Novel Peptide‐Heterocycle Hybrids: Synthesis and Preliminary Studies on Calpain Inhibition

New peptidic compounds, having peptide chains linked to bi‐ and tricyclic heterocycles (peptide‐heterocycle hybrids), have been synthesized. The heterocyclic components are derivatives of partially reduced isoquinoline and pyrido[1,2‐b]isoquinoline bearing α,β‐unsaturated carbonyl functionalities. The heterocyclic compounds have been used as acylating agents in coupling reactions with short N‐unprotected peptides. Based on our interest on potential calpain inhibitors, we have used short (2–4 amino acids) peptides with hydrophobic amino acids of the two enantiomeric series. We report preliminary studies on the inhibition of calpain, with some compounds having IC₅₀ values in the nanomolar range.     

Formation versus Hydrolysis of the Peptide Bond from a Quantum-mechanical Viewpoint: The Role of Mineral Surfaces and Implications for the Origin of Life

The condensation (polymerization by water elimination) of molecular building blocks to yield the first active biopolymers (e.g. of amino acids to form peptides) during primitive Earth is an intriguing question that nowadays still remains open since these processes are thermodynamically disfavoured in highly dilute water solutions. In the present contribution, formation and hydrolysis of glycine oligopeptides occurring on a cluster model of sanidine feldspar (001) surface have been simulated by quantum mechanical methods. Results indicate that the catalytic interplay between Lewis and Brønsted sites both present at the sanidine surface, in cooperation with the London forces acting between the biomolecules and the inorganic surface, plays a crucial role to: i) favour the condensation of glycine to yield oligopeptides as reaction products; ii) inhibit the hydrolysis of the newly formed oligopeptides. Both facts suggest that mineral surfaces may have helped in catalyzing, stabilizing and protecting from hydration the oligopeptides formed in the prebiotic era.     

Synthesis of silk sericin peptides–L‐asparaginase bioconjugates and their characterization

The natural silk sericin, recovered from Bombyx mori silk waste by degumming and degrading, is a water‐soluble peptide with different molecular masses, ranging from 20 to 60 kDa. It is composed of 15 sorts of amino acids, among which the polar amino acids with hydroxyl, carboxyl and amino groups such as aspartic acid, serine and lysine account for 72%. The covalent attachment of the silk sericin peptides to L ‐asparaginase (ASNase) produces silk sericin peptides–L ‐asparaginase (SS–ASNase) bioconjugates that are active, stable, have a lower immune response, and have extended half‐lives in vitro in human serum. The modified enzyme coupled with sericin protein retains 55.8% of the original activity of the native enzyme. The optimal pH of SS–ASNase derivatives shifts considerably, to 5.0 in comparison with pH 6.0–8.0 of the native form. The thermostability and resistance to trypsin digestion of the modified enzyme are greatly enhanced as compared with ASNase alone. The Michaelis constant (K_m) of SS–ASNase is 65 times lower than that of the enzyme alone. This suggests that the affinity of the enzyme to its substrate L ‐asparagine greatly increases when bioconjugated with silk sericin. The in vivo experiments also show that the silk sericin peptides have no immunogenicity, and the antigenicity of the enzyme is obviously decreased when coupled covalently with the silk sericin peptides. Copyright © 2005 Society of Chemical Industry     

Ultrasmall peptides self-assemble into diverse nanostructures: morphological evaluation and potential implications

In this study, we perform a morphological evaluation of the diverse nanostructures formed by varying concentration and amino acid sequence of a unique class of ultrasmall self-assembling peptides. We modified these peptides by replacing the aliphatic amino acid at the C-aliphatic terminus with different aromatic amino acids. We tracked the effect of introducing aromatic residues on self-assembly and morphology of resulting nanostructures. Whereas aliphatic peptides formed long, helical fibers that entangle into meshes and entrap >99.9% water, the modified peptides contrastingly formed short, straight fibers with a flat morphology. No helical fibers were observed for the modified peptides. For the aliphatic peptides at low concentrations, different supramolecular assemblies such as hollow nanospheres and membrane blebs were found. Since the ultrasmall peptides are made of simple, aliphatic amino acids, considered to have existed in the primordial soup, study of these supramolecular assemblies could be relevant to understanding chemical evolution leading to the origin of life on Earth. In particular, we propose a variety of potential applications in bioengineering and nanotechnology for the diverse self-assembled nanostructures.     

Probing the Role of Chirality in Phospholipid Membranes

Nucleotides, amino acids, sugars, and lipids are almost ubiquitously homochiral within individual cells on Earth. While oligonucleotides and proteins exist as one natural chirality throughout the tree of life, two stereoisomers of phospholipids have separately emerged in archaea and bacteria, an evolutionary divergence known as “the lipid divide”. Within this review, we focus on the emergence of phospholipid homochirality and compare the stability of synthetic homochiral and heterochiral membranes in vitro. We discuss chemical probes designed to study the stereospecific interactions of lipid membranes in vitro. Overall, we aim to highlight studies that help elucidate the determinants of stereospecific interactions between lipids, peptides, and small molecule ligands. Continued work in understanding the drivers of favorable interactions between chiral molecules and biological membranes will lead to the design of increasingly selective chemical tools for bioorthogonal labeling of lipid membranes and safer membrane-associating pharmaceuticals.     

A Cactus‐Derived Toxin‐Like Cystine Knot Peptide with Selective Antimicrobial Activity

Naturally occurring cystine knot peptides show a wide range of biological activity, and as they have inherent stability they represent potential scaffolds for peptide‐based drug design and biomolecular engineering. Here we report the discovery, sequencing, chemical synthesis, three‐dimensional solution structure determination and bioactivity of the first cystine knot peptide from Cactaceae (cactus) family: Ep‐AMP1 from Echinopsis pachanoi. The structure of Ep‐AMP1 (35 amino acids) conforms to that of the inhibitor cystine knot (or knottin) family but represents a novel diverse sequence; its activity was more than 500 times higher against bacterial than against eukaryotic cells. Rapid bactericidal action and liposome leakage implicate membrane permeabilisation as the mechanism of action. Sequence homology places Ec‐AMP1 in the plant C6‐type of antimicrobial peptides, but the three dimensional structure is highly similar to that of a spider neurotoxin.     

Improved Identification of Small Open Reading Frames Encoded Peptides by Top-Down Proteomic Approaches and De Novo Sequencing

Small open reading frames (sORFs) have translational potential to produce peptides that play essential roles in various biological processes. Nevertheless, many sORF-encoded peptides (SEPs) are still on the prediction level. Here, we construct a strategy to analyze SEPs by combining top-down and de novo sequencing to improve SEP identification and sequence coverage. With de novo sequencing, we identified 1682 peptides mapping to 2544 human sORFs, which were all first characterized in this work. Two-thirds of these new sORFs have reading frame shifts and use a non-ATG start codon. The top-down approach identified 241 human SEPs, with high sequence coverage. The average length of the peptides from the bottom-up database search was 19 amino acids (AA); from de novo sequencing, it was 9 AA; and from the top-down approach, it was 25 AA. The longer peptide positively boosts the sequence coverage, more efficiently distinguishing SEPs from the known gene coding sequence. Top-down has the advantage of identifying peptides with sequential K/R or high K/R content, which is unfavorable in the bottom-up approach. Our method can explore new coding sORFs and obtain highly accurate sequences of their SEPs, which can also benefit future function research.     

Efficient Synthesis of 2’-O-Methoxyethyl Oligonucleotide-Cationic Peptide Conjugates

Single-stranded phosphorothioate (PS) oligonucleotide drugs have shown potential for the treatment of several rare diseases. However, a barrier to their widespread use is that they exhibit activity in only a narrow range of tissues. One way to circumvent this constraint is to conjugate them to cationic cell-penetrating peptides (CPPs). Although there are several examples of morpholino and peptide nucleic acids conjugated with CPPs, there are noticeably few examples of PS oligonucleotide-CPP conjugates. This is surprising given that PS oligonucleotides presently represent the largest class of approved RNA-based drugs, including Nusinersen, that bears the 2’-O-methoxyethyl (MOE)-chemistry. In this work, we report a method for in-solution conjugation of cationic, hydrophobic peptides or human serum albumin to a 22-nucleotide MOE-PS oligonucleotide. Conjugates were obtained in high yields and purities. Our findings pave the way for their large-scale synthesis and testing in vivo.     

Peptoid–Peptide Hybrid Backbone Architectures

Peptidomimetic oligomers and foldamers have received considerable attention for over a decade, with β‐peptides and the so‐called peptoids (N‐alkylglycine oligomers) representing prominent examples of such architectures. Lately, hybrid or mixed backbones consisting of both α‐ and β‐amino acids (α/β‐peptides) have been investigated in some detail as well. The present Minireview is a survey of the literature concerning hybrid structures of α‐amino acids and peptoids, including β‐peptoids (N‐alkyl‐β‐alanine oligomers), and is intended to give an overview of this area of research within the field of peptidomimetic science.     

Alpha‐Linolenic Acid,but Not Palmitic Acid,Negatively Impacts Survival,Asexual Reproductive Rate,and Clonal Offspring Size in Hydra oligactis

Hydra, as sit‐and‐wait predators with limited food selectivity, could serve as model organisms for the analysis of the effect of a particular dietary component on growth and reproduction. We investigated the effect of food quality and of diets enriched with palmitic (PAM) or α‐linolenic acid (ALA) on the life history traits of two hydra species: Hydra oligactis and Hydra vulgaris. We tested the hypothesis that a diet enriched with polyunsaturated fatty acids (PUFA) can stimulate growth and reproduction in simple metazoans with a sit‐and‐wait type of predatory strategy. Our results revealed that a diet based on Artemia nauplii, which are not a natural food for freshwater hydra, stimulated growth, asexual reproduction, and survival in hydra. Artemia nauplii were characterized by the highest lipid content of all used food sources. The analysis of the fatty acid content of hydra indicated the domination the n‐6 fatty acids over n‐3 (eicosapentaenoic acid [EPA], docosahexaenoic acid [DHA], and ALA). Arachidonic acid appeared to be the dominant PUFA in Hydra, irrespective of diet supplementation with palmitic acid or ALA. The dietary supplementation of ALA negatively affected the survival, asexual reproductive rate, and size of clonal offspring of H. oligactis and had no effect on the life history traits of H. vulgaris. Our results also suggest that the hydras are not able to efficiently convert ALA into other essential fatty acids, such as EPA and DHA. To our knowledge, this is the first report about the adverse effects of n‐3 fatty acid supplementation in primitive metazoans such as hydra.     

Designed RNAs with Two Peptide‐Binding Units as Artificial Templates for Native Chemical Ligation of RNA‐Binding Peptides

The template effect plays important roles not only in modern synthetic and enzymatic catalysis but also in the ancient “RNA‐polypeptide (RNP) world,” which has been postulated to be a crucial stage in the origin of life. To mimic primitive template catalysis of peptide ligations by RNAs, we previously reported the design and synthesis of a ternary RNP complex in which the ligation of two peptides was significantly facilitated by a template RNA with two peptide‐binding units. However, RNA molecules also promoted the ligation reaction in a nonspecific manner through electrostatic interactions between RNA and basic peptides. In this study, we suppressed this effect by reducing the length of the original template derived from the Tetrahymena intron RNA. This modification, however, decreased the template ability for the specific reaction. As an alternative RNA that was as effective as the original template, we found that a self‐dimerizing RNA was a promising template for peptide ligation without a nonspecific effect.     

New Antimicrobial Hexapeptides: Synthesis,Antimicrobial Activities,Cytotoxicity, and Mechanistic Studies

Synthetic antimicrobial peptides have recently emerged as promising candidates against drug‐resistant pathogens. We identified a novel hexapeptide, Orn‐D ‐Trp‐D ‐Phe‐Ile‐D ‐Phe‐His(1‐Bzl)‐NH₂, which exhibits broad‐spectrum antifungal and antibacterial activity. A lead optimization was undertaken by conducting a full amino acid scan with various proteinogenic and non‐proteinogenic amino acids depending on the hydrophobic or positive‐charge character of residues at various positions along the sequence. The hexapeptide was also cyclized to study the correlation between the linear and cyclic structures and their respective antimicrobial activities. The synthesized peptides were found to be active against the fungus Candida albicans and Gram‐positive bacteria such as methicillin‐resistant Staphylococcus aureus and methicillin‐resistant Staphylococcus epidermidis, as well as the Gram‐negative bacterium Escherichia coli; MIC values for the most potent structures were in the range of 1–5 μg mL^?1 (IC₅₀ values in the range of 0.02–2 μg mL^?1). Most of the synthesized peptides showed no cytotoxic effects in an MTT assay up to the highest test concentration of 200 μg mL^?1. A tryptophan fluorescence quenching study was performed in the presence of negatively charged and zwitterionic model membranes, mimicking bacterial and mammalian membranes, respectively. The results of the fluorescence study demonstrate that the tested peptides are selective toward bacterial over mammalian cells; this is associated with a preferential interaction between the peptides and the negatively charged phospholipids of bacterial cells.     

Prebiotic Membranes and Micelles Do Not Inhibit Peptide Formation During Dehydration

Cycles of dehydration and rehydration could have enabled formation of peptides and RNA in otherwise unfavorable conditions on the early Earth. Development of the first protocells would have hinged upon colocalization of these biopolymers with fatty acid membranes. Using atomic force microscopy, we find that a prebiotic fatty acid (decanoic acid) forms stacks of membranes after dehydration. Using LC-MS-MS (liquid chromatography-tandem mass spectrometry) with isotope internal standards, we measure the rate of formation of serine dipeptides. We find that dipeptides form during dehydration at moderate temperatures (55 °C) at least as fast in the presence of decanoic acid membranes as in the absence of membranes. Our results are consistent with the hypothesis that protocells could have formed within evaporating environments on the early Earth.     

Kinetic Analysis of PRMT1 Reveals Multifactorial Processivity and a Sequential Ordered Mechanism

Arginine methylation is a prevalent post‐translational modification in eukaryotic cells. Two significant debates exist within the field: do these enzymes dimethylate their substrates in a processive or distributive manner, and do these enzymes operate using a random or sequential method of bisubstrate binding? We revealed that human protein arginine N‐methyltransferase 1 (PRMT1) enzyme kinetics are dependent on substrate sequence. Further, peptides containing an Nη‐hydroxyarginine generally demonstrated substrate inhibition and had improved K_M values, which evoked a possible role in inhibitor design. We also revealed that the perceived degree of enzyme processivity is a function of both cofactor and enzyme concentration, suggesting that previous conclusions about PRMT sequential methyl transfer mechanisms require reassessment. Finally, we demonstrated a sequential ordered Bi–Bi kinetic mechanism for PRMT1, based on steady‐state kinetic analysis. Together, our data indicate a PRMT1 mechanism of action and processivity that might also extend to other functionally and structurally conserved PRMTs.     

α‐Complementation in an Artificial Genome Replication System in Liposomes

Genome size is considered one of the limiting factors for the replication of primitive life forms. However, the relationship between genome size and replication efficiency has not been tested experimentally. In this study, we examined the effect of genome size on genome replication by using an artificial cell model: a self‐replicating RNA genome encapsulated in a liposome. For the reduced genome size we used α‐complementation of the lacZ gene. We first characterized α‐complementation in the purified translation system and then applied α‐complementation to the genome replication system. The reduction in the genome size together with the addition of ω‐fragment increased the replication efficiency approximately eightfold. This result provides experimental evidence that genome size can be a limiting factor for primitive self‐replication systems; it also implies that this artificial cell model could be a useful experimental model to identify possible mechanisms of genome enlargement.     

Biosynthetic Gene Cluster of Linaridin Peptides Contains Epimerase Gene

Salinipeptins belong to the type-A linaridin class of ribosomally synthesized and post-translationally modified peptides (RiPPs) comprising 22 amino acid residues with multiple D -amino acids. Although chirality of other type-A linaridins, such as grisemycin and cypemycin, has not been reported, the biosynthetic gene clusters of type-A linaridins have identical gene organization. Here, we report heterologous expression of grisemycin biosynthetic gene cluster (grm) and show that grisemycin contains multiple D -amino acids, similar to salinipeptins. The heterologous expression experiments also confirm the involvement of a novel peptide epimerase in grisemycin biosynthesis. Gene-deletion experiments indicate that grmL, a single gene with unknown function, is indispensable for grisemycin production. We also show that the presence of D -amino acids is likely a common feature of linaridin natural products by analyzing two other type-A linaridin clusters.     

The composition and organization of cytoplasm in prebiotic cells

This article discusses the hypothesized composition and organization of cytoplasm in prebiotic cells from a theoretical perspective and also based upon what is currently known about bacterial cytoplasm. It is unknown if the first prebiotic, microscopic scale, cytoplasm was initially contained within a primitive, continuous, semipermeable membrane, or was an uncontained gel substance, that later became enclosed by a continuous membrane. Another possibility is that the first cytoplasm in prebiotic cells and a primitive membrane organized at the same time, permitting a rapid transition to the first cell(s) capable of growth and division, thus assisting with the emergence of life on Earth less than a billion years after the formation of the Earth. It is hypothesized that the organization and composition of cytoplasm progressed initially from an unstructured, microscopic hydrogel to a more complex cytoplasm, that may have been in the volume magnitude of about 0.1-0.2 μm(3) (possibly less if a nanocell) prior to the first cell division.     

Rapid Determination of the Amino Acid Configuration of Xenotetrapeptide

An E. coli strain with deletions in five transaminases (ΔaspC ΔilvE ΔtyrB ΔavtA ΔybfQ) was constructed to be unable to degrade several amino acids. This strain was used as an expression host for the analysis of the amino acid configuration of nonribosomally synthesized peptides, including the novel peptide “xenotetrapeptide” from Xenorhabdus nematophila, by using a combination of labeling experiments and mass spectrometry. Additionally, the number of D ‐amino acids in the produced peptide was assigned following simple cultivation of the expression strain in D₂O.     

N‐3 fatty acids for human nutrition: stability considerations

Currently there is great interest in dietary n‐3 fatty acids to promote health. The food industry aims to produce food products enriched in α‐linolenic acid (Ln), eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) to reduce some of the physiological effects of linoleic acid (L), the major polyunsaturated fatty acid in our diet. However, the goal is hampered by the susceptibility of the n‐3 fatty acids to oxidation. As a result the sensory and nutritional quality of such foods deteriorates. Lipid scientists therefore have to find a way to stabilise these fatty acids. Innovative technologies to protect n‐3 polyunsaturates using antioxidants, adequate preparation, refining and packaging of the oil are needed. In this paper we review the inherent stability and the stabilisation of these nutritionally valuable polyunsaturated fatty acids.     

On the Chiral Homogeneity of Nature: From Atoms to Small Molecules

The genesis of enantiopurity in nature, as exemplified by terrestrial amino acids and sugars, has fascinated scientists for more than 150 years. This is a field where we know what we do not know. Starting from theoretical treatments postulated more than 50 years ago, numerous hypotheses, theories, and experiments have been formulated to claim niches of logic and truth. Such arguments vary from purely speculative, often bizarre, to sound enough in terms of reproducibility and feasible generation of mirror symmetry breaking. Although we cannot exclude a biotic origin (i.e,. homochirality was an a posteriori event where one enantiomer tripped and the other survived and evolved, in close resemblance to a Darwinian-like process), most data and models available are consistent with an abiotic origin that amplified small and stochastic imbalances to produce enantiopure homogeneity prior to life. This analysis revisits recent theoretical background and experiments, which appear to be prebiotically credible, and focuses on primordial molecules such as amino acids. This by no means implies a recreation of the origin of life on Earth, as this task will likely be a surmise forever. These strategies reveal a high degree of robustness and prove how home-made enantiopurity can be accomplished. Such routes should, in addition, be of enormous practical interest for the production of chiral materials and drugs.