Position Of Peptide Research Articles

Nanopore-Based High Resolution Detection of Multiple Post-Translational Modifications in Proteins.

Protein post-translational modifications (PTMs) play crucial roles in various cellular processes. Despite their significance, only a few PTMs have been extensively studied at the proteome level, primarily due to the scarcity of reliable, convenient, and low-cost sensing methods. Here, we present a straightforward and effective strategy for detecting PTMs on short peptides through host-guest interaction-assisted nanopore sensing. Our results demonstrate that the identity of 13 types of PTMs in a specific position of a phenylalanine-containing peptide could be determined via current blockage during translocation of the peptide through α-hemolysin nanopores in the presence of cucurbit[7]uril. Furthermore, we extend this strategy by incorporating a short peptide into the probe, enabling the discrimination of various PTMs, positional isomers, and even multiple PTMs on the target peptide. With ongoing improvements, our method holds promise for practical applications in sensing PTMs in biologically relevant samples, offering an efficient alternative to traditional mass spectrometry approaches.

Thermal Responses and Cell Adhesive Properties of Glycosylated Jigsaw‐Shaped Self‐Assembling Peptides

ABSTRACTAs represented by the posttranslational modifications of proteins, the chemical modifications of side chains of peptide residues enable to regulate their physicochemical properties, conformation, and biological activities. Glycosylation is a main post‐translational modification, which allows for recognition by partner proteins to exert diverse function including increase in the conformational stability. Here, we studied the effects of glucose modification, which is a representative glycosylation sugar chains, to different positions of an amphiphilic peptide having a jigsaw‐shaped hydrophobic part (JigSAP). JigSAP forms a β‐sheet structure to self‐assemble into fibrous objects providing a hydrogel that can be used as a matrix for regenerative medicine. JigSAPs modified with glucose at the N‐terminus and middle portion were synthesized, and both of them self‐assemble in water to form hydrogels. The hydrogel composed of JigSAP containing the glucose group attached to the N‐terminus undergoes a gel‐to‐sol macroscopic phase transition upon heating, accompanied by a conformational change of JigSAP from the β‐sheet to an α‐helix structure. On the other hand, JigSAP with the glucose group in the middle, retained its β‐sheet structure even at high temperatures maintaining the gel state. Both glucose‐modified JigSAP fibers showed cell adhesion activity similar to that of non‐glycosylated JigSAP. This suggests that the glycosylation is effective in regulating the thermal stability of JigSAP fibers while maintaining their extracellular matrix properties depending on the modification site.

Tyrosine-modified tilapia skin antioxidant peptides and their hydroxyl radical quenching activities.

In an antioxidant peptide study, the number and position of active amino acid sites, as well as the peptides' conformation, are found to be crucial for scavenging hydroxyl radicals (˙OH). Herein, ˙the OH scavenging activity of tilapia pentapeptide (P1, YGDQY) and its analogs including P2 (YYYGDQY), P3 (YYGDQYY) and P4 (YYGPDQYY) was investigated. The results showed that the tyrosine's amount, location and the peptides' conformation played important roles in determining peptides' scavenging activity (34.1 ± 0.8%, 45.1 ± 0.9%, 58.6 ± 1.3% and 48.4 ± 0.96% for P1, P2, P3, and P4, respectively). Density functional theory simulation showed that only the tyrosine sites located within the effective diffusion distance of ˙OH could scavenge the radical. The peptides did not cause cytotoxicity in Caco-2 cells. And the peptide-treated group could increase the activities of glutathione peroxidase (GSH-PX), catalase (CAT) and superoxide dismutase (SOD), and reduced malondialdehyde (MDA) levels. This work may contribute to designing more active antioxidant peptides based on natural peptides' analogs.

B12-Dependent Radical SAM Enzymes Catalyze C-Fluoromethylation via a CH2F-Cobalamin Intermediate.

Fluorine and fluorine-containing functional groups play important roles in drugs and agrochemicals. Recently, SAM-dependent methyltransferases and several SAM analogues have been reported for fluoromethyl transfer through a nucleophilic mechanism. However, fluoromethylation of unactivated carbon centers is very challenging, and their substitution usually involves a radical mechanism. To date, no biocatalysts have been developed for fluoromethylation of unactivated carbon centers. In this study, we found that the B12-dependent radical SAM methyltransferase (B12-RSMT) QCMT can fluoromethylate the glutamine Cα position of peptides with fluorinated SAM (F-SAM) generated in situ by the enzyme AclHMT. QCMT can cleave F-SAM to produce the 5'-dA radical. The significant reaction intermediate CH2FCbI was characterized by HR-MS, 19F NMR spectroscopy and X-ray crystallography. In addition, B12-RSMTs CysS and GenD1 can also transfer fluoromethyl groups onto natural products. We also found that F-SAM is not compulsory. The reduced B12-RSMTs can directly generate CH2FCbI with CH2FI and transfer the CH2F group when SAM is used as the radical initiator. Our results demonstrate a radical-mediated enzymatic strategy for fluoromethylation with abiological cofactors and expand radical SAM enzymes to the field of fluorine chemistry.

Engineering GID4 for use as an N-terminal proline binder via directed evolution.

Nucleic acid sequencing technologies have gone through extraordinary advancements in the past several decades, significantly increasing throughput while reducing cost. To create similar advancement in proteomics, numerous approaches are being investigated to advance protein sequencing. One of the promising approaches uses N-terminal amino acid binders (NAABs), also referred to as recognizers, that selectively can identify amino acids at the N-terminus of a peptide. However, there are only a few engineered NAABs currently available that bind to specific amino acids and meet the requirements of a biotechnology reagent. Therefore, additional NAABs need to be identified and engineered to enable confident identification and, ultimately, de novo protein sequencing. To fill this gap, a human protein GID4 was engineered to create a NAAB for N-terminal proline (Nt-Pro). While native GID4 binds Nt-Pro, its binding is weak (µmol/L) and greatly influenced by the identity of residues following the Nt-Pro. Through directed evolution, yeast-surface display, and fluorescence-activated cell sorting, we identified sequence variants of GID4 with increased binding response to Nt-Pro. Moreover, variants with an A252V mutation showed a reduced influence from residues in the second and third positions of the target peptide when binding to Nt-Pro. The workflow outlined here is shown to be a viable strategy for engineering NAABs, even when starting from native Nt-binding proteins whose binding is strongly impacted by the identity of residues following Nt-amino acid.

The Wheel of Fortune: Helical Wheel Alanine Scanning of a Spider Venom Antimicrobial Peptide Reveals Residues Involved in Antimicrobial and Cytotoxic Activity.

A preference for several amino acids is observed to occur at particular positions of cationic α-helical antimicrobial peptides (AMPs), which ensures the formation of amphipathic regions once they assume their correct secondary structure in membranes or membrane-mimicking environments and makes them active against pathogens. This study determined the effect of alanine mutations on the secondary structure and bioactivity of lyp1987 (GRLQAFLAKMKEIAAQTL-NH2), a cationic α-helical AMP obtained from the venom of Lycosa poonaensis which exhibits broad range activity against Gram-positive and Gram-negative bacteria with micromolar minimum inhibitory concentrations (MIC). CD spectroscopy revealed no significant difference in the secondary structure, with all alanine-substituted analogs exhibiting predominantly α-helical structure in buffered 2,2,2-trifluoroethanol solution. Alanine substitution at Glu12 and Thr17 increased the activity of lyp1987 against Gram-positive and -negative bacteria, while alanine substitution at Lys9 increased its selectivity against Gram-positive bacteria. Further investigation can be done to determine positions and substitutions that will give less cytotoxic analogs.

Modulation of SpyCatcher Ligation Kinetics by SpyTag Thioamide Substitution.

Thioamide substitutions have been shown to impart valuable properties on peptides for biophysical experiments as well as cell or in vivo studies, but a rational understanding of thioamide effects on protein structure and protein-protein interactions is lacking. To elucidate their effects in β-sheet structures, we have used SpyCatcher003-SpyTag003 as a host-guest system to study individual thioamide incorporation at eight different positions in the SpyTag peptide. We have demonstrated that incorporating thioamides into SpyTag at specific positions can result in a ∼2-fold faster ligating complex, as well as >2000-fold slower ligating complex. Biophysical analysis and structural modeling provide a reasonable explanation for most of the thioamide effects, altering hydrogen bond networks as well as modulating an n→π* interaction within the SpyTag peptide. Our findings have important implications for potential applications of thioamide SpyTag variants, where the thioamide could impart protease stability in cells while also controlling the rate of ligation to SpyCatcher. These SpyCatcher-SpyTag host-guest experiments will also help to build a database for predicting thioamide effects on protein structure and function.

Residue profiles of peptides with cholesterol esterase and pancreatic lipase inhibitory activities through virtual screening and sequence analysis

The detailed characterization of the structural features of peptides targeting cholesterol esterase (CEase) or pancreatic lipase (PPL) will benefit the management of hyperlipidemia and obesity. This study employed the Glide SP (standard precision)-peptide method to predict the binding modes of 202 dipeptides and 203 tripeptides to these targets, correlating residue composition and position with binding energy. Strong preferences for Trp, Phe, and Tyr were observed at all positions of potential inhibitory peptides, whereas negatively charged residues Glu and Asp were disfavored. Notably, Arg and aromatic rings significantly influenced the peptide conformation at the active site. Tripeptide IWR demonstrated the high efficacy, with IC50 values of 0.214 mg/mL for CEase and 0.230 mg/mL for PPL. Five novel IWR scaffold-tetrapeptides exhibited promising inhibitory activity. Non-covalent interactions and energy contributions dominated the formation of stable complexes. Our results provide insights for the development of new sequences or peptide-like molecules with enhanced inhibitory activity.

Postassembly Modification of Peptides by Histidine-Directed β-C(sp3)-H Arylation of Alanine at the Internal Positions: Overcoming the Inhibitory Effect of Peptide Bonds.

Peptide modification by C(sp3)-H functionalization of residues at the internal positions remains underdeveloped due to the inhibitory effect of backbone amides. In this study, using histidine (His) as an endogenous directing group, we developed a novel method for the β-C(sp3)-H functionalization of alanine (Ala) at diverse positions of peptides. Through this approach, a wide range of linear peptides were modified on the side-chain of Ala adjacent to His to afford the functionalized peptides in moderate to good yield and excellent position selectivity. Furthermore, conjugation of peptides with functional molecules such as glucuronide, oleanolic acid, dipeptide, and fluorophore derivatives was achieved.

General Installation of (4H)-Imidazolone cis-Amide Bioisosteres Along the Peptide Backbone.

Imidazolones represent an important class of heterocycles present in a wide range of pharmaceuticals, metabolites, and bioactive natural products and serve as the active chromophore in green fluorescent protein. Recently, imidazolones have received attention for their ability to act as a nonaromatic amide bond bioisotere which improves pharmacological properties. Herein, we present a tandem amidine installation and cyclization with an adjacent ester to yield (4H)-imidazolone products. Using amino acid building blocks, we can access the first examples of α-chiral imidazolones that have been previously inaccessible. Additionally, our method is amenable to on-resin installation which can be seamlessly integrated into existing solid-phase peptide synthesis protocols. Finally, we show that peptide imidazolones are potent cis-amide bond surrogates that preorganize linear peptides for head-to-tail macrocyclization. This work represents the first general approach to the backbone and side-chain insertion of imidazolone bioisosteres at various positions in linear and cyclic peptides.

Lineage-specific gene duplication and expansion of DUF1216 gene family in Brassicaceae.

Proteins containing domain of unknown function (DUF) are prevalent in eukaryotic genome. The DUF1216 proteins possess a conserved DUF1216 domain resembling to the mediator protein of Arabidopsis RNA polymerase II transcriptional subunit-like protein. The DUF1216 family are specifically existed in Brassicaceae, however, no comprehensive evolutionary analysis of DUF1216 genes have been performed. We performed a first comprehensive genome-wide analysis of DUF1216 proteins in Brassicaceae. Totally 284 DUF1216 genes were identified in 27 Brassicaceae species and classified into four subfamilies on the basis of phylogenetic analysis. The analysis of gene structure and conserved motifs revealed that DUF1216 genes within the same subfamily exhibited similar intron/exon patterns and motif composition. The majority members of DUF1216 genes contain a signal peptide in the N-terminal, and the ninth position of the signal peptide in most DUF1216 is cysteine. Synteny analysis revealed that segmental duplication is a major mechanism for expanding of DUF1216 genes in Brassica oleracea, Brassica juncea, Brassica napus, Lepidium meyneii, and Brassica carinata, while in Arabidopsis thaliana and Capsella rubella, tandem duplication plays a major role in the expansion of the DUF1216 gene family. The analysis of Ka/Ks (non-synonymous substitution rate/synonymous substitution rate) ratios for DUF1216 paralogous indicated that most of gene pairs underwent purifying selection. DUF1216 genes displayed a specifically high expression in reproductive tissues in most Brassicaceae species, while its expression in Brassica juncea was specifically high in root. Our studies offered new insights into the phylogenetic relationships, gene structures and expressional patterns of DUF1216 members in Brassicaceae, which provides a foundation for future functional analysis.

Dual FGFR-targeting and pH-activatable ruthenium-peptide conjugates for targeted therapy of breast cancer.

Dysregulation of Fibroblast Growth Factor Receptors (FGFRs) signaling has been associated with breast cancer, yet employing FGFR-targeted delivery systems to improve the efficacy of cytotoxic agents is still sparsely exploited. Herein, we report four new bi-functional ruthenium-peptide conjugates (RuPCs) with FGFR-targeting and pH-dependent releasing abilities, envisioning the selective delivery of cytotoxic Ru complexes to FGFR(+)-breast cancer cells, and controlled activation at the acidic tumoral microenvironment. The antiproliferative potential of the RuPCs and free Ru complexes was evaluated in four breast cancer cell lines with different FGFR expression levels (SKBR-3, MDA-MB-134-VI, MCF-7, and MDA-MB-231) and in human dermal fibroblasts (HDF), at pH 6.8 and pH 7.4 aimed at mimicking the tumor microenvironment and normal tissues/bloodstream pHs, respectively. The RuPCs showed higher cytotoxicity in cells with higher level of FGFR expression at acidic pH. Additionally, RuPCs showed up to 6-fold higher activity in the FGFR(+) breast cancer lines compared to the normal cell line. The release profile of Ru complexes from RuPCs corroborates the antiproliferative effects observed. Remarkably, the cytotoxicity and releasing ability of RuPCs were shown to be strongly dependent on the conjugation of the peptide position in the Ru complex. Complementary molecular dynamic simulations and computational calculations were performed to help interpret these findings at the molecular level. In summary, we identified a lead bi-functional RuPC that holds strong potential as a FGFR-targeted chemotherapeutic agent.

Virtual screening and structure optimization of xanthine oxidase inhibitory peptides from whole protein sequences of Pacific white shrimp via molecular docking.

Xanthine oxidase (XO) inhibitory peptides are safer than conventional pharmacological therapy in relieving hyperuricemia. However, traditional enzymatic hydrolysis, separation, and purification techniques for bio-active peptide preparation are time-consuming, inefficient, and labor-intensive. In this study, molecular docking and BLAST were used to virtually screen XO inhibitory peptides from whole protein sequences of Pacific white shrimp according to the bio-active peptides database, and the structure of peptides was optimized based on the structure-effective relationship. Seven new XO inhibitory peptides were virtual screened rapidly from Pacific white shrimp, and YNITGW (IC50=9.78±0.13mM) showed the strongest activity. The results of YNITGW optimization showed that the insertion of Trp residue in the middle position of peptides could effectively enhance the activity. This study revealed that screening and optimizing peptides by molecular docking were a novel and feasible method to obtain bio-active peptides.

Electrostatics-Based Computational Design and Experimental Analysis of Buforin II Antimicrobial Peptide Variants with Increased DNA Affinities.

Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics in the treatment of bacterial infections in part due to their targeting of generic bacterial structures that make it more difficult to develop drug resistance. In this study, we introduce and implement a design workflow to develop more potent AMPs by improving their electrostatic interactions with DNA, which is a putative intracellular target. Using the existing membrane-translocating AMP buforin II (BF2) as a starting point, we use a computational workflow that integrates electrostatic charge optimization, continuum electrostatics, and molecular dynamics simulations to suggest peptide positions at which a neutral BF2 residue could be substituted with arginine to increase DNA-binding affinity either significantly or minimally, with the latter choice done to determine whether AMP binding affinity depends on charge distribution and not just overall monopole. Our analyses predicted that T1R and L8R BF2 variants would yield substantial and minimal increases in DNA-binding affinity, respectively. These predictions were validated with experimental peptide-DNA binding assays with additional computational analyses providing structural insights. Additionally, experimental measurements of antimicrobial potency showed that a design to increase DNA binding can also yield greater potency. As a whole, this study takes initial steps to support the idea that (i) a design strategy aimed to increase AMP binding affinity to DNA by focusing only on electrostatic interactions can improve AMP potency and (ii) the effect on DNA binding of increasing the overall peptide monopole via arginine substitution depends on the position of the substitution. More broadly, this design strategy is a novel way to increase the potency of other membrane-translocating AMPs that target nucleic acids.

Leader peptide removal in lasso peptide biosynthesis based on penultimate isoleucine residue.

Lasso peptides are ribosomally synthesized peptides that undergo post-translational modifications including leader peptide removal by B (or the segregated B1 and B2) proteins and core peptide macrolactamization by C proteins to form a unique lariat topology. A conserved threonine residue at the penultimate position of leader peptide is hitherto found in lasso peptide precursors and shown to be a critical recognition element for effective enzymatic processing. We identified a lasso peptide biosynthetic gene cluster (bsf) from Bradymonas sediminis FA350, a Gram-negative and facultatively prey-dependent bacterium that belongs to a novel bacterial order Bradymonadales in the class Deltaproteobacteria. The kinase BsfK specifically catalyzes the phosphorylation of the precursor peptide BsfA on the Ser3 residue. BsfB1 performs dual functions to accelerate the post-translational phosphorylation and assist BsfB2 in leader peptide removal. Most importantly, the penultimate residue of leader peptide is an isoleucine rather than the conserved threonine and this isoleucine has a marked impact on the phosphorylation of Ser3 as well as leader peptide removal, implying that BsfB1 and BsfB2 exhibit a new substrate selectivity for leader peptide binding and excision. This is the first experimentally validated penultimate isoleucine residue in a lasso peptide precursor to our knowledge. In silico analysis reveals that the leader peptide Ile/Val(-2) residue is rare but not uncommon in phosphorylated lasso peptides, as this residue is also discovered in Acidobacteriaceae and Sphingomonadales in addition to Bradymonadales.

Design of Multicomponent Peptide Fibrils with Ordered and Programmable Compositional Patterns.

Advanced applications of biomacromolecular assemblies require a stringent degree of control over molecular arrangement, which is a challenge to current synthetic methods. Here we used a neighbor-controlled patterning strategy to build multicomponent peptide fibrils with an unprecedented capacity to manipulate local composition and peptide positions. Eight peptides were designed to have regulable nearest neighbors upon co-assembly, which, by simulation, afforded 412 different patterns within fibrils, with varied compositions and/or peptide positions. The fibrils with six prescribed patterns were experimentally constructed with high accuracy. The controlled patterning also applies to functionalities appended to the peptides, as exemplified by arranging carbohydrate ligands at nanoscale precision for protein recognition. This study offers a route to molecular editing of inner structures of peptide assemblies, prefiguring the uniqueness and richness of patterning-based material design.

Design of Multicomponent Peptide Fibrils with Ordered and Programmable Compositional Patterns

AbstractAdvanced applications of biomacromolecular assemblies require a stringent degree of control over molecular arrangement, which is a challenge to current synthetic methods. Here we used a neighbor‐controlled patterning strategy to build multicomponent peptide fibrils with an unprecedented capacity to manipulate local composition and peptide positions. Eight peptides were designed to have regulable nearest neighbors upon co‐assembly, which, by simulation, afforded 412 different patterns within fibrils, with varied compositions and/or peptide positions. The fibrils with six prescribed patterns were experimentally constructed with high accuracy. The controlled patterning also applies to functionalities appended to the peptides, as exemplified by arranging carbohydrate ligands at nanoscale precision for protein recognition. This study offers a route to molecular editing of inner structures of peptide assemblies, prefiguring the uniqueness and richness of patterning‐based material design.

Computational prediction of MHC anchor locations guides neoantigen identification and prioritization.

Neoantigens are tumor-specific peptide sequences resulting from sources such as somatic DNA mutations. Upon loading onto major histocompatibility complex (MHC) molecules, they can trigger recognition by T cells. Accurate neoantigen identification is thus critical for both designing cancer vaccines and predicting response to immunotherapies. Neoantigen identification and prioritization relies on correctly predicting whether the presenting peptide sequence can successfully induce an immune response. Because most somatic mutations are single-nucleotide variants, changes between wild-type and mutated peptides are typically subtle and require cautious interpretation. A potentially underappreciated variable in neoantigen prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patient's specific MHC molecules. Whereas a subset of peptide positions are presented to the T cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T cell responses. We computationally predicted anchor positions for different peptide lengths for 328 common HLA alleles and identified unique anchoring patterns among them. Analysis of 923 tumor samples shows that 6 to 38% of neoantigen candidates are potentially misclassified and can be rescued using allele-specific knowledge of anchor positions. A subset of anchor results were orthogonally validated using protein crystallography structures. Representative anchor trends were experimentally validated using peptide-MHC stability assays and competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, we hope to formalize, streamline, and improve the identification process for relevant clinical studies.

Additive energetic contributions of multiple peptide positions determine the relative promiscuity of viral and human sequences for PDZ domain targets.

Protein-protein interactions that involve recognition of short peptides are critical in cellular processes. Protein-peptide interaction surface areas are relatively small and shallow, and there are often overlapping specificities in families of peptide-binding domains. Therefore, dissecting selectivity determinants can be challenging. PDZ domains are a family of peptide-binding domains located in several intracellular signaling and trafficking pathways. These domains are also directly targeted by pathogens, and a hallmark of many oncogenic viral proteins is a PDZ-binding motif. However, amidst sequences that target PDZ domains, there is a wide spectrum in relative promiscuity. For example, the viral HPV16 E6 oncoprotein recognizes over double the number of PDZ domain-containing proteins as the cystic fibrosis transmembrane conductance regulator (CFTR) in the cell, despite similar PDZ targeting-sequences and identical motif residues. Here, we determine binding affinities for PDZ domains known to bind either HPV16 E6 alone or both CFTR and HPV16 E6, using peptides matching WT and hybrid sequences. We also use energy minimization to model PDZ-peptide complexes and use sequence analyses to investigate this difference. We find that while the majority of single mutations had marginal effects on overall affinity, the additive effect on the free energy of binding accurately describes the selectivity observed. Taken together, our results describe how complex and differing PDZ interactomes can be programmed in the cell.

Alanine-based spacers promote an efficient antigen processing and presentation in neoantigen polypeptide vaccines

Neoantigens are tumor-specific antigens that are mostly particular for each patient. Since the immune system is able to mount a specific immune response against these neoantigens, they are a promising tool for the development of therapeutic personalized cancer vaccines. Neoantigens must be presented to T cells by antigen presenting cells (APC) in the context of MHC-I or MHC-II molecules. Therefore, the strategy of vaccine delivery may have a major impact on the magnitude and quality of T cell responses. Neoantigen-based vaccines are frequently administered as a pool of individual synthetic peptides that induce mainly CD4+ T cell responses. MHC-I-mediated presentation and the elicitation of CD8+ T cell responses may be improved using DNA or RNA sequences that code for a unique long polypeptide that concatenates the different neoantigens spaced by linker sequences. When administered this way, the selection of the spacer between neoantigens is of special interest, as it might influence the processing and presentation of the right peptides by APCs. Here, we evaluate the impact of such linker regions on the MHC-I-dependent antigen presentation using an in vitro assay that assesses the MHC-I presentation of SIINFEKL, a H-2 Kb-restricted OVA peptide. Our results show that spacers used to generate epitope concatenates have a large impact on the efficiency of neoantigen processing and presentation by MHC-I molecules; in contrast, the peptide position and the flanking regions have a minimal impact. Moreover, linkers based on alanine residues promote a more efficient peptide presentation than the commonly used GGGS linker.