous hydration studies of model peptides for the adhesive protein of the mussel,Mytilus edulis L

Conformations of model peptides of the adhesive protein of the mussel, Mytilus edulis L were investigated using molecular mechanics. The protein structure was represented as the repeat of a 10-residue unit. This decamer, and di- and tri-decamers of it, were considered in the modeling. Incorporation of the unusual dopamine residue in the decamer repeat may be explained by its hydrogen bond forming ability via its 3-OH group to a proline carbonyl oxygen. This bond contributes to maintaining a double reverse β-turn structure in the decamer. This conformation was found more stable than 3₁ and α helical conformations. Adjacent reverse β-turn structures are connected by short segments (2 to 3 residues) having little conformational preference. Thus, the overall protein can possess a significant random nature, yet have a highly ordered embedded conformational component. Hydrophilic character is in line with the larger number of OH groups on the phenyl ring for residue 9 (the site of the Dopa residue). The dehydration free energy of the (3-OH)-Phe as compared to the Dopa derivative is less by 1.4kcal per decamer unit. This amounts to more than 100kcal energy gain in the dehydration process for the total protein.     

Design and NMR studies of cyclic peptides targeting the N-terminal domain of the protein tyrosine phosphatase YopH

We report on the design and evaluation of novel cyclic peptides targeting the N-terminal domain of the protein tyrosine phosphatase YopH from Yersinia. Cyclic peptides have been designed based on a short sequence from the protein SKAP-HOM [DE(pY)DDPF (pY=phosphotyrosine)], and they all contain the motif DEZXDPfK (where Z is a phosphotyrosine or a non-hydrolyzable phosphotyrosine mimetic, X is an aspartic acid or a leucine and f is a d-phenylalanine). These peptides present a 'head to tail' architecture, enabling cyclization through formation of an amide bond in between the side chains of the first aspartic acid and the lysine residues. Chemical shift perturbation studies have been carried out to monitor the binding of these peptides to the N-terminal domain of YopH. Peptides containing a phosphotyrosine moiety exhibit binding affinities in the low micromolar range; substitution of the phosphotyrosine with one of its non-hydrolyzable derivatives dramatically reduces the binding affinities. These preliminary studies may pave the way for the discovery of more potent and selective peptide-based ligands of the YopH N-terminal domain which could be further investigated for their ability to inhibit Yersiniae infections.     

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val-Gly-Val-Lys, corresponding to sequence 296–316 of thermolysin and thus encompassing the COOH-termi-nal helical segment 301–312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296–316 was then cleaved with trypsin at Lys³⁰⁷ and Staphylococcus aureus V8 protease at Glu³⁰², producing the additional fragments 296–307, 308–316, 296–302, and 303–316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296–316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301–312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296–316 attains in 90% aqueous trifluoroethanol the same percentage (～58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296–316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule     

The effects of peptides on tolerance to the cataleptic and hypothermic effects of morphine in the rat

The effects of melanotropin release inhibiting factor (MIF) and a cyclic analog, cycio (Leu-Gly) derived from MIF on the development of tolerance to the cataleptic and hypothermic effects of morphine in the rat were investigated. The rats were made tolerant to morphine by the subcutaneous implantation of four morphine pellets over a 3-day period. Each pellet contained 75 mg of morphine-free base. Following morphine pellet implantation, tolerance developed to the cataleptic and hypothermic effects of morphine. A dose of morphine (50 mg/kg) which produced hypothermia in placebo-pelleted rats, produced hyperthermia in morphine-pelleted rats. Administration of MIF and cycio (Leu-Gly) prior to and during morphine pellet implantation inhibited the development of tolerance to the cataleptic effect of morphine. Similarly the hyperthermic effect of morphine in morphine-tolerant rats was prevented by both the peptides. Previously, the present authors reported Ithat these peptides also inhibited tolerance to the analgesic effect of morphine. It is concluded that tolerance to analgesic, cataleptic and hypothermic effects of morphine may share common features in its genesis.     

Snake venom disintegrins: evolution of structure and function

Disintegrins represent a family of polypeptides present in the venoms of various vipers that selectively block the function of integrin receptors. Here, we review our current view and hypothesis on the emergence and the structural and functional diversification of disintegrins by accelerated evolution and the selective loss of disulfide bonds of duplicated genes. Research on disintegrins is relevant for understanding the biology of viper venom toxins, but also provides information on new structural determinants involved in integrin recognition that may be useful in basic and clinical research. The role of the composition, conformation, and dynamics of the integrin inhibitory loop acting in concert with the C-terminal tail in determining the selective inhibition of integrin receptors is discussed.