Artificial Peptide Research Articles

Electrosynthesis of Artificial Peptides and Nucleosides via the Nitromethane–Lithium Perchlorate Electrolyte System: A Review

AbstractRemarkable advances have recently been achieved in the application of electrochemical methodology in synthetic chemistry area. In the past few decades, various oxidative electrochemical reactions have reportedly been enhanced using a nitromethane–lithium perchlorate electrolyte system. Herein, we summarise our efforts toward the skeletal modification of proline residue and nitrogen‐containing nucleoside analogues (azanucleosides) via electrosynthesis.

Design of an Artificial Peptide Inspired by Transmembrane Mitochondrial Protein for Escorting Exogenous DNA into the Mitochondria to Restore their Functions by Simultaneous Multiple Gene Expression

AbstractMitochondria are vital organelles regulating essential cellular functions. Human mitochondrial DNA (mtDNA) consists of 37 genes, 13 of which encode mitochondrial proteins, and the remaining 24 genes encode two ribosomal RNAs and 22 transfer RNAs needed for the translation of the mtDNA‐encoded 13 proteins. However, mtDNA often impairs the expression and function of these genes due to various mutations, ultimately causing mitochondrial dysfunction. To recover from this desperate condition, developing the technology to supply all mitochondrial proteins encoded by mtDNA at once is an urgent task, but there is no established strategy for this purpose. In this study, a simple yet effective mitochondrial gene delivery system is proposed comprising an artificial peptide inspired by a transmembrane mitochondrial membrane protein. The designed mitochondria‐targeting peptides presented on the carrier surface effectively guide the encapsulated plasmid to the mitochondria, facilitating mitochondrial uptake and gene expression. The developed system successfully delivers exogenous mtDNA to mtDNA‐depleted cells and leads to simultaneous multigene expression, ultimately restoring mitochondrial functions, including the mitochondrial respiration rate. The established multiple gene expression system in each mitochondrion is a game‐changing technology that can accelerate the development of mitochondrial engineering technologies as well as clinical applications for mitochondrial diseases.

Rational design of supramolecular self-assembly sensor for living cell imaging of HDAC1 and its application in high-throughput screening

Supramolecular chemistry offers new insights in bioimaging, but specific tracking of enzyme in living cells via supramolecular host-guest reporter pair remains challenging, largely due to the interference caused by the complex cellular environment on the binding between analytes and hosts. Here, by exploiting the principle of supramolecular tandem assay (STA) and the classic host-guest reporter pair (p-sulfonatocalix[4]arene (SC4A) and lucigenin (LCG)) and rationally designing artificial peptide library to screen sequence with high affinity of the target enzyme, we developed a “turn-on” fluorescent sensing system for intracellular imaging of histone deacetylase 1 (HDAC1), which is a potential therapeutic target for various diseases, including cancer, neurological, and cardiovascular diseases. Based on computational simulations and experimental validations, we verified that the deacetylated peptide by HDAC1 competed LCG, freeing it from the SC4A causing fluorescence increase. Enzyme kinetics experiments were further conducted to prove that this assay could detect HDAC1 specifically with high sensitivity (the LOD value is 0.015 μg/mL, ten times lower than the published method). This system was further applied for high-throughput screening of HDAC1 inhibitors over a natural compound library containing 147 compounds, resulting in the identification of a novel HDAC1 down-regulator (Ginsenoside RK3). Our results demonstrated the sensitivity and robustness of the assay system towards HDAC1. It should serve as a valuable tool for biochemical studies and drug screening.

Chemical catalysis by biological amyloids.

Toxic aggregation of proteins and peptides into amyloid fibers is the basis of several human diseases. In each disease, a particular peptide noncovalently assembles into long thin structures with an overall cross-β fold. Amyloids are not only related to disease: functional amyloids are found in many biological systems and artificial peptide amyloids are developed into novel nanomaterials. Amyloid fibers can act as template for the generation of more amyloids but are considered nonreactive in chemical catalysis. The perception of amyloids as chemically inert species was recently challenged by in vitro work on three human amyloid systems. With the use of model substrates, amyloid-β, α-synuclein and glucagon amyloids were found to catalyze biologically relevant chemical reactions. The detected catalytic activity was much less than that of 'real' enzymes, but like that of designed (synthetic) catalytic amyloids. I here describe the current knowledge around this new activity of natural amyloids and the putative connection to metabolic changes in amyloid diseases. These pioneering studies imply that catalytic activity is an unexplored gain-of-function activity of disease amyloids. In fact, all biological amyloids may harbor intrinsic catalytic activity, tuned by each amyloid's particular fold, that await discovery.

Nanoparticles Carrying Conserved Regions of Influenza A Hemagglutinin, Nucleoprotein, and M2 Protein Elicit a Strong Humoral and T Cell Immune Response and Protect Animals from Infection.

Current influenza vaccines are mainly strain-specific and have limited efficacy in preventing new influenza A strains. Efficient control of infection can potentially be achieved through the development of broad-spectrum vaccines based on conserved antigens. A combination of several such antigens, including the conserved region of the second subunit of the hemagglutinin (HA2), the extracellular domain of the M2 protein (M2e), and epitopes of nucleoprotein (NP), which together can elicit an antibody- and cell-mediated immune response, would be preferred for vaccine development. In this study, we obtained recombinant virus-like particles formed by an artificial self-assembling peptide (SAP) carrying two epitopes from NP, tandem copies of M2e and HA2 peptides, along with a T helper Pan DR-binding epitope (PADRE). Fusion proteins expressed in Escherichia coli self-assembled in vitro into spherical particles with a size of 15-35 nm. Immunization of mice with these particles induced strong humoral immune response against M2e and the entire virus, and lead to the formation of cytokine-secreting antigen-specific CD4+ and CD8+ effector memory T cells. Immunization provided high protection of mice against the lethal challenge with the influenza A virus. Our results show that SAP-based nanoparticles carrying conserved peptides from M2, HA, and NP proteins of the influenza A virus, as well as T helper epitope PADRE, can be used for the development of universal flu vaccines.

Discovery of Antimicrobial Peptides That Can Accelerate Culture Diagnostics of Slow-Growing Mycobacteria Including Mycobacterium tuberculosis.

Antimicrobial peptides (AMPs) can directly kill Gram-positive bacteria, Gram-negative bacteria, mycobacteria, fungi, enveloped viruses, and parasites. At sublethal concentrations, some AMPs and also conventional antibiotics can stimulate bacterial response increasing their resilience, also called the hormetic response. This includes stimulation of growth, mobility, and biofilm production. Here, we describe the discovery of AMPs that stimulate the growth of certain mycobacteria. Peptide 14 showed a growth stimulating effect on Mycobacteria tuberculosis (MTB), M. bovis, M. avium subsp. paratuberculosis (MAP), M. marinum, M. avium-intracellulare, M. celatum, and M. abscessus. The effect was more pronounced at low bacterial inocula. The peptides induce a faster transition from the lag phase to the log phase and keep the bacteria longer in the log phase before entering stationary phase when compared to nontreated controls. In some cases, an increase in the division rate was observed. An initial screen using MAP and a collection of 75 peptides revealed 13 peptides with a hormetic effect. For MTB, a collection of 25 artificial peptides were screened and 13 were found to reduce the time to positivity (TTP) by at least 5%, improving growth. A screen of 43 naturally occurring peptides, 11 fragments of naturally occurring peptides and 5 designed peptides, all taken from the database APD3, identified a further 44 peptides that also lowered TTP by at least 5%. Lasioglossin LL-III (Bee) and Ranacyclin E (Frog) were the most active natural peptides, and the human cathelicidin LL37 fragment GF-17 and a porcine cathelicidin protegrin-1 fragment were the most active fragments of naturally occurring peptides. Peptide 14 showed growth-stimulating activity between 10 ng/mL and 10 µg/mL, whereas the stability-optimised Peptide 14D had a narrow activity range of 0.1-1 µg/mL. Peptides identified in this study are currently in commercial use to improve recovery and culture for the diagnostics of mycobacteria in humans and animals.

Guidelines for Optimizing Type S Nonribosomal Peptide Synthetases.

Bacterial biosynthetic assembly lines, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), play a crucial role in the synthesis of natural products that have significant therapeutic potential. The ability to engineer these biosynthetic assembly lines offers opportunities to produce artificial nonribosomal peptides, polyketides, and their hybrids with improved properties. In this study, we introduced a synthetic NRPS variant, termed type S NRPS, which simplifies the engineering process and enables biocombinatorial approaches for generating nonribosomal peptide libraries in a parallelized high-throughput manner. However, initial generations of type S NRPSs exhibited a bottleneck that led to significantly reduced production yields. To address this challenge, we employed two optimization strategies. First, we truncated SYNZIPs from the N- and/or C-terminus of the NRPS. SYNZIPs comprise a large set of well-characterized synthetic protein interaction reagents. Second, we incorporated a structurally flexible glycine-serine linker between the NRPS protein and the attached SYNZIP, aiming to improve dynamic domain-domain interactions. Through an iterative optimization process, we achieved remarkable improvements in production yields, with titer increases of up to 55-fold compared to the nonoptimized counterparts. These optimizations successfully restored production levels of type S NRPSs to those observed in wild-type NRPSs and even surpassed them. Overall, our findings demonstrate the potential of engineering bacterial biosynthetic assembly lines for the production of artificial nonribosomal peptides. In addition, optimizing the SYNZIP toolbox can have valuable implications for diverse applications in synthetic biology, such as metabolic engineering, cell signaling studies, or engineering of other multienzyme complexes, such as PKSs.

Chemical Evolution of Amphiphilic Xenopeptides for Potentiated Cas9 Ribonucleoprotein Delivery.

The introduction of the CRISPR/Cas9 system in the form of Cas9/sgRNA ribonucleoproteins (RNP) is an efficient, straightforward strategy for genome editing, and potent RNP carriers are in high demand. Here, we report a series of artificial peptides based on novel ionizable amino acids that are able to deliver Cas9 RNP into cells very efficiently. Systematic variation of hydrophobic properties revealed a relationship between the xenopeptide logD7.4 and genome editing potency. By correlating the physicochemical properties with biological activity, individual optima were found for different xenopeptide sequence architectures. The optimized amphiphilic carriers enable ∼88% eGFP knockout at an RNP dose of only 1 nM and up to 40% homology-directed repair (HDR) in eGFP/BFP switchable reporter cells by co-delivery with an ssDNA template. Mechanistic studies demonstrated that hydrophobically balanced xenopeptides are more resistant to ionic stress as well as concentration-dependent dissociation and promote endocytosis by both clathrin- and macropinocytosis-mediated pathways. The systematic study develops a versatile and adjustable carrier platform and highlights impactful structure-activity relationships, providing a new chemical guide for the design and optimization of nonviral Cas9 RNP nanocarriers.

Plant-Produced Nanoparticles Based on Artificial Self-Assembling Peptide Bearing the Influenza M2e Epitope.

Despite advances in vaccine development, influenza remains a persistent global health threat and the search for a broad-spectrum recombinant vaccine against influenza continues. The extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is highly conserved and can be used to develop a universal vaccine. M2e is a poor immunogen by itself, but it becomes highly immunogenic when linked to an appropriate carrier. Here, we report the transient expression of a recombinant protein comprising four tandem copies of M2e fused to an artificial self-assembling peptide (SAP) in plants. The hybrid protein was efficiently expressed in Nicotiana benthamiana using the self-replicating potato virus X-based vector pEff. The protein was purified using metal affinity chromatography under denaturing conditions. The hybrid protein was capable of self-assembly in vitro into spherical particles 15-30 nm in size. The subcutaneous immunization of mice with M2e-carrying nanoparticles induced high levels of M2e-specific IgG antibodies in serum and mucosal secretions. Immunization provided mice with protection against a lethal influenza A virus challenge. SAP-based nanoparticles displaying M2e peptides can be further used to develop a recombinant "universal" vaccine against influenza A produced in plants.

Peptide Stapling with Boronate Esters- A Reversible Folding of (Artificial) Peptide Chain to α-Helix.

Stabilization of a peptide conformation via stapling strategy may be realized by the reversible or more often irreversible connection of side chains being in mutually appropriate geometry. An incorporation of phenylboronic acid and sugar residues (fructonic or galacturonic acid), attached to two lysine side chains via amide bonds and separated by 2, 3, or 6 other residues in the C-terminal fragment of RNase A introduces the intramolecular interaction stabilizing the α-helical organization. The boronate ester stapling is stabilized in mild basic conditions and may be switched off by acidification leading to unfolded organization of the peptide chain. We investigated the possibility of using switchable stapling by mass spectrometry, NMR and UV-CD spectroscopies, and DFT calculations.

Refinement of the Fusion Tag PagP for Effective Formation of Inclusion Bodies in Escherichia coli.

Methods for efficient insoluble protein production require further exploration. PagP, an Escherichia coli outer membrane protein with high β-sheet content, could function as an efficient fusion partner for inclusion body-targeted expression of recombinant peptides. The primary structure of a given polypeptide determines to a large extent its propensity to aggregate. Herein, aggregation "hot spots" (HSs) in PagP were analyzed using the web-based software AGGRESCAN, leading to identification of a C-terminal region harboring numerous HSs. Moreover, a proline-rich region was found in the β-strands. Substitution of these prolines by residues with high β-sheet propensity and hydrophobicity significantly improved its ability to form aggregates. Consequently, the absolute yields of recombinant antimicrobial peptides Magainin II, Metchnikowin, and Andropin were increased significantly when expressed in fusion with this refined version of PagP. We describe separation of recombinant target proteins expressed in inclusion bodies fused with the tag. An artificial NHT linker peptide with three motifs was implemented for separation and purification of authentic recombinant antimicrobial peptides. IMPORTANCE Fusion tag-induced formation of inclusion bodies provides a powerful means to express unstructured or toxic proteins. For a given fusion tag, how to enhance the formation of inclusion bodies remains to be explored. Our study illustrated that the aggregation HSs in a fusion tag played important roles in mediating its insoluble expression. Efficient production of inclusion bodies could also be implemented by refining its primary structure to form a more stable β-sheet with higher hydrophobicity. This study provides a promising method for improvement of the insoluble expression of recombinant proteins.

An Artificial Peptide-Based Bifunctional HIV-1 Entry Inhibitor That Interferes with Viral Glycoprotein-41 Six-Helix Bundle Formation and Antagonizes CCR5 on the Host Cell Membrane.

Human immunodeficiency virus type 1 (HIV-1) is characterized by high variability and drug resistance. This has necessitated the development of antivirals with a new chemotype and therapy. We previously identified an artificial peptide with non-native protein sequence, AP3, with the potential to inhibit HIV-1 fusion through targeting hydrophobic grooves on the N-terminal heptad repeat trimer of viral glycoprotein gp41. Here, a small-molecule HIV-1 inhibitor targeting chemokine coreceptor CCR5 on the host cell was integrated into the AP3 peptide, producing a novel dual-target inhibitor with improved activity against multiple HIV-1 strains including those resistant to the currently used anti-HIV-1 drug enfuvirtide. Its superior antiviral potency in comparison with the respective pharmacophoric moieties is in consonance with the dual binding of viral gp41 and host factor CCR5. Therefore, our work provides a potent artificial peptide-based bifunctional HIV-1 entry inhibitor and highlights the multitarget-directed ligands approach in the development of novel therapeutic anti-HIV-1 agents.

Suppression of TDP-43 aggregation by artificial peptide binder targeting to its low complexity domain

TAR DNA-binding protein 43 (TDP-43), aggregation prone protein, is a potential target of drug discovery for amyotrophic lateral sclerosis. The molecular binders, targeting the disordered low complexity domain (LCD) relevant to the aggregation, may suppress the aggregation. Recently, Kamagata et al. developed a rational design of peptide binders targeting intrinsically disordered proteins based on contact energies between residue pairs. In this study, we designed 18 producible peptide binder candidates to TDP-43 LCD by using this method. Fluorescence anisotropy titration and surface plasmon resonance assays demonstrated that one of the designed peptides bound to TDP-43 LCD at 30 μM. Thioflavin-T fluorescence and sedimentation assays showed that the peptide binder suppressed the aggregation of TDP-43. In summary, this study highlights the potential applicability of peptide binder design for aggregation prone proteins.

Clean Production of l-Alanyl-l-glutamine by an Efficient Yeast Biocatalyst Expressing α-Amino Acid Ester Acyltransferase without N-Glycosylation.

l-Alanyl-l-glutamine (Ala-Gln) is a widely used value-added dipeptide whose production relies heavily upon an efficient biocatalyst. The currently available yeast biocatalysts that express α-amino acid ester acyltransferase (SsAet) possess relatively low activity, which may be attributed to glycosylation. Here, to promote SsAet activity in yeast, we identified the N-glycosylation site as the Asn residue at position 442 and subsequently eliminated the negative effect of N-glycosylation on SsAet by removing artificial and native signal peptides to obtain K3A1, a novel yeast biocatalyst with significantly improved activity. Additionally, the optimal reaction conditions of strain K3A1 were determined (25 °C, pH 8.5, AlaOMe/Gln = 1:2), resulting in a maximum molar yield and productivity of approximately 80% and 1.74 g·(L·min)-1, respectively. Therefore, we developed a promising system to cleanly produce Ala-Gln in a safe, efficient, and sustainable manner, which may contribute to the future industrial production of Ala-Gln.

Rational design of phase separating peptides based on phase separating protein sequence of p53

Artificial phase-separating (PS) peptides can be used in various applications such as microreactors and drug delivery; however, the design of artificial PS peptides remains a challenge. This can be attributed to the limitation of PS-relevant residues that drive phase separation by interactions of their pairs in short peptides and the difficulty in the design involving interaction with target PS proteins. In this study, we propose a rational method to design artificial PS peptides that satisfy the requirements of liquid droplet formation and co-phase separation with target PS proteins based on the target PS protein sequence. As a proof of concept, we designed five artificial peptides from the model PS protein p53 using this method and confirmed their PS properties using differential interference contrast and fluorescence microscopy. Single-molecule fluorescent tracking demonstrated rapid diffusion of the designed peptides in their droplets compared to that of p53 in p53 droplets. In addition, size-dependent uptake of p53 oligomers was observed in the designed peptide droplets. Large oligomers were excluded from the droplet voids and localized on the droplet surface. The uptake of high-order p53 oligomers into the droplets was enhanced by the elongated linker of the designed peptides. Furthermore, we found that the designed peptide droplets recruited p53 to suppress gel-like aggregate formation. Finally, we discuss aspects that were crucial in the successful design of the artificial PS peptides.

Self-assembling Nanomaterials from Artificial Peptides Bearing Various Aromatic Groups at <i>N</i>-Termini

タンパク質やペプチドなどの生体高分子の生み出す精緻な構造や機能，高度な分子集合システムを手本としたナノバイオ材料の開発が精力的に進められている。本稿では，自己組織化ユニットとしての人工ペプチドの魅力について述べるとともに，筆者らが構築した三重らせん構造をモチーフとした芳香族環修飾コラーゲン様ペプチドの階層的自己組織化により生み出される多彩なナノ組織体について紹介したい。とくに分子構造と自己集合形態（高次構造）との相関性，光／温度などの外部刺激応答性やそれに基づく蛍光発光特性などを中心に述べる。

Fluorescence labeling of anchor-modified Mart-1 peptide for increasing its affinity for HLA-A*0201: Hit two targets with one arrow.

One of the most successful strategies in designing peptide-based cancer vaccines is modifying natural epitope peptides to increase their binding strength to human leukocyte antigens (HLAs). Anchor-modified Mart-1 peptide (ELAGIGILTV) is among the artificial epitope peptides with the highest binding affinity for HLA-A*0201. In this study, by fluorescence labeling of its either C- or N-terminus with Nε -(5-carboxyfluorescein)-l-lysine, we not only made it traceable but also drastically increased its binding strength to HLA-A*0201. HLA streptamer, for the first time, is introduced for measuring the binding constants (Ka ) of the labeled peptides. The affinity of the labeled peptides for the HLA-A*201 of the MCF-7 cells was extraordinarily high and co-incubating them with the highest possible amount of the unlabeled peptide, as a competitor, did not significantly prohibit them from binding to the HLA. The reproducibility of the obtained results was confirmed by using the T2 cell line. The HLA-deficient K562 cell line was used as the negative control. With in silico simulations, we found two hydrophobic pockets on both sides of HLA-A*0201 for anchoring the C- or N-terminal 5-carboxyfluorescein probe, which can explain the extraordinary affinity of the labeled peptides for the HLA-A*0201.

Functional mimicry of sea urchin biomineralization proteins with CaCO3-binding peptides selected by phage display.

The intricate process of biomineralization, e.g. in sea urchins, involves the precise interplay of highly regulated mineralization proteins and the spatiotemporal coordination achieved through compartmentalization. However, the investigation of biomineralization effector molecules, e.g. proteins, is challenging, due to their very low abundance. Therefore, we investigate the functional mimicry in the bioinspired precipitation of calcium carbonate (CaCO3) with artificial peptides selected from a peptide library by phage display based on peptide-binding to calcite and aragonite, respectively. The structure-directing effects of the identified peptides were compared to those of natural protein mixes isolated from skeletal (test) structures of two sea urchin species (Arbacia lixula and Paracentrotus lividus). The calcium carbonate samples deposited in the absence or presence of peptides were analyzed with a set of complementary techniques with regard to morphology, polymorph, and nanostructural motifs. Remarkably, some of the CaCO3-binding peptides induced morphological features in calcite that appeared similar to those obtained in the presence of the natural protein mixes. Many of the peptides identified as most effective in exerting a structure-directing effect on calcium carbonate crystallization were rich in basic amino acid residues. Hence, our in vitro mineralization study further highlights the important, but often neglected, role of positively charged soluble organic matrices associated with biological and bioinspired CaCO3 deposition.

Targeting β-catenin and PD-L1 simultaneously by a racemic supramolecular peptide for the potent immunotherapy of hepatocellular carcinoma.

Objective: The low clinical utility of immune checkpoint inhibitors (ICIs) against PD-1 or PD-L1 has recently been associated with the activation of the Wnt/β-catenin signaling pathway in hepatocellular carcinoma (HCC), which promotes tumor immune escape and resistance to anti-PD-1/PD-L1 therapy. Hence, we aimed to fabricate a supramolecular peptide which could target the Wnt/β-catenin signaling pathway coupled with ICIs blockage therapy for optimizing HCC immunotherapy. Methods: A racemic spherical supramolecular peptide termed sBBI&PDP nanoparticle was constructed by hierarchical self-assembly, comprising an L-enantiomeric peptide as an inhibitor of BCL9 and β-catenin (sBBI) and a D-enantiomeric peptide as an inhibitor of PD-1/PD-L1 (PDP). Results: sBBI&PDP nanoparticle potently suppressed the hyperactivated Wnt/β-catenin signaling pathway in vitro and in vivo, while blocking endogenous PD-L1 effectively. Furthermore, sBBI&PDP increased the infiltration and action of CD8+ T cells at tumor sites. Notably, compared with the original sBBI and commercial Anti-PD-L1 inhibitors, the designed sBBI&PDP showed stronger antitumor efficacy in an orthotopic homograft mice model of HCC and a PDX HCC model in Hu-PBMC-NSG mice. Moreover, sBBI&PDP possessed a favorable biosafety profile. Conclusion: The successful implementation of this strategy could revitalize ICIs blockage therapy and promote the discovery of artificial peptides for HCC immunotherapy.

Conformational landscapes of artificial peptides predicted by various force fields: are we ready to simulate β-amino acids?

With the introduction of artificial peptides as antimicrobial agents and organic catalysts, numerous efforts have been made to design foldamers with desirable structures and functions. Computational tools are a helpful proxy for revealing the dynamic structures at atomic resolution and understanding foldamer's complex structure-function relationships. However, the performance of conventional force fields in predicting the structures of artificial peptides has not been systematically evaluated. In this study, we critically assessed three popular force fields, AMBER ff14SB, CHARMM36m, and OPLS-AA/L, in predicting conformational propensities of a β-peptide foldamer at monomer and hexamer levels. Simulation results were compared to those obtained from quantum chemistry calculations and experimental data. We also utilised replica exchange molecular dynamics simulations to investigate the energy landscape of each force field and assess the similarities and differences between force fields. We compared different solvent systems in the AMBER ff14SB and CHARMM36m frameworks and confirmed the unanimous role of hydrogen bonds in shaping energy landscapes. We anticipate that our data will pave the way for further improvements to force fields and for understanding the role of solvents in peptide folding, crystallisation, and engineering.