Showing papers on "Peptide sequence published in 2019"

SignalP 5.0 improves signal peptide predictions using deep neural networks

Abstract: Signal peptides (SPs) are short amino acid sequences in the amino terminus of many newly synthesized proteins that target proteins into, or across, membranes. Bioinformatic tools can predict SPs from amino acid sequences, but most cannot distinguish between various types of signal peptides. We present a deep neural network-based approach that improves SP prediction across all domains of life and distinguishes between three types of prokaryotic SPs.

Deep learning enables de novo peptide sequencing from data-independent-acquisition mass spectrometry.

Abstract: We present DeepNovo-DIA, a de novo peptide-sequencing method for data-independent acquisition (DIA) mass spectrometry data. We use neural networks to capture precursor and fragment ions across m/z, retention-time, and intensity dimensions. They are then further integrated with peptide sequence patterns to address the problem of highly multiplexed spectra. DIA coupled with de novo sequencing allowed us to identify novel peptides in human antibodies and antigens. A deep-learning-based tool, DeepNovo-DIA, uses data-independent-acquisition mass spectrometry data to sequence peptides without using a database.

Is protein context responsible for peptide-mediated interactions?

Abstract: Many cell signaling pathways are orchestrated by the weak, transient, and reversible protein-protein interactions that are mediated by the binding of a short peptide segment in one protein (parent protein) to a globular domain in another (partner protein), known as peptide-mediated interactions (PMIs). Previous studies normally had an implicit hypothesis that a PMI is functionally equivalent or analogous to the protein-peptide interaction (PTI) involved in the PMI system, while ignoring parent context contribution to the peptide binding. Here, we perform a systematic investigation on the reasonability and applicability of the hypothesis at structural, energetic and dynamic levels. It is revealed that the context impacts PMIs primarily through conformational constraint of the peptide segments, which can (i) reduce the peptide flexibility and disorder in an unbound state, (ii) help the peptide conformational selection to fit the active pocket of partner proteins, and (iii) enhance the peptide packing tightness against the partners. Long, unstructured and/or middle-located peptide segments seem to be more vulnerable to their context than short, structured and/or terminal ones. The context is found to moderately or considerably improve both the binding affinity and specificity of PMIs as compared to their PTI counterparts; with the context support a peptide segment can contribute to ∼30-60% total binding energy of the whole PMI system, whereas the contribution is reduced to ∼5-50% when the context constraint is released. In addition, we also observe that peptide selectivity is largely impaired or even reversed upon stripping of their parent context (global selectivity decreases from 34.2 to 1.7-fold), by examining the crystal structures of full-length Src family kinases in an autoinhibitory state. Instead of the direct interaction and desolvation that are primarily concerned in traditional studies, peptide flexibility and the entropy penalty should also play a crucial role in the context effect on PMIs. Overall, we suggest that the context factor should not be ignored in most cases, particularly those with peptide segments that are long, highly disordered, and/or located at the middle region of their parent proteins.

Why do eukaryotic proteins contain more intrinsically disordered regions

Abstract: Intrinsic disorder is more abundant in eukaryotic than prokaryotic proteins. Methods predicting intrinsic disorder are based on the amino acid sequence of a protein. Therefore, there must exist an underlying difference in the sequences between eukaryotic and prokaryotic proteins causing the (predicted) difference in intrinsic disorder. By comparing proteins, from complete eukaryotic and prokaryotic proteomes, we show that the difference in intrinsic disorder emerges from the linker regions connecting Pfam domains. Eukaryotic proteins have more extended linker regions, and in addition, the eukaryotic linkers are significantly more disordered, 38% vs. 12-16% disordered residues. Next, we examined the underlying reason for the increase in disorder in eukaryotic linkers, and we found that the changes in abundance of only three amino acids cause the increase. Eukaryotic proteins contain 8.6% serine; while prokaryotic proteins have 6.5%, eukaryotic proteins also contain 5.4% proline and 5.3% isoleucine compared with 4.0% proline and ≈ 7.5% isoleucine in the prokaryotes. All these three differences contribute to the increased disorder in eukaryotic proteins. It is tempting to speculate that the increase in serine frequencies in eukaryotes is related to regulation by kinases, but direct evidence for this is lacking. The differences are observed in all phyla, protein families, structural regions and type of protein but are most pronounced in disordered and linker regions. The observation that differences in the abundance of three amino acids cause the difference in disorder between eukaryotic and prokaryotic proteins raises the question: Are amino acid frequencies different in eukaryotic linkers because the linkers are more disordered or do the differences cause the increased disorder?

Self-assembly of mitochondria-specific peptide amphiphiles amplifying lung cancer cell death through targeting the VDAC1-hexokinase-II complex.

Abstract: Mitochondria-targeting peptides represent an emergent tool for cancer inhibition. Here supramolecular assemblies of novel amphiphilic cell-penetrating peptides for targeting cancer cell mitochondria are reported. The employed strategy aims at amplifying the apoptotic stimuli by weakening the mitochondrial VDAC1 (voltage-dependent anion channel-1)-hexokinase-II (HK-II) interaction. Peptide engineering is performed with the N-terminus of the HK-II protein, which binds to VDAC1. First, a designed positively charged segment (pKV) is anchored to the specific 15 amino acid sequence (MIASHLLAYFFTELN) to yield a cell-penetrating peptide (pHK-pKV). Second, a lipid chain (Pal) is conjugated to the N-terminus of pHK-pKV in order to enhance the intracellular delivery of the HK-II scaffold. The self-assembly properties of these two synthetic peptides are investigated by synchrotron small-angle X-ray scattering (BioSAXS) and cryogenic transmission electron (cryo-TEM) imaging, which evidence the formation of nanoassemblies of ellipsoid-like shapes. Circular dichroism (CD) spectroscopy demonstrates the induction of partial α-helical structures in the amphiphilic peptides. Confocal microscopy reveals the specific mitochondrial location of Pal-pHK-pKV assemblies in human non-small cell lung cancer (NSCLC) A549 cells. The cytotoxicity and apoptotic studies indicate the enhanced bioactivity of Pal-pHK-pKV self-assembled reservoirs, which cause massive A549 cell death with regard to pHK-pKV. Of significance, Pal-pHK-pKV treatment of non-cancerous NCM460 cells resulted in substantially lower cytotoxicity. The results demonstrate the potential of self-assembled lipo-peptide (HK-II-derived) conjugates as a promising strategy in cancer therapy.

International union of basic and clinical pharmacology. CVII. structure and pharmacology of the apelin receptor with a recommendation that elabela/toddler is a second endogenous peptide ligand

Abstract: The predicted protein encoded by the APJ gene discovered in 1993 was originally classified as a class A G protein-coupled orphan receptor but was subsequently paired with a novel peptide ligand, apelin-36 in 1998. Substantial research identified a family of shorter peptides activating the apelin receptor, including apelin-17, apelin-13, and [Pyr1]apelin-13, with the latter peptide predominating in human plasma and cardiovascular system. A range of pharmacological tools have been developed, including radiolabeled ligands, analogs with improved plasma stability, peptides, and small molecules including biased agonists and antagonists, leading to the recommendation that the APJ gene be renamed APLNR and encode the apelin receptor protein. Recently, a second endogenous ligand has been identified and called Elabela/Toddler, a 54-amino acid peptide originally identified in the genomes of fish and humans but misclassified as noncoding. This precursor is also able to be cleaved to shorter sequences (32, 21, and 11 amino acids), and all are able to activate the apelin receptor and are blocked by apelin receptor antagonists. This review summarizes the pharmacology of these ligands and the apelin receptor, highlights the emerging physiologic and pathophysiological roles in a number of diseases, and recommends that Elabela/Toddler is a second endogenous peptide ligand of the apelin receptor protein.

Chemoselective Peptide Cyclization and Bicyclization Directly on Unprotected Peptides.

Abstract: Cyclic peptides are drawing wide attention as potential medium-sized modulators of biomolecular interactions with large binding surfaces. Simple but effective peptide cyclization methods are needed to construct cyclic peptide libraries by both peptide and nonpeptide chemists. Herein, we report a highly chemoselective and operation-simple method directly cyclizing unprotected peptides, in which ortho-phthalaldehyde (OPA) is found to react with the lysine/N-terminus and cysteine within one unprotected peptide sequence effectively to form the isoindole-bridged cyclic peptides. This reaction is carried out in the aqueous buffer and features tolerance of diverse functionalities, rapid and clean transformation, and operational simplicity. In addition, OPA peptide cyclization can also be combined with native chemical ligation-mediated cyclization to generate bicyclic peptides. Furthermore, the OPA peptide cyclization product can further react with the N-maleimide moiety in a one-pot manner to introduce additional functional motifs, like a fluorophore probe, biomolecules (e.g., glycan, peptide, or DNA). This OPA-cyclization method extends the toolbox for integrating postcyclization modification and bioconjugation into peptide cyclization with an all-in-one manner strategy.

Human NK cell receptor KIR2DS4 detects a conserved bacterial epitope presented by HLA-C.

Abstract: Natural killer (NK) cells have an important role in immune defense against viruses and cancer. Activation of human NK cell cytotoxicity toward infected or tumor cells is regulated by killer cell immunoglobulin-like receptors (KIRs) that bind to human leukocyte antigen class I (HLA-I). Combinations of KIR with HLA-I are genetically associated with susceptibility to disease. KIR2DS4, an activating member of the KIR family with poorly defined ligands, is a receptor of unknown function. Here, we show that KIR2DS4 has a strong preference for rare peptides carrying a Trp at position 8 (p8) of 9-mer peptides bound to HLA-C*05:01. The complex of a peptide bound to HLA-C*05:01 with a Trp at p8 was sufficient for activation of primary KIR2DS4+ NK cells, independent of activation by other receptors and of prior NK cell licensing. HLA-C*05:01+ cells that expressed the peptide epitope triggered KIR2DS4+ NK cell degranulation. We show an inverse correlation of the worldwide allele frequency of functional KIR2DS4 with that of HLA-C*05:01, indicative of functional interaction and balancing selection. We found a highly conserved peptide sequence motif for HLA-C*05:01-restricted activation of human KIR2DS4+ NK cells in bacterial recombinase A (RecA). KIR2DS4+ NK cells were stimulated by RecA epitopes from multiple human pathogens, including Helicobacter, Chlamydia, Brucella, and Campylobacter. We predict that over 1,000 bacterial species could activate NK cells through KIR2DS4, and propose that human NK cells also contribute to immune defense against bacteria through recognition of a conserved RecA epitope presented by HLA-C*05:01.

Systems structural biology measurements by in vivo cross-linking with mass spectrometry.

Abstract: This protocol describes a workflow for utilizing large-scale cross-linking with mass spectrometry (XL-MS) to make systems-level structural biology measurements in complex biological samples, including cells, isolated organelles, and tissue samples. XL-MS is a structural biology technique that provides information on the molecular structure of proteins and protein complexes using chemical probes that report the proximity of probe-reactive amino acids within proteins, typically lysine residues. Information gained through XL-MS studies is often complementary to more traditional methods, such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy. The use of MS-cleavable cross-linkers, including protein interaction reporter (PIR) technologies, enables XL-MS studies on protein structures and interactions in extremely complex biological samples, including intact living cells. PIR cross-linkers are designed to contain chemical bonds at specific locations within the cross-linker molecule that can be selectively cleaved by collision-induced dissociation or UV light. When broken, these bonds release the intact peptides that were cross-linked, as well as a reporter ion. Conservation of mass dictates that the sum of the two released peptide masses and the reporter mass equals the measured precursor mass. This relationship is used to identify cross-linked peptide pairs. Release of the individual peptides permits accurate measurement of their masses and independent amino acid sequence determination by tandem MS, allowing the use of standard proteomics search engines such as Comet for peptide sequence assignment, greatly simplifying data analysis of cross-linked peptide pairs. Search results are processed with XLinkProphet for validation and can be uploaded into XlinkDB for interaction network and structural analysis. Cross-linking with mass spectrometry (XL-MS) can reveal the topology of protein complexes. This protocol describes how to synthesize a cleavable cross-linker and use it to map protein structures and interactions within intact cells and animal tissues.

Single-cell proteomics.

Abstract: New technologies bring single-cell proteomics closer to reality.

Site-Specific Sequential Protein Labeling Catalyzed by a Single Recombinant Ligase.

Abstract: Protein ligases of defined substrate specificity are versatile tools for protein engineering. Upon completion of the reaction, the products of currently reported protein ligases contain the amino acid sequence that is recognized by that same ligase, resulting in repeated cycles of ligation and hydrolysis as competing reactions. Thus, previous efforts to sequentially label proteins at distinct positions required ligases of orthogonal specificity. A recombinant Oldenlandia affinis asparaginyl endopeptidase, OaAEP1, is promiscuous for incoming nucleophiles. This promiscuity enabled us to define a nucleophile composed of natural amino acids that is ligated efficiently to the substrate yet yields a product that is poorly recognized by OaAEP1. Proteins modified with an efficient recognition module could be readily modified to yield a defined product bearing a cleavage-resistant motif, whereas proteins containing this inferior recognition motif remained essentially unmodified. We demonstrate the versatility of the N- or C-terminal protein modifications obtainable with this approach and modify the N- and C-termini of a single substrate protein in a sequential, site-specific manner in excellent yield.

Bioactive peptide with antioxidant and anticancer activities from black soybean [Glycine max (L.) Merr.] byproduct: isolation, identification and molecular docking study

Abstract: In this study, a novel peptide was isolated from the black soybean byproduct using bioactive guided isolation technique. Initially, the hydrolysate of black soybean protein was fractionated and separated sequentially by ultrafiltration, Sephadex G 25 chromatography and reverse-phase HPLC techniques. The peptide fractions that collected in each step were tested for their antioxidant capacity and anticancer activities against human liver (HepG2), lung (MCF-7) and cervical (Hela) cancer cell lines. The most active fraction was subjected to further purification. The final purified peptide fraction (F2-c) with the molecular weight of 455.0 Da showed highest DPPH free radical scavenging and hydroxyl radical scavenging activity with the IC50 values of 0.12 and 0.037 µM, respectively. Moreover, it showed high cytotoxic potential against HepG2, MCF-7 and Hela cells with the IC50 values of 0.22, 0.15 and 0.32 µM, respectively. The amino acid sequence of F2-c was identified as Leu/Ile-Val-Pro-Lys (L/I-VPK). Molecular docking studies revealed that the purified peptides effectively bound with four apoptosis related key proteins (XIAP, caspase-3, caspase-7, and Bcl-2) via hydrophobic effects and hydrogen bonds, amongst them, the peptide-caspase-3 binding showed the strongest binding energy. All the results suggested that the peptide fraction Leu/Ile-Val-Pro-Lys from black soybean byproduct with significant antioxidant and anticancer activities could be a good candidate for functional foods or related drugs.

O-Methyltransferase-Mediated Incorporation of a β-Amino Acid in Lanthipeptides.

Abstract: Lanthipeptides represent a large class of cyclic natural products defined by the presence of lanthionine (Lan) and methyllanthionine (MeLan) cross-links. With the advances in DNA sequencing technologies and genome mining tools, new biosynthetic enzymes capable of installing unusual structural features are continuously being discovered. In this study, we investigated an O-methyltransferase that is a member of the most prominent auxiliary enzyme family associated with class I lanthipeptide biosynthetic gene clusters. Despite the prevalence of these enzymes, their function has not been established. Herein, we demonstrate that the O-methyltransferase OlvSA encoded in the olv gene cluster from Streptomyces olivaceus NRRL B-3009 catalyzes the rearrangement of a highly conserved aspartate residue to a β-amino acid, isoaspartate, in the lanthipeptide OlvA(BCSA). We elucidated the NMR solution structure of the GluC-digested peptide, OlvA(BCSA)GluC, which revealed a unique ring topology comprising four interlocking rings and positions the isoaspartate residue in a solvent exposed loop that is stabilized by a MeLan ring. Gas chromatography-mass spectrometry analysis further indicated that OlvA(BCSA) contains two dl-MeLan rings and two Lan rings with an unusual ll-stereochemistry. Lastly, in vitro reconstitution of OlvSA activity showed that it is a leader peptide-independent and S-adenosyl methionine-dependent O-methyltransferase that mediates the conversion of a highly conserved aspartate residue in a cyclic substrate into a succinimide, which is hydrolyzed to generate an Asp or isoAsp containing peptide. This overall transformation converts an α-amino acid into a β-amino acid in a ribosomally synthesized peptide, via an electrophilic intermediate that may be the intended product.

Heterologous and in Vitro Reconstitution of Fuscanodin, a Lasso Peptide from Thermobifida fusca.

Abstract: Lasso peptides are a class of ribosomally derived natural products typified by their threaded rotaxane structure. The conversion of a linear precursor peptide into a lasso peptide structure requires two enzymatic activities: cleavage of the precursor via a cysteine protease and cyclization via isopeptide bond formation. In vitro studies of lasso peptide enzymology have been hampered by difficulties in obtaining pure, soluble enzymes. We reasoned that thermophilic bacteria would be a good source for well-behaved lasso peptide biosynthetic enzymes. The genome of the thermophilic actinobacterium Thermobifida fusca encodes for a lasso peptide with an unprecedented Trp residue at its N-terminus, a peptide we have named fuscanodin. Here we reconstitute fuscanodin biosynthesis in vitro with purified components, establishing a minimal fuscanodin synthetase. These experiments have allowed us to probe the kinetics of lasso peptide biosynthesis for the first time, and we report initial rates of fuscanodin biosynthesis. The fuscanodin biosynthetic enzymes are insensitive to substrate concentration and operate in a near single-turnover regime in vitro. While lasso peptides are often touted for their stability to both chaotropic and thermal challenges, fuscanodin is found to undergo a conformational change consistent with lasso peptide unthreading in organic solvents at room temperature.

Ribosomal Synthesis of Backbone-Cyclic Peptides Compatible with In Vitro Display

Abstract: Backbone-cyclic peptides are an attractive class for therapeutic development. However, in vitro display technologies coupled with ribosomal synthesis are intrinsically inapplicable to such “phenotypes” because of loss of the C-terminal peptide region linking to “genotype”. Here, we report a methodology enabling the display of backbone-cyclic peptides. To achieve this, genetic code reprogramming was utilized to implement a rearrangement strategy involving the ribosomal incorporation of a designer initiator containing a thiazolidine-protected cysteine and 2-chloroacetoamide (ClAc) side chain, followed by an α-thio acid and cysteine at downstream positions. Upon expression of the linear peptide, spontaneous thioester rearrangement occurs between the α-thioester and the thiol group of the cysteine, liberating the α-thio group and resulting in cross-linking to the upstream ClAc side-chain group. Then selective deprotection of the thiazolidine-protected cysteine immediately promotes intramolecular native chemic...

Characterization of Collagen Derived From Tropical Freshwater Carp Fish Scale Wastes and Its Amino Acid Sequence

Abstract: Collagen from fish scale waste is currently being studied as a promising biological material to replace collagen from animals because of advantages such as safe, fat-free, not suffering from commun...

Positive Charge Patterning and Hydrophobicity of Membrane-Active Antimicrobial Peptides as Determinants of Activity, Toxicity, and Pharmacokinetic Stability.

Abstract: Natural α-helical cationic antimicrobial peptide (CAP) sequences are predominantly amphipathic, with only ca. 2% containing four or more consecutive positively charged amino acids (Lys/Arg). We have designed synthetic CAPs that deviate from these natural sequences, as typified by the charge-clustered peptide KKKKKKAAFAAWAAFAA-NH2, (termed 6K-F17), which displays high antimicrobial activity with no toxicity to mammalian cells. We created a series of peptides varying in charge patterning, increasing the amphipathic character of 6K-F17 to mimic the design of natural CAPs (e.g., KAAKKFAKAWAKAFAA-NH2). Amphipathic sequences displayed increased antimicrobial activity against bacteria but were significantly more toxic to mammalian cells and more susceptible to protease degradation than their corresponding charge-clustered variants, suggesting that amphipathic sequences may be desirable in nature to allow for more versatile functions (i.e., antibacterial, antifungal, antipredator) and rapid clearance from vulnerable host cells. Our approach to clustering of charges may therefore allow for specialization against bacteria, in concert with prolonged peptide half-life.

A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity

Abstract: Autoreactivity to myeloperoxidase (MPO) causes anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), with rapidly progressive glomerulonephritis. Here, we show that a Staphylococcus aureus peptide, homologous to an immunodominant MPO T-cell epitope (MPO409-428), can induce anti-MPO autoimmunity. The peptide (6PGD391-410) is part of a plasmid-encoded 6-phosphogluconate dehydrogenase found in some S. aureus strains. It induces anti-MPO T-cell autoimmunity and MPO-ANCA in mice, whereas related sequences do not. Mice immunized with 6PGD391-410, or with S. aureus containing a plasmid expressing 6PGD391-410, develop glomerulonephritis when MPO is deposited in glomeruli. The peptide induces anti-MPO autoreactivity in the context of three MHC class II allomorphs. Furthermore, we show that 6PGD391-410 is immunogenic in humans, as healthy human and AAV patient sera contain anti-6PGD and anti-6PGD391-410 antibodies. Therefore, our results support the idea that bacterial plasmids might have a function in autoimmune disease.

Unusual Metabolism and Hypervariation in the Genome of a Gracilibacterium (BD1-5) from an Oil-Degrading Community.

Abstract: The candidate phyla radiation (CPR) comprises a large monophyletic group of bacterial lineages known almost exclusively based on genomes obtained using cultivation-independent methods. Within the CPR, Gracilibacteria (BD1-5) are particularly poorly understood due to undersampling and the inherent fragmented nature of available genomes. Here, we report the first closed, curated genome of a gracilibacterium from an enrichment experiment inoculated from the Gulf of Mexico and designed to investigate hydrocarbon degradation. The gracilibacterium rose in abundance after the community switched to dominance by Colwellia Notably, we predict that this gracilibacterium completely lacks glycolysis, the pentose phosphate and Entner-Doudoroff pathways. It appears to acquire pyruvate, acetyl coenzyme A (acetyl-CoA), and oxaloacetate via degradation of externally derived citrate, malate, and amino acids and may use compound interconversion and oxidoreductases to generate and recycle reductive power. The initial genome assembly was fragmented in an unusual gene that is hypervariable within a repeat region. Such extreme local variation is rare but characteristic of genes that confer traits under pressure to diversify within a population. Notably, the four major repeated 9-mer nucleotide sequences all generate a proline-threonine-aspartic acid (PTD) repeat. The genome of an abundant Colwellia psychrerythraea population has a large extracellular protein that also contains the repeated PTD motif. Although we do not know the host for the BD1-5 cell, the high relative abundance of the C. psychrerythraea population and the shared surface protein repeat may indicate an association between these bacteria.IMPORTANCE CPR bacteria are generally predicted to be symbionts due to their extensive biosynthetic deficits. Although monophyletic, they are not monolithic in terms of their lifestyles. The organism described here appears to have evolved an unusual metabolic platform not reliant on glucose or pentose sugars. Its biology appears to be centered around bacterial host-derived compounds and/or cell detritus. Amino acids likely provide building blocks for nucleic acids, peptidoglycan, and protein synthesis. We resolved an unusual repeat region that would be invisible without genome curation. The nucleotide sequence is apparently under strong diversifying selection, but the amino acid sequence is under stabilizing selection. The amino acid repeat also occurs in a surface protein of a coexisting bacterium, suggesting colocation and possibly interdependence.

DeepGOPlus: Improved protein function prediction from sequence

Abstract: Protein function prediction is one of the major tasks of bioinformatics that can help in wide range of biological problems such as understanding disease mechanisms or finding drug targets. Many methods are available for predicting protein functions from sequence based features, protein–protein interaction networks, protein structure or literature. However, other than sequence, most of the features are difficult to obtain or not available for many proteins thereby limiting their scope. Furthermore, the performance of sequence-based function prediction methods is often lower than methods that incorporate multiple features and predicting protein functions may require a lot of time. We developed a novel method for predicting protein functions from sequence alone which combines deep convolutional neural network (CNN) model with sequence similarity based predictions. Our CNN model scans the sequence for motifs which are predictive for protein functions and combines this with functions of similar proteins. We evaluate the performance of DeepGOPlus on the CAFA3 dataset and significantly improve the performance of predictions of biological processes and cellular components with Fmax of 0.47 and 0.70, respectively, using only the amino acid sequence of proteins as input. DeepGOPlus can annotate around 40 protein sequences per second, thereby making fast and accurate function predictions available for a wide range of proteins.

Molecular biology of trophoblast interferons and studies of their effects in vivo

Abstract: Southern blotting of bovine genomic DNA indicated the presence of at least 3 bovine tIFN genes. The full DNA sequence of one of these genes, thought to be expressed in trophoblast, has been determined, including 193 bp of 5' non-coding region. The inferred amino acid sequence of bovine tIFN is more similar to ovine tIFN (80%) than to bovine IFN-alpha II (70%). The 5' flanking sequence has some similarity with bovine IFN-alpha II, and may contain a viral response element. A recombinant bovine alpha I interferon (Ciba Geigy; brIFN), resembling tIFN, extended oestrous cycle length in sheep when administered by intrauterine infusion over the period, Days 12-15 after oestrus, when maternal recognition of pregnancy occurs. Intramuscular injection was only effective at the doses used if given over a longer period (i.e. Days 9-15). Our experiments indicate that both tIFN and brIFN inhibit luteolysis by preventing a rise in endometrial oxytocin receptor concentrations, and suggest that tIFN achieves this by extending the time for which progesterone suppresses oxytocin receptor development. Further studies are required to confirm this hypothesis and to elucidate the interaction of the effects of progesterone and tIFN in endometrial cells.

Purification and Characterization of a Novel Pentadecapeptide from Protein Hydrolysates of Cyclina sinensis and Its Immunomodulatory Effects on RAW264.7 Cells

Abstract: In the present study, peptide fractions of Cyclina sinensis hydrolysates, with molecular weight (MW) < 3 kDa and highest relative proliferation rate of murine macrophage cell line RAW 264.7, were purified by a series of chromatographic purification methods, to obtain peptide fractions with immunomodulatory activity. The amino acid sequence of the peptide was identified to be Arg-Val-Ala-Pro-Glu-Glu-His-Pro-Val-Glu-Gly-Arg-Tyr-Leu-Val (RVAPEEHPVEGRYLV) with MW of 1750.81 Da, and the novel pentadecapeptide (named SCSP) was synthesized for subsequent immunomodulatory activity experiments. Results showed the SCSP enhanced macrophage phagocytosis, increased productions of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), and up-regulated the protein level of inducible nitric oxide synthase (iNOS), nuclear factor κB (NF-κB), and NOD-like receptor protein 3 (NLRP3) in RAW 264.7 cells. Furthermore, the expression of inhibitor of nuclear factor κB-α (IκB-α) was down-regulated. These findings suggest that SCSP might stimulate macrophage activities by activating the NF-κB signaling pathway and can be used as a potential immunomodulatory agent in functional food or medicine.

Enhancement of siRNA transfection by the optimization of fatty acid length and histidine content in the CPP.

Abstract: Extracellular synthetic nucleic acids, such as siRNAs, are unable to reach their intended targets efficiently. Therefore, delivery methods such as cell-penetrating peptides (CPP), which increase their transport, could enhance the potency of siRNA as therapeutic agents. The CPP NickFect55 (NF55) is an efficient peptide-based delivery vector, which has been previously used to deliver plasmid DNA into cells in vivo. To achieve higher intracellular delivery and bioactivity from the delivered cargo, we designed a series of histidine-containing peptides by optimizing pH-sensitivity, net charge, hydrophobicity, and charge distribution in the sequence of the CPP NF55. In the current work, we have applied a strategy where we have replaced amino acids in the C-terminus of the peptide in order to distribute hydrophobic and hydrophilic amino acids into distinct regions along the alpha-helical projection, including histidine amino acids into the sequence at the N-terminus, and optimizing the N-terminal fatty acid modification to suit the specific peptide sequence. We tested the CPPs based on the transfection efficacy, CPP/siRNA complex stability, and the properties of the CPPs, such as hemolytic activity, buffering capability and cell toxicity. As a result, we have introduced a new peptide with a completely redesigned N-terminus that displays adaptive response to its physical environment. This peptide – NickFect70 (NF70) – efficiently condenses siRNA, protects the cargo against degradation and effectively mediates target gene knockdown both in mammalian cell culture and in vivo, in a mouse model.

Molecular Driving Forces in Peptide Adsorption to Metal Oxide Surfaces.

Abstract: Molecular recognition between peptides and metal oxide surfaces is a fundamental process in biomineralization, self-assembly, and biocompatibility. Yet, the underlying driving forces and dominant mechanisms remain unclear, bringing obstacles to understand and control this process. To elucidate the mechanism of peptide/surface recognition, specifically the role of serine phosphorylation, we employed molecular dynamics simulation and metadynamics-enhanced sampling to study five artificial peptides, DDD, DSS, DpSpS, DpSpSGKK, and DpSKGpSK, interacting with two surfaces: rutile TiO2 and quartz SiO2. On both surfaces, we observe that phosphorylation increases the binding energy. However, the interfacial peptide conformation reveals a distinct binding mechanism on each surface. We also study the impact of peptide sequence to binding free energy and interfacial conformation on both surfaces, specifically the impact on the behavior of phosphorylated serine. Finally, the results are discussed in context of prior studies investigating the role of serine phosphorylation in peptide binding to silica.

Peptide and protein mimetics by retro and retroinverso analogs

Abstract: Retroinverso analog of a natural polypeptide can sometimes mimic the structure and function of the natural peptide. The additional advantage of using retroinverso analog is that it is resistant to proteolysis. The retroinverso analogs have peptide sequence in reverse direction with respect to natural peptide and also have chirality of amino acid inverted from L to D. The D amino acids cannot be recognized by common proteases of the body; therefore, these peptides will not be degraded easily and have a longer-lasting effect as vaccine and inhibitor drugs. There have been many contested propositions about the geometric relationship between a peptide and its retro, inverso, or retroinverso analog. A retroinverso analog sometimes fails to adopt the structure that can mimic the function of the natural peptide. In such cases, partial retroinverso analog and other modifications can help in achieving the desired structure and function. Here, we review the theory, major experimental attempts, prediction methods, and alternative strategies related to retroinverso peptidomimetics.

Proline- and Arginine-Rich Peptides as Flexible Allosteric Modulators of Human Proteasome Activity.

Abstract: Proline- and arginine-rich peptide PR11 is an allosteric inhibitor of 20S proteasome. We modified its sequence inter alia by introducing HbYX, RYX, or RHbX C-terminal extensions (Hb, hydrophobic moiety; R, arginine; Y, tyrosine; X, any residue). Consequently, we were able to improve inhibitory potency or to convert inhibitors into strong activators: the former with an aromatic penultimate Hb residue and the latter with the HbYX motif. The PR peptide activator stimulated 20S proteasome in vitro to efficiently degrade protein substrates, such as α-synuclein and enolase, but also activated proteasome in cultured fibroblasts. The positive and negative PR modulators differently influenced the proteasome conformational dynamics and affected opening of the substrate entry pore. The resolved crystal structure showed PR inhibitor bound far from the active sites, at the proteasome outer face, in the pocket used by natural activators. Our studies indicate the opportunity to tune proteasome activity by allosteric reg...

Discovery of novel fungal RiPP biosynthetic pathways and their application for the development of peptide therapeutics

Abstract: Bioactive peptide natural products are an important source of therapeutics. Prominent examples are the antibiotic penicillin and the immunosuppressant cyclosporine which are both produced by fungi and have revolutionized modern medicine. Peptide biosynthesis can occur either non-ribosomally via large enzymes referred to as non-ribosomal peptide synthetases (NRPS) or ribosomally. Ribosomal peptides are synthesized as part of a larger precursor peptide where they are posttranslationally modified and subsequently proteolytically released. Such peptide natural products are referred to as ribosomally synthesized and posttranslationally modified peptides (RiPPs). Their biosynthetic pathways have recently received a lot of attention, both from a basic and applied research point of view, due to the discoveries of several novel posttranslational modifications of the peptide backbone. Some of these modifications were so far only known from NRPSs and significantly increase the chemical space covered by this class of peptide natural products. Latter feature, in combination with the promiscuity of the modifying enzymes and the genetic encoding of the peptide sequence, makes RiPP biosynthetic pathways attractive for synthetic biology approaches to identify novel peptide therapeutics via screening of de novo generated peptide libraries and, thus, exploit bioactive peptide natural products beyond their direct use as therapeutics. This review focuses on the recent discovery and characterization of novel RiPP biosynthetic pathways in fungi and their possible application for the development of novel peptide therapeutics.

A New Searching Strategy for the Identification of O-Linked Glycopeptides.

Abstract: For the analysis of homogeneous post-translational modifications such as protein phosphorylation and acetylation, setting a variable modification on the specific residue(s) is applied to identify the modified peptides for database searching. However, this approach is often not applicable to identify intact mucin-type O-glycopeptides due to the high microheterogeneity of the glycosylation. Because there is virtually no carbohydrate-related tag on the peptide fragments after the O-glycopeptides are dissociated in HCD, we find it is unnecessary to set the variable mass tags on the Ser/Thr residues to identify the peptide sequences. In this study, we present a novel approach, termed as O-Search, for the interpretation of O-glycopeptide HCD spectra. Instead of setting the variable mass tags on the Ser/Thr residues, we set variable mass tags on the peptide level. The precursor mass of the MS/MS spectrum was deducted by every possible summed mass of O-glycan combinations on at most three S/T residues. All the sp...