Search

Background: To assess the relationship of chosen salivary proteins and peptides levels with the occurrence of caries in children. Methods: PubMed, MEDLINE and EMBASE databases from 2000 to 2018 were researched for original observational studies published in English. The risk of bias and quality of the included papers were assessed regarding the guidelines by Fowkes and Fulton. Results: Twenty‐two studies were included in the review, from which the issue of glycoproteins (including immunoglobulins), AMPs and salivary enzymes was discussed. The research involved primary dentition (13 papers), as well as mixed (7) and permanent dentition (5). Caries assessment included visual inspection, dmft/s and DMFT/S indexed; quantity of Streptococcus mutans and Lactobacillus spp. bacteria; and caries risk assessment. Discussion: The results of studies regarding the connection between salivary peptides and proteins and caries development in children are promising; however, further investigations should be undertaken. The majority of studies included are case–control and cross‐sectional; however, it is necessary to conduct more cohort studies with adequate follow‐up prior to considering this as markers for caries risk assessment. [ABSTRACT FROM AUTHOR]

Rationale: Protein citrullination (deimination) is a post-translational modification of proteins converting arginine(s) into citrulline(s). "Overcitrullination" could be associated with severe pathological conditions. Mass spectrometric analysis of modified proteins is hindered by several problems. A comprehensive study of the fragmentation of deiminated peptides is not yet available. In this paper we have made an attempt to describe the characteristics of these processes, based on the studies of epitope model oligopeptides derived from clinically relevant proteins. Methods: Solutions of purified model peptides containing either one or two citrulline residues as well as their native variants were injected directly into the electrospray source of a high accuracy and resolution quadrupole-time-of-flight instrument and were analysed by tandem mass spectrometry using low-energy collision-induced dissociation. Results: Loss of isocyanic acid from citrulline residues is a preferred fragmentation route for deiminated peptides, which yields ornithine residues in the sequence. However, simultaneous detection of both the isocyanic acid loss and sequence fragments is often compromised. A preferential cleavage site was observed between citrulline and any other following amino acids yielding intensive complementary b- and y-type ions. Also, citrulline positioned at the C-termini displays a preferential cleavage N-terminal to this residue yielding characteristic y1 ions. These phenomena are described here for the first time and are referred to as the "citrulline effect". Conclusions: We found that the citrulline effect is very pronounced and could be used as a complementary tool for the confirmation of modification sites in addition to losses of isocyanic acids from the protonated molecules or from fragment ions. Low collision energy applied to peptide ions having partially mobile protons reveals the site of modification by generating specific and intensive fragments of the sequence. On the other hand, fragmenting precursor ions with mobile protons usually allows full sequence coverage, although citrulline-specific fragments may exhibit lower intensities compared to other fragments. [ABSTRACT FROM AUTHOR]

The assembly line biosynthesis of the powerful anticancer‐antiviral didemnin cyclic peptides is proposed to follow a prodrug release mechanism in Tristella bacteria. This strategy commences with the formation of N‐terminal prodrug scaffolds and culminates in their cleavage during the cellular export of the mature products. In this study, a comprehensive exploration of the genetic and biochemical aspects of the enzymes responsible for both the assembly and cleavage of the acylated peptide prodrug scaffolds is provided. This process involves the assembly of N‐acyl‐polyglutamine moieties orchestrated by the nonribosomal peptide synthetase DidA and the cleavage of these components at the post‐assembly stage by DidK, a transmembrane CAAX hydrolase homolog. The findings not only shed light on the complex prodrug mechanism that underlies the synthesis and secretion of didemnin compounds but also offer novel insights into the expanded role of CAAX hydrolases in microbes. Furthermore, this knowledge can be leveraged for the strategic design of genome mining approaches aimed at discovering new bioactive natural products that employ similar prodrug biochemical strategies. [ABSTRACT FROM AUTHOR]

Herein we provide a guide for adapting the tools developed for protein X-ray crystallography to study the structures and supramolecular assembly of peptides. Peptide crystallography involves selecting a suitable peptide, crystallizing the peptide, collecting X-ray diffraction data, processing the diffraction data, determining the crystallographic phases and generating an electron density map, building and refining models, and depositing the crystallographic structure in the Protein Data Bank (PDB). Advances in technology make this process easy for a newcomer to adopt. This paper describes techniques for determining the X-ray crystallographic structures of peptides: incorporation of amino acids containing heavy atoms for crystallographic phase determination, commercially available kits to crystallize peptides, modern techniques for X-ray crystallographic data collection, and free user-friendly software for data processing and producing a crystallographic structure. [ABSTRACT FROM AUTHOR]

The oral delivery of peptides and proteins has been hampered by an array of obstacles. However, several promising novel oral delivery systems have been developed. This paper reviews the most advanced oral formulation technologies, and highlights key lessons and implications from studies undertaken to date with these oral formulations. Special interest is given to oral salmon calcitonin ( CT), glucagon-like peptide-1 ( GLP-1), insulin, PYY-(3-36), recombinant human parathyroid hormone ( rhPTH(1-31)- NH2) and PTH(1-34), by different technologies. The issues addressed include (i) interaction with water, (ii) interaction with food, (iii) diurnal variation, (iv) inter- and intra-subject variability, (v) correlation between efficacy and exposure and (vi) key deliverables of different technologies. These key lessons may aid research in the development of other oral formulations. [ABSTRACT FROM AUTHOR]

Background: ADAMTS (a disintegrin-like and metalloproteinase with thrombospondin) are a family of proteinases that are structurally similar to the family of matrix metalloproteinases with critical roles in damage and repair of the extracellular matrix. Their functions are closely related to inflammation, hypoxia, and vascularization. Our aim was to determine levels of ADAMTS-1 in gingival crevicular fluid (GCF) in patients with advanced periodontal diseases and identify their association with hypoxia-inducible factor-1alpha (HIF-1α), vascular endothelial growth factor (VEGF-A), and clinical parameters of periodontitis.Methods: The study consisted of three groups: healthy individuals (control; n = 20), generalized chronic periodontitis (CP; n = 21), and generalized aggressive periodontitis (GAgP; n = 20). Clinical parameters were measured. Levels of ADAMTS-1, VEGF-A, and HIF-1α in GCF and serum were quantified by enzyme-linked immunosorbent assay (ELISA) and reported as total amounts and concentration.Results: ADAMTS-1 total amount in GCF were significantly higher in patients with CP and GAgP compared with healthy individuals (P < 0.05). HIF-1α total amount in GCF were also higher in periodontitis groups compared with the control group (P < 0.05). GCF total VEGF-A content was significantly higher in the GAgP group compared with the CP and the controls (respectively; P = 0.023, P = 0.003). There was a significant correlation between ADAMTS-1, VEGF-A, and HIF-1α levels in the GCF and clinical periodontal parameters (probing depth [PD], bleeding on probing [BOP], and clinical attachment loss (CAL); P < 0.05).Conclusion: ADAMTS-1 may play a role in advanced periodontal disease pathogenesis in correlation with tissue hypoxia and vascularization. [ABSTRACT FROM AUTHOR]

Nanofabrication based on biological templates has attracted considerable interest because of its applications in materials science and biomedicine. Herein, a facile method is reported for the synthesis of well‐defined TiO2 nanofibers by using a simple N‐(9‐fluorenylmethoxycarbonyl)‐protected phenylalanine–phenylalanine–aspartic acid tripeptide (Fmoc‐Phe–Phe–Asp‐OH, Fmoc‐FFD) as the template. Compared with other synthetic methods of inorganic nanomaterials, these self‐assembling peptides could control the structure and the catalytic activity of the synthesized nanomaterials produced under mild synthetic conditions. The as‐synthesized peptide‐TiO2 hybrid nanofibers and the calcined TiO2 nanofibers were characterized by using transmission electron microscopy (TEM), scanning electron microscopy (SEM), circular dichroism (CD) spectroscopy, and X‐ray diffraction (XRD). The results show that well‐defined TiO2 nanofibers could form when the pH of the peptide solution was 5–7, whereas nanoparticles formed when the pH was 8. Moreover, the peptide‐templated TiO2 nanofibers showed improved photocatalytic activity for the degradation of methyl orange (MO). Finally, the photocatalytic activity of the TiO2 could be controlled by adjusting the pH of the peptide solution during the synthetic process. Peptide template: TiO2 nanomaterials with tunable photocatalytic activity were prepared by using N‐(9‐fluorenylmethoxycarbonyl)‐protected phenylalanine—aspartic acid tripeptide (Fmoc‐FFD) as the template. By incorporating titanium diisopropoxide bis(acetyl‐acetonate) into the peptide solutions, the TiO2 precursors could mineralize and condense with the peptide assemblages. After removing the peptide templates by calcination, a series of TiO2 nanostructures with enhanced photocatalytic activity were formed. [ABSTRACT FROM AUTHOR]

Peptide asparaginyl ligases (PALs) are precision tools for peptide cyclization, cell‐surface labelling, protein semisynthesis and protein conjugation. PALs are expressed as inactive proenzymes requiring low pH activation. During activation, a large portion of the cap domain of the proenzyme that covers the substrate binding site is proteolytically removed, exposing the active site to solvent and releasing a population of heterogenous active enzymes. The availability of a readily active ligase not requiring acid activation and subsequent purification of active forms would facilitate manufacturing and streamline applications. Here, we engineered the OaAEP1b‐C247A hyperactive ligase via serial truncations along the linker connecting the cap and core domain of the proenzyme. The recombinant expression of the truncated constructs was carried out in Escherichia coli. Following a solubilization/refolding protocol, one truncated construct termed 'OaAEP1b‐C247A‐∆351' could be overexpressed in the insoluble fraction, purified, and displayed a level of ligase activity comparable to the acid‐activated OaAEP1b‐C247A enzyme. This constitutively active protein can be stored for up to 2 years at −80 °C and readily used for peptide cyclization and protein conjugation. We were able to express and purify a stable constitutively active asparaginyl ligase that can be stored for months without significant activity loss. The removal of the low pH proenzyme activation step eliminates the heterogeneity introduced by this procedure. The yield of purified recombinant active ligase that can be routinely obtained per 100 mL of E. coli cell culture is about 0.9 mg. This recombinant active ligase can be used to carry out protein conjugation. [ABSTRACT FROM AUTHOR]

The application of thiol-yne/thiol-ene reactions to synthesize mono- and bicyclic-stapled peptides and proteins is reported. First, a thiol-ene-based peptide-stapling method in aqueous conditions was developed. This method enabled the efficient stapling of recombinantly expressed coil-coiled proteins. The resulting stapled protein demonstrated higher stability in its secondary structure than the unstapled version. Furthermore, a thiol-yne coupling was performed by using an α,ω-diyne to react with two cysteine residues to synthesize a stapled peptide with two vinyl sulfide groups. The stapled peptide could further react with another biscysteine peptide to yield a bicyclic stapled peptide with enhanced properties. For example, the cell permeability of a stapled peptide was further increased by appending an oligoarginine cell-penetrating peptide. The robustness and versatility of thiol-yne/thiol-ene reactions that can be applied to both synthetic and expressed peptides and proteins were demonstrated. [ABSTRACT FROM AUTHOR]

Stimuli‐responsive peptides and proteins are an exciting class of smart biomaterials for various applications and have received significant attention over the past decades. A wide variety of stimuli such as temperature, pH, ions, enzymes, magnetic field, redox, etc., are explored. This article provides a review of five intensively studied types of stimuli‐responsive peptides and proteins, their design principles and applications, including temperature‐, pH‐, light‐, metal ion‐, and enzyme‐responsive with an emphasis on the key design concepts and switch function. Moreover, typical examples of their applications are discussed to provide a better understanding of the design concept and underlying methodology. This review will facilitate and inspire future innovation toward new peptide‐ and protein‐based materials and their diverse applications. [ABSTRACT FROM AUTHOR]

This work tested the hypothesis that infection causes unexplained production of anti‐centromere protein antibodies (ACA) via autoimmune cross‐reactivity. To further examine the clinical origin of ACA, the overlapped peptides between human pathogens, including viruses, bacteria and fungi and centromere proteins (CENP‐A, CENP‐B and CENP‐C) were assessed. We found a broad overlap of pathogenetic peptides with human centromere proteins. These data indicate potential immune cross‐reactivity between pathogens and human centromere proteins. Additionally, the current findings corroborate a molecular and mechanistic framework for autoimmune disorders related to infection. Moreover, preliminary evidence for a potential role of infection in ACA‐related autoimmune diseases was presented. [ABSTRACT FROM AUTHOR]

Self‐assembled peptides are an emerging family of biomaterials that show great promise for a range of biomedical and biotechnological applications. Introducing and tuning the pH‐responsiveness of the assembly is highly desirable for improving their biological activities. Inspired by proteins with internal ionizable residues, we report a simple but effective approach to constructing pH‐responsive peptide assembly containing unnatural ionic amino acids with an aliphatic tertiary amine side chain. Through a combined experimental and computational investigation, we demonstrate that these residues can be accommodated and stabilized within the internal hydrophobic compartment of the peptide assembly. The hydrophobic microenvironment shifts their pKa significantly from a basic pH typically found for free amines to a more biologically relevant pH in the weakly acidic range. The pH‐induced ionization and ionization‐dependent self‐assembly and disassembly are thoroughly investigated and correlated with the biological activity of the assembly. This new approach has unique advantages in tuning the pH‐responsiveness of self‐assembled peptides across a large pH range in a complex biological environment. We anticipate the ionizable amino acids developed here can be widely applicable to the synthesis and self‐assembly of many amphiphilic peptides with endowed pH‐responsive properties to enhance their biological activities toward applications ranging from targeted therapeutic delivery to proton transport. [ABSTRACT FROM AUTHOR]

We synthesized a lysine-linked auxiliary-derived building block, e-N-2-mercapto-1-(2,4-dimethoxyphenyl)ethyl lysine, in 20% isolated yield over a six-step route. The new building block is well compatible with Fmoc solid-phase peptide synthesis (SPPS) and hydrazide-based native chemical ligation (NCL). Furthermore, we successfully incorporated the new lysine-linked auxiliary handle into a protein segment and performed the chemical synthesis of K48-linked diubiquitin with stepwise NCL. [ABSTRACT FROM AUTHOR]

We synthesized a lysine-linked auxiliary-derived building block, ϵ- N-2-mercapto-1-(2,4-dimethoxyphenyl)ethyl lysine, in 20 % isolated yield over a six-step route. The new building block is well compatible with Fmoc solid-phase peptide synthesis (SPPS) and hydrazide-based native chemical ligation (NCL). Furthermore, we successfully incorporated the new lysine-linked auxiliary handle into a protein segment and performed the chemical synthesis of K48-linked diubiquitin with stepwise NCL. [ABSTRACT FROM AUTHOR]

The inhibition of the G protein-coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41-residue and three-disulfide cross-bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for Nα-deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca2+ ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production. [ABSTRACT FROM AUTHOR]

Background and Purpose: cGMP underpins the bioactivity of NO and natriuretic peptides and is key to cardiovascular homeostasis. cGMP‐driven responses are terminated primarily by PDEs, but cellular efflux via multidrug resistance proteins (MRPs) might contribute. Herein, the effect of pharmacological blockade of MRPs on cGMP signalling in the heart and vasculature was investigated in vitro and in vivo. Experimental Approach Proliferation of human coronary artery smooth muscle cells (hCASMCs), vasorelaxation of murine aorta and reductions in mean arterial BP (MABP) in response to NO donors or natriuretic peptides were determined in the absence and presence of the MRP inhibitor MK571. The ability of MRP inhibition to reverse morphological and contractile deficits in a murine model of pressure overload‐induced heart failure was also explored. Key Results: MK571 attenuated hCASMC growth and enhanced the anti‐proliferative effects of NO and atrial natriuretic peptide (ANP). MRP blockade caused concentration‐dependent relaxations of murine aorta and augmented responses to ANP (and to a lesser extent NO). MK571 did not decrease MABP per se but enhanced the hypotensive actions of ANP and improved structural and functional indices of disease severity in experimental heart failure. These beneficial actions of MRP inhibition were associated with a greater intracellular:extracellular cGMP ratio in vitro and in vivo. Conclusions and Implications: MRP blockade promotes the cardiovascular functions of natriuretic peptides in vitro and in vivo, with more modest effects on NO. MRP inhibition may have therapeutic utility in cardiovascular diseases triggered by dysfunctional cGMP signalling, particularly those associated with altered natriuretic peptide bioactivity. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc [ABSTRACT FROM AUTHOR]

This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited. [ABSTRACT FROM AUTHOR]

Vicinal-sulfydryl-containing peptides/proteins (VSPPs) play a crucial role in human pathologies. Fluorescent probes that are capable of detecting intracellular VSPPs in vivo would be useful tools to explore the mechanisms of some diseases. In this study, by regulating the spatial separation of two maleimide groups in a fluorescent dye to match that of two active cysteine residues contained in the conserved amino acid sequence (-CGPC-) of human thioredoxin, two active-site-matched fluorescent probes, o-Dm-Ac and m-Dm-Ac, were developed for real-time imaging of VSPPs in living cells. As a result, the two probes can rapidly respond to small peptide models and reduced proteins, such as WCGPCK (W-6), WCGGPCK (W-7), and WCGGGPCK (W-8), reduced bovine serum albumin (rBSA), and reduced thioredoxin (rTrx). Moreover, o-Dm-Ac displays a higher binding sensitivity with the above-mentioned peptides and proteins, especially with W-7 and rTrx. Furthermore, o-Dm-Ac was successfully used to rapidly and directly detect VSPPs both in vitro and in living cells. Thus, a novel probe-design strategy was proposed and the synthesized probe applied successfully in imaging of target proteins in situ. [ABSTRACT FROM AUTHOR]

BACKGROUND The pine wilt disease (PWD) caused by Bursaphelenchus xylophilus is a devastating forest disease and its pathogenesis remains unclear. Secreted enzymes and proteins are important pathogenicity determinants and Bx‐FAR‐1 is an important pathogenic protein involved in the interaction between pine and B. xylophilus. However, the function of the Bx‐FAR‐1 protein in monitoring and prevention PWD remains unknown. RESULTS: We found a small peptide of B. xylophilus effector Bx‐FAR‐1 is sufficient for immunosuppression function in Nicotiana benthamiana. Transient expression of Bx‐FAR‐1 in N. benthamiana revealed that nuclear localization is required for its function. The results of the ligand binding test showed that Bx‐FAR‐1 protein had the ability to bind fatty acid and retinol. We demonstrated that Bx‐FAR‐1 targeted to the nuclei of Pinus thunbergii using the polyclonal antibody by immunologic approach. The content of jasmonic acid (JA) was significantly increased in P. thunbergii infected with B. xylophilus when Bx‐FAR‐1 was silenced. We identified an F‐box protein as the host target of Bx‐FAR‐1 by yeast two‐hybrid and co‐immunoprecipitation. Moreover, we found that Pt‐F‐box‐1 was up‐regulated during B. xylophilus infection and the expression of Pt‐F‐box‐1 was increased in Bx‐FAR‐1 double‐stranded RNA (dsRNA)‐treated host pines. CONCLUSION: This study illustrated that Bx‐FAR‐1 might mediate the JA pathway to destroy the immune system of P. thunbergii, indicating that PWN likely secretes effectors to facilitate parasitism and promote infection, which could better reveal the pathogenesis mechanisms of B. xylophilus and would be beneficial for developing disease control strategies. [ABSTRACT FROM AUTHOR]

This work reports a novel chlorooxime mediated modification of native peptides and proteins under physiologic conditions. This method features fast reaction kinetics (apparent k2=306±4 M−1s−1 for GSH) and exquisite selectivity for cysteine residues. This cysteine conjugation reaction can be carried out with just single‐digit micromolar concentrations of the labeling reagent. The conjugates show high stability towards acid, base, and external thiol nucleophiles. A nitrile oxide species generated in situ is likely involved as the key intermediate. Furthermore, a bis‐chlorooxime reagent is synthesized to enable facile Cys‐Cys stapling in native peptides and proteins. This highly efficient cysteine conjugation and stapling was further implemented on bacteriophage to construct chemically modified phage libraries. [ABSTRACT FROM AUTHOR]

Keywords: Circular dichroism; Copper; Cotton effect; Peptides; Prion protein EN Circular dichroism Copper Cotton effect Peptides Prion protein 1 1 1 02/01/22 20220118 NES 220118 B The Front Cover b shows how the Cotton effect of the ligand field transitions in the CD spectra of the 4N Cu(II)-PrP(92-99) complex with G94 I L i A (blue) is reversed by the G94 I D i A substitution (red). Front Cover: Reversible Stereoisomer-Specific Cotton Effect of the Ligand Field Transitions at a Copper(II) Binding Site of the Prion Protein (Eur. J. Inorg. Circular dichroism, Copper, Cotton effect, Peptides, Prion protein. [Extracted from the article]

Summary: Vegetative storage proteins (VSPs) are known to serve as nitrogen reserves in many dicot plants but remain undiscovered in grasses, most widely grown group of crops globally. We identified and characterized a VSP in maize and demonstrated that its overexpression improved drought tolerance. Nitrogen supplementation selectively induced a mesophyll lipoxygenase (ZmLOX6), which was targeted to chloroplasts by a novel N‐terminal transit peptide of 62 amino acids. When ectopically expressed under the control of various tissue‐specific promoters, it accumulated to a fivefold higher level upon expression in the mesophyll cells than the wild‐type plants. Constitutive expression or targeted expression specifically to the bundle sheath cells increased its accumulation by less than twofold. The overexpressed ZmLOX6 was remobilized from the leaves like other major proteins during grain development. Evaluated in the field over locations and years, transgenic hybrids overexpressing ZmLOX6 in the mesophyll cells significantly outyielded nontransgenic sibs under managed drought stress imposed at flowering. Additional storage of nitrogen as a VSP in maize leaves ameliorated the effect of drought on grain yield. [ABSTRACT FROM AUTHOR]

The aldehyde tag is appropriate to selectively label proteins, prepare antibody‐drug conjugates or to immobilize enzymes or antibodies for biotechnological and medical applications. The cysteine within the consensus sequence CxPxR of the aldehyde tag is specifically oxidized by the formylglycine‐generating enzyme (FGE) to the non‐canonical and electrophilic amino acid Cα‐formylglycine (FGly). Subsequent reductive amination is a common method for site‐directed immobilization, which usually results in poor immobilization efficiency due to the reaction conditions. Here, we introduce a new solid support like agarose modified with an aryl substituted pyrazolone (Knoevenagel reagent) that was obtained in a facile and efficient 2‐step synthesis. The modified agarose allowed the site‐selective and efficient immobilization of aldehyde‐containing small molecules, peptides and proteins – in particular enzymes – at physiological pH (6.2–8.2) without any additive or catalyst needed. In comparison to reductive amination, higher loadings and activities were achieved in various buffers at different concentrations and temperatures. [ABSTRACT FROM AUTHOR]

The C‐terminal domain (CTD) of RNA polymerase II serves as a binding platform for numerous enzymes and transcription factors involved in nascent RNA processing and the transcription cycle. The S2, S5‐phosphorylated CTD is recognized by the transcription factor SCAF4, which functions as a transcription anti‐terminator by preventing early mRNA transcript cleavage and polyadenylation. Here, we measured the binding affinities of differently modified CTD peptides by hSCAF4 and solved the complex structure of the hSCAF4‐CTD‐interaction domain (CID) bound to a S2, S5‐quadra‐phosphorylated CTD peptide. Our results revealed that the S2, S5‐quadra‐phosphorylated CTD peptide adopts a trans conformation and is located in a positively charged binding groove of hSCAF4‐CID. Although hSCAF4‐CID has almost the same binding pattern to the CTD as other CID‐containing proteins, it preferentially binds to the S2, S5‐phosphorylated CTD. Our findings provide insight into the regulatory mechanism of hSCAF4 in transcription termination. [ABSTRACT FROM AUTHOR]

Linear or branched 1,3‐diketone‐linked thymidine 5′‐O‐mono‐ and triphosphate were synthesized through CuAAC click reaction of diketone‐alkynes with 5‐azidomethyl‐dUMP or ‐dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5‐dioxohexyl group (HDO) efficiently reacted with arginine‐containing peptides to form stable pyrimidine‐linked conjugates, whereas the branched 2‐acetyl‐3‐oxo‐butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross‐linking with Arg‐containing peptides or proteins (e.g. histones). [ABSTRACT FROM AUTHOR]

Cytosolic protein delivery techniques are of great importance for cell biology, biotechnology and protein drug development. The design of carriers with robust efficiency in cytosolic protein delivery is challenging. This account provides a progress report of polymeric carriers for this purpose in our group. During the past years, we have developed several types of functionalized polymers for cytosolic protein and peptide delivery by engineering polymers with ligands such as guanidinium, boronate, coordination ligands and fluoroalkyls. The designed polymers showed improved protein/peptide binding affinities, and successfully delivered various cargo proteins into the cytosol of living cells, while maintaining their bioactivity. In addition, the polymers showed potent efficiencies in the delivery of tumor antigens, therapeutic peptides, toxins and antioxidant proteins in vivo. We hope these polymers could be translated for protein delivery in the treatment of various diseases in the future. What is the most favorite and original chemistry developed in your research group? Fluorinated polymers for cytosolic biomolecule delivery. How do you get into this specific field? Could you please share some experiences with our readers? I get into this field by unexpected discoveries when designing polymeric gene delivery systems. Usually, modification of polymers with hydrophobic ligands yields more efficient gene carriers. Fluoroalkyl ligands were proposed as one type of hydrophobic candidates in our experiments. Interestingly, the fluorous ligand modified polymers showed unprecedented efficiency in gene delivery. More importantly, the polymers exhibited excellent serum resistance and could achieve high efficiency at extremely low polymer dose. These unexpected discoveries motivated us to investigate the fluorine effect of fluorinated materials in gene, protein and peptide delivery. [ABSTRACT FROM AUTHOR]

Objective: To perform a comparative analysis of saliva protein profile of patients with early childhood caries at different levels of severity and caries‐free individuals. Materials and methods: Stimulated saliva samples were collected from 126 children (2–6 years old), classified according to the ICDAS II, and divided into 3 groups (n = 42): caries‐free (CF), enamel caries (EC), and dentine caries (DC). Samples were digested and analyzed by nanoUPLC coupled with a mass spectrometry. Data analyses were conducted with Progenesis QI for Proteomics Software v2.0. Gene Ontology (GO) terms and protein–protein interaction analysis were obtained. Results: A total of 306 proteins (≈6 peptides) were identified. Among them, 122 were differentially expressed in comparisons among children with different caries status. Out of the 122 proteins, the proteins E2AK4 and SH3L2 were exclusively present in groups CF and EC, respectively, and 8 proteins (HAUS4, CAH1, IL36A, IL36G, AIMP1, KLHL8, KLH13, and SAA1) were considered caries‐related proteins when compared to caries‐free children; they were up‐regulated proteins in the caries groups (EC and DC). Conclusion: The identification of exclusive proteins for caries‐free or carious‐related conditions may help in understanding the mechanisms of caries and predicting risk as well as advancing in caries control or anti‐caries approaches. [ABSTRACT FROM AUTHOR]

Summary: The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural 'green' revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. Azotobacter vinelandii nitrogen fixation genes, nifH, M, U and S, were re‐designed for protein accumulation in tobacco cells. Targeting to the chloroplast was optimized by screening and identifying minimal length transit peptides performing properly for each specific Nif protein. Putative peptidyl‐prolyl cis‐trans isomerase NifM proved necessary for NifH solubility in the stroma. Purified NifU, a protein involved in the biogenesis of NifH [4Fe‐4S] cluster, was found functional in NifH reconstitution assays. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of nifU and nifS co‐expression. These results support the suitability of chloroplasts to host functional nitrogenase proteins, paving the way for future studies in the engineering of nitrogen fixation in higher plant plastids and describing an optimization pipeline that could also be used in other organisms and in the engineering of new metabolic pathways in plastids. [ABSTRACT FROM AUTHOR]

Cover Feature: Reductant-Induced Free Radical Fluoroalkylation of Nitrogen Heterocycles and Innate Aromatic Amino Acid Residues in Peptides and Proteins (Chem. Eur. J. 69/2019) The follow-up click reaction by using the pink reagent carrying a payload delivers the conjugate of choice. Realistic depiction of hypervalent iodine reagents includes the T-shape geometry on the iodine atom and the azido-containing fluoroalkyl group. [Extracted from the article]

Aim: The appendix testis (AT) is a vestigial remnant of Müller's paramesonephric duct. Insulin-like 3 hormone (INSL3) is produced in the Leydig cells of the testis. We investigated the possible correlation between AT length and plasma INSL3 concentrations in patients with congenital cryptorchidism (CCO) and patients with hydrocele, who served as controls.Methods: A total of 40 patients with CCO and 34 patients with hydrocele and orthotopic testes were investigated. Sixteen patients presented high cryptorchidism and 24 low cryptorchidism. During surgery, AT was identified in 34 patients with CCO (high cryptorchidism:15, low cryptorchidism:19) and 28 controls. Plasma INSL3 levels were measured with a spectrophotometry enzyme immunoassay Elisa sandwich technique.Results: AT was present in 85.0% of the boys with CCO and 82.4% of the controls. A significant positive correlation was found between the AT length and INSL3 concentrations in CCO patients.Conclusions: A longer AT may reflect better testicular function in boys with CCO, since it is correlated with higher INSL3 concentrations. [ABSTRACT FROM AUTHOR]

The Ras superfamily of small GTPases are guanine‐nucleotide‐dependent switches essential for numerous cellular processes. Mutations or dysregulation of these proteins are associated with many diseases, but unsuccessful attempts to target the small GTPases directly have resulted in them being classed as "undruggable". The GTP‐dependent signaling of these proteins is controlled by their regulators; guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and in the Rho and Rab subfamilies, guanine nucleotide dissociation inhibitors (GDIs). This review covers the recent small molecule and biologics strategies to target the small GTPases through their regulators. It seeks to critically re‐evaluate recent chemical biology practice, such as the presence of PAINs motifs and the cell‐based readout using compounds that are weakly potent or of unknown specificity. It highlights the vast scope of potential approaches for targeting the small GTPases in the future through their regulatory proteins. [ABSTRACT FROM AUTHOR]

Long non‐coding RNAs have been implicated in the regulation of a plethora of biological processes, yet it has been challenging to verify that they are truly not coding for proteins. Terminal differentiation‐induced non‐coding RNA (TINCR) is a 3.7‐kilobase mRNA that is highly abundant in epidermal keratinocytes prior to cornification. Here, we report the presence of an evolutionarily conserved open reading frame in TINCR and the identification of peptides derived from this open reading frame in the proteome of human stratum corneum. Our results demonstrate that TINCR is a protein‐coding RNA and suggest that the TINCR‐encoded protein is involved in keratinocyte cornification. [ABSTRACT FROM AUTHOR]

Quinoa protein has been paid more and more attention because of its nutritional properties and beneficial effects. With the development of bioinformatics, bioactive peptide database and computer‐assisted simulation provide an efficient and time‐saving method for the theoretical estimation of potential bioactivities of protein. Therefore, the potential of quinoa protein sequences for releasing bioactive peptides was evaluated using the BIOPEP database, which revealed that quinoa protein, especially globulin, is a potential source of peptides with dipeptidyl peptidase‐IV (DPP‐IV) and angiotensin‐I‐converting enzyme (ACE) inhibitory activities. Three plant proteases, namely papain, ficin, and stem bromelain, were employed for the in silico proteolysis of quinoa protein. Furthermore, four tripeptides (MAF, NMF, HPF, and MCG) were screened as novel promising bioactive peptides by PeptideRanker. The bioactivities of selected peptides were confirmed using chemical synthesis and in vitro assay. The present work suggests that quinoa protein can serve as a good source of bioactive peptides, and in silico approach can provide theoretical assistance for investigation and production of functional peptides. [ABSTRACT FROM AUTHOR]

The advent of bioorthogonal chemistry has revolutionized the common practices in protein bioconjugation and contributed to a large extent to the development of chemical biology, a discipline aimed at studying biological/biochemical processes/events in their natural setting (living cells, whole organisms) using dedicated chemical tools. This minireview intends to provide an up‐to‐date overview on the various bioorthogonal strategies implemented for the conjugation of transition organometallic entities to peptides, peptide nucleic acids and proteins with a focus on targeted applications, i.e. fluorescence‐ or radio‐labeling for imaging, controlled delivery of therapeutic agents and bioanalysis. [ABSTRACT FROM AUTHOR]

A simple and efficient strategy for the selective modification of the peptide N terminus with an unnatural amino acid is described. A peptide having a SUMO‐HisTag‐TEV sequence (SUMO: small ubiquitin‐related modifier, TEV: tobacco etch virus) preceding the N terminus of the target peptide was designed. Recombinant expression in E. coli and subsequent SUMO protease cleavage yielded the HisTag‐TEV‐target peptide. Partial protection of the lysine side chains of this peptide with d‐glucopyranosyloxycarbonyl and removal of the HisTag‐TEV sequence by TEV protease yielded the partially protected peptide with a free N‐terminal amine. Coupling of selenocysteine selectively at the N terminus and subsequent acidic deprotection of the carbohydrate protecting groups yielded a modified peptide that can be used for native chemical ligation (NCL). As a proof of concept, the modification of a longer recombinant peptide with selenocysteinylserine (GalNAc) at the N terminus was demonstrated. [ABSTRACT FROM AUTHOR]

Background and Purpose: A cholecystokinin (CCK) system has been identified in white adipose tissue (WAT). Nevertheless, the endocrine actions of CCK on WAT remain unknown. Our goal was to investigate the role of CCK in regulating the production of adiponectin, an adipokine expressed in WAT, which is pivotal in preserving energy homeostasis.Experimental Approach: The effect of the bioactive CCK fragment CCK-8 on adiponectin production was studied both in vivo and in vitro. CCK-8 effects were characterized in rats treated with selective CCK1 and CCK2 receptor antagonists as well as in pre-adipocytes carrying the selective silencing of either CCK1 or CCK2 receptors. The influence of insulin on CCK-8 responses was also analysed.Key Results: In WAT, CCK-8 increased plasma adiponectin levels and the expression of the adiponectin gene (Adipoq). In pre-adipocytes, CCK-8 up-regulated adiponectin production. CCK-8 effects were abolished by L-365,260, a selective CCK2 receptor antagonist. CCK2 receptor knockdown also abolished the effects of CCK-8 in pre-adipocytes. Moreover, in vitro CCK-8 effects were blocked by triciribine, a specific inhibitor of protein kinase B (Akt) and by the PPARγ antagonist T0070907. Silencing the expression of the insulin receptor inhibited CCK-8-induced Adipoq expression in pre-adipocytes. Furthermore, insulin potentiated the effect of CCK-8.Conclusion and Implications: CCK-8 stimulates adiponectin production in WAT by acting on CCK2 receptors, through a mechanism involving both Akt and PPARγ. Moreover, CCK-8 actions are only observed in the presence of insulin. Our results could have translational value in the design of new insulin-sensitizing therapies. [ABSTRACT FROM AUTHOR]

Summary: To identify novel antioxidant peptides from egg‐white protein and investigate antioxidant mechanism, the hydrolysate of egg‐white protein was purified by ultrafiltration and a Sephadex G‐15 column. The peptides VYLPR, EVYLPR, VEVYLPR and VVEVYLPR were identified from the fraction with the highest antioxidant activity. The Results showed that the peptide VYLPR exhibited the strongest protective effect on HEK‐293 cells. The viability of cells recovered to 97.45 ± 1.98% with pre‐treatment of 20 μm VYLPR. We further investigated antioxidant mechanism. The results showed VYLPR could inhibit lipid peroxidation process, maintain cell membrane integrity, inhibit intracellular LDH activity, reduce MDA content, and improve the activity of antioxidant enzyme T‐SOD and GSH‐Px. This work could help researchers in understanding antioxidant mechanism of peptides and also contribute to developing functional egg‐white product. [ABSTRACT FROM AUTHOR]

Background and Purpose: Inflammasome-mediated pyroptosis is an important neuronal cell death mechanism. Previous studies reported that activation of melanocortin MC4 receptor exerted neuroprotection in several neurological diseases. Here, we have investigated the role of MC4 receptor activation with RO27-3225 in suppressing neuronal pyroptosis after experimental intracerebral haemorrhage (ICH) and the underlying mechanism.Experimental Approach: One hundred and sixty-nine male CD1 mice were used. ICH was induced by injection of bacterial collagenase into the right-side basal ganglia. RO27-3225, a selective agonist of MC4 receptor, was injected intraperitoneally at 1 hr after ICH. To elucidate the underlying mechanism, we used the specific MC4 receptor antagonist HS024 and NQDI-1, a specific inhibitor of the apoptosis signalling-regulating kinase 1 (ASK1). Neurological tests, Western blot, Fluoro-Jade C, TUNEL, and immunofluorescence staining were conducted.Key Results: Expression of MC4 receptor and the NOD-like receptor family, pyrin domain containing 1 (NLRP1) inflammasome in brain were increased after ICH. RO27-3225 treatment decreased neuronal pyroptosis and neurobehavioural deficits at 24 and 72 hr after ICH. RO27-3225 reduced the expression of p-ASK1, p-JNK, p-p38 MAPK, NLRP1 inflammasome, cleaved caspase-1, and IL-1β after ICH. HS024 pretreatment prevented the effects of RO27-3225. Similar to RO27-3225, NQDI-1 alone improved neurological functions and down-regulated ASK1/JNK/p38MAPK expression after ICH.Conclusions and Implications: RO27-3225 suppressed NLRP1-dependent neuronal pyroptosis and improved neurological function, possibly mediated by activation of MC4 receptor and inhibition of ASK1/JNK/p38 MAPK signalling pathways, after experimental ICH in mice. The MC4 receptor may be a promising therapeutic target for the management of ICH. [ABSTRACT FROM AUTHOR]

Ligand‐based control of protein functional motions can provide novel opportunities in the study of fundamental biological mechanisms and in the development of novel therapeutics. In this work we addressed the ligand‐based modulation of integrin functions. Inhibitors of integrin αvβ3 are interesting anticancer agents but their molecular mechanisms are still unclear: Peptides and peptidomimetics characterized by the Arg‐Gly‐Asp (RGD) or isoAsp‐Gly‐Arg (isoDGR) binding motifs have shown controversial agonist/antagonist effects. We have investigated the differential mechanisms of integrin activation/deactivation by three distinct ligands (cyclo‐RGDf(NMe)V (Cilengitide), cyclo[DKP3‐RGD], cyclo[DKP3‐isoDGR]; DKP=diketopiperazine) through a comparative analysis of ligand‐controlled protein internal dynamics: Although RGD facilitates the onset of dynamic states leading to activation, isoDGR induces a diffuse rigidification of the complex consistent with antagonist activities. Computational predictions have been experimentally probed by showing that the antibody AP5, which is capable of recognizing the active form of integrin, binds specifically to the RGD complexes and not to the isoDGR complex, which supports opposite functional roles of the two motifs targeting the same binding site. [ABSTRACT FROM AUTHOR]

Background: Amylin is a calcitonin gene-related peptide family member expressed by nociceptors. Amylin's expression is down-regulated following nerve damage, and studies suggested it affects nociception. We aimed at clarifying amylin's effects on chronic neuropathic pain and investigating its site of action.Methods: Chronic neuropathic pain was induced in rats by spared nerve injury (SNI) surgery. Mechanical allodynia/hyperalgesia and cold allodynia/hyperalgesia were assessed by the von Frey, pinprick, acetone and cold plate behavioural tests, respectively. Amylin, amylin-receptor antagonist (AC187) or vehicle solutions were delivered chronically, by a subcutaneous (SC) mini-osmotic pump, or acutely, by SC or intrathecal (IT) injections. Cellular and fibre markers were used to detect spinal cord alterations in SNI rats after chronic amylin administration.Results: Continuous subcutaneous amylin administration aggravated cold allodynia in SNI animals, possibly via amylin-receptors (AmyR) in supraspinal areas. Acute intrathecal administration of amylin attenuated mechanical hyperalgesia, whereas AC187 reduced mechanical allodynia, suggesting distinct roles of endogenous amylin and of pharmacological amylin doses when targeting spinal cord amylin receptors. Chronic amylin administration promoted c-Fos activation only in the dorsal horn neurons of SHAM animals, suggesting a distinctive role of amylin in the activation of the spinal neuronal circuitry under neuropathic and physiological conditions. ERK1/2 phosphorylation increased in the dorsal horn neurons of SNI rats chronically treated with amylin. This ERK1/2 cascade activation may be related to amylin's effect on the aggravation of cold allodynia in SNI rats.Conclusions: Amylin's nociceptive effects seem to depend on the treatment duration and route of administration by acting at different levels of the nervous system.Significance: Amylin modulated neuropathic pain by acting at different levels of the nervous system. Whereas supraspinal areas may be involved in amylin's induced pronociception, modulation of spinal cord amylin receptors by endogenous or pharmacological amylin doses triggers both pro- and antinociceptive effects. [ABSTRACT FROM AUTHOR]

Background: We have recently reported that the spinal angiotensin (Ang) converting enzyme (ACE)/Ang II/AT1 receptor axis and downstream p38 MAPK phosphorylation are activated in streptozotocin (STZ)-induced diabetic mice and lead to tactile hypersensitivity. Moreover, our previous results suggested that the intrathecal (i.t.) administration of Ang (1-7), an N-terminal fragment of Ang II, may attenuate the Ang II-induced nociceptive behaviour through the inhibition of p38 MAPK phosphorylation via Mas receptors. Here, we investigated whether the i.t. administration of Ang (1-7) can attenuate STZ-induced diabetic neuropathic pain.Methods: Tactile and thermal hypersensitivities were determined using the von Frey filament and Hargreaves tests, respectively. The protein expression of ACE2, Mas receptors and phospho-p38 MAPK was measured by western blotting. Spinal ACE2 activity was determined using ACE2 activity assay kit.Results: The i.t. administration of Ang (1-7) significantly reduced the tactile and thermal hypersensitivities on day 14 after STZ injection, and these effects were significantly prevented by the Mas receptor antagonist A779. The expression of ACE2 and Mas receptors in the plasma membrane fraction of the lumbar dorsal spinal cord was both significantly decreased in STZ mice. Spinal ACE2 activity was also decreased while p38 MAPK phosphorylation was increased in the lumbar dorsal region of these mice. This phosphorylation was attenuated by the injection of Ang (1-7), whose effect was reversed by A779.Conclusions: Our data demonstrate that Ang (1-7) attenuates STZ-induced diabetic neuropathic pain and that this occurs through a mechanism involving spinal Mas receptors and he inhibition of p38 MAPK phosphorylation.Significance: The ACE2/Ang (1-7)/Mas receptor axis was down-regulated in the spinal cord of STZ mice and the i.t. administration of Ang (1-7) attenuated the STZ-induced diabetic neuropathic pain via Mas receptors. Therefore, the activation of this axis could be an effective therapeutic target to alleviate the neuropathic pain in diabetic patients. [ABSTRACT FROM AUTHOR]

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. Once a field dominated by small molecule radiotracers, position emission tomography (PET) has seen a shift towards the use of radiolabelled affinity peptide and proteins (APP). Monoclonal antibodies are important examples, yet a preference for smaller APPs, exhibiting faster pharmacokinetics, has become apparent. This review article seeks to assess and compare the diverse approaches taken to radiolabel this emerging class with 18F, with the intention of highlighting trends within this expanding field. [ABSTRACT FROM AUTHOR]

Alcohol consumption is considered a major risk factor for disease and mortality worldwide. In the absence of effective treatments in alcohol use disorders, it is important to find new biological targets that could modulate alcohol consumption. We tested the role of the N-terminal galanin fragment (1-15) [GAL(1-15)] in voluntary ethanol consumption in rats using the two-bottle choice paradigm as well as compare the effects of GAL(1-15) with the whole molecule of GAL. We describe for the first time that GAL(1-15), via central mechanisms, induces a strong reduction in preference and ethanol consumption in rats. These effects were significantly different than GAL. GAL receptor (GALR) 2 was involved in these effects, because the specific GALR2 antagonist M871 blocked GAL(1-15) mediated actions in preference and ethanol intake. Importantly, the mechanism of this action involves changes in GALR expression and also in immediate-early gene C-Fos and receptors-internalization-related gene Rab5 in the striatum. The relevance of the striatum as a target for GAL(1-15) was supported by the effect of GAL(1-15) on the locomotor activity of rats after ethanol administration. These results may give the basis for the development of novel therapeutics strategies using GAL(1-15) analogues for the treatment of alcohol use disorders in humans. [ABSTRACT FROM AUTHOR]

The development of synthetic methods to prepare conformationally constrained peptides and peptide–polyketide hybrids remain an important chemical challenge. It is known that structural rigidity correlates with the specificity, bioactivity, and stability of these peptide systems, thus rigid systems are particularly attractive leads for development of potent biopharmaceuticals. Herein we provide an overview of recent developments in the syntheses of naturally derived constrained peptides and peptide–polyketide hybrids, with a particular emphasis on those systems containing an ene‐like bond. Peptide rigidification: Recent developments in the chemical syntheses of highly constrained natural derived cyclopeptides containing ene‐like linkages has opened the door to the potential addition of clinical tools to treat otherwise intractable ailments. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Lupin seeds are rich in proteins, which are utilized in the food industry. There is an increased interest in lupin research due to its association with health‐related benefits, such as reduction of hypertension and hyperglycemia. However, studies on the peptides derived from lupin proteins are rare. RESULTS: Lupin protein hydrolysates (LPHs) were prepared by proteolysis using alcalase, trypsin and pepsin, respectively. All the hydrolysates demonstrated higher antioxidant and angiotensin I‐converting enzyme (ACE) inhibitory activities compared to lupin proteins. The hydrolysates were fractionated into three fractions based on molecular weight (MW), and the peptides with MW < 3 kDa (LPH3) had the highest antioxidant and ACE inhibitory activities compared to other fractions. Cell model study revealed that LPH3 fraction had the highest protection against the generation of reactive oxygen species in HepG2 cells, which was associated with increased activities of superoxide dismutase and glutathione peroxidase through upregulation of SOD1, GPX1, GCLM, SLC7A11 and SRXN1 expression. CONCLUSIONS: The analysis of amino acid composition indicated that the peptides were characterized with high content of hydrophobic amino acids, which may be responsible for the greatest antioxidant activity. This study highlights the promising potential of lupin peptides as a functional ingredient in healthy foods. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Exosomes are nanovesicles (40–100 nm) containing various RNAs and different proteins. Exosomes are involved in intracellular communication and immune system function. Exosomes from different sources are usually isolated using standard methods—centrifugation and ultracentrifugations. Exosomes isolated by these procedures were reported to contain from a few dozen to thousands of different proteins. Here crude vesicle preparations from five placentas (normal pregnancy) were first obtained using standard centrifugation procedures. According to electron‐microscopic studies, these preparations contained vesicles of different size (30–225 nm), particles of round shape of average electron density ("nonvesicles" 20–40 nm) (A), structured clusters of associated proteins and shapeless aggregations (B), as well as ring‐shaped 10–14 nm structures formed by ferritin (C). After additional purification of the vesicle preparations by gel filtration on Sepharose 4B, the main part of protein structures was removed; however, the preparations still contained small admixtures of components A–C. Further purification of the preparations by affinity chromatography on Sepharose bearing immobilized antibodies against exosome surface protein CD81 led to isolation of highly purified exosomes (40–100 nm). These exosomes according to electron microscopy data contained tetraspanin embedded in the membrane, which was stained with antibodies against CD81 conjugated with 10–12 nm gold nanoparticles. SDS‐PAGE and MALDI MS and MS/MS mass spectrometry of tryptic hydrolysates of proteins contained in these exosomes revealed eleven major proteins (>10 kDa): hemoglobin subunits, CD81, interleukin‐1 receptor, annexin A5, cytoplasmic actin, alpha‐actin‐4, alkaline phosphatase, human serum albumin, serotransferrin, and lactotrasferrin. Using MALDI mass analysis of the highly purified exosomes, we for the first time found that in addition to the large proteins (>10 kDa), exosomes having affinity to CD81 contain more than 27 different peptides and small proteins of 2–10 kDa. This finding can be useful for revealing biological functions of pure exosomes. © 2018 IUBMB Life, 70(11):1144–1155, 2018 [ABSTRACT FROM AUTHOR]

Background and Purpose: The nociceptin/orphanin FQ opioid peptide (NOP) receptor system plays a significant role in the regulation of pain. This system functions differently in the spinal cord and brain. The mechanism by which the NOP receptor agonists regulate pain transmission in these regions is not clearly understood. Here, we investigate the peripheral and spinal NOP receptor distribution and antinociceptive effects of intrathecal nociceptin/orphanin FQ (N/OFQ) in chronic neuropathic pain.Experimental Approach: We used immunohistochemistry to determine changes in NOP receptor distribution triggered by spinal nerve ligation (SNL) using NOP-eGFP knock-in mice. Antinociceptive effects of intrathecal N/OFQ on SNL-mediated allodynia and heat/cold hyperalgesia were assessed in wild-type mice.Key Results: NOP-eGFP immunoreactivity was decreased by SNL in the spinal laminae I and II outer, regions that mediate noxious heat stimuli. In contrast, immunoreactivity of NOP-eGFP was unchanged in the ventral border of lamina II inner, which is an important region for the development of allodynia. NOP-eGFP expression was also decreased in a large number of primary afferents in the L4 dorsal root ganglion (DRG) of SNL mice. However, SNL mice showed increased sensitivity, compared to sham animals to the effects of i.t administered N/OFQ with respect to mechanical as well as thermal stimuli.Conclusions and Implications: Our findings suggest that the spinal NOP receptor system attenuates injury-induced hyperalgesia by direct inhibition of the projection neurons in the spinal cord that send nociceptive signals to the brain and not by inhibiting presynaptic terminals of DRG neurons in the superficial lamina. [ABSTRACT FROM AUTHOR]

To simulate full multidimensional nuclear magnetic resonance spectra of peptides and proteins in a reasonable time frame, a strategy for separating the time-consuming full-density matrix calculations from the chemical shift prediction and calculation of coupling patterns is presented. The simulation setup uses SIMulation Program for SOlid-state NMR (SIMPSON) to calculate total correlation spectroscopy transfer amplitudes and average distances as a source for nuclear Overhauser effect spectroscopy transfer amplitudes. Simulated 1H 1D, 2D total correlation spectroscopy, and 2D nuclear Overhauser effect spectroscopy nuclear magnetic resonance spectra of peptides with sequence Pro─Ala─Gly─Tyr─Asn and Asn─Phe─Gly─Ala─Ile─Leu and of ubiquitin are presented. In all cases, the simulations lasted from a few seconds to tens of seconds on a normal laptop computer. [ABSTRACT FROM AUTHOR]

Abstract: In contrast with the common belief that all the amino acid residues in higher organisms are l‐forms, d‐amino acid residues have been recently detected in various aging tissues. Aspartic acid (Asp) residues are known to be the most prone to stereoinvert via cyclic imide intermediate. Although the glutamic acid (Glu) is similar in chemical structure to Asp, little has been reported to detect d‐Glu residues in human proteins. In this study, we investigated the mechanism of the Glu‐residue stereoinversion catalyzed by water molecules using B3LYP/6‐31+G(d,p) density functional theory calculations. We propose that the Glu‐residue stereoinversion proceeds via a cyclic imide intermediate, i.e., glutarimide (GI). All calculations were performed by using a model compound in which a Glu residue was capped with acetyl and methylamino groups on the N‐ and C‐termini, respectively. We found that two water molecules catalyze the three steps involved in the GI formation: iminolization, cyclization, and dehydration. The activation energy required for the Glu residue to form a GI intermediate was estimated to be 32.3 kcal mol−1, which was higher than that of the experimental Asp‐residue stereoinversion. This calculation result suggests that the Glu‐residue stereoinversion is not favored under the physiological condition. [ABSTRACT FROM AUTHOR]