LimF is a versatile prenyltransferase catalyzing histidine-C-geranylation on diverse nonnatural substrates

Prenylation plays an important role in diversifying structure and function of secondary metabolites. Although several cyanobactin prenyltransferases have been characterized, their modes of action are mainly limited to the modification of electron-rich hetero atoms. Here we report a unique prenyltransferase originating from Limnothrix sp. CACIAM 69d, referred to as LimF, which catalyzes an unprecedented His-C-geranylation. Interestingly, LimF executes the geranylation on not only its native peptide substrate but also a wide range of exotic peptides, including thioether-closed macrocycles. We have also serendipitously uncovered an ability of Tyr-O-geranylation as the secondary function of LimF, indicating it is an unusual bifunctional prenyltransferase. Crystallographic analysis of LimF complexed with a pentapeptide substrate and a prenyl donor analog provides structural basis for its unique His recognition and its bifunctionality. Lastly, we show the LimF’s prenylation ability on various bioactive molecules containing an imidazole group, highlighting its potential as a versatile biocatalyst for site-specific geranylation.

Computational study, synthesis and evaluation of active peptides derived from Parasporin-2 and spike protein from Alphacoronavirus against colon cancer cells

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 KDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and β-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer –therapeutic agents.

Semisynthesis of functional transmembrane proteins in GUVs

Cellular transmembrane (TM) proteins are essential sentries of the cell facilitating cell-cell communication, internal signaling, and solute transport. Reconstituting functional TM proteins into model membranes remains a challenge due to the difficulty of expressing hydrophobic TM domains and the required use of detergents. Herein, we use a intein-mediated ligation strategy to semisynthesize bitopic TM proteins in synthetic membranes. We have adapted the trans splicing capabilities of split inteins for a native peptide ligation between a synthetic TM peptide embedded in the membrane of giant unilamellar vesicles (GUVs) and an expressed soluble protein. We demonstrate that the extracellular domain of programmed cell death protein 1 (PD-1), a mammalian transmembrane immune checkpoint receptor, retains its function for binding its ligand PD-L1 at a reconstituted membrane interface after ligation to a synthetic TM peptide in GUV membranes. We envision that the construction of full-length TM proteins using orthogonal split intein-mediated semisynthetic protein ligations will expand applications of membrane protein reconstitution in pharmacology, biochemistry, biophysics, and artificial cell development.

Synthesis, Physicochemical Characterization, In Vitro 2D/3D Human Cell Culture, and In Vitro Aerosol Dispersion Performance of Advanced Spray Dried and Co-Spray Dried Angiotensin (1—7) Peptide and PNA5 with Trehalose as Microparticles/Nanoparticles for Targeted Respiratory Delivery as Dry Powder Inhalers

The peptide hormone Angiotensin (1—7), Ang (1—7) or (Asp-Arg-Val-Tyr-Ile-His-Pro), is an essential component of the renin–angiotensin system (RAS) peripherally and is an agonist of the Mas receptor centrally. Activation of this receptor in the CNS stimulates various biological activities that make the Ang (1—7)/MAS axis a novel therapeutic approach for the treatment of many diseases. The related O-linked glycopeptide, Asp-Arg-Val-Tyr-Ile-His-Ser-(O-β-D-Glc)-amide (PNA5), is a biousian revision of the native peptide hormone Ang (1—7) and shows enhanced stability in vivo and greater levels of brain penetration. We have synthesized the native Ang (1—7) peptide and the glycopeptide, PNA5, and have formulated them for targeted respiratory delivery as inhalable dry powders. Solid phase peptide synthesis (SPPS) successfully produced Ang (1—7) and PNA5. Measurements of solubility and lipophilicity of raw Ang (1—7) and raw PNA5 using experimental and computational approaches confirmed that both the peptide and glycopeptide have high-water solubility and are amphipathic. Advanced organic solution spray drying was used to engineer the particles and produce spray-dried powders (SD) of both the peptide and the glycopeptide, as well as co-spray-dried powders (co-SD) with the non-reducing sugar and pharmaceutical excipient, trehalose. The native peptide, glycopeptide, SD, and co-SD powders were comprehensively characterized, and exhibited distinct glass transitions (Tg) consistent with the amorphous glassy state formation with Tgs that are compatible with use in vivo. The homogeneous particles displayed small sizes in the nanometer size range and low residual water content in the solid-state. Excellent aerosol dispersion performance with a human DPI device was demonstrated. In vitro human cell viability assays showed that Ang (1—7) and PNA5 are biocompatible and safe for different human respiratory and brain cells.

Semisynthetic head-to-tail cyclized peptides obtained by combining protein trans-splicing and intramolecular expressed protein ligation

A dual-intein approach for the preparation of head-to-tail macrocyclic peptides is reported, where synthetic and genetically encoded fragments are ligated by two native peptide bonds. A split intein ligates the...

Receptor-free Discrimination of Peptide Native Bound Conformations Suggests Limited Transferability of the “cen_std+score4l” Rosetta Energy Function From Proteins to Peptides

One of the strategies of peptide–protein docking is to pregenerate an ensemble of peptide conformations in the absence of the receptor, and then dock them as rigid bodies onto its surface. Success of this strategy requires that the scoring function that drives the pregeneration step be able to discriminate in favor of conformations that resemble the native bound conformation. Here we present a study on the discrimination of peptide native bound conformations as achieved without receptor by the “cen_std+score4L” Rosetta energy function, a low-resolution scoring function equivalent to one chosen for other tasks where the modeling of solvent effects is of special importance. The cen_std+score4L function was able to assign, on average, lower energies to native-like than to non-native decoy conformations for only 3 of our 18 test peptides; it also ranked one or more native-like decoys in the top 1% for only 2 peptides. However, by optimizing the weights of the energy terms that define the cen_std+score4L function, native discrimination improved substantially: Native-like decoys were assigned lower energies than non-native decoys for 16 peptides, with a discrimination signal larger than noise for 9 peptides, that is, 50% of the test set. And for 9 peptides, too, native-like decoys ranked in the top 1%. An ensuing energetic analysis of native-like versus non-native decoys suggests that native peptide conformations have solvation and non-local electrostatics that poorly recapitulate those of native protein conformations. Native peptide conformations are also characterized by few backbone–backbone H-bonds and by lack of compactness, presumably to optimize interaction with the receptor. Overall, this study lays groundwork for pregenerating dockable peptide conformations with Rosetta, whether the subsequent docking will be performed by Rosetta or some other software.

Abstract P102: Generation Of A Novel Peptoid-peptide Analog Of Alpha-calcitonin Gene Related Peptide (α-CGRP) For The Treatment And Prevention Of Cardiovascular Diseases

Alpha-calcitonin gene related peptide (α-CGRP) is a 37-amino acid cardioprotective neuropeptide. Studies carried out in our laboratory and others establish α-CGRP as a potential therapeutic agent against a variety of cardiovascular diseases. However, the short half-life of α-CGRP limits its use in any long-term treatment regime. The goal of the present study is to develop an α-CGRP agonist analog with extended bioavailability using peptoid chemistry. A peptoid is a N -substituted glycine peptidomimetic molecule and is identical to α-amino acid except that the side chain in a peptoid is attached on the nitrogen rather than the α-carbon atom. Inclusion of a peptoid makes the native peptide protease-resistant and thus biostable in vivo . Using a solid-phase submonomer method, we synthesized a novel human α-CGRP analog containing two monomers of N -methoxyethylglycine (NMEG) peptoid at the N-terminus. Electrospray mass spectrometry (MALDI-TOF) analysis showed that the molecular mass of synthesized peptoid-peptide hybrid, NMEG-α-CGRP, was 4044 that is ~6.7% more than native peptide. An in vitro trypan blue cell exclusion assay demonstrated that incubation of NMEG-α-CGRP (5 μM) for 7 days did not affect the viability of rat H9C2 and mouse HL-1 cardiac cells. To evaluate the biological activity of NMEG-α-CGRP, a subcutaneous injection of human α-CGRP (10 μg/mice) and NMEG-α-CGRP (1, 3, 10, and 30 μg/mice) were given in 9-week-old C57BL6 mice (n=2 mice/dose), and blood pressure (BP) was measured using a tail-cuff method. A dose response curve showed that NMEG-α-CGRP decreased BP in mice in a time-dependent manner. Beginning with and injection of 3 μg of NMEG-α-CGRP, a dip in BP (85 ± 1 mmHg; in ±SD) was observed at 10 min after injection, and BP returned to baseline (125 mmHg) by 6 h, 18 h, and 24 h when injected with 3, 10, and 30 μg doses, respectively. Moreover, 10 μg of human-α-CGRP and NMEG-α-CGRP lowered BP from baseline for 2 h and 18 h, respectively, suggesting that NMEG addition increased stability, and thus bioavailability, of α-CGRP in vivo . In summary, our results show that a NMEG based α-CGRP modification is an effective approach to increase stability and, thus, bioavailability of α-CGRP in vivo making α-CGRP a viable therapeutic drug to treat cardiovascular diseases.

T Cells Expressing a TCR-Like Antibody Selected Against the Heteroclitic Variant of a Shared MAGE-A Epitope Do Not Recognise the Cognate Epitope

Antibodies-recognising peptides bound to the major histocompatibility complex (pMHC) represent potentially valuable and promising targets for chimeric antigen receptor (CAR) T cells to treat patients with cancer. Here, a human phage-Fab library has been selected using HLA-A2 complexed with a heteroclitic peptide variant from an epitope shared among multiple melanoma-associated antigens (MAGEs). DNA restriction analyses and phage ELISAs confirmed selection of unique antibody clones that specifically bind to HLA-A2 complexes or HLA-A2-positive target cells loaded with native or heteroclitic peptide. Antibodies selected against heteroclitic peptide, in contrast to native peptide, demonstrated significantly lower to even negligible binding towards native peptide or tumour cells that naturally expressed peptides. The binding to native peptide was not rescued by phage panning with antigen-positive tumour cells. Importantly, when antibodies directed against heteroclitic peptides were engineered into CARs and expressed by T cells, binding to native peptides and tumour cells was minimal to absent. In short, TCR-like antibodies, when isolated from a human Fab phage library using heteroclitic peptide, fail to recognise its native peptide. We therefore argue that peptide modifications to improve antibody selections should be performed with caution as resulting antibodies, either used directly or as CARs, may lose activity towards endogenously presented tumour epitopes.

Addition of K22 Converts Spider Venom Peptide Pme2a from an Activator to an Inhibitor of NaV1.7

Spider venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (NaV). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/δ-theraphotoxin-Pme2a, two novel peptides from the venom of the Gooty Ornamental tarantula Poecilotheria metallica that modulate NaV channels. Pme1a is a 35 residue peptide that inhibits NaV1.7 peak current (IC50 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V1/2 activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits NaV1.7 peak current (EC50 > 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at NaV1.7 (IC50 5.6 ± 1.1 μM) and removed the effect of the native peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of spider venom-derived peptides with improved potency and selectivity at NaV channels in the future.