Peptide Segments Research Articles

Novel Covalent Modifier-Induced Local Conformational Changes within the Intrinsically Disordered Region of the Androgen Receptor.

Intrinsically disordered regions (IDRs) of transcription factors play an important biological role in liquid condensate formation and gene regulation. It is thus desirable to investigate the druggability of IDRs and how small-molecule binders can alter their conformational stability. For the androgen receptor (AR), certain covalent ligands induce important changes, such as the neutralization of the condensate. To understand the specificity of ligand-IDR interaction and potential implications for the mechanism of neutralizing liquid-liquid phase separation (LLPS), we modeled and performed computer simulations of ligand-bound peptide segments obtained from the human AR. We analyzed how different covalent ligands affect local secondary structure, protein contact map, and protein-ligand contacts for these protein systems. We find that effective neutralizers make specific interactions (such as those between cyanopyrazole and tryptophan) that alter the helical propensity of the peptide segments. These findings on the mechanism of action can be useful for designing molecules that influence IDR structure and condensate of the AR in the future.

Mirror-Image Single-Domain Antibody for a Novel Nonimmunogenic Drug Scaffold.

Immunogenic responses by protein therapeutics often lead to reduced therapeutic effects and/or adverse effects via the generation of neutralizing antibodies and/or antidrug antibodies (ADA). Mirror-image proteins of the variable domain of the heavy chain of the heavy chain antibody (VHH) are potential novel protein therapeutics with high-affinity binding to target proteins and reduced immunogenicity because these mirror-image VHHs (d-VHHs) are less susceptible to proteolytic degradation in antigen-presenting cells (APCs). In this study, we investigated the preparation protocols of d-VHHs and their biological properties, including stereoselective target binding and immunogenicity. Initially, we established a facile synthetic process of two model VHHs [anti-GFP VHH and PMP12A2h1 (monomeric VHH of caplacizumab)] and their mirror-image proteins by three-step native chemical ligations (NCLs) from four peptide segments. The folded synthetic VHHs (l-anti-GFP VHH and l-PMP12A2h1) bound to the target proteins (EGFP and vWF-A1 domain, respectively), while their mirror-image proteins (d-anti-GFP VHH and d-PMP12A2h1) showed no binding to the native proteins. For biodistribution studies, l-VHH and d-VHH with single radioactive indium diethylenetriamine-pentaacid (111In-DTPA) labeling at the C-terminus were designed and synthesized by the established protocol. The distribution profiles were essentially similar between l-VHH and d-VHH, in which the probes accumulated in the kidney within 15 min after intravenous administration in mice, because of the small molecular size of VHHs. Comparative assessment of the immunogenicity responses revealed that d-VHH-induced levels of ADA generation were significantly lower than those of native VHH, regardless of the peptide sequences and administration routes. The resulting scaffold investigated should be applicable in the design of d-VHHs with various C-terminal CDR3 sequences, which can be identified by screening using display technologies.

Antihypertensive Effect, ACE Inhibitory Activity, and Stability of Umami Peptides from Yeast Extract.

Bioactive peptides from foods have garnered considerable attention as viable supplements for hypertensive patients. Herein, the antihypertensive effect and mechanism of umami peptides from yeast extract were investigated based on the pharmacophore model, simulated digestion, spontaneously hypertensive rat (SHR) model, and molecular docking. Notably, umami peptide LLLLPKP exhibited favorable angiotensin I-converting enzyme (ACE) inhibitory activity (IC50 = 10.22 μM) in vitro and regulated blood pressure in the SHR model with excellent durability. Remarkably, LLLLPKP showed the highest Fitvalue (4.022) of the pharmacophore model, indicating its similar pharmacological effects as ACE inhibitors. During the simulated gastrointestinal digestion, the ACE inhibition rate of LLLLPKP was merely reduced by 5.89%, but it was enzymatically cleaved into 14 peptide segments. The C-terminal sequence comprising L (4), P (5), K (6), and P (7) exhibited robust stability and a notable presence within the peptide segments postdigestion. Meanwhile, according to molecular docking, these four residues within LLLLPKP were responsible for all interactions with key sites within active pockets S1 and S2 and the active pocket of Zn2+. In light of these findings, LLLLPKP is a highly promising antihypertensive peptide. Developing this umami peptide with antihypertensive effects holds substantial importance for the long-term treatment of hypertension.

Peptiligase, an enzyme for efficient chemo-enzymatic synthesis of aviptadil

Increasing attention to peptides as prospective therapeutics has created a renaissance in searching for new alternatives to the current peptide synthetic approaches as well as their modification. In this context, it is necessary to develop different approaches for peptide ligation. Using enzymes as a novel strategy and powerful tool for the peptide and protein ligation has recently received a lot of attention. We here designed a fully convergent chemo-enzymatic peptide synthesis (CEPS) process for the synthesis of aviptadil a 28-mer therapeutic peptide with potential therapeutic effects in various medical contexts specially in the treatment of acute respiratory distress syndrome (ARDS) by coupling two peptide segments with four different peptiligase variants in aqueous environments. Our study reveals that peptiligase variants are capable of ligation reaction in 15 min. The overall time of ligation is shorter than those peptides with similar lengths and hinderance to aviptadil which reported for conventional synthesis by full solid-phase peptide synthesis. Yields ranged from 54 % to 76 %.

Construction of Tumor Microenvironment‐Responsive Gene Carriers

AbstractPeptide‐ and polypeptide‐based self‐assembling gene delivery systems have received considerable attention owing to their inherent biocompatibility and bioactivity. Gene carriers based on elastin‐like polypeptides (ELPs) have been extensively studied because of their controllability and unique temperature responsiveness. The (EK)10‐PLGLAG‐Tat polypeptide sequence was selected for tumor gene delivery, with ELP serving as the hydrophobic core. In this sequence, a hydration layer can be formed on the surface of the carrier using the zwitterionic peptide segment (EK)10, which helps prevent the nonspecific adsorption of plasma proteins. Additionally, the MMP‐2 enzyme‐responsive PLGLAG peptide segment is responsible for exposing the cell‐penetrating peptide Tat specifically near tumor cells, facilitating the penetration of tumor cells. To introduce (EK)10‐PLGLAG‐Tat into the self‐assembling carrier while ensuring its bioactivity, a leucine zipper ZR/ZE with opposite charges was used to link it to the ELP. Because of its high specificity and low systemic toxicity, the carrier was named environmentally responsive gene carrier (ERGV). Experimental results demonstrated that the ERGV effectively removed (EK)10 in MMP‐2 overexpressed environments, altering the surface charge from negative to positive and facilitating ssDNA delivery into tumor cells. These findings highlight the potential of ERGVs as a safe and efficient method for targeted gene delivery to tumors.

N-substituting perturbation on the interaction affinity and recognition specificity between rheumatic immune-related Abl SH3 domain and its peptoid ligands

Abl is a nonreceptor tyrosine kinase involved in a variety of disease pathways such as rheumatic immune. Full-length Abl protein consists of a catalytic tyrosine kinase (TK) domain as well as two regulatory Src homology domains 2 and 3 (SH2 and SH3, respectively); the latter recognizes and binds to those natural proline-rich peptide segments containing a PxxP motif on the protein surface of its interacting partners. However, natural peptides cannot bind effectively to the modular domain in high affinity and strong selectivity due to their small size and broad specificity. Here, a synthetic proline-rich peptide p41 was used as template; its structural diversity was extended by combinationally replacing the Pro0 and Pro+3 residues with a number of N-substituted amino acids. Consequently, peptide affinity change upon the replacement was derived to create a systematic N-substituting perturbation profile, from which we identified several N-substitution combinations at the Pro0 and Pro+3 residues of p41 PxxP motif that may moderately or significantly improve the peptide binding potency to Abl; they represent potent peptoid binders of Abl SH3 domain, with affinity improved considerably relative to p41. More significantly, the designed potent peptoids were also found to exhibit a good SH3-selectivity for their cognate Abl over other noncognate nonreceptor tyrosine kinases, with S = 9.7-fold.

Development of novel isobaric tags enables accurate and sensitive multiplexed proteomics using complementary ions.

High-throughput quantitative analysis of the cells' proteomes across multiple conditions such as various perturbations and different time points is essential for gaining insights into treatment-induced biological responses or disease pathological states. The advancements in mass spectrometry instrumentation and isobaric labeling methods provided useful tools to help address such demands. However, the current widely adopted isobaric labeling methods such as tandem mass tag (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ) are based on low-mass reporter ions, which are indistinguishable among different peptide analytes, to achieve relative quantification. Therefore, these methods intrinsically suffer from severe ratio distortion when analyzing complex samples due to peptide coelution and cofragmentation. Here, we developed a novel set of isobaric tags named dimethylated leucine complementary ion (DiLeuC) and relied on complementary ions for relative quantification, in which the complementary ions are the remanent peptide segments after fragmentation in the high-mass range. Since those residual peptide fragments are precursor-specific, they retain the relative abundance information in an interference-free manner even in a complex matrix environment. The quantification accuracy of our method was validated in a two-proteome model where the yeast proteome was spiked with a strong background human proteome as interference. In addition, we also applied this strategy to single-cell proteome analysis, demonstrating its potential utility for sensitive high-throughput quantitative proteomics.

Polymorphic amyloid nanostructures of hormone peptides involved in glucose homeostasis display reversible amyloid formation

A large group of hormones are stored as amyloid fibrils in acidic secretion vesicles before they are released into the bloodstream and readopt their functional state. Here, we identify an evolutionarily conserved hexapeptide sequence as the major aggregation-prone region (APR) of gastrointestinal peptides of the glucagon family: xFxxWL. We determine nine polymorphic crystal structures of the APR segments of glucagon-like peptides 1 and 2, and exendin and its derivatives. We follow amyloid formation by CD, FTIR, ThT assays, and AFM. We propose that the pH-dependent changes of the protonation states of glutamate/aspartate residues of APRs initiate switching between the amyloid and the folded, monomeric forms of the hormones. We find that pH sensitivity diminishes in the absence of acidic gatekeepers and amyloid formation progresses over a broad pH range. Our results highlight the dual role of short aggregation core motifs in reversible amyloid formation and receptor binding.

Fragments of local symmetry in a sequence of amino acids: Does one can use for QSPR/QSAR of peptides?

Most of the protein interactions rely on small domains binding to short peptides. However, neither the number of potential interactions mediated by each domain nor the degree of affinity at a whole proteome level has been studied. Peptide segments involved in 14–3–3 domain-medicated cellular signalling networks were collected from sequence-based datasets of domain-peptide interaction affinities. The affinities peptides measured represented by the Boehringer light units (logBLU) considered as the endpoint for the development of peptide quantitative structure-property/activity relationships (p-QSPR/QSARs). The sequences of amino acids are examined as the structure of the peptide. This approach allows the rational simulate interaction of proteomic systems via amino acid configurations in proteins. Adding the contributions of local symmetry and chaos extracted from the amino acid sequences described here increases the value of the average (over seven different splits into training and control) determination coefficient for the external validation set and reduces its dispersion.

Inhibitor Design Strategy for Myostatin: Dynamics and Interaction Networks Define the Affinity and Release Mechanisms of the Inhibited Complexes.

Myostatin, an important negative regulator of muscle mass, is a therapeutic target for muscle atrophic disorders such as muscular dystrophy. Thus, the inhibition of myostatin presents a strategy to treat these disorders. It has long been established that the myostatin prodomain is a strong inhibitor of the mature myostatin, and the minimum peptide of the prodomain-corresponding to the α1-helix of its lasso-region-responsible for the inhibitory efficiency was defined and characterized as well. Here we show that the minimum peptide segment based on the growth differentiation factor 11 (GDF11), which we found to be more helical in its stand-alone solvated stfate than the similar segment of myostatin, is a promising new base scaffold for inhibitor design. The proposed inhibitory peptides in their solvated state and in complex with the mature myostatin were analyzed by in silico molecule modeling supplemented with the electronic circular dichroism spectroscopy measurements. We defined the Gaussian-Mahalanobis mean score to measure the fraction of dihedral angle-pairs close to the desired helical region of the Ramachandran-plot, carried out RING analysis of the peptide-protein interaction networks and characterized the internal motions of the complexes using our rigid-body segmentation protocol. We identified a variant-11m2-that is sufficiently ordered both in solvent and within the inhibitory complex, forms a high number of contacts with the binding-pocket and induces such changes in its internal dynamics that lead to a rigidified, permanently locked conformation that traps this peptide in the binding site. We also showed that the naturally evolved α1-helix has been optimized to simultaneously fulfill two very different roles: to function as a strong binder as well as a good leaving group. It forms an outstanding number of non-covalent interactions with the mature core of myostatin and maintains the most ordered conformation within the complex, while it induces independent movement of the gate-keeper β-hairpin segment assisting the dissociation and also results in the least-ordered solvated form which provides extra stability for the dissociated state and discourages rebinding.

Backbone-Installed Split Intein-Assisted Ligation for the Chemical Synthesis of Mirror-Image Proteins.

Membrane-associated D-proteins are an important class of synthetic molecules needed for D-peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a Backbone-Installed Split Intein-Assisted Ligation (BISIAL) method for the synthesis of these proteins, wherein the native L-forms of the N- and C-intein fragments of the unique consensus-fast (Cfa) (i.e. L-CfaN and L-CfaC) are separately installed onto the two D-peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (μM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D-proteins without leaving any "ligation scar" on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D-enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D-protein targets.

Backbone‐Installed Split Intein‐Assisted Ligation for the Chemical Synthesis of Mirror‐Image Proteins

AbstractMembrane‐associated D‐proteins are an important class of synthetic molecules needed for D‐peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a Backbone‐Installed Split Intein‐Assisted Ligation (BISIAL) method for the synthesis of these proteins, wherein the native L‐forms of the N‐ and C‐intein fragments of the unique consensus‐fast (Cfa) (i.e. L–CfaN and L–CfaC) are separately installed onto the two D‐peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (μM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D‐proteins without leaving any “ligation scar” on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D‐enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D‐protein targets.

Biomimetic Sequence-Templating Approach toward a Multiscale Modulation of Chromogenic Polymer Properties.

Precision control via molecular structure over adaptive conjugated polymer properties in aqueous environments is critical for realizing their biomedical applications. Here, we unravel the dependence of amphiphilic peptide-polydiacetylene (PDA) conjugate properties on the characteristic steric and hydrophobic contributions within peptide segments that serve as a biomimetic template for diacetylene polymerization in water. We investigated the functional impacts of molecular volume and polarity changes brought by dipeptide substitution domains on the following peptide-PDA material properties at multiple length scales: supramolecular assembly behavior, chain conformation-dependent photophysical properties, cell-material interfacing, and for the first time, bulk electrical properties of their films processed in water. A library of peptide-PDAs with systematically varied sequences show that the contributions of steric effects predominantly influence the electronic structure and resulting trends in photophysical properties, while the interplay between size and hydrophobicity of individual residues becomes more significant for higher-order assemblies affecting bulk properties. This work demonstrates sequence-tunable molecular volume and polarity as synthetic handles to rationally modulate PDA material properties across length scales, providing insights into the programmability of biomimetic conjugated polymers with adaptive functionalities.

Hydrocarbon Stapling-Improved Coupled Folding-Upon-Binding of Peptide-Mediated Interaction between the Nucleocapsid and Phosphoprotein of Human Orthopneumovirus

Human orthopneumovirus (OPV) is a common respiratory virus that mostly causes respiratory illness in children. Development of therapeutic strategies to treat OPV infection has attracted considerable interest in the medicinal and biological communities. Over the past decades, significant effects have been addressed on disrupting membrane fusion events by targeting viral fusogenic glycoprotein (F-protein) with chemical small-molecule fusion inhibitors. Here, we focus on the rational design of biologic peptide inhibitors to disrupt the peptide-mediated interaction (PMI) between phosphoprotein (P-protein) and nucleoprotein (N-protein) involved in OPV genomic ribonucleoprotein complex. It is revealed that the core 9-mer peptide segment (C-peptide) derived from the C-terminal tail of P-protein is intrinsically disordered in an unbound state but would be structured into an ordered helical conformation when bound to N-protein, representing a biophysical process known as coupled folding-upon-binding. We demonstrated that the process is a compromise between the favorable enthalpy contribution and unfavorable entropy penalty, thus rendering the weak and transient nature of PMI, which could therefore be readily disrupted by competitive agents. Hydrocarbon stapling strategy was used to constrain the helical conformation of C-peptide in an unbound state by chemically introducing an all-hydrocarbon bridge spanning two [Formula: see text] residues of C-peptide. The stapling is observed to considerably enhance the helical propensity of peptide in an unbound state and to effectively improve the binding affinity of peptide to N-protein. Structural modeling analysis suggests that the stapled counterparts can bind to the active site of N-protein in a similar mode with unstapled C-peptide, where the stapled peptide is folded into a helical-like conformation, its anchor residue Phe241 deeply roots in a small druggable pocket of the active site, and the all-hydrocarbon bridge, as designed, is out of N–C complex interface to avoid disrupting the complex interaction.

Rapid Chemical Synthesis of Serine Protease Inhibitor Kazal-type 13 (SPINK13) Glycoform by a Combined Method with Glycan Insertion Strategy and Fast-Flow Fmoc SPPS.

Serine protease inhibitor Kazal type 13 (SPINK13) is a secreted protein that has been recently studied as a therapeutic drug and an interesting biomarker for cancer cells. Although SPINK13 has a consensus sequence (Pro-Asn-Val-Thr) for N-glycosylation, the existence of N-glycosylation and its functions are still unclear. In addition to this, the preparation of glycosylated SPINK 13 has not been examined by both the cell expression method and chemical synthesis. Herein we report the chemical synthesis of the scarce N-glycosylated form of SPINK13 by a rapid synthetic method combined with the chemical glycan insertion strategy and a fast-flow SPPS method. Glycosylated asparagine thioacid was designed to chemoselectively be inserted between two peptide segments where is the sterically bulky Pro-Asn(N-glycan)-Val junction by two coupling reactions which consist of diacyl disulfide coupling (DDC) and thioacid capture ligation (TCL). This insertion strategy successfully afforded the full-length polypeptide of SPINK13 within two steps from glycosylated asparagine thioacid. Because the two peptides used for this synthesis were prepared by a fast-flow SPPS, the total synthetic time of glycoprotein was considerably shortened. This synthetic concept enables us to repetitively synthesize a target glycoprotein easily. Folding experiments afforded well-folded structure confirmed by CD and disulfide bond map. Invasion assays of glycosylated SPINK13 and non-glycosylated SPINK13 with pancreatic cancer cells showed that non-glycosylated SPINK-13 was more potent than that of glycosylated SPINK13.

Computational Characterization of the Binding Properties of the HIV1-Neutralizing Antibody PG16 and Design of PG16-Derived CDRH3 Peptides.

PG16 is a broadly neutralizing antibody that binds to the gp120 subunit of the HIV-1 Env protein. The major interaction site is formed by the unusually long complementarity determining region (CDR) H3. The CDRH3 residue Tyr100H is known to represent a tyrosine sulfation site; however, this modification is not present in the experimental complex structure of PG16 with full-length HIV-1 Env. To investigate the role of sulfation for this complex, we modeled the sulfation of Tyr100H and compared the dynamics and energetics of the modified and unmodified complex by molecular dynamics simulations at the atomic level. Our results show that sulfation does not affect the overall conformation of CDRH3, but still enhances gp120 interactions both at the site of modification and for the neighboring residues. This stabilization affects not only protein-protein contacts, but also the interactions between PG16 and the gp120 glycan shield. Furthermore, we also investigated whether PG16-CDRH3 is a suitable template for the development of peptide mimetics. For a peptide spanning residues 93-105 of PG16, we obtained an experimental EC50 value of 3nm for the binding of gp120 to the peptide. This affinity can be enhanced by almost one order of magnitude by artificial disulfide bonding between residues 99 and 100F. In contrast, any truncation results in significantly lower affinity, suggesting that the entire peptide segment is involved in gp120 recognition. Given their high affinity, it should be possible to further optimize the PG16-derived peptides as potential inhibitors of HIV invasion.

Development of Convergent Hybrid Phase Ligation for Efficient and Convenient Total Synthesis of Proteins.

Simple and efficient total synthesis of homogeneous and chemically modified protein samples remains a significant challenge. Here, we report development of a convergent hybrid phase native chemical ligation (CHP-NCL) strategy for facile preparation of proteins. In this strategy, proteins are split into ~100-residue blocks, and each block is assembled on solid support from synthetically accessible peptide fragments before ligated together into full-length protein in solution. With the new method, we increase the yield of CENP-A synthesis by 2.5-fold compared to the previous hybrid phase ligation approach. We further extend the new strategy to the total chemical synthesis of 212-residue linker histone H1.2 in unmodified, phosphorylated, and citrullinated forms, each from eight peptide segments with only one single purification. We demonstrate that fully synthetic H1.2 replicates the binding interactions of linker histones to intact mononucleosomes, as a proxy for the essential function of linker histones in the formation and regulation of higher order chromatin structure.

Semisynthesis of homogeneous misfolded glycoprotein interleukin-8

To uncover how cells distinguish between misfolded and correctly-folded glycoproteins, homogeneous misfolded glycoproteins are needed as a probe for analysis of their structure and chemical characteristic nature. In this study, we have synthesized misfolded glycosyl interleukin-8 (IL-8) by combining E. coli expression and chemical synthesis to improve the synthetic efficiency. In order to prepare N-terminal peptide-thioester segment (1-33), we prepared an E. coli expressed peptide and then activated the C-terminal Cys by using an intramolecular N-to-S acyl shift reaction, followed by trans-thioesterification of the Cys-thioester with an external bis(2-sulfanylethyl)amine (SEA). The glycopeptide segment (34-49) was prepared by solid phase peptide synthesis and the C-terminal peptide (50-72) was prepared in E. coli. These peptide and glycopeptide segments were successfully coupled by sequential native chemical ligation. To obtain homogeneous misfolded glycoproteins by shuffling the disulfide bond pattern, folding conditions were optimized to maximize the yield of individual homogeneous misfolded glycoproteins.

Antibacterial and immunoregulatory activity of an antimicrobial peptide hepcidin in loach (Misgurnus anguillicaudatus)

Antimicrobial peptides (AMPs) are members of humoral immunity and particpate in resisting microbial invasion. In this study, an AMP gene hepcidin was obtained from the oriental loach Misgurnus anguillicaudatus and named Ma-Hep. This Ma-Hep encodes a peptide of 90 amino acids, with a predicted active peptide segment (Ma-sHep) of 25 amino acids at C terminus. Stimulation by a bacterial pathogen Aeromonas hydrophila resulted in significant up-regulation of Ma-Hep transcripts in loach midgut, head kidney, and gill. Ma-Hep and Ma-sHep proteins were expressed in Pichia pastoris and their antibacterial activity was examined. Results showed that Ma-sHep possessed stronger antibacterial activity against various Gram-positive and Gram-negative bacteria, compared to Ma-Hep. Scanning electron microscopy showed that Ma-sHep might kill bacteria by destroying bacterial cell membranes. Moreover, we found that Ma-sHep had an inhibitory effect on blood cell apoptosis induced by A. hydrophila and facilitated the bacterial phagocytosis and clearance in loach. Histopathological analysis indicated Ma-sHep could protect liver and gut of loach from bacterial infection. Ma-sHep has high thermal stability and PH stability, which is conducive to further feed addition. Feed supplemented with Ma-sHep expressing yeast improved the intestinal flora of loach by increasing the dominant bacteria and decreasing the harmful bacteria. Feed supplemented with Ma-sHep expressing yeast also regulated the expression of inflammatory related factors in various tissues of loach and reduced the mortality of loach upon bacterial infection. These findings show that the antibacterial peptide Ma-sHep is involved in the antibacterial defense of loach and can be used as a candidate for new antimicrobial agents in aquaculture.

Inhibition of tumor immune escape by blocking PD-1/PD-L1 engagement with dual-targeting molecularly imprinted polymer layer

Blocking the binding of PD-1/PD-L1 has become an effective strategy in inhibition of tumor immune escape. At present, it mainly depends on the employment of macromolecular antibodies, which target PD-1/PD-L1 protein through binding one of PD-1 or PD-L1 domains. In this study, we present a different strategy, an aptamer modified molecularly imprinted polymer layer (APD–PD-L1–MIPL), to break PD-1/PD-L1 binding for the inhibition of tumor immune escape. The APD–PD-L1–MIPL is prepared by a MIP layer on the surface of CaCO3 nanospheres using the peptide segment of the PD-L1 protein as a template. The subsequent removal of CaCO3 nanospheres core formats the MIP layer, to ensure high specifically matching capacity and short equilibrium time. A PD-L1 antagonistic DNA aptamer, is modified into the MIP layer to enhance recognition capacity, resulting in dual-targeting functionality. The APD–PD-L1–MIPL is able to bind PD-L1 and allow suppressing the engagement of PD-L1 with PD-1, inducing to block of the downstream signaling pathways and, therefore, restore T cell function and inhibition of cancer growth. The APD–PD-L1–MIPL can quantitatively detect the bound proteins and the LOD of APD–PD-L1–MIPL is 0.003 mg mL−1. This strategy enables provide a new idea for tumor immunotherapy.