Single Peptide Research Articles

A novel peptide isolated from Catla skin collagen acts as a self-assembling scaffold promoting nucleation of calcium-deficient hydroxyapatite nanocrystals.

Catla collagen hydrolysate (CH) was fractionated by chromatography and each fraction was subjected to HA nucleation, with the resultant HA-fraction composites being scored based on the structural and functional group of the HA formed. The process was repeated till a single peptide with augmented HA nucleation capacity was obtained. The peptide (4.6kDa), exhibited high solubility, existed in polyproline-II conformation and displayed a dynamic yet stable hierarchical self-assembling property. The 3D modelling of the peptide revealed multiple calcium and phosphate binding sites and a high propensity to self-assemble. Structural analysis of the peptide-HA crystals revealed characteristic diffraction planes of HA with mineralization following the (002) plane, retention of the self-assembled hierarchy of the peptide and intense ionic interactions between carboxyl groups and calcium. The peptide-HA composite crystals were mostly of 25-40nm dimensions and displayed 79% mineralization, 92% crystallinity, 39.25% porosity, 12GPa Young's modulus and enhanced stability in physiological pH. Cells grown on peptide-HA depicted faster proliferation rates and higher levels of osteogenic markers. It was concluded that the prerequisite for HA nucleation by a peptide included: a conserved sequence with a unique charge topology allowing calcium chelation and its ability to form a dynamic self-assembled hierarchy for crystal propagation.

Characterization of a new potent and long-lasting single chain peptide agonist of RXFP1 in cells and in vivo translational models

Despite beneficial effects in acute heart failure, the full therapeutic potential of recombinant relaxin-2 has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. A multiparametric optimization of the relaxin B-chain led to the identification of single chain lipidated peptide agonists of RXFP1 like SA10SC-RLX with subcutaneous bioavailability and extended half-life. SA10SC-RLX has sub nanomolar activity on cells expressing human RXFP1 and molecular modeling associated with the study of different RXFP1 mutants was used to decipher the mechanism of SA10SC-RLX interaction with RXFP1. Telemetry was performed in rat where SA10SC-RLX was able to engage RXFP1 after subcutaneous administration without tachyphylaxis after repeated dosing. Renal blood flow was then used as a translational model to evaluate RXFP1 activation. SA10SC-RLX increased renal blood flow and decreased renal vascular resistance in rats as reported for relaxin in humans. In conclusion, SA10SC-RLX mimics relaxin activity in in vitro and in vivo models of acute RXFP1 engagement. SA10SC-RLX represents a new class of long-lasting RXFP1 agonist, suitable for once daily subcutaneous administration in patients and potentially paving the way to new treatments for chronic fibrotic and cardiovascular diseases.

Assembly and Biomineralization of Polymorphic Gold–Peptide Superstructures Using Tyrosine‐Rich Short Peptides

AbstractPeptides show tremendous promise in synthesizing metal–peptide superstructures with tailored shapes and functions. However, bottom‐up control of the polymorphs of these superstructures using a single short peptide without molecular modification has not been mechanistically clarified. Therefore, an approach to constructing gold–peptide superstructures with unprecedented structural diversity using a tyrosine‐rich short peptide is developed, based on the assembling and mineralizing attributes of tyrosine. One‐step UV irradiation of peptide/gold‐salt systems enables on‐site mineralization of gold ions, permitting controlled solvent‐dependent fabrication of various superstructures. 0D colloids, 2D sheets, 3D superspheres, and 3D hollow capsules are produced via one‐pot reactions in a pH‐10 buffer, at the interface of a toluene–water biphasic system, in water, and in toluene‐in‐water emulsions, respectively, whereas 1D rod/fibril structures are produced using rapidly assembling peptides (A, F, I, L, N, V, Y, and D as X in YY‐X‐YY) in a two‐step process. Several peptide derivatives, which also exhibit assembling and biomineralizing abilities and form various superstructures, validate the functionality of the tyrosine‐rich peptide. This study sheds light on the design and development of diverse gold‐peptide superstructures for applications including catalysis, sensing, imaging, and photothermal therapy.

Sars-Cov-2-Specific CD4+ and CD8+ T Cell Responses Can Originate from Cross-Reactive CMV-Specific T Cells

Introduction: SARS-CoV-2-specific CD4+ and CD8+ T cells play an important role in COVID-19 pathogenesis. In addition to de novo SARS-CoV-2-specific T cell responses in infected individuals, SARS-CoV-2-specific T cells have also been identified in samples frozen down before the pandemic. So far, pre-existing SARS-CoV-2 specific T cell responses has mostly been ascribed to cross-reactive T cells that were initially primed by other coronaviruses. The presence of these cross-reactive T cells is not surprising considering these human coronaviruses share a relatively high sequence homology. However, T cells are also able to cross-react towards dissimilar pathogens, called heterologous immunity. Given the high prevalence and magnitudes of pre-pandemic SARS-CoV-2-specific T cells, we hypothesized that pre-pandemic SARS-CoV-2 T cell immunity could be explained by cross-reactive cytomegalovirus (CMV)-specific T cells. Methods & Results: PBMCs from CMV seropositive and seronegative donors that were cryopreserved before the pandemic were incubated with SARS-CoV-2 peptide pools. To detect SARS-CoV-2-specific T cells using flow cytometry, CD4+ T cell responses were identified by upregulation of CD154 and CD137 whereas CD8+ T cell responses were identified by upregulation of CD137 and IFN-γ production. In the CMV seropositive group, clear CD4+ T cell responses against SARS-CoV-2 membrane and CD8+ T cell responses against SARS-CoV-2 spike were detected whilst this was not observed in CMV seronegative donors. To investigate whether these T cells were reactive against both SARS-CoV-2 and CMV, the SARS-CoV-2 reactive T cells were single cell sorted, clonally expanded, and tested in co-culture assays in which stimulator cells expressing the relevant human leukocyte antigen (HLA) alleles were pulsed with overlapping peptide pools, peptide libraries or single peptides. The results demonstrated that the pre-pandemic SARS-CoV-2 membrane-specific CD4+ T cells recognized both SARS-CoV-2 membrane and CMV-pp65 peptide pools in HLA-DRB3*02:02, and specifically recognized AGILRNLVPM peptide of CMV-pp65. The pre-pandemic SARS-CoV-2 spike-specific CD8+ T cells recognized spike peptide FVSNGTHWF (FVS) and, interestingly, dissimilar CMV-pp65 peptide IPSINVHHY (IPS) both presented by HLA-B*35:01. Importantly, these FVS/IPS-cross-reactive T cells displayed a higher avidity for CMV compared to SARS-CoV-2, which illustrates that these cells were originally primed against CMV since repeated antigen exposure results in a selection for high avidity T cell clonotypes. This selection cannot have occurred in response to SARS-CoV-2-specific peptides in SARS-CoV-2-unexposed individuals. PBMCs from pre-pandemic CMV-seropositive donors that express HLA-B*35:01 were screened for the presence of these dual IPS/FVS-specific CD8+ T cells by peptide-HLA tetramer technology. In multiple donors these cross-reactive T cells were detected, and isolation followed by direct T cell receptor (TCR) sequencing of the IPS/FVS-tetramer positive cells showed that these T cells express an amino acid-identical TCR, illustrating that this SARS-CoV-2/CMV cross-reactivity of the IPS/FVS T cells is caused by a public TCR. To investigate the potential of the IPS/FVS-specific T cells to kill virus-infected cells, and thereby reducing spreading of SARS-CoV-2, the T cells were incubated with SARS-CoV-2-infected airway epithelial Calu-3 cells. Intracellular SARS-CoV-2 RNA was reduced at low virus titers in IPS/FVS-specific T cell co-cultures, indicating that the cross-reactive T cells are killing SARS-CoV-2-infected cells and thereby reduce spreading of SARS-CoV-2 during initial infection. Circulating IPS/FVS-specific T cells were not phenotypically activated in two patients with advanced COVID-19, as measured by flow cytometry analysis of CD38 and HLA-DR expression, indicating that cross-reactive T cells may more likely play a role during early stage of infection. Conclusion: CMV-specific T cells cross-react with SARS-CoV-2, despite low sequence homology between the two viruses, and may contribute to the pre-pandemic immunity against SARS-CoV-2. Pre-print link: https://www.biorxiv.org/content/10.1101/2022.07.31.502203v1

Abstract C004: TCRs directed to mutant KRAS display distinct antigen recognition motifs and minimal cross-reactivity to non-cognate antigens

Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most immune-resistant tumor types. Emerging evidence suggests improved clinical outcomes for subsets of PDAC patients with neoantigen-specific T cell immunity. A low-moderate tumor mutational burden limits antigenic targets in PDAC tumors, but recurrent missense mutations within the KRAS proto-oncogene drive tumorigenesis in >90% of cases. We and others have validated mutant KRAS (mKRAS) epitopes as immunological targets of T cell receptor (TCR)-based therapies. TCRs directed against mKRAS offer broad therapeutic potential. However, TCR degeneracy poses a major challenge to the clinical development of therapeutic TCRs given the risk of unforeseen off-target immunotoxicity due to peptide cross-reactivity. Here, we investigate the specificity and cross-reactivity of four TCRs specific for mKRAS G12V restricted to the HLA-A3 superfamily of HLA class I alleles. Of the four TCRs, one is restricted to HLA-A*03:01 (TCRA3V) and three are restricted to HLA-A*11:01 (TCRA11Va, TCRA11Vb, TCRA11Vc). All TCRs exhibit reactivity to the decamer peptide VVVGAVGVGK, while TCRA11Vc also exhibits reactivity to the nonamer peptide VVGAVGVGK. In-depth characterization of each TCR peptide recognition motif using alanine/glycine scanning libraries revealed TCRA3V and TCRA11Va-c demonstrate unique antigen binding motifs with a range of 3-6 critical amino acid residues, suggesting that substantial changes within the peptide:TCR interface may be tolerated for epitope recognition in the context of HLA-A*03:01 or HLA-A*11:01. TCR fingerprinting via combinatorial peptide libraries identified peptide repertoires capable of inducing TCR activation. Candidate cross-reactive peptides encoded in the human proteome were identified using the UnitProtKB database and filtered by computational epitope binding (NetMHC 4.0), setting a predicted peptide binding threshold of ≤500 nM. Using this approach, we identified a range of 2-35 candidate cross-reactive peptides for TCRA3V and TCRA11Va-c. Candidate cross-reactive peptides were synthesized and screened for their ability to induce TCR activation, which identified a single potential off-target peptide recognized by TCRA3V. Evaluation of tumor cell lines expressing high endogenous levels of this candidate cross-reactive antigen failed to elicit TCR activation, suggesting limited natural processing and presentation of this epitope. No potential off-target peptides were identified for TCRA11Va-c. Overall, this study serves as a platform to identify TCRs with high therapeutic potency and limited off-target potential for the clinical development of TCR therapy trials. Citation Format: Rebecca B. Nadler, Adham Bear, Robert H. Vonderheide, Gerald P. Linette, Beatriz M. Carreno. TCRs directed to mutant KRAS display distinct antigen recognition motifs and minimal cross-reactivity to non-cognate antigens [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C004.

DDEL-12. QUADRUPLE CARBON DOT NANO MODEL FOR ENHANCED TUMOR TARGETING AND DUAL DRUG DELIVERY FOR THE TREATMENT OF PEDIATRIC HIGH-GRADE GLIOMAS

Abstract High-grade gliomas remain among the most lethal neoplasms. Nanotechnology aims to Improve drug targeting and efficacy. Here we developed a quadruple nano-model (QNM) that specifically targets pediatric high-grade glioma cells and delivers chemotherapies to the cell nucleus. This carbon dot based QNM were fabricated by covalently attaching two drugs (epirubicin and temozolomide) and two targeting peptides. ShPep-1 peptide binds to the IL-13Rα2 receptors allowing for cellular import while lnPep-1h peptide delivers the DNA damaging drugs to the nucleus. Despite demonstrating the lowest measurable drug content (23.3%), the dual peptide linked QNM induced significantly greater cell death than single peptide linked conjugates suggesting enhanced cell uptake. Greatest effect was observed in glioblastoma (SJ-GBM2) and diffuse intrinsic pontine glioma (NP53) cells with IC50s of approximately 60 nM, 3-6-fold lower than single peptide conjugates. Imaging studies using FITC-conjugated carbon dots confirmed the dual peptide conjugate demonstrated greatest cellular uptake and nuclear localization.

The long multi-epitope peptide vaccine combined with adjuvants improved the therapeutic effects in a glioblastoma mouse model.

Emerging data have suggested that single short peptides have limited success as a cancer vaccine; however, extending the short peptides into longer multi-epitope peptides overcame the immune tolerance and induced an immune response. Moreover, the combination of adjuvants such as lenalidomide and anti-programmed cell death protein 1 (PD1) with a peptide vaccine showed potential vaccine effects in previous studies. Therefore, the effects of a long multi-epitope peptide vaccine in combination with lenalidomide and anti-PD1 were analyzed in this study. Long multi-epitope peptides from two MHCI peptides (BIRC597-104 and EphA2682-689) and the pan-human leukocyte antigen-DR isotype (HLA-DR) binding epitope (PADRE) were synthesized. The therapeutic effects of long multi-epitope peptides in combination with lenalidomide and anti-PD1 were confirmed in the murine GL261 intracranial glioma model. Immune cells' distribution and responses to the long multi-epitope peptides in combination with these adjuvants were also estimated in the spleens, lymph nodes, and tumor tissues. The difference between long multi-epitope peptides and a cocktail of multi-epitope peptides combined with lenalidomide and anti-PD1 was also clarified. As a result, long multi-epitope peptides combined with lenalidomide and anti-PD1 prolonged the survival of mice according to the suppression of tumor growth in an intracranial mouse model. While long multi-epitope peptides combined with these adjuvants enhanced the percentages of activated and memory effector CD8+ T cells, the increase in percentages of regulatory T cells (Tregs) was observed in a cocktail of multi-epitope peptides combined with lenalidomide and anti-PD1 group in the tumors. Long multi-epitope peptides combined with these adjuvants also enhanced the function of immune cells according to the enhanced pro-inflammatory cytokines and cytotoxicity against GL261 cells in ex vivo. In conclusion, long multi-epitope peptides composed of MHCI peptides, BIRC5 and EphA2, and the MHCII peptide, PADRE, in combination with lenalidomide and anti-PD1 has the potential to improve the therapeutic effects of a vaccine against GBM.

The SWGEDWGEIW from Soybean Peptides Reduce Oxidative Damage-Mediated Apoptosis in PC-12 Cells by Activating SIRT3/FOXO3a Signaling Pathway.

The goal of the investigation was to study the protective effects of the SWGEDWGEIW (the single peptide, TSP) from soybean peptides (SBP) on hydrogen peroxide (H2O2)-induced apoptosis together with mitochondrial dysfunction in PC-12 cells and their possible implications to protection mechanism. Meanwhile, the SBP was used as a control experiment. The results suggested that SBP and TSP significantly (p < 0.05) inhibited cellular oxidative damage and ROS-mediated apoptosis. In addition, SBP and TSP also enhanced multiple mitochondrial biological activities, decreased mitochondrial ROS levels, amplified mitochondrial respiration, increased cellular maximal respiration, spare respiration capacity, and ATP production. In addition, SBP and TSP significantly (p < 0.05) raised the SIRT3 protein expression and the downstream functional gene FOXO3a. In the above activity tests, the activity of TSP was slightly higher than that of SBP. Taken together, our findings suggested that SBP and TSP can be used as promising nutrients for oxidative damage reduction in neurons, and TSP is more effective than SBP. Therefore, TSP has the potential to replace SBP and reduce neuronal oxidative damage.

Peptide-capped Au and Ag nanoparticles: Detection of heavy metals and photochemical core/shell formation

We present a short peptide of only six amino acids that can be used in ambient conditions to simultaneously reduce either Au3+ or Ag+ ions, forming nanoparticles, and function as a stabilizing capping agent. At acidic pH, Hg2+ ions oxidize the silver nanoparticles and Fe2+ ions promote the aggregation of the gold nanoparticles. At alkaline conditions, Mn2+ ions induce the aggregation of the silver nanoparticles. Through the absorbance changes of these processes, these peptide-capped nanoparticles demonstrated a fast, selective, and sensitive pH-dependent detection system. The limit of detection of Hg2+, Mn2+, and Fe2+ was 319 nм, 184 nм, and 320 nм, respectively. Furthermore, the formed gold nanoparticles were successfully enveloped by a silver shell in a peptide-mediated photoreduction process. These bimetallic Au@Ag core/shell nanoparticles were characterized using UV–vis spectroscopy, high-resolution scanning transmission electron microscopy (HR-STEM), and energy dispersive X-ray spectroscopy (EDS). While prior studies used peptides as ligands for nanoparticles, the versatile abilities of the novel peptide presented in this study display the promising potential of using peptides for nanoparticles synthesis. This is because a single peptide can be used in a single-step one-pot synthesis to prepare and stabilize AuNPs, AgNPs, and Au@Ag core/shell nanoparticles, while also allowing to selectively probe different metal ions.

RF04 | PSUN295 Evolution of Monomeric Multi-Agonists of GLP-1 and NPY Receptors

Abstract Current treatments for type 2 diabetes (T2D) and obesity do not reliably achieve long-term weight-loss and up to 50% of patients experience nausea and vomiting. Thus, there is a critical need for obesity medications that provide glycemic control with enhanced hypophagic response without nausea/emesis. Based on rational design and in silico modelling of the glucagon-like peptide 1-receptor agonist (GLP1RA) exendin-4 (Ex4) and the neuropeptide Y2 receptor (Y2R) agonist peptide YY (PYY) 3-36, we have developed and screeened several new monomeric chimeric peptides that display dual agonism of the anorectic Y2R and the glucoregulatory GLP1R. In addition, we explored a third agonistic behavior at the neuropeptide Y1 receptor (Y1R), which potentially relates to pancreatic beta-cell protection. Our current lead, GEP44, binds the Y2R, Y1R, and GLP1R. We tested effects of daily injections of these chimeric peptides in adult Sprague-Dawley rats on food intake (FI), body weight (BW) changes, blood glucose levels and an indicator of nausea (kaolin intake). GEP44 produced profound reduction in FI (2-d average GEP44 at 20 nmol/kg FI -71%; Ex-4 20 nmol/kg FI -40%). Anorectic doses of GEP44 did not trigger a pica response assessed by kaolin consumption in treated rats, while in Ex-4 treated rats, kaolin consumption accounted for 28% of total daily solid intake, indicating a clear nausea response. In addition, we assayed for emetic response in the musk shrew, with little to no such emesis displayed (n=8) for GEP44 (1/8), but emesis noted across all animals tested for Ex4. During 11 d of treatment with GEP44, FI was consistently reduced resulting in a significantly stronger reduction of BW compared to Ex4 at the end of treatment (GEP44 -7.6%, Ex4 -3.7%, p<0.05). Furthermore, 5-day injections of GEP44 showed greater reduction of glucose levels than for Ex4 in diet induced obese rats receiving ip glucose. In conclusion, simultaneously targeting serial anorectic pathways with single molecule chimeric peptides can address multiple coexisting conditions to more efficaciously reduce FI, BW and blood glucose levels, devoid of side-effects to improve patient patient and quality of life. Presentation: Saturday, June 11, 2022 1:00 p.m. - 1:05 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

LY3437943, a novel triple GIP, GLP-1, and glucagon receptor agonist in people with type 2 diabetes: a phase 1b, multicentre, double-blind, placebo-controlled, randomised, multiple-ascending dose trial

Treating hyperglycaemia and obesity in individuals with type 2 diabetes using multi-receptor agonists can improve short-term and long-term outcomes. LY3437943 is a single peptide with agonist activity for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide 1 (GLP-1) receptors that is currently in development for the treatment of type 2 diabetes and for the treatment of obesity and associated comorbidities. We investigated the safety, pharmacokinetics, and pharmacodynamics of multiple weekly doses of LY3437943 in people with type 2 diabetes in a 12-week study. In this phase 1b, proof-of-concept, double-blind, placebo-controlled, randomised, multiple-ascending dose trial, adults (aged 20-70 years) with type 2 diabetes for at least 3 months, a glycated haemoglobin A1c (HbA1c) value of 7·0-10·5%, body-mass index of 23-50 kg/m2, and stable bodyweight (<5% change in previous 3 months) were recruited at four centres in the USA. Using an interactive web-response system, participants were randomly assigned to receive once-weekly subcutaneous injections of LY3437943, placebo, or dulaglutide 1·5 mg over a 12-week period. Five ascending dose cohorts were studied, with randomisation in each cohort such that a minimum of nine participants received LY3437943, three received placebo, and one received dulaglutide 1·5 mg within each cohort. The top doses in the two highest dose cohorts were attained via stepwise dose escalations. The primary outcome was to investigate the safety and tolerability of LY3437943, and characterising the pharmacodynamics and pharmacokinetics were secondary outcomes. Safety was analysed in all participants who received at least one dose of study drug, and pharmacodynamics and pharmacokinetics in all participants who received at least one dose of study drug and had evaluable data. This trial is registered at ClinicalTrials.gov, NCT04143802. Between Dec 18, 2019, and Dec 28, 2020, 210 people were screened, of whom 72 were enrolled, received at least one dose of study drug, and were included in safety analyses. 15 participants had placebo, five had dulaglutide 1·5 mg and, for LY3437943, nine had 0·5 mg, nine had 1·5 mg, 11 had 3 mg, 11 had 3/6 mg, and 12 had 3/6/9/12 mg. 29 participants discontinued the study prematurely. Treatment-emergent adverse events were reported by 33 (63%), three (60%), and eight (54%) participants who received LY3437943, dulaglutide 1·5 mg, and placebo, respectively, with gastrointestinal disorders being the most frequently reported treatment-emergent adverse events. The pharmacokinetics of LY3437943 were dose proportional and its half-life was approximately 6 days. At week 12, placebo-adjusted mean daily plasma glucose significantly decreased from baseline at the three highest dose LY3437943 groups (least-squares mean difference -2·8 mmol/L [90% CI -4·63 to -0·94] for 3 mg; -3·1 mmol/L [-4·91 to -1·22] for 3/6 mg; and -2·9 mmol/L [-4·70 to -1·01] for 3/6/9/12 mg). Placebo-adjusted sHbA1c also decreased significantly in the three highest dose groups (-1·4% [90% CI -2·17 to -0·56] for 3 mg; -1·6% [-2·37 to -0·75] for 3/6 mg; and -1·2% [-2·05 to -0·45] for 3/6/9/12 mg). Placebo-adjusted bodyweight reduction with LY3437943 appeared to be dose dependent (up to -8·96 kg [90% CI -11·16 to -6·75] in the 3/6/9/12 mg group). In this early phase study, LY3437943 showed an acceptable safety profile, and its pharmacokinetics suggest suitability for once-weekly dosing. This finding, together with the pharmacodynamic findings of robust reductions in glucose and bodyweight, provides support for phase 2 development. Eli Lilly and Company.

Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device.

Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect posttranslational modifications with single-molecule sensitivity. Here, we demonstrate single-molecule protein sequencing using a dynamic approach in which single peptides are probed in real time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. We annotate amino acids and identify the peptide sequence by measuring fluorescence intensity, lifetime, and binding kinetics on an integrated semiconductor chip. Our results demonstrate the kinetic principles that allow recognizers to identify multiple amino acids in an information-rich manner that enables discrimination of single amino acid substitutions and posttranslational modifications. With further development, we anticipate that this approach will offer a sensitive, scalable, and accessible platform for single-molecule proteomic studies and applications.

Reproductive roles of the vasopressin/oxytocin neuropeptide family in teleost fishes.

The vertebrate nonapeptide families arginine vasopressin (AVP) and oxytocin (OXT) are considered to have evolved from a single vasopressin-like peptide present in invertebrates and termed arginine vasotocin in early vertebrate evolution. Unprecedented genome sequence availability has more recently allowed new insight into the evolution of nonapeptides and especially their receptor families in the context of whole genome duplications. In bony fish, nonapeptide homologues of AVP termed arginine vasotocin (Avp) and an OXT family peptide (Oxt) originally termed isotocin have been characterized. While reproductive roles of both nonapeptide families have historically been studied in several vertebrates, their roles in teleost reproduction remain much less understood. Taking advantage of novel genome resources and associated technological advances such as genetic modifications in fish models, we here critically review the current state of knowledge regarding the roles of nonapeptide systems in teleost reproduction. We further discuss sources of plasticity of the conserved nonapeptide systems in the context of diverse reproductive phenotypes observed in teleost fishes. Given the dual roles of preoptic area (POA) synthesized Avp and Oxt as neuromodulators and endocrine/paracrine factors, we focus on known roles of both peptides on reproductive behaviour and the regulation of the hypothalamic-pituitary-gonadal axis. Emphasis is placed on the identification of a gonadal nonapeptide system that plays critical roles in both steroidogenesis and gamete maturation. We conclude by highlighting key research gaps including a call for translational studies linking new mechanistic understanding of nonapeptide regulated physiology in the context of aquaculture, conservation biology and ecotoxicology.

Label-Free Detection of β-Sheet Polymorphism.

The ability to detect and characterize multiple secondary structures or polymorphs within peptide and protein aggregates is crucial to treatment and prevention of amyloidogenic diseases, production of novel biomaterials, and many other applications. Here we report a label-free method to distinguish multiple β-sheet configurations within a single peptide aggregate using two-dimensional infrared spectroscopy. By calculating the transition dipole strength (TDS) spectrum from the ratio of linear and two-dimensional signals, we can extract maximum TDS values which provide higher sensitivity to vibrational coupling, and thus specifics of protein structure, than vibrational frequency alone. TDS spectra of AcKFE8 aggregates reveal two distinct β-sheet structures within fibers that appear homogeneous by other techniques. Furthermore, TDS spectra taken during early stages of aggregation show additional peaks that may indicate the presence of more weakly coupled β-sheet structures. These results demonstrate a unique and powerful spectroscopic method capable of distinguishing multiple oligomeric and polymorphic motifs throughout the aggregation using only native vibrational modes.

3D Blockage Mapping for Identifying Familial Point Mutations in Single Amyloid-β Peptides with a Nanopore.

Accurate discrimination of amyloid-β (Aβ) peptides containing familial point mutations would advance the knowledge of their roles in early-onset Alzheimer's disease. Herein, we simultaneously identified the mutant A21G, E22G, E22Q, and the wild-type (WT) Aβ18-26 peptides with aerolysin nanopore using a 3D blockage mapping strategy. The standard deviation of current blockade fluctuations (σb ) was proposed as a new supplement to current blockage (Ib /I0 ) and duration time (tD ) to profile the blockage characteristics of single molecules. Although the WT and A21G Aβ18-26 are indistinguishable in a traditional Ib /I0 -tD 2D description, ∼87 % of the blockade events can be accurately classified with half reduction of false identification using a combination of Ib /I0 , tD, and σb . This work offers an easy and reliable strategy to promote nanopore sensitivity of peptide mutants, leading to a more precise analysis of pathogenic mutations for developing effective diagnosis and treatment.

Characterising salivary peptidome across diurnal dynamics and variations induced by sampling procedures.

This study aimed to characterise diurnal dynamics of salivary peptidome and variations induced by sampling procedures. A supervised short-term longitudinal study was conducted amongst ten healthy participants. Saliva samples were collected by different procedures (stimulated/unstimulated conditions, forepart/midstream segments) on three consecutive days. The peptidome compositions of saliva samples were analysed using matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF MS). The salivary peptidome exhibited a stable trend generally, even though some diurnal dynamics happened in aspects of both overall structure and certain single peptides. The results indicated saliva samples collected under unstimulated and stimulated conditions have significantly different structures of peptidome, whilst the peptidome profile of stimulated saliva was more abundant than that of unstimulated saliva. It was also indicated that the midstream segment effect might exist in the segmented process of saliva sampling. In summary, salivary peptidome was able to maintain stability though some dynamic changes might happen within a short-term period. Stimulated and unstimulated saliva samples had significantly different peptidome profiles, whilst the stimulated whole saliva was a larger pool of low molecular weight peptides. The stability of the salivary peptidome highlights the reliability of salivary peptidome as a source of diagnostic biomarker. We recommend keeping one collection condition (stimulated/unstimulated) consistently within one study on salivary peptidome. Stimulated whole saliva would be preferred if more abundant peptidome profile is needed.

The Structures of Heterogeneous Membranes and Their Interactions with an Anticancer Peptide: A Molecular Dynamics Study.

We conduct molecular dynamics simulations of model heterogeneous membranes and their interactions with a 24-amino acid peptide—NAF-144–67. NAF-144–67 is an anticancer peptide that selectively permeates and kills malignant cells; it does not permeate normal cells. We examine three membranes with different binary mixtures of lipids, DOPC–DOPA, DOPC–DOPS, and DOPC–DOPE, with a single peptide embedded in each as models for the diversity of biological membranes. We illustrate that the peptide organization in the membrane depends on the types of nearby phospholipids and is influenced by the charge and size of the head groups. The present study sheds light on early events of permeation and the mechanisms by which an amphiphilic peptide crosses from an aqueous solution to a hydrophobic membrane. Understanding the translocation mechanism is likely to help the design of new permeants.

Simulation Study of the Effect of Antimicrobial Peptide Associations on the Mechanism of Action with Bacterial and Eukaryotic Membranes.

Antimicrobial peptides (AMPs) can be directed to specific membranes based on differences in lipid composition. In this study, we performed atomistic and coarse-grained simulations of different numbers of the designed AMP adepantin-1 with a eukaryotic membrane, cytoplasmic Gram-positive and Gram-negative membranes, and an outer Gram-negative membrane. At the core of adepantin-1’s behavior is its amphipathic α-helical structure, which was implemented in its design. The amphipathic structure promotes rapid self-association of peptide in water or upon binding to bacterial membranes. Aggregates initially make contact with the membrane via positively charged residues, but with insertion, the hydrophobic residues are exposed to the membrane’s hydrophobic core. This adaptation alters the aggregate’s stability, causing the peptides to diffuse in the polar region of the membrane, mostly remaining as a single peptide or pairing up to form an antiparallel dimer. Thus, the aggregate’s proposed role is to aid in positioning the peptide into a favorable conformation for insertion. Simulations revealed the molecular basics of adepantin-1 binding to various membranes, and highlighted peptide aggregation as an important factor. These findings contribute to the development of novel anti-infective agents to combat the rapidly growing problem of bacterial resistance to antibiotics.

Cooperativity in Bacterial Membrane Association Controls the Synergistic Activities of Antimicrobial Peptides.

Antimicrobial peptides (AMPs), or defence peptides, are compounds naturally produced during immune responses of living organisms against bacterial infections that are currently actively considered as promising alternatives to antibiotics. Recent experimental studies uncovered that in many situations, combinations of different AMPs are much more successful in eliminating the bacterial pathogens than single peptide species. However, the microscopic origin of such synergistic activities remains not fully understood. We present and investigate a possible mechanism of synergy between AMPs. It is based on the idea that due to inter-molecular interactions, the presence of an AMP of one type stimulates the association of an AMP of another type, and this accelerates the overall association to the membrane, eventually killing the bacteria. This approach allows us to fully quantify the synergistic activities of AMPs, and it is successfully applied for several experimental systems. It is found that strong cooperativity can be achieved for relatively weak inter-molecular interactions, suggesting that the application of combinations of AMPs can be further rationally optimized to make it a powerful antibacterial treatment.

Stereoselective Interaction of Helix-Hairpin-Helix Motif Peptides with DNA Structure

The helix-hairpin-helix (HhH) motif in prokaryote and eukaryote DNA binding proteins is involved in non-sequence-specific DNA binding. Several studies have examined various HhH binding interactions, but the effect of stereochemistry on non-sequence-specific DNA binding by HhH peptides remains unresolved. Here, we analyzed stereochemistry-specific interactions between DNA structures and HhH motif peptides derived from NAD+-dependent DNA ligase, which is a promising target for developing new antibiotics. We synthesized l- and d-forms of single HhH motif peptides and found that both the native l-HhH-N peptide and d-HhH-N peptide bind bubble DNA and the peptides were changed to α-helical structure. Moreover, the d-HhH-N peptide showed different interaction properties against single-stranded and double-stranded DNA when compared with the l-HhH-N peptide. This study provides insights into non-sequence-specific DNA binding of d-peptides for developing d-peptide drugs.