Dimeric Peptide Research Articles

Peptide Dimerization as a Strategy for the Development of Antileishmanial Compounds.

Leishmaniasis is recognized as a serious public health problem in Brazil and around the world. The limited availability of drugs for treatment, added to the diversity of side effects and the emergence of resistant strains, shows the importance of research focused on the development of new molecules, thus contributing to treatments. Therefore, this work aimed to identify leishmanicidal compounds using a peptide dimerization strategy, as well as to understand their mechanisms of action. Herein, it was demonstrated that the dimerization of the peptide TSHa, (TSHa)2K, presented higher potency and selectivity than its monomeric form when evaluated against Leishmania mexicana and Leishmania amazonensis. Furthermore, these compounds are capable of inhibiting the parasite cysteine protease, an important target explored for the development of antileishmanial compounds, as well as to selectively interact with the parasite membranes, as demonstrated by flow cytometry, permeabilization, and fluorescence microscopy experiments. Based on this, the identified molecules are candidates for use in in vivo studies with animal models to combat leishmaniasis.

9198 Dimeric [Cys25]PTH(1-34) Induces Differential Transcriptional Responses Versus Monomeric PTH(1-34)

Abstract Disclosure: M. Noh: None. H.J. Kim: None. J. Kim: None. M. Kang: None. S. Lee: None. H. Lee: None. S. Lee: None. Introduction: Patients with hypoparathyroidism caused by the homozygous mutation that changes arginine to cysteine at position 25 in parathyroid hormone (PTH) exhibit high bone densities, which suggests a potential influence of the mutant hormone on bone formation. We also have found that [Cys25]PTH forms homodimers, and that the dimeric peptide exhibits altered functionality versus monomeric PTH. Dimeric [Cys25]PTH(1-34) (Dimer) thus displays selective binding to the G protein-coupled PTH1R conformation (RG) in vitro and markedly diminished cAMP-inducing capability in vivo. Despite these alterations, Dimer induced bone anabolic effects in both calvarial injection assays and after injection in ovariectomized osteoporotic mice, closely mimicking the effects of PTH(1-34) (wtPTH). Our current objective is to explore the downstream signaling pathways activated by Dimer vs wtPTH to help elucidate the bone anabolic effects of the Dimeric analog and its therapeutic potential. Methods: A phospho-protein antibody array assay developed for G protein-coupled receptor (GPCR) downstream target proteins was employed in HEK293/hPTH1R (GP-2.3) cells following treatment with chemically synthesized wtPTH or Dimer. We further conducted RNA-seq analysis of transcriptome changes in primary cells collected from calvarial bones of mice treated with wtPTH or Dimer at 6, 12, and 24-hours post-injection. Finally, we used quantitative reverse transcription PCR (qRT-PCR) to analyze changes in specific marker genes in tibiae and kidney isolated from mice after treatment with Dimer vs. wtPTH. Results: The number of phosphorylated proteins significantly regulated by PTH1R upon exposure to Dimer was remarkably enhanced, relative to the number regulated upon exposure to wtPTH. RNA-seq analysis revealed that the early effects of wtPTH and Dimer on transcriptional regulation were similar, whereas marked differences in expression levels became apparent over time. qRT-PCR analyses of RNA expression levels in kidneys versus tibiae from mice treated with wtPTH or Dimer revealed in bone that the expression of genes associated with bone catabolism, particularly RANKL, was significantly lower in response to Dimer as compared to wtPTH treatment, while in kidney there was an overall reduced response to Dimer as compared to wtPTH. Conclusions: Our study supports the hypothesis that as compared to PTH(1-34), dimeric [Cys25]PTH (1-34) induces altered signaling responses in vivo and has a lower effect on bone catabolism. Dimeric [Cys25]PTH(1-34) thus holds interest as a potential bone anabolic agent that utilizes a distinct mechanism of action. Presentation: 6/3/2024

8279 Dimeric [Cys25]PTH(1-34) Induces Differential Transcriptional Responses Versus Monomeric PTH(1-34)

Abstract Disclosure: M. Noh: None. H.J. Kim: None. J. Kim: None. M. Kang: None. S. Lee: None. H. Lee: None. S. Lee: None. Introduction: Patients with hypoparathyroidism caused by the homozygous mutation that changes arginine to cysteine at position 25 in parathyroid hormone (PTH) exhibit high bone densities, which suggests a potential influence of the mutant hormone on bone formation. We also have found that [Cys25]PTH forms homodimers, and that the dimeric peptide exhibits altered functionality versus monomeric PTH. Dimeric [Cys25]PTH(1-34) (Dimer) thus displays selective binding to the G protein-coupled PTH1R conformation (RG) in vitro and markedly diminished cAMP-inducing capability in vivo. Despite these alterations, Dimer induced bone anabolic effects in both calvarial injection assays and after injection in ovariectomized osteoporotic mice, closely mimicking the effects of PTH(1-34) (wtPTH). Our current objective is to explore the downstream signaling pathways activated by Dimer vs wtPTH to help elucidate the bone anabolic effects of the Dimeric analog and its therapeutic potential. Methods: A phospho-protein antibody array assay developed for G protein-coupled receptor (GPCR) downstream target proteins was employed in HEK293/hPTH1R (GP-2.3) cells following treatment with chemically synthesized wtPTH or Dimer. We further conducted RNA-seq analysis of transcriptome changes in primary cells collected from calvarial bones of mice treated with wtPTH or Dimer at 6, 12, and 24-hours post-injection. Finally, we used quantitative reverse transcription PCR (qRT-PCR) to analyze changes in specific marker genes in tibiae and kidney isolated from mice after treatment with Dimer vs. wtPTH. Results: The number of phosphorylated proteins significantly regulated by PTH1R upon exposure to Dimer was remarkably enhanced, relative to the number regulated upon exposure to wtPTH. RNA-seq analysis revealed that the early effects of wtPTH and Dimer on transcriptional regulation were similar, whereas marked differences in expression levels became apparent over time. qRT-PCR analyses of RNA expression levels in kidneys versus tibiae from mice treated with wtPTH or Dimer revealed in bone that the expression of genes associated with bone catabolism, particularly RANKL, was significantly lower in response to Dimer as compared to wtPTH treatment, while in kidney there was an overall reduced response to Dimer as compared to wtPTH. Conclusions: Our study supports the hypothesis that as compared to PTH(1-34), dimeric [Cys25]PTH (1-34) induces altered signaling responses in vivo and has a lower effect on bone catabolism. Dimeric [Cys25]PTH(1-34) thus holds interest as a potential bone anabolic agent that utilizes a distinct mechanism of action. Presentation: 6/3/2024

Structural Characterization of Disulfide-Linked p53-Derived Peptide Dimers

Structural Characterization of Disulfide-Linked p53-Derived Peptide Dimers

Energy landscapes for clusters of hexapeptides.

We present the results for energy landscapes of hexapeptides obtained using interfaces to the Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) program. We have used basin-hopping global optimization and discrete path sampling to explore the landscapes of hexapeptide monomers, dimers, and oligomers containing 10, 100, and 200 monomers modeled using a residue-level coarse-grained potential, Mpipi, implemented in LAMMPS. We find that the dimers of peptides containing amino acid residues that are better at promoting phase separation, such as tyrosine and arginine, have melting peaks at higher temperature in their heat capacity compared to phenylalanine and lysine, respectively. This observation correlates with previous work on the same uncapped hexapeptide monomers modeled using atomistic potential. For oligomers, we compare the variation in monomer conformations with radial distance and observe trends for selected angles calculated for each monomer. The LAMMPS interfaces to the GMIN and OPTIM programs for landscape exploration offer new opportunities to investigate larger systems and provide access to the coarse-grained potentials implemented within LAMMPS.

The dimer effect: A refinement approach towards skin sensitization assessment in-chemico using Amino acid Derivative Reactivity Assay.

Skin sensitization is a key endpoint for safety assessment, especially for cosmetics and personal care products. The adverse outcome pathway for skin sensitization and the chemical and biological events driving the induction of human skin sensitization are now well understood. Several non-animal test methods have been developed to predict sensitizer potential by measuring the impact of chemical sensitizers on these key events. In this work, we have focused on Key Event 1 (the molecular initiating step), which is based on formation of a covalent adduct between skin sensitizers and endogenous proteins and/or peptides in the skin. There exists three in-chemico assays approved by the Organization for Economic Co-operation and Development-(1) Direct Peptide Reactivity Assay (DPRA), (2) Amino Acid Derivative Reactivity Assay (ADRA), and (3) Kinetic Direct Peptide Reactivity Assay (kDPRA) to quantify peptide/amino acid derivative depletion after incubation with test chemicals. However, overestimated depletion of the cysteine-based peptide/amino acid derivatives is known in such assays because of the dimerization of the thiol group. In this present work, we report the synthesis and structural confirmation of the dimer of N-(2-[1-naphthyl]acetyl)-L-cysteine (NAC) from the ADRA assay to allow simultaneous determination of (a) peptide depletion by quantifying NAC monomer and (b) peptide dimerization by quantifying NAC dimer thereby eliminating the overestimation. We present a case study with three chemicals to demonstrate the importance of this approach. Thus, this simultaneous assay gives a more informed view of the peptide reactivity of chemicals to better identify skin sensitizers.

Structural Characterization of Disulfide-Linked p53-Derived Peptide Dimers.

Disulfide bonds provide a convenient method for chemoselective alteration of peptide and protein structure and function. We previously reported that mild oxidation of a p53-derived bisthiol peptide (CTFANLWRLLAQNC) under dilute non-denaturing conditions led to unexpected disulfide-linked dimers as the exclusive product. The dimers were antiparallel, significantly α-helical, resistant to protease degradation, and easily reduced back to the original bisthiol peptide. Here we examine the intrinsic factors influencing peptide dimerization using a combination of amino acid substitution, circular dichroism (CD) spectroscopy, and X-ray crystallography. CD analysis of peptide variants suggests critical roles for Leu6 and Leu10 in the formation of stable disulfide-linked dimers. The 1.0 Å resolution crystal structure of the peptide dimer supports these data, revealing a leucine-rich LxxLL dimer interface with canonical knobs-into-holes packing. Two levels of higher-order oligomerization are also observed in the crystal: an antiparallel "dimer of dimers" mediated by Phe3 and Trp7 residues in the asymmetric unit and a tetramer of dimers mediated by Trp7 and Leu10. In CD spectra of Trp-containing peptide variants, minima at 227 nm provide evidence for the dimer of dimers in dilute aqueous solution. Importantly, and in contrast to the original dimer model, the canonical leucine-rich core and robust dimerization of most peptide variants suggests a tunable molecular architecture to target various proteins and evaluate how folding and oligomerization impact various properties, such as cell permeability.

Synthesis, Characterization, and Study of the Antimicrobial Potential of Dimeric Peptides Derived from the C-Terminal Region of Lys49 Phospholipase A2 Homologs.

Currently, the search for new alternatives to conventional antibiotics to combat bacterial resistance is an urgent task, as many microorganisms threaten human health due to increasing bacterial resistance to traditional medicines. Thus, new molecules such as antimicrobial peptides have emerged as promising alternatives because of their low induction of resistance and broad spectrum of action. In this context, in the past few years, our research group has synthesized and characterized a peptide derived from the C-terminal region of the Lys49 PLA2-like BthTX-I, named p-BthTX-I. After several studies, the peptide (p-BthTX-I)2K was proposed as the molecule with the most considerable biotechnological potential. As such, the present work aimed to evaluate whether the modifications made on the peptide (p-BthTX-I)2K can be applied to other molecules originating from the C-terminal region of PLA2-like Lys49 from snake venoms. The peptides were obtained through the solid-phase peptide synthesis technique, and biochemical and functional characterization was carried out using dichroism techniques, mass spectrometry, antimicrobial activity against ESKAPE strains, hemolytic activity, and permeabilization of lipid vesicles. The antimicrobial activity of the peptides was promising, especially for the peptides (p-AppK)2K and (p-ACL)2K, which demonstrated activity against all strains that were tested, surpassing the model molecule (p-BthTX-I)2K in most cases and maintaining low hemolytic activity. The modifications initially proposed for the (p-BthTX-I)2K peptide were shown to apply to other peptides derived from Lys49 PLA2-like from snake venoms, showing promising results for antimicrobial activity. Future assays comparing the activity of the dimers obtained through this strategy with the monomers of these peptides should be carried out.

Development and characterization of the mode-of-action of inhibitory and agonist peptides targeting the voltage-gated sodium channel SCN1B beta-subunit

Cardiac arrhythmia treatment is a clinical challenge necessitating safer and more effective therapies. Recent studies have highlighted the role of the perinexus, an intercalated disc nanodomain enriched in voltage-gated sodium channels including both Nav1.5 and β1 subunits, adjacent to gap junctions. These findings offer insights into action potential conduction in the heart. A 19-amino acid SCN1B (β1/β1B) mimetic peptide, βadp1, disrupts VGSC beta subunit-mediated adhesion in cardiac perinexii, inducing arrhythmogenic changes. We aimed to explore βadp1's mechanism and develop novel SCN1B mimetic peptides affecting β1-mediated adhesion. Using patch clamp assays in neonatal rat cardiomyocytes and electric cell substrate impedance sensing (ECIS) in β1-expressing cells, we observed βadp1 maintained inhibitory effects for up to 5 h. A shorter peptide (LQLEED) based on the carboxyl-terminus of βadp1 mimicked this inhibitory effect, while dimeric peptides containing repeated LQLEED sequences paradoxically promoted intercellular adhesion over longer time courses. Moreover, we found a link between these peptides and β1-regulated intramembrane proteolysis (RIP) - a signaling pathway effecting gene transcription including that of VGSC subunits. βadp1 increased RIP continuously over 48 h, while dimeric agonists acutely boosted RIP for up to 6 h. In the presence of DAPT, an RIP inhibitor, βadp1's effects on ECIS-measured intercellular adhesion was reduced, suggesting a relationship between RIP and the peptide's inhibitory action. In conclusion, novel SCN1B (β1/β1B) mimetic peptides are reported with the potential to modulate intercellular VGSC β1-mediated adhesion, potentially through β1 RIP. These findings suggest a path towards the development of anti-arrhythmic drugs targeting the perinexus.

Computational analysis of hydrogen bonds network dynamics in the water layers surrounding Aβ(1-42) and Aβ(1-40) peptide dimers

The amyloid-β peptide (Aβ) dimer is involved in the development of Alzheimer’s disease through forming amyloid plaques. Aβ(1-40) and Aβ(1-42) are the most common and poisonous species causing Alzheimer’s disease. They form homogeneous dimers containing two similar peptides and heterogeneous dimers containing different ones. The low molecular weight, fast dynamics, and varying compositions of these dimers make it difficult to study them by conventional experimental methods. Hence, we clarify the effect of the diversity of the Aβ dimers on the structure of the water molecules around the peptides by the molecular dynamics (MD) simulation method in this study. MD simulations of the Aβ(1-40) and Aβ(1-42) monomers are also performed for the comparison. The calculations are repeated in the presence of dimethyl sulfoxide (DMSO). The water molecules positioned at distances of 0.5, 0.75, 1, 1.25, and 1.5 nm from the protein were chosen to investigate the distribution and dynamics of hydrogen bonds in the layers surrounding the peptide. The results show that the heterogeneity of the dimer system increases the percentage of hydrogen (H) atoms not involved in any water-water hydrogen bonds around the peptides in the different layers. While it decreases the percentage of H atoms involved in one and two water-water hydrogen bonds. The DMSO presence reduces the percentage of H atoms involved in two hydrogen bonds in the systems containing a mixture of water and DMSO. The average interaction energy of the dimers demonstrates the enhanced interactions of the two peptides in water in comparison with that in the mixture of water and DMSO. The behavior of the water molecules around the heterogeneous dimer is very similar to that around the Aβ(1-42) homogeneous dimer.

A Novel Dimeric Short Peptide Derived from α-Defensin-Related Rattusin with Improved Antimicrobial and DNA-Binding Activities.

Rattusin, an α-defensin-related antimicrobial peptide isolated from the small intestine of rats, has been previously characterized through NMR spectroscopy to elucidate its three-dimensional structure, revealing a C2 homodimeric scaffold stabilized by five disulfide bonds. This study aimed to identify the functional region of rattusin by designing and synthesizing various short analogs, subsequently leading to the development of novel peptide-based antibiotics. The analogs, designated as F1, F2, F3, and F4, were constructed based on the three-dimensional configuration of rattusin, among which F2 is the shortest peptide and exhibited superior antimicrobial efficacy compared to the wild-type peptide. The central cysteine residue of F2 prompted an investigation into its potential to form a dimer at neutral pH, which is critical for its antimicrobial function. This activity was abolished upon the substitution of the cysteine residue with serine, indicating the necessity of dimerization for antimicrobial action. Further, we synthesized β-hairpin-like analogs, both parallel and antiparallel, based on the dimeric structure of F2, which maintained comparable antimicrobial potency. In contrast to rattusin, which acts by disrupting bacterial membranes, the F2 dimer binds directly to DNA, as evidenced by fluorescence assays and DNA retardation experiments. Importantly, F2 exhibited negligible cytotoxicity up to 515 μg/mL, assessed via hemolysis and MTT assays, underscoring its potential as a lead compound for novel peptide-based antibiotic development.

Inducing α-Helicity in Peptides by Silver Coordination to Cysteine.

Short peptide sequences consisting of two cysteine residues separated by three other amino acids display complete change from random coil to α-helical secondary structure in response to addition of Ag+ ions. The folded CXXXC/Ag+ complex involves formation of multinuclear Ag+ species and is stable in a wide pH range from below 3 to above 8. The complex is stable through reversed-phase HPLC separation as well as towards a physiological level of chloride ions, based on far-UV circular dichroism spectroscopy. In electrospray MS under acidic conditions a peptide dimer with four Ag+ ions bound was observed, and modelling based on potentiometric experiments supported this to be the dominating complex at neutral pH together with a peptide dimer with 3 Ag+ and one proton at lower pH. The complex was demonstrated to work as a N-terminal nucleation site for inducing α-helicity into longer peptides. This type of silver-mediated peptide assembly and folding may be of more general use for stabilizing not only peptide folding but also for controlling oligomerization even under acidic conditions.

Abstract 6037: Copper-67 as a next-generation radioconjugate warhead for cancer therapy

Abstract Background and Purpose: Antibody drug conjugates (ADCs) have shown promise as cancer treatments. However, the efficacy of ADCs has been limited in solid tumors and can come with significant toxicities. Radioconjugates utilize various radionuclides that are chelated and can be conjugated to small molecules, peptides and antibodies. We propose Targeted Copper Theranostics (TCTs) which use copper-64 to image tumors and beta-emitting copper-67 to irradiate and kill cancer cells using various molecular targets of oncological relevance. We have evaluated the efficacy of copper-67 based therapy in three formats; peptides, antibodies, and pre-targeting of antibody conjugates in relevant tumor mouse models. Benefits of copper-67 [half-life, 61.8 h; 100% β- energy in the range of 0.18-0.56 MeV] include high purity, high specific activity, manufacturing in a centralized distribution model at high yields on electron accelerators, and half-life suitable to various targeting formats. Copper-64 and copper-67 provide multiple logistical benefits relative to other radionuclides including Ga-68, F-18 and Lu-177. Methods: We evaluated three tumor-targeting moieties conjugated to a sarcophagine bifunctional chelator (SAR), including: a peptide dimer targeting prostate specific membrane antigen (SAR-bisPSMA); an antibody 5B1, targeting the PDAC-specific biomarker carbohydrate antigen-19-9 (CA19-9); and a Tz-TCO IDEEA huA33 antibody/sarcophagine tetrazine conjugate targeting transmembrane glycoprotein A33. Various doses were administered of [67Cu]Cu-SAR-bisPSMA in LNCAP xenografts; [67Cu]Cu-SAR-5B1 (~10 μg) in FC1245 Ft2Ab PDAC tumor-bearing C57BL/6 mice; and [67Cu]Cu-MeCOSAR-Tz in athymic nude mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts 72 hours after administration of huA33-TCO. Results: Administration of either 7.5, 15 or 30 MBq of [67Cu]Cu-SAR-bisPSMA increased overall survival relative to the control group. Similarly, after administration of 4, 11, 18 MBq of [67Cu]Cu-5B1, treated groups enhanced overall survival to 32 days compared to control (14 days). Pre-targeting using ~18.5, 37.0 or 55.5 MBq or fractionated dosing of two 27.8 MBq doses of [67Cu]Cu-MeCOSAR-Tz showed efficient tumor growth reduction with enhanced overall survival for treated mice pre-administered huA33-TCO, from 68 days to >200 days (dose dependent) compared to control (21 days). Conclusion: We report the inhibition of tumor growth with minimal radiotoxicity and each format demonstrated significantly improved overall survival compared to controls. Collectively our results demonstrated that TCTs using the sarcophagine chelator with copper-67 are effective radioconjugates across multiple formats with an acceptable safety profile. Copper-67-based TCTs are being translated in multiple theranostic clinical trials in the USA and Australia. Citation Format: Matt Harris, Kurt Gehlsen, Lachlan McInnes, Jason Lewis. Copper-67 as a next-generation radioconjugate warhead for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6037.

Cysteine-Specific Multifaceted Bioconjugation of Peptides and Proteins Using 5-Substituted 1,2,3-Triazines.

Peptide and protein postmodification have gained significant attention due to their extensive impact on biomolecule engineering and drug discovery, of which cysteine-specific modification strategies are prominent due to their inherent nucleophilicity and low abundance. Herein, the study introduces a novel approach utilizing multifunctional 5-substituted 1,2,3-triazine derivatives to achieve multifaceted bioconjugation targeting cysteine-containing peptides and proteins. On the one hand, this represents an inaugural instance of employing 1,2,3-triazine in biomolecular-specific modification within a physiological solution. On the other hand, as a powerful combination of precision modification and biorthogonality, this strategy allows for the one-pot dual-orthogonal functionalization of biomolecules utilizing the aldehyde group generated simultaneously. 1,2,3-Triazine derivatives with diverse functional groups allow conjugation to peptides or proteins, while bi-triazines enable peptide cyclization and dimerization. The examination of the stability of bi-triazines revealed their potential for reversible peptide modification. This work establishes a comprehensive platform for identifying cysteine-selective modifications, providing new avenues for peptide-based drug development, protein bioconjugation, and chemical biology research.

Modification of a Selective NTRK2 Agonist and Confirmation of Activity in a Glaucoma-on-a-Chip Model.

RNYK is a selective agonist of the neurotrophic tyrosine kinase receptor type 2 (NTRK2) which has been screened from a phage-displayed peptide library. Its sequence is SGVYKVAYDWQH, similar to a native NTRK2 ligand, that is, brain-derived neurotrophic factor (BDNF). The current study was performed to recognize and confirm critical residues for RNYK activity in a glaucoma-on-a-chip model. We designed a modified RNYK (mRNYK) peptide based on hotspots of the RNYK sequence identified by alanine scanning. The critical residues consisted of tyrosine, valine, aspartic acid, and tryptophan (YVDW); however, lysine and glutamine were also maintained in the final sequence (YKVDWQ) for forming amide bonds and peptide dimerization. The affinity of mRNYK binding was confirmed by testing against NTRK2 receptors on the surface of ATRA-treated SH-SY5Y cells. The neuroprotective effect of mRNYK was also evaluated in cell culture after elevated pressure insult in a glaucoma-on-a-chip model. The primary amine on the lysine side-chain from one sequence (YKVDWQ) reacted with a γ-carboxamide group of glutamine from the other sequence, forming dimeric mRNYK. In silico, molecular dynamic simulations of the mRNYK-NTRK2 complex showed more stable and stronger interactions as compared to the RNYK-NTRK2 complex. In vitro, mRNYK demonstrated a neuroprotective effect on SH-SY5Y cells under normal and elevated pressure comparable to RNYK. The 50% effective concentration (logEC50) for mRNYK was 0.7009, which was better than RNYK with a logEC50 of 0.8318. The modified peptide studied herein showed improved stability over the original peptide (RNYK) and demonstrated potential for use as a BDNF agonist with neuroprotective properties for treatment of neurodegenerative disorders such as glaucoma.

Binding and dimerization of PGLa peptides in anionic lipid bilayer studied by replica exchange molecular dynamics

The 21-residue PGLa peptide is well known for antimicrobial activity attributed to its ability to compromize bacterial membranes. Using all-atom explicit solvent replica exchange molecular dynamics with solute tempering, we studied PGLa binding to a model anionic DMPC/DMPG bilayer at the high peptide:lipid ratio that promotes PGLa dimerization (a two peptides per leaflet system). As a reference we used our previous simulations at the low peptide:lipid ratio (a one peptide per leaflet system). We found that the increase in the peptide:lipid ratio suppresses PGLa helical propensity, tilts the bound peptide toward the bilayer hydrophobic core, and forces it deeper into the bilayer. Surprisingly, at the high peptide:lipid ratio PGLa binding induces weaker bilayer thinning, but deeper water permeation. We explain these effects by the cross-correlations between lipid shells surrounding PGLa that leads to a much diminished efflux of DMPC lipids from the peptide proximity at the high peptide:lipid ratio. Consistent with the experimental data the propensity for PGLa dimerization was found to be weak resulting in coexistence of monomers and dimers with distinctive properties. PGLa dimers assemble via apolar criss-cross interface and become partially expelled from the bilayer residing at the bilayer-water boundary. We rationalize their properties by the dimer tendency to preserve favorable electrostatic interactions between lysine and phosphate lipid groups as well as to avoid electrostatic repulsion between lysines in the low dielectric environment of the bilayer core. PGLa homedimer interface is predicted to be distinct from that involved in PGLa-magainin heterodimers.

Heterologous expression of the novel dimeric antimicrobial peptide LIG in Pichia pastoris

The antimicrobial peptide (AMP) LI is a fusion product of antimicrobial peptide LL37 produced by human neutrophils and Indolicidin secreted by bovine neutrophils. LI retained the antimicrobial activity of the parental peptides and showed high cell selectivity. In this study, the flexible linker Gly-Ser-Gly (G-S-G) was used to ligate LI into dimeric LIG, and constructed the Pichia pastoris (P. pastoris) expression vector pPIC9K-6×His-3×FLAG-LIG. The total protein expression of P. pastoris GS115 reached the highest level (189.6 mg/L) after 96 h induction with 3 % methanol at the initial pH value of 7.0. Finally, 5.9 mg/L of recombinant LIG (rLIG) was obtained after enterokinase digestion and purification. The rLIG had high antimicrobial activity and low hemolytic activity. Compared with monomer LI, GSG linked dimeric LIG, which had no significant change in antimicrobial activity and had good salt ions stability. In this study, the dimeric antimicrobial peptide LIG was successfully expressed, which provided a new idea for the expression of AMPs in the P. pastoris expression system, and had important significance for the application of AMPs.

Silver nanoparticles alter the dimerization of Aβ42 studied by REMD simulations.

The aggregation of amyloid beta (Aβ) peptides is associated with the development of Alzheimer's disease (AD). However, there has been a growing belief that the oligomerization of Aβ species in different environments has a neurotoxic effect on the patient's brain, causing damage. It is necessary to comprehend the compositions of Aβ oligomers in order to develop medications that may effectively inhibit these neurotoxic forms that affect the nervous system of AD patients. Thus, dissociation or inhibition of Aβ aggregation may be able to prevent AD. To date, the search for traditional agents and biomolecules has largely been unsuccessful. In this context, nanoparticles have emerged as potential candidates to directly inhibit the formation of Aβ oligomers. The oligomerization of the dimeric Aβ peptides with or without the influence of a silver nanoparticle was thus investigated using temperature replica-exchange molecular dynamics (REMD) simulations. The physical insights into the dimeric Aβ oligomerization were clarified by analyzing intermolecular contact maps, the free energy landscape of the dimeric oligomer, secondary structure terms, etc. The difference in obtained metrics between Aβ with or without a silver nanoparticle provides a picture of the influence of silver nanoparticles on the oligomerization process. The underlying mechanisms that are involved in altering Aβ oligomerization will be discussed. The obtained results may play an important role in searching for Aβ inhibitor pathways.

Unexpected diselenide metathesis in selenocysteine-substituted biologically active peptides.

Substitution of disulfide bonds with a diselenide bonds in peptides and proteins is an often-used strategy to increase the stability of naturally occurring peptides and proteins. In this paper, diselenide metathesis between model diselenide dimer peptides, as well as that in diselenide(s)-substituted biologically active peptides, were analyzed. Surprisingly, depending on the tertiary structure of the peptides, we observed that the metathesis reaction occurs under physiological conditions even in the absence of reducing agents, light and heating.

HDL mimetics protect Alzheimer’s patients carrying APOE ε4 from SARS-CoV-2 invasion

Patients with Alzheimer’s disease (AD) inheriting Apolipoprotein (APOE) ε4 are susceptible to COVID-19, but the underlying mechanism of how such a neurodegenerative disease promotes respiratory vulnerability to viral infections remains insufficiently understood. The uncovering of genetic basis of COVID-19 outcome holds the potential to establish disease models for therapeutic development. Here, using human iPSC-derived type II alveolar epithelial cells (hAECII) from AD patients with APOE ε4 and healthy individuals, we showed that AECII of AD had a greater level of SARS-CoV-2 invasion but not replication, along with increased expressions of viral receptor and co-receptors, while reduced pulmonary surfactant proteins. Since low serum HDL-C levels have been implicated in the onset of both AD and COVID-19, we further revealed that HDL mimetics, including 4 F dimeric peptide and its phospholipid conjugate pHDL, were effective in protecting AECII bearing APOE ε4 against SARS-CoV-2 invasion. In AD, concomitant with improved AD pathological phenotypes, HDL mimetics specifically restored the defective basal SP-D, a pulmonary innate immune lipoprotein that targets glycans of the spike protein to neutralize invading viruses. Moreover, HDL mimetics demonstrated strong SP-D-independent virucidal effects, which additionally restricted the entry of coronaviral particles into non-AD AECII models and lungs of human ACE2-transgenic mice in response to Alpha and Omicron variants of SARS-CoV-2. Our work offers critical insights into the respiratory sensitivity of AD patients carrying APOE ε4 to viral infection and repurpose HDL-based therapeutics as potential preventive interventions in respiratory viral pandemics.