C-terminal Amide Research Articles

Conformational Flexibility and Net Charge are Key Determinants for the Antimicrobial Activity of Peptide Uy234 Against Multidrug-resistant Bacteria

BackgroundThe antimicrobial activity of two peptides, Uy234 derived from the venom of the scorpion Urodacus yaschenkoi and a consensus peptide QnCs-Buap, was evaluated. We tested different pathogenic bacteria: Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella enterica, Bacillus subtilis, Enterococcus spp. and Staphylococcus aureus, including one methicillin resistant (MRSA) and two multidrug resistant (MDR) clinical isolates. In contrast to the QnCs-Buap peptide, Uy234 showed relevant growth inhibitory activity on A. baumannii and B. subtilis, and mostly on S. aureus strains.ObjectiveThe present research focused on elucidating the mechanism for this antibacterial activity.MethodologyWe carried out an in-depth analysis of the composition, structure, flexibility, and physicochemical properties of both peptides.ResultsWe found a crucial role of the C-terminal amide and composition in favoring the formation of a dense H-bond network in the Uy234 peptide. This H-bonding network slightly stiffens the peptide and keeps it in a preordered conformation in the aqueous phase.ConclusionsWe hypothesize that, given that Uy234 is a very short peptide (18 aa), it could have a destabilizing effect and favor micellization phenomena instead forming pores. In contrast, the QnCs-Buap peptide (13 aa), having only the positive charge at the N-terminal end and being significantly more hydrophobic and rigid, is not capable of overcoming the energy barrier to disturb the membrane. We propose that Uy234 peptide can be a scaffold to develop new derivatives with high potential against infections caused by diverse multidrug-resistant bacteria.Graphical

Molecular mechanism of prolactin-releasing peptide recognition and signaling via its G protein-coupled receptor

Prolactin-releasing peptide (PrRP) is an RF-amide neuropeptide that binds and activates its cognate G protein-coupled receptor, prolactin-releasing peptide receptor (PrRPR), also known as GPR10. PrRP and PrRPR are highly conserved across mammals and involved in regulating a range of physiological processes, including stress response, appetite regulation, pain modulation, cardiovascular function, and potentially reproductive functions. Here we present cryo-electron microscopy structures of PrRP-bound PrRPR coupled to Gq or Gi heterotrimer, unveiling distinct molecular determinants underlying the specific recognition of the ligand’s C-terminal RF-amide motif. We identify a conserved polar pocket that accommodates the C-terminal amide shared by RF-amide peptides. Structural comparison with neuropeptide Y receptors reveals both similarities and differences in engaging the essential RF/RY-amide motifs. Our findings demonstrate the general mechanism governing RF-amide motif recognition by PrRPR and RF-amide peptide receptors, and provide a foundation for elucidating activation mechanisms and developing selective drugs targeting this important peptide–receptor system.

Redox-Active Conopeptide Li520 Has Evolved to Catalyze Oxidative Folding of Conotoxins.

The evolution of miniature conopeptide Li520 (COWC*, *: C-terminal amidation) to exhibit the disulfide isomerase activity was probed using structure, function, disulfide conformation, and the precursor gene sequence. The peptides Li520, Li504, [O2A]Li520, [W3A]Li520, and Grx506, homologues active-site motif of glutaredoxin, were chemically synthesized and assessed for their disulfide reduction potential, intrinsic folding of disulfides, and disulfide isomerization activity on α-conotoxin ImI. The reduction potential of the disulfide of peptides varies from -189 to -344 mV, which is within the range observed for the redox family of proteins that modulates the folding of protein disulfides. The oxidative folding studies confirm the significance of the tryptophan residue in engaging Li520 in disulfide-exchange reactions and the role of proline hydroxylation in extending the lifetime of Li520 in a reduced free thiol state. Studies of quenching of tryptophan fluorescence by the disulfide in situ folding reaction in conjunction with the optimized structures by density functional theory (DFT) confirm the difference in conformation of disulfides between the native and mutant peptides. Interestingly, the native peptide Li520/Li504 shares a similar disulfide conformation of (-,-)AntiRHHook with the redox family of proteins known to modulate disulfides, particularly in lieu of the tetrapeptide of glutaredoxin, deviating from its disulfide conformation compared to its naive protein. Analysis of the precursor gene sequences of M-superfamily conotoxins revealed the presence of Li520 in different cone snail species with distinct food habits and possible modes of evolution through the diversification of cysteine motifs. The results of the report suggest that the short redox conopeptide Li520 has evolved to facilitate the oxidative folding of conotoxins and may be useful to develop as reagents for the synthesis of therapeutically important cysteine-rich peptides.

Isolation and characterization of NGFFYamide neuropeptide from Patiria pectinifera pyloric caeca extract

Neuropeptides are small molecules that mediate intercellular signaling and regulate physiological processes. Starfish possess various myoactive neuropeptides, including starfish myorelaxant peptide (SMP) and a calcitonin-type peptide with apical muscle relaxing properties. In this study, we report the purification of a neuropeptide from starfish (Patiria pectinifera) pyloric caeca extract using high-performance liquid chromatography (HPLC) and an in vitro bioassay to screen for fractions and peptides with relaxing effects on P. pectinifera apical muscle preparations. A series of HPLC steps using reversed-phase and cation-exchange columns yielded a purified peptide with muscle-relaxing effects. The purified peptide's structure was determined by LC-MS and Edman degradation, revealing a pentapeptide with an amidated C-terminus (NGFFYamide) and a molecular mass of 646.2930 Da. This is the first report of NGFFYamide purification from P. pectinifera through biochemical methods. The nucleotide sequence encoding the NGFFYamide precursor was determined, showing the presence of a conserved neurophysin domain in the C-terminal region. RT-qPCR results confirmed high expression in radial nerves cord, consistent with previous findings on NG peptides in echinoderms. In vitro pharmacological studies on muscle preparations from P. pectinifera and Asterias amurensis revealed differential relaxing activity of NGFFYamide on apical muscles, while its effects on tube foot preparations were similar in both species. NGFFYamide also induced potent contraction in P. pectinifera cardiac stomach. Comparison of three NG peptides (NGFFYamide, NGFFFamide, and NGIWYamide) on P. pectinifera cardiac stomach revealed varying potency, suggesting class-specific receptor interactions. Tachyphylaxis was observed in P. pectinifera apical muscle but not in A. amurensis, warranting further investigation. Based on these results, it is plausible that NGFFYamide could be involved in regulating locomotion and feeding behavior in P. pectinifera, consistent with findings in Asterias rubens.

Biomimetic Trypsin-Responsive Structure-Bridged Mesoporous Organosilica Nanomedicine for Precise Treatment of Acute Pancreatitis.

Developing strategies to target injured pancreatic acinar cells (PACs) in conjunction with primary pathophysiology-specific pharmacological therapy presents a challenge in the management of acute pancreatitis (AP). We designed and synthesized a trypsin-cleavable organosilica precursor bridged by arginine-based amide bonds, leveraging trypsin's ability to selectively identify guanidino groups on arginine via Asp189 at the active S1 pocket and cleave the carboxy-terminal (C-terminal) amide bond via catalytic triads. The precursors were incorporated into the framework of mesoporous silica nanoparticles (MSNs) for encapsulating the membrane-permeable Ca2+ chelator BAPTA-AM with a high loading content (∼43.9%). Mesenchymal stem cell membrane coating and surface modification with PAC-targeting ligands endow MSNs with inflammation recruitment and precise PAC-targeting abilities, resulting in the highest distribution at 3 h in the pancreas with 4.7-fold more accumulation than that of naked MSNs. The outcomes transpired as follows: After bioinspired MSNs' skeleton biodegradation by prematurely and massively activated trypsin, BAPTA-AM was on-demand released in injured PACs, thereby effectively eliminating intracellular calcium overload (reduced Ca2+ level by 81.3%), restoring cellular redox status, blocking inflammatory cascades, and inhibiting cell necrosis by impeding the IκBα/NF-κB/TNF-α/IL-6 and CaMK-II/p-RIP3/p-MLKL/caspase-8,9 signaling pathways. In AP mice, a single dose of the formulation significantly restored pancreatic function (lipase and amylase reduced more by 60%) and improved the survival rate from 50 to 91.6%. The formulation offers a potentially effective strategy for clinical translation in AP treatment.

Two C-terminal isoforms of Aplysia tachykinin–related peptide receptors exhibit phosphorylation-dependent and phosphorylation-independent desensitization mechanisms

Diversity, a hallmark of G protein-coupled receptor (GPCR) signaling, partly stems from alternative splicing of a single gene generating more than one isoform for a receptor. Additionally, receptor responses to ligands can be attenuated by desensitization upon prolonged or repeated ligand exposure. Both phenomena have been demonstrated and exemplified by the deuterostome tachykinin (TK) signaling system, although the role of phosphorylation in desensitization remains a subject of debate. Here, we describe the signaling system for tachykinin-related peptides (TKRPs) in a protostome, mollusk Aplysia. We cloned the Aplysia TKRP precursor, which encodes three TKRPs (apTKRP-1, apTKRP-2a, and apTKRP-2b) containing the FXGXR-amide motif. In situ hybridization and immunohistochemistry showed predominant expression of TKRP mRNA and peptide in the cerebral ganglia. TKRPs and their post-translational modifications were observed in extracts of CNS ganglia using mass spectrometry. We identified two Aplysia TKRP receptors (TKRPRs), named apTKRPR-A and apTKRPR-B. These receptors are two isoforms generated through alternative splicing of the same gene and differ only in their intracellular C-termini. Structure-activity relationship analysis of apTKRP-2b revealed that both C-terminal amidation and conserved residues of the ligand are critical for receptor activation. C-terminal truncates and mutants of apTKRPRs suggested that there is a C-terminal phosphorylation-independent desensitization for both receptors. Moreover, apTKRPR-B also exhibits phosphorylation-dependent desensitization through the phosphorylation of C-terminal Ser/Thr residues. This comprehensive characterization of the Aplysia TKRP signaling system underscores the evolutionary conservation of the TKRP and TK signaling systems, while highlighting the intricacies of receptor regulation through alternative splicing and differential desensitization mechanisms.

Scalable preparation of green C-terminal amidation peptide-synthesis TAGs and the optimized TAG-assisted liquid-phase synthesis of eptifibatide

Eptifibatide is a cycloheptapeptide with C-terminal amidation and head-to-tail disulfide bridging, which has been approved as an antithrombotic drug. Currently, both at the laboratory scale and industrial level, the synthesis of eptifibatide predominantly relies on solid-phase peptide synthesis (SPPS), which is not regarded as a green sustainable and cost-effective approach to prepare eptifibatide. Herein, we have investigated the strategy employing tag-assisted liquid-phase peptide synthesis (TAG-Assisted LPPS) technology that is scalable to multi-gram scale manufacturing for eptifibatide preparation. We successfully obtained soluble Rink-Amide TAGs by esterifying organophosphorus TAG-OH with Rink-Amide, thereby facilitating the transition from synthesizing C-terminal carboxyl peptides to C-terminal amidation peptides. The products obtained during the tag-assisted coupling and Fmoc deprotection of eptifibatide intermediates can be purified through precipitation processes, eliminating the need for complex chromatographic operations. Accordingly, we developed an optimized laboratory-scale process (300 mmol) for the preparation of TAG-Rink Amide TAG, which was subsequently upscaled successfully to a 50 mmol batch synthesis of eptifibatide. The subsequent work also explores the processes of peptide cleavage and disulfide bond oxidative cyclization in eptifibatide preparation.

Synthesis and Evaluation of the First 68Ga-Labeled C-Terminal Hydroxamate-Derived Gastrin-Releasing Peptide Receptor-Targeted Tracers for Cancer Imaging with Positron Emission Tomography.

Gastrin-releasing peptide receptor (GRPR), overexpressed in many solid tumors, is a promising imaging marker and therapeutic target. Most reported GRPR-targeted radioligands contain a C-terminal amide. Based on the reported potent antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH, we synthesized C-terminal hydroxamate-derived [68Ga]Ga-LW02075 ([68Ga]Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH) and [68Ga]Ga-LW02050 ([68Ga]Ga-DOTA-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH), and compared them with the closely related and clinically validated [68Ga]Ga-SB3 ([68Ga]Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt). Binding affinities (Ki) of Ga-SB3, Ga-LW02075, and Ga-LW02050 were 1.20 ± 0.31, 1.39 ± 0.54, and 8.53 ± 1.52 nM, respectively. Both Ga-LW02075 and Ga-LW02050 were confirmed to be GRPR antagonists by calcium release assay. Imaging studies showed that PC-3 prostate cancer tumor xenografts were clearly visualized at 1 h post injection by [68Ga]Ga-SB3 and [68Ga]Ga-LW02050 in PET images, but not by [68Ga]Ga-LW02075. Ex vivo biodistribution studies conducted at 1 h post injection showed that the tumor uptake of [68Ga]Ga-LW02050 was comparable to that of [68Ga]Ga-SB3 (5.38 ± 1.00 vs. 6.98 ± 1.36 %ID/g), followed by [68Ga]Ga-LW02075 (3.97 ± 1.71 %ID/g). [68Ga]Ga-SB3 had the highest pancreas uptake (37.3 ± 6.90 %ID/g) followed by [68Ga]Ga-LW02075 (17.8 ± 5.24 %ID/g), while the pancreas uptake of [68Ga]Ga-LW02050 was only 0.53 ± 0.11 %ID/g. Our data suggest that [68Ga]Ga-LW02050 is a promising PET tracer for detecting GRPR-expressing cancer lesions.

P18: Novel Anticancer Peptide from Induced Tumor-Suppressing Cells Targeting Breast Cancer and Bone Metastasis.

The skeletal system is a common site for metastasis from breast cancer. In our prior work, we developed induced tumor-suppressing cells (iTSCs) capable of secreting a set of tumor-suppressing proteins. In this study, we examined the possibility of identifying anticancer peptides (ACPs) from trypsin-digested protein fragments derived from iTSC proteomes. The efficacy of ACPs was examined using an MTT-based cell viability assay, a Scratch-based motility assay, an EdU-based proliferation assay, and a transwell invasion assay. To evaluate the mechanism of inhibitory action, a fluorescence resonance energy transfer (FRET)-based GTPase activity assay and a molecular docking analysis were conducted. The efficacy of ACPs was also tested using an ex vivo cancer tissue assay and a bone microenvironment assay. Among the 12 ACP candidates, P18 (TDYMVGSYGPR) demonstrated the most effective anticancer activity. P18 was derived from Arhgdia, a Rho GDP dissociation inhibitor alpha, and exhibited inhibitory effects on the viability, migration, and invasion of breast cancer cells. It also hindered the GTPase activity of RhoA and Cdc42 and downregulated the expression of oncoproteins such as Snail and Src. The inhibitory impact of P18 was additive when it was combined with chemotherapeutic drugs such as Cisplatin and Taxol in both breast cancer cells and patient-derived tissues. P18 had no inhibitory effect on mesenchymal stem cells but suppressed the maturation of RANKL-stimulated osteoclasts and mitigated the bone loss associated with breast cancer. Furthermore, the P18 analog modified by N-terminal acetylation and C-terminal amidation (Ac-P18-NH2) exhibited stronger tumor-suppressor effects. This study introduced a unique methodology for selecting an effective ACP from the iTSC secretome. P18 holds promise for the treatment of breast cancer and the prevention of bone destruction by regulating GTPase signaling.

Novel Scorpion Toxin ω-Buthitoxin-Hf1a Selectively Inhibits Calcium Influx via CaV3.3 and CaV3.2 and Alleviates Allodynia in a Mouse Model of Acute Postsurgical Pain.

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 μM) and CaV3.3 (IC50 = 0.49 μM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.

Efficient production of peptidylglycine α-hydroxylating monooxygenase in yeast for protein C-terminal functionalization

Peptidylglycine α-hydroxylating monooxygenase (PHM) is pivotal for C-terminal amidation of bioactive peptides in animals, offering substantial potential for customized protein synthesis. However, efficient PHM production has been hindered by the complexity of animal cell culture and the absence of glycosylation in bacterial hosts. Here, we demonstrate the recombinant expression of Caenorhabditis elegans PHM in the yeast Pichia pastoris, achieving a remarkable space-time yield of 28.8 U/L/day. This breakthrough surpasses prior PHM production rates and eliminates the need for specialized cultivation equipment or complex transfection steps. Mass spectrometry revealed N-glycosylation at residue N182 of recombinant CePHM, which impacts the enzyme's activity as indicated by biochemical experiments. To showcase the utility of CePHM, we performed C-terminal amidation on ubiquitin at a substrate loading of 30 g/L, a concentration meeting the requirements for pharmaceutical peptide production. Overall, this work establishes an efficient PHM production method, promising advancements in scalable manufacturing of C-terminally modified bioactive peptides and probe proteins.

A novel bioinformatic method for the identification of antimicrobial peptides in metagenomes.

This study aimed to develop a new bioinformatic approach for the identification of novel antimicrobial peptides (AMPs), which did not depend on sequence similarity to known AMPs held within databases, but on structural mimicry of another antimicrobial compound, in this case an ultrashort, synthetic, cationic lipopeptide (C12-OOWW-NH2). When applied to a collection of metagenomic datasets, our outlined bioinformatic method successfully identified several short (8-10aa) functional AMPs, the activity of which was verified via disk diffusion and minimum inhibitory concentration assays against a panel of 12 bacterial strains. Some peptides had activity comparable to, or in some cases, greater than, those from published studies that identified AMPs using more conventional methods. We also explored the effects of modifications, including extension of the peptides, observing an activity peak at 9-12aa. Additionally, the inclusion of a C-terminal amide enhanced activity in most cases. Our most promising candidate (named PB2-10aa-NH2) was thermally stable, lipid-soluble, and possessed synergistic activity with ethanol but not with a conventional antibiotic (streptomycin). While several bioinformatic methods exist to predict AMPs, the approach outlined here is much simpler and can be used to quickly scan huge datasets. Searching for peptide sequences bearing structural similarity to other antimicrobial compounds may present a further opportunity to identify novel AMPs with clinical relevance, and provide a meaningful contribution to the pressing global issue of AMR.

Computational Enzyme Redesign Enhances Tolerance to Denaturants for Peptide C-Terminal Amidation.

The escalating demand for biocatalysts in pharmaceutical and biochemical applications underscores the critical imperative to enhance enzyme activity and durability under high denaturant concentrations. Nevertheless, the development of a practical computational redesign protocol for improving enzyme tolerance to denaturants is challenging due to the limitations of relying solely on model-driven approaches to adequately capture denaturant-enzyme interactions. In this study, we introduce an enzyme redesign strategy termed GRAPE_DA, which integrates multiple data-driven and model-driven computational methods to mitigate the sampling biases inherent in a single approach and comprehensively predict beneficial mutations on both the protein surface and backbone. To illustrate the methodology's effectiveness, we applied it to engineer a peptidylamidoglycolate lyase, resulting in a variant exhibiting up to a 24-fold increase in peptide C-terminal amidation activity under 2.5 M guanidine hydrochloride. We anticipate that this integrated engineering strategy will facilitate the development of enzymatic peptide synthesis and functionalization under denaturing conditions and highlight the role of engineering surface residues in governing protein stability.

Antimicrobial activity and properties of de novo design of short synthetic lipopeptides

The aim of this article is to introduce the topic of newly designed peptides as well as their biological activity. We designed nine encoded peptides composed of six amino acids. All these peptides were synthesized with C-terminal amidation. To investigate the importance of increased hydrophobicity at the amino end of the peptides, all of them were subsequently synthesized with palmitic or lithocholic acid at the N-terminus. Antimicrobial activity was tested on Gram-positive and Gram-negative bacteria and fungi. Cytotoxicity was measured on HepG2 and HEK 293 T cell cultures. Peptides bearing a hydrophobic group exhibited the best antimicrobial activity. Lipopeptides with palmitic or lithocholic acid (PAL or LCA peptides) at the N-terminus and with C-terminal amidation were highly active against Gram-positive bacteria, especially against strains of Staphylococcus aureus and Candida tropicalis. The LCA peptide SHP 1.3 with the sequence LCA-LVKRAG-NH2, had high efficiency on HepG2 human liver hepatocellular carcinoma cells (97%).

On the importance of being amidated: Analysis of the role of the conserved C-terminal amide of amylin in amyloid formation and cytotoxicity

The polypeptide hormone Amylin (also known as islet amyloid polypeptide) plays a role in regulation of glucose metabolism, but forms pancreatic islet amyloid deposits in type 2 diabetes. The process of islet amyloid formation contributes to β-cell dysfunction and the development of the disease. Amylin is produced as a pro-from and undergoes processing prior to secretion. The mature hormone contains an amidated C-terminus. Analysis of an alignment of vertebrate amylin sequences reveals that the processing signal for amidation is strictly conserved. Furthermore, the enzyme responsible for C-terminal amidation is found in all of these organisms. Comparison of the physiologically relevant amidated form to a variant with a free C-terminus (Amylin-COO−) shows that replacement of the C-terminal amide with a carboxylate slows, but does not prevent amyloid formation. Pre-fibrillar species produced by both variants are toxic to cultured β-cells, although hAmylin-COO− is moderately less so. Amyloid fibrils produced by either peptide are not toxic. Prior work (ACS Pharmacol. Translational. Sci. 1, 132–49 (2018)) shows that Amylin- COO− exhibits a 58-fold reduction in activation of the Amylin1 receptor and 20-fold reduction in activation of the Amylin3 receptor. Thus, hAmylin-COO− exhibits significant toxicity, but significantly reduced activity and offers a reagent for studies which aim to decouple hAmylin's toxic effects from its activity. The different behaviours of free and C-terminal amidated Amylin should be considered when designing systems to produce the polypeptide recombinantly.

Structure-function relationship study for sulfated protein therapeutics using hydrophobic interaction chromatography and mass spectrometry

Protein tyrosine sulfation is a post-translational modification (PTM) that is rarely reported in recombinant therapeutic proteins. However, when sulfation does occur, the additional negative charge from the modification can influence intermolecular interactions and antigen-binding activity, making it a critical quality attribute that necessitates stringent control. In this study, we developed a unique hydrophobic interaction chromatography (HIC) method for the separation and quantification of a therapeutic bispecific antibody with varying degrees of sulfation. Despite the increased surface hydrophilicity of sulfated species, the HIC method provides enhanced retention. Baseline resolution was attained based on the degree of sulfation, independent of other PTMs such as C-terminal amidation and forced deamidation. Further structure-function relationship studies of enriched sulfated bispecific antibody species were conducted using mass spectrometry and fluorescence-linked immunosorbent assay (FLISA). These studies revealed that the tyrosine sulfation modification, which occurs in the complementarity-determining region (CDR), is a critical quality attribute and can adversely impact the antibody’s binding to its cognate antigen. The evaluation of sulfation assay using HIC method confirmed it is an effective means for controlling this critical quality attribute.

Antimicrobial activities and mechanisms of truncated and amino-acid-substituted peptides derived from bacteriocin PZJ5

Truncation and amino acid residue substitution are common methods to optimise the design of antimicrobial peptides (AMPs). In the present work, seven truncated and residue-substituted derivatives of Plantaricin ZJ5 (PZJ5) were designed and synthesised. PZJ5-5 was a truncation that simultaneously contained three substituted amino-acid residues, with enhanced antimicrobial activity and low haemolytic activity. The effects of PZJ5-5 on Escherichia coli microstructure were investigated using scanning and transmission electron microscopy, which indicated that its antibacterial mechanism was similar to PZJ5. C-terminal amidation of PZJ5-5 (PZJ5-7) was deleterious, and resulted in a dramatic reduction in potency against E. coli and Listeria monocytogenes, with no potency against the other three indicator bacteria. Truncation and residue substitution of bacteriocin PZJ5 changed its antimicrobial activities and specificities, which provided a rationale for bacteriocin design.

A class of secreted mammalian peptides with potential to expand cell-cell communication

Peptide hormones and neuropeptides are signaling molecules that control diverse aspects of mammalian homeostasis and physiology. Here we provide evidence for the endogenous presence of a sequence diverse class of blood-borne peptides that we call “capped peptides.” Capped peptides are fragments of secreted proteins and defined by the presence of two post-translational modifications – N-terminal pyroglutamylation and C-terminal amidation – which function as chemical “caps” of the intervening sequence. Capped peptides share many regulatory characteristics in common with that of other signaling peptides, including dynamic physiologic regulation. One capped peptide, CAP-TAC1, is a tachykinin neuropeptide-like molecule and a nanomolar agonist of mammalian tachykinin receptors. A second capped peptide, CAP-GDF15, is a 12-mer peptide cleaved from the prepropeptide region of full-length GDF15 that, like the canonical GDF15 hormone, also reduces food intake and body weight. Capped peptides are a potentially large class of signaling molecules with potential to broadly regulate cell-cell communication in mammalian physiology.

PAM variants in patients with thyrotrophinomas, cyclical Cushing's disease and prolactinomas.

Germline loss-of-function variants in PAM, encoding peptidylglycine α-amidating monooxygenase (PAM), were recently discovered to be enriched in conditions of pathological pituitary hypersecretion, specifically: somatotrophinoma, corticotrophinoma, and prolactinoma. PAM is the sole enzyme responsible for C-terminal amidation of peptides, and plays a role in the biosynthesis and regulation of multiple hormones, including proopiomelanocortin (POMC). We performed exome sequencing of germline and tumour DNA from 29 individuals with functioning pituitary adenomas (12 prolactinomas, 10 thyrotrophinomas, 7 cyclical Cushing's disease). An unfiltered analysis was undertaken of all PAM variants with population prevalence <5%. We identified five coding, non-synonymous PAM variants of interest amongst seven individuals (six germline, one somatic). The five variants comprised four missense variants and one truncating variant, all heterozygous. Each variant had some evidence of pathogenicity based on population prevalence, conservation scores, in silico predictions and/or prior functional studies. The yield of predicted deleterious PAM variants was thus 7/29 (24%). The variants predominated in individuals with thyrotrophinomas (4/10, 40%) and cyclical Cushing's disease (2/7, 29%), compared to prolactinomas (1/12, 8%). This is the second study to demonstrate a high yield of suspected loss-of-function, predominantly germline, PAM variants in individuals with pathological pituitary hypersecretion. We have extended the association with corticotrophinoma to include the specific clinical entity of cyclical Cushing's disease and demonstrated a novel association between PAM variants and thyrotrophinoma. PAM variants might act as risk alleles for pituitary adenoma formation, with a possible genotype-phenotype relationship between truncating variants and altered temporal secretion of cortisol.

Design of novel analogues of t-DPH1 with reduced cytotoxicity, taking the three conserved characteristics of the dermaseptin family as the feasible starting point

In recent years, there has been growing scientific interest in balancing the bioactivity and cytotoxicity of antimicrobial peptides (AMPs) for more comprehensive research and application. In this study, we focused on t-DPH1, a representative member of the dermaseptin family, as a template. We synthesized five analogues were synthesised to to assess the impact of three conserved features of the dermaseptin family (1. the tryptophan residue at 3rd position, 2. the consensus motif at mid-region, and 3. C-terminal amidation) on the bioactivity of t-DPH1 and their potential to reduce cytotoxicity. Our results demonstrated that analogues lacking an amino group at the C-terminus, specifically t-DPH1-NH2, exhibited potent antimicrobial activity against Gram-negative bacteria, including Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae). Furthermore, t-DPH1-NH2 retained its robust antiproliferative activity against the human non-small lung cancer cell line H157. Regarding cytotoxicity, t-DPH1-NH2 displayed lower levels of haemolytic activity and cytotoxicity against two normal cell lines. Notably, we employed C. elegans as an in vivo model to assess the cytotoxicity of peptides, and the results revealed no induction of in vivo toxicity by t-DPH1-NH2. Collectively, t-DPH1-NH2 provides valuable insights for further investigation as promising bioactive peptide templates in the development of antibiotic and anticancer prototype drugs. The modifications implemented based on the conserved structure of the peptide family offer a new direction for reducing the cytotoxicity of peptides.