РАЗРАБОТКА ПЕПТИДА, УСТОЙЧИВОГО К ПРОТЕОЛИЗУ, С ПОСЛЕДУЮЩИМ СИНТЕЗОМ ПЛАЗМИДЫ, КОДИРУЮЩЕЙ ЕГО ПОСЛЕДОВАТЕЛЬНОСТЬ ДЛЯ ЭКСПРЕССИИ В БИООБЪЕКТЕ

Thrombosis is a major cause of mortality from cardiovascular disease (CVD). The use of known antithrombin drugs is limited by the presence of side effects and complications. Peptides may be promising antithrombin agents. A peptide having the amino acid sequence QLSNGLFLFVDYLWW, designated as QW-13, was designed, synthesized, and used as a research object to evaluate the efficiency of the algorithm. The solid-phase Fmoc (SPPS) method, followed by purification by high-performance liquid chromatography (HPLC) was used for synthesis. The molecular weight distribution of the peptide was estimated by mass spectrometry. Peptide identification was performed using the MALDI-TOF MS Ultraflex method. Mass spectra were analyzed using the Mascot program. To confirm the efficiency of the algorithm, the antithrombin effect of the peptide was studied, which was evaluated by the activated partial thromboplastin time (APTT) of human citrate blood plasma coagulation. A decision tree algorithm was developed to predict antithrombin activity and bioavailability of oral peptides. The top-down inductions of decision tree algorithms were used to create the algorithm. The decision tree is pruned if there is a mismatch in the peptide under study. Algorithm criteria or descriptors include amino acid sequence, number of amino acids, molecular weight, clinical potency, body distribution and metabolism index, plasma clearance, and half-life. According to the algorithm, if the result of "Antithrombin Peptide" is positive, it is concluded that it is bioavailable and effective for clinical use. To validate and evaluate the efficiency of the algorithm, a peptide containing antithrombin amino acid sequences was synthesized. The algorithm found the peptide to be antithrombin and bioavailable. The results of the biological activity of the peptide were confirmed in an in vitro experiment. Employing a decision tree method to assess the antithrombin activity and bioavailability of peptides can facilitate the development of effective oral peptides, hence minimizing the development time and the quantity of in vitro and in vivo studies required to validate efficacy.

Recombinant peptides targeted to the radiation-inducible response in tumor microvasculature

Recombinant peptides targeted to the radiation-inducible response in tumor microvasculature

Unlocking the bioactivity of meat proteins: Comparison of meat and meat hydrolysate via simulated gastrointestinal digestion

Mannan-binding protein (MBP) is a C-type serum lectin that is an important constituent of the innate immune defense because it activates the complement system via the lectin pathway. While the pig has been proposed to be an attractive source of xenotransplantable tissues and organs, little is known about porcine MBP. In our previous studies, phosphomannan, but not mannan, was found to be an effective inhibitor of the C1q-independent bactericidal activity of newborn piglet serum against some rough strains of Gram-negative bacteria. In contrast, the inhibitory activities of phosphomannan and mannan were very similar in the case of MBP-dependent bactericidal activity against rough strains of Escherichia coli K-12 and S-16. Based on these findings, we inferred that an MBP-like lectin with slightly or completely different carbohydrate binding specificity might exist in newborn piglet serum and be responsible for the C1q-independent bactericidal activity. Herein we report that a novel phosphomannan-binding lectin (PMBL) of 33 kDa under reducing conditions was isolated from both newborn and adult porcine serum and characterized. Porcine PMBL functionally activated the complement system via the lectin pathway triggered by binding with both phosphomannan (P-mannan) and mannan, which, unlike MBP, was effectively inhibited by mannose 6-phosphate- or galatose-containing oligosaccharides. Our observations suggest that porcine PMBL plays a critical role in the innate immune defense from the newborn stage to adult-hood, and the establishment of a newborn piglet experimental model for the innate immune system studies is a valuable step toward elucidation of the physiological function and molecular mechanism of lectin pathway.

Cyclic peptides are a promising class of therapeutics due to their attractive drug qualities such as increased structural stability, cell permeability, and resistance to proteolytic degradation. With recent advancements in cyclic peptide backbone generation models like CyclicCAE and RFPeptide, generating cyclic peptide backbones can be done more rapidly compared to traditional algorithm or physics based approaches. However, designing energetically favorable cyclic peptide sequences to fit generated backbones using only canonical amino acids is nontrivial. We fine-tuned the state-of-the-art deep learning model for protein sequence design, ProteinMPNN, using a combination of X-ray crystal structures from the Protein Data Bank and in silico generated cyclic peptides. Our approach surpasses ProteinMPNN in cyclic peptide sequence design, producing energetically stable sequences with a higher success rate of folding into the generated cyclic peptide backbones. We show that CyclicMPNN can be used as a motif-inpainting strategy and in de novo sequence design tasks. We propose that CyclicMPNN will enable the rapid design of energetically stable cyclic peptide sequences, increasing the success rate of therapeutic cyclic peptide development.

Identification of an unknown substance without any information remains a daunting challenge despite advances in chemistry and mass spectrometry. However, an unknown cyclic peptide in a sample with very limited volume seized at a Pennsylvania racetrack has been successfully identified. The unknown sample was determined by accurate mass measurements to contain a small unknown peptide as the major component. Collision-induced dissociation (CID) of the unknown peptide revealed the presence of Lys (not Gln, by accurate mass), Phe, and Arg residues, and absence of any y-type product ion. The latter, together with the tryptic digestion results of the unusual deamidation and absence of any tryptic cleavage, suggests a cyclic structure for the peptide. Electron-transfer dissociation (ETD) of the unknown peptide indicated the presence of Gln (not Lys, by the unusual deamidation), Phe, and Arg residues and their connectivity. After all the results were pieced together, a cyclic tetrapeptide, cyclo[Arg-Lys-N(C(6)H(9))Gln-Phe], is proposed for the unknown peptide. Observations of different amino acid residues from CID and ETD experiments for the peptide were interpreted by a fragmentation pathway proposed, as was preferential CID loss of a Lys residue from the peptide. ETD was used for the first time in sequencing of a cyclic peptide; product ions resulting from ETD of the peptide identified were categorized into two types and named pseudo-b and pseudo-z ions that are important for sequencing of cyclic peptides. The ETD product ions were interpreted by fragmentation pathways proposed. Additionally, multi-stage CID mass spectrometry cannot provide complete sequence information for cyclic peptides containing adjacent Arg and Lys residues. The identified cyclic peptide has not been documented in the literature, its pharmacological effects are unknown, but it might be a "designer" drug with athletic performance-enhancing effects.

A group of 16-kDa proteins, synthesized and secreted by rat pancreatic acinar cells and composed of pancreatic stone protein (PSP/reg) and isoforms of pancreatitis-associated protein (PAP), show structural homologies, including conserved amino acid sequences, cysteine residues, and highly sensitive N-terminal trypsin cleavage sites, as well as conserved functional responses in conditions of pancreatic stress. Trypsin activation of recombinant stress proteins or counterparts contained in rat pancreatic juice (PSP/reg, PAP I and PAP III) resulted in conversion of 16-kDa soluble proteins into 14-kDa soluble isoforms (pancreatic thread protein and pancreatitis-associated thread protein, respectively) that rapidly polymerize into insoluble sedimenting structures. Activated thread proteins show long lived resistance to a wide spectrum of proteases contained in pancreatic juice, including serine proteases and metalloproteinases. In contrast, PAP II, following activation with trypsin or pancreatic juice, does not form insoluble structures and is rapidly digested by pancreatic proteases. Scanning and transmission electron microscopy indicate that activated thread proteins polymerize into highly organized fibrillar structures with helical configurations. Through bundling, branching, and extension processes, these fibrillar structures form dense matrices that span large topological surfaces. These findings suggest that PSP/reg and PAP I and III isoforms consist of a family of highly regulated soluble secretory stress proteins, which, upon trypsin activation, convert into a family of insoluble helical thread proteins. Dense extracellular matrices, composed of helical thread proteins organized into higher ordered matrix structures, may serve physiological functions within luminal compartments in the exocrine pancreas.

Platelet-activating factor (PAF)-dependent transacetylase (TA) is an enzyme that transfers an acetyl group from PAF to acceptor lipids such as lysophospholipids and sphingosine. This enzyme is distributed in membrane and cytosol of the cells. We previously revealed that TA purified from rat kidney membrane showed an amino acid sequence similarity to that of bovine PAF-acetylhydrolase (AH) (II). In the present study, we purified TA from the rat kidney cytosol and analyzed its amino acid sequence. The amino acid sequence of the cytosolic TA is similar to that of bovine PAF-AH (II) and membrane TA. To clarify the relationship between TA and PAF-AH (II), we isolated cDNA of rat PAF-AH (II). The predicted amino acid sequence of rat PAF-AH (II) from isolated cDNA included all the sequences found in TAs purified from the membrane and cytosolic TAs. In addition, monoclonal antibody to recombinant PAF-AH (II) cross-reacted with both cytosolic and membrane TAs. Consistent with sequence identity, recombinant PAF-AH (II) showed TA activity, whereas recombinant PAF-AH Ib, which is a different subtype of intracellular PAF-AHs, did not possess TA activity. Analysis of a series of site-directed mutant PAF-AH (II) proteins showed that TA activity was decreased, whereas PAF-AH activity was not affected in C120S and G2A mutant proteins. Thus, Cys¹²⁰ and Gly² are implicated in the catalysis of TA reaction in this enzyme. Furthermore, the transfer of acetate from PAF to endogenous acceptor lipids was significantly increased in a time-dependent manner in CHO-K1 cells transfected with PAF-AH (II) gene. These results demonstrate that PAF-AH (II) can function, as a TA in intact cells, and PAF-AH (II) and TA are the same enzyme.

Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, regulates the biological function of various proteins. We identified novel low-abundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing selectively excluded mass screening analysis for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight tandem mass spectrometry, in conjunction with MODi and MODmap algorithm. We observed unexpected mass shifts (Δm=-16, -34, +64, +87, and +103 Da) at redox-active cysteine residue in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of -16, -34, and +64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO2-SH respectively. To determine the plausible pathways to the formation of these products, we prepared model compounds and examined the hydrolysis and hydration of thiosulfonate (Cys-S-SO2-Cys) either to DHA (Δm=-34 Da) or serine along with Cys-SO2-SH (Δm=+64 Da). We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da). These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO2-SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.

Human milk (HM), donor HM, and/or infant formulas (IFs) are the diets of most neonates. Bioactive peptides are known to be formed during gastrointestinal digestion of milk proteins, but the extent to which this occurs is not clear. HM, pasteurized HM (simulating donor HM), standard IF, and hypo‐allergenic IFs were subjected to in vitro digestion, followed by LC‐MS/MS analysis for identifying bioactive peptides. HM, where some peptides were present prior to digestion, released practically the same pattern of bioactive peptides with and without pasteurization, suggesting that provision of donor HM may be similar to breast‐feeding in terms of bioactive peptides, especially anti‐oxidative and ACE‐inhibitory peptides. Sequences of bioactive peptides were different between HM and standard IF, which may result in bioactivities with different intensity. Bioactive peptides formed from digestion of hypo‐allergenic IFs were less diverse than those from standard IF, indicating that industrial hydrolysis of cow’s milk protein for hypo‐allergenicity may impair the ‘encrypted’ bioactivities of the milk proteins. Further investigations in infants are needed to evaluate the bioactivities of the peptides.

This study used four different apricot (Prunus armeniaca) kernels cultivated in Malatya during two consecutive years. The varieties were Hacihaliloglu, Hasanbey, Kabaasi, and Zerdali. The physicochemical properties of the kernels were determined, and the bioactive content of the kernels was evaluated using kernel hydrolysates prepared using trypsin. With regard to the physicochemical properties of the kernels, the dry matter ratio and protein content were the highest in the Hacihaliloglu variety; the ash ratio was the highest in the Kabaasi variety, and the free oil ratio was the highest in the Hasanbey variety. The bioactive compound content changed according to kernel variety. Angiotensin-converting enzyme inhibitors activity was found to be the highest in the Hacihaliloglu and Hasanbey varieties, which had the lowest amygdalin content, and Zerdali had the highest amygdalin content. The antioxidant and antimicrobial effects of the kernels varied, with Hasanbey and Kabaasi generally having the highest content in both analyses. Moreover, a concentration of 20 mg mL-1 of the hydrolysate was determined to have a destructive effect for the microorganisms used in this study. The storage protein of the kernels, except Hacihaliloglu, was found to be Prunin 1, with the longest matching protein chain in the kernels being R.QQQGGQLMANGLEETFCSLRLK.E. The results suggest that the peptide sequences identified in the kernels could have antihypertensive, antioxidative, and Dipeptidyl peptidase IV (DPP-IV)inhibitory effects. Consequently, apricot kernels show potential for use in the production of functional food products. Of the kernels evaluated in this study, Hacihaliloglu and Hasanbey were deemed the most suitable varieties due to their higher bioactive content and lower amygdalin content. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Sulfhydryl oxidase (SOx) from mouse epidermis: molecular cloning, nucleotide sequence, and expression of recombinant protein in the cultured cells

Objective To clone and express the hexon protein of three prevalent human adenovi -rus strains causing respiratory disease and analyze the antigenic characteristics of the recombinant proteins . Methods The full length genes encoding hexon protein of human adenovirus serotype 3（HAdV3）, serotype 4（HAdV4） and serotype 7（HAdV7） were cloned by PCR and sequenced , respectively.The alignment anal-ysis was performed by using hexon gene sequences from GenBank .The major antigenic regions of hexon pro-tein of the three serotypes were expressed in E.coli and purified.The antigenicity, immunogenicity and cross reactivity of the recombinant proteins were determined by ELISA and Western blot assay .Results The full length gene sequence encoding hexon protein of human adenovirus serotype 4 was firstly reported in China , which showed more than 99%homology in both nucleotide and amino acid sequences with the human adeno-virus type 4 NHRC3 strain.The partial hexon protein sequence of HAdV 3, HAdV4 and HAdV7 containing all of the 7 hyperviriable regions （ HVRs） were expressed in E.coli, respectively .The purified recombinant proteins could be recognized by antiserum of the three serotypes of adenovirus .The antiserum samples against the three recombinant proteins could cross-react with particles of the three serotypes of adenovirus . The possible type-and species-specific epitopes were predicted .Conclusion The major antigenic regions of hexon protein of the three serotypes were successfully expressed .The purified recombinant proteins contai-ning both intertypes and type-specific epitopes showed a strong immunogenicity . Key words: Human respiratory adenovirus; Hexon; Recombinant expression; Antigenicity; Epitope

Structural insights into the inhibition of bacterial RecA by naphthalene polysulfonated compounds.