Novel Cell Permeable Polymers of N-Substituted L-2,3-Diaminopropionic Acid (DAPEGs) and Cellular Consequences of Their Interactions with Nucleic Acids

The present study aimed to synthesize novel polycationic polymers composed of N-substituted L-2,3-diaminopropionic acid residues (DAPEGs) and investigate their cell permeability, cytotoxicity, and DNA-binding ability. The most efficient cell membrane-penetrating compounds (O2Oc-Dap(GO2)n-O2Oc-NH2, where n = 4, 6, and 8) showed dsDNA binding with a binding constant in the micromolar range (0.3, 3.4, and 0.19 µM, respectively) and were not cytotoxic to HB2 and MDA-MB-231 cells. Selected compounds used in the transfection of a GFP plasmid showed high transfection efficacy and minimal cytotoxicity. Their interaction with plasmid DNA and the increasing length of the main chain of tested compounds strongly influenced the organization and shape of the flower-like nanostructures formed, which were unique for 5/6-FAM-O2Oc-[Dap(GO2)]8-O2Oc-NH2 and typical for large proteins.

Bringing BOS to light: Uncovering the key enzyme in the biosynthesis of the neurotoxin β-ODAP in Grass Pea (Lathyrus sativus L.)

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in parts of Asia and Africa for food and forage. While being a highly nutritious and robust crop, able to survive both drought and floods, it produces a neurotoxic compound, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which can cause a severe neurological disorder if consumed as a main diet component. So far, the enzyme that catalyzes the formation of β-ODAP has not been identified. By combining protein purification and enzymatic assays with transcriptomic and proteomic analyses, we were able to identify the enzyme β-ODAP synthetase (BOS) from grass pea. We show that BOS is an HXXXD-type acyltransferase of the BAHD superfamily and that its crystal structure is highly similar to that of plant hydroxycinnamoyl transferases. The identification of BOS, more than 50 years after it was proposed, paves the way towards the generation of non-toxic grass pea cultivars safe for human and animal consumption.

Interaction of the neutral amino acid transporter ASCT2 with basic amino acids

Glutamine transport across cell membranes is performed by a variety of transporters, including the alanine serine cysteine transporter 2 (ASCT2). The substrate-binding site of ASCT2 was proposed to be specific for small amino acids with neutral side chains, excluding basic substrates such as lysine. A series of competitive inhibitors of ASCT2 with low µM affinity were developed previously, on the basis of the 2,4-diaminobutyric acid (DAB) scaffold with a potential positive charge in the side chain. Therefore, we tested whether basic amino acids with side chains shorter than lysine can interact with the ASCT2 binding site. Molecular docking of L-1,3-diaminopropionic acid (L-DAP) and L-DAB suggested that these compounds bind to ASCT2. Consistent with this prediction, L-DAP and L-DAB, but not ornithine, lysine or D-DAP, elicited currents when applied to ASCT2-expressing cells. The currents were carried by anions and showed the hallmark properties of ASCT2 currents induced by transported substrates. The L-DAP response could be eliminated by a competitive ASCT2 inhibitor, suggesting that binding occurs at the substrate binding site. The KM for L-DAP was weakly voltage dependent. Furthermore, the pH dependence of the L-DAP response showed that the compound can bind in several protonation states. Together, these results suggest that the ASCT2 binding site is able to recognize L-amino acids with short, basic side chains, such as the L-DAP derivative β-N-methylamino-l-Alanine (BMAA), a well-studied neurotoxin. Our results expand the substrate specificity of ASCT2 to include amino acid substrates with positively charged side chains.

A Peptidomimetic Fluorescent Probe to Detect the Trypsin β2 Subunit of the Human 20S Proteasome

This work describes the chemical synthesis, combinatorial selection, and enzymatic evaluation of peptidomimetic fluorescent substrates specific for the trypsin-like (β2) subunit of the 20S human proteasome. After deconvolution of a library comprising nearly 6000 compounds composed of peg substituted diaminopropionic acid DAPEG building blocks, the sequence ABZ–Dap(O2(Cbz))–Dap(GO1)–Dap(O2(Cbz))–Arg–ANB–NH2, where ABZ is 2-aminobenzoic acid, and ANB- 5 amino 2- nitro benzoic acid was selected. Its cleavage followed sigmoidal kinetics, characteristic for allosteric enzymes, with Km = 3.22 ± 0.02 μM, kcat = 245 s−1, and kcat/Km = 7.61 × 107 M−1 s−1. This process was practically halted when a selective inhibitor of the β2 subunit of the 20S human proteasome was supplemented to the reaction system. Titration of the substrate resulting in decreased amounts of proteasome 20S produced a linear signal up to 10−11 M. Using this substrate, we detected human proteasome 20S in human urine samples taken from the bladders of cancer patients. This observation could be useful for the noninvasive diagnosis of this severe disease.

Biochemical Composition of Lathyrus L. Seeds: Antioxidant Activities, Phenolic Profiles, β-ODAP and Protein Contents

Lathyrus taxa are used in different areas including nutritive, agricultural areas, and they are seen as the source of both protein and phenolics. Lathyrus taxa seeds contain a neurotoxic substance called β-N-oxalyl-l-β,β-diaminopropionic acid (β-ODAP) in different amounts. Lathyrus species grown in Turkey is reported to have low β-ODAP and high protein content. In this study, antioxidant activities, phenolic profiles, β-ODAP and protein contents of six Lathyrus taxa were investigated. Total phenolic content (TPC) in Lathyrus taxa used was ranging from 0.17±0.05 to 5.10±0.02. The antioxidant activities were observed in a wide interval that IC50 values were between 7.05±0.11 to 1.15±0.08 mg/mL. The highest TPC and antioxidant activity were recorded for L. clymenum. In HPLC analysis, gallic, p-hydroxybenzoic, caffeic, chlorogenic acid and epicatechin in all of the extracts were determined. Also, β-ODAP and protein contents in seeds of the Lathyrus taxa were found between 0.20–1.18 mg/g and 22.66–29.74%, respectively. β-ODAP contents of investigated Layhyrus taxa were within the safe consumption range in terms of health (plessthan2.00 mg/g). The investigated Lathyrus taxa were found to be excellent protein sources with low β-ODAP content and contain natural antioxidants. As a result, this study has provided important data for wild Lathyrus taxa to be used as a cheap protein source and functional food for promoting health.

The L-alanosine gene cluster encodes a pathway for diazeniumdiolate biosynthesis

N-nitroso-containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate (N-nitrosohydroxylamine) group display a wide range of bioactivities ranging from cytotoxicity to metal chelation. Despite the importance of this structural motif, knowledge of its biosynthesis is limited. Herein, we describe the discovery of a biosynthetic gene cluster in Streptomyces alanosinicus ATCC 15710 responsible for producing the diazeniumdiolate natural product L-alanosine. Gene disruption and stable isotope feeding experiments identified essential biosynthetic genes and revealed the nitrogen source of the N-nitroso group. Additional biochemical characterization of the biosynthetic enzymes revealed that the non-proteinogenic amino acid L-2,3-diaminopropionic acid (L-Dap) is synthesized and loaded onto a peptidyl carrier protein (PCP) domain in L-alanosine biosynthesis, which we propose may be a mechanism of handling unstable intermediates generated en route to the diazeniumdiolate. This research framework will facilitate efforts to determine the biochemistry of diazeniumdiolate formation.

An Improved HILIC HPLC-MS/MS Method for the Determination of β-ODAP and Its α Isomer in Lathyrus sativus

β-N-Oxalyl-l-α,β-diaminopropionic acid (β-ODAP) is a non-protein amino acid present in Lathyrus sativus (grass pea) and other Lathyrus species, in parallel with its nontoxic isomer, α-ODAP. When consuming grass pea for several months as staple food, β-ODAP may cause neurolathyrism, a motor neuron degeneration syndrome. Therefore, the independent quantification of both ODAP isomers instead of only the total amount in grass pea allows the identification of less toxic varieties and the development of tools to support breeding for improving grass pea quality. In this work, a simple and fast HPLC-MS/MS method was developed without sample derivatization, using a hydrophilic interaction chromatography (HILIC) column and an isocratic gradient of eluents for 18 min, which allowed the determination of both α- and β-ODAP. The proposed method was fully validated and applied to the determination of α- and β-ODAP contents in a diverse collection of 107 grass pea accessions representative of the main grass pea-growing geographical regions in the world, with the prompt identification of contrasting accessions. β-ODAP content in the analyzed grass pea samples ranged from 0.45 ± 0.02 to 6.04 ± 0.45 mg g−1. The moderate correlation found between α- and β-ODAP contents (0.65) in this collection reinforces the importance of the independent quantification of both ODAP isomers.

Selective incorporation of proteinaceous over nonproteinaceous cationic amino acids in model prebiotic oligomerization reactions

Numerous long-standing questions in origins-of-life research center on the history of biopolymers. For example, how and why did nature select the polypeptide backbone and proteinaceous side chains? Depsipeptides, containing both ester and amide linkages, have been proposed as ancestors of polypeptides. In this paper, we investigate cationic depsipeptides that form under mild dry-down reactions. We compare the oligomerization of various cationic amino acids, including the cationic proteinaceous amino acids (lysine, Lys; arginine, Arg; and histidine, His), along with nonproteinaceous analogs of Lys harboring fewer methylene groups in their side chains. These analogs, which have been discussed as potential prebiotic alternatives to Lys, are ornithine, 2,4-diaminobutyric acid, and 2,3-diaminopropionic acid (Orn, Dab, and Dpr). We observe that the proteinaceous amino acids condense more extensively than these nonproteinaceous amino acids. Orn and Dab readily cyclize into lactams, while Dab and Dpr condense less efficiently. Furthermore, the proteinaceous amino acids exhibit more selective oligomerization through their α-amines relative to their side-chain groups. This selectivity results in predominantly linear depsipeptides in which the amino acids are α-amine−linked, analogous to today’s proteins. These results suggest a chemical basis for the selection of Lys, Arg, and His over other cationic amino acids for incorporation into proto-proteins on the early Earth. Given that electrostatics are key elements of protein−RNA and protein−DNA interactions in extant life, we hypothesize that cationic side chains incorporated into proto-peptides, as reported in this study, served in a variety of functions with ancestral nucleic acid polymers in the early stages of life.