A fluorometric assay to determine labile copper(II) ions in serum.

Labile copper(II) ions (Cu2+) in serum are considered to be readily available for cellular uptake and to constitute the biologically active Cu2+ species in the blood. It might also be suitable to reflect copper dyshomeostasis during diseases such as Wilson's disease (WD) or neurological disorders. So far, no direct quantification method has been described to determine this small Cu2+ subset. This study introduces a fluorometric high throughput assay using the novel Cu2+ binding fluoresceine-peptide sensor FP4 (Kd of the Cu2+-FP4-complex 0.38 pM) to determine labile Cu2+ in human and rat serum. Using 96 human serum samples, labile Cu2+was measured to be 0.14 ± 0.05 pM, showing no correlation with age or other serum trace elements. No sex-specific differences in labile Cu2+ concentrations were noted, in contrast to the total copper levels in serum. Analysis of the effect of drug therapy on labile Cu2+ in the sera of 19 patients with WD showed a significant decrease in labile Cu2+ following copper chelation therapy, suggesting that labile Cu2+ may be a specific marker of disease status and that the assay could be suitable for monitoring treatment progress.

Gene amplifications cause high-level resistance against albicidin in gram-negative bacteria.

Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

One Size Fits All-Venomics of the Iberian Adder (Vipera seoanei, Lataste 1878) Reveals Low Levels of Venom Variation across Its Distributional Range.

European vipers (genus Vipera) are medically important snakes displaying considerable venom variation, occurring at different levels in this group. The presence of intraspecific venom variation, however, remains understudied in several Vipera species. Vipera seoanei is a venomous snake endemic to the northern Iberian Peninsula and south-western France, presenting notable phenotypic variation and inhabiting several diverse habitats across its range. We analysed the venoms of 49 adult specimens of V. seoanei from 20 localities across the species' Iberian distribution. We used a pool of all individual venoms to generate a V. seoanei venom reference proteome, produced SDS-PAGE profiles of all venom samples, and visualised patterns of variation using NMDS. By applying linear regression, we then assessed presence and nature of venom variation between localities, and investigated the effect of 14 predictors (biological, eco-geographic, genetic) on its occurrence. The venom comprised at least 12 different toxin families, of which five (i.e., PLA2, svSP, DI, snaclec, svMP) accounted for about 75% of the whole proteome. The comparative analyses of the SDS-PAGE venom profiles showed them to be remarkably similar across the sampled localities, suggesting low geographic variability. The regression analyses suggested significant effects of biological and habitat predictors on the little variation we detected across the analysed V. seoanei venoms. Other factors were also significantly associated with the presence/absence of individual bands in the SDS-PAGE profiles. The low levels of venom variability we detected within V. seoanei might be the result of a recent population expansion, or of processes other than directional positive selection.

Hydroxy Acid Activation in Fungal Non-Ribosomal Peptide Synthesis Assessed by Site-Directed Mutagenesis.

The fungal cyclodepsipeptides (CDPs) enniatin, beauvericin, bassianolide, and PF1022 consist of alternating N-methylated l-amino and d-hydroxy acids. They are synthesized by non-ribosomal peptide synthetases (NRPS). The amino acid and hydroxy acid substrates are activated by adenylation (A) domains. Although various A domains have been characterized thus giving insights into the mechanism of substrate conversion, little is known about the utilization of hydroxy acids in NRPSs. Therefore, we used homology modelling and molecular docking of the A1 domain of enniatin synthetase (EnSyn) to gain insights into the mechanism of hydroxy acid activation. We introduced point mutations into the active site and used a photometric assay to study the substrate activation. The results suggest that the hydroxy acid is selected by interaction with backbone carbonyls rather than by a specific side chain. These insights enhance the understanding of non-amino acid substrate activation and could contribute to the engineering of depsipeptide synthetases.

Peptidomics.

Peptides are biopolymers, typically consisting of 2-50 amino acids. They are biologically produced by the cellular ribosomal machinery or by non-ribosomal enzymes and, sometimes, other dedicated ligases. Peptides are arranged as linear chains or cycles, and include post-translational modifications, unusual amino acids and stabilizing motifs. Their structure and molecular size render them a unique chemical space, between small molecules and larger proteins. Peptides have important physiological functions as intrinsic signalling molecules, such as neuropeptides and peptide hormones, for cellular or interspecies communication, as toxins to catch prey or as defence molecules to fend off enemies and microorganisms. Clinically, they are gaining popularity as biomarkers or innovative therapeutics; to date there are more than 60 peptide drugs approved and more than 150 in clinical development. The emerging field of peptidomics comprises the comprehensive qualitative and quantitative analysis of the suite of peptides in a biological sample (endogenously produced, or exogenously administered as drugs). Peptidomics employs techniques of genomics, modern proteomics, state-of-the-art analytical chemistry and innovative computational biology, with a specialized set of tools. The complex biological matrices and often low abundance of analytes typically examined in peptidomics experiments require optimized sample preparation and isolation, including in silico analysis. This Primer covers the combination of techniques and workflows needed for peptide discovery and characterization and provides an overview of various biological and clinical applications of peptidomics.

Transcription activation by the resistance protein AlbA as a tool to evaluate derivatives of the antibiotic albicidin.

The rising numbers of fatal infections with resistant pathogens emphasizes the urgent need for new antibiotics. Ideally, new antibiotics should be able to evade or overcome existing resistance mechanisms. The peptide antibiotic albicidin is a highly potent antibacterial compound with a broad activity spectrum but also with several known resistance mechanisms. In order to assess the effectiveness of novel albicidin derivatives in the presence of the binding protein and transcription regulator AlbA, a resistance mechanism against albicidin identified in Klebsiella oxytoca, we designed a transcription reporter assay. In addition, by screening shorter albicidin fragments, as well as various DNA-binders and gyrase poisons, we were able to gain insights into the AlbA target spectrum. We analysed the effect of mutations in the binding domain of AlbA on albicidin sequestration and transcription activation, and found that the signal transduction mechanism is complex but can be evaded. Further demonstrating AlbA's high level of specificity, we find clues for the logical design of molecules capable of avoiding the resistance mechanism.

Discovery of the Lanthipeptide Curvocidin†and Structural Insights into its Trifunctional Synthetase CuvL.

Lanthipeptides are ribosomally-synthesized natural products from bacteria featuring stable thioether-crosslinks and various bioactivities. Herein, we report on a new clade of tricyclic class-IV lanthipeptides with curvocidin from Thermomonospora curvata as its first representative. We obtained crystal structures of the corresponding lanthipeptide synthetase CuvL that showed a circular arrangement of its kinase, lyase and cyclase domains, forming a central reaction chamber for the iterative substrate processing involving nine catalytic steps. The combination of experimental data and artificial intelligence-based structural models identified the N-terminal subdomain of the kinase domain as the primary site of substrate recruitment. The ribosomal precursor peptide of curvocidin employs an amphipathic α-helix in its leader region as an anchor to CuvL, while its substrate core shuttles within the central reaction chamber. Our study thus reveals general principles of domain organization and substrate recruitment of class-IV and class-III lanthipeptide synthetases.

Molecular mechanism of topoisomerase poisoning by the peptide antibiotic albicidin.

The peptide antibiotic albicidin is a DNA topoisomerase inhibitor with low-nanomolar bactericidal activity towards fluoroquinolone-resistant Gram-negative pathogens. However, its mode of action is poorly understood. We determined a 2.6 Å resolution cryoelectron microscopy structure of a ternary complex between Escherichia coli topoisomerase DNA gyrase, a 217 bp double-stranded DNA fragment and albicidin. Albicidin employs a dual binding mechanism where one end of the molecule obstructs the crucial gyrase dimer interface, while the other intercalates between the fragments of cleaved DNA substrate. Thus, albicidin efficiently locks DNA gyrase, preventing it from religating DNA and completing its catalytic cycle. Two additional structures of this trapped state were determined using synthetic albicidin analogues that demonstrate improved solubility, and activity against a range of gyrase variants and E. coli topoisomerase IV. The extraordinary promiscuity of the DNA-intercalating region of albicidins and their excellent performance against fluoroquinolone-resistant bacteria holds great promise for the development of last-resort antibiotics.

Class II two-peptide lanthipeptide proteases: exploring LicTP for biotechnological applications.

The enzymatic machinery involved in the biosynthesis of lantibiotic is an untapped source of proteases with different specificities. Lanthipeptide biosynthesis requires proteolysis of specific target sequences by known proteases, which are encoded by contiguous genes. Herein, the activity of lichenicidin A2 (LicA2) trimming proteases (LicP and LicT) was investigated in vivo. Firstly, the impact of some residues and the size of the peptide were evaluated. Then followed trials in which LicA2 leader was evaluated as a tag to direct production and secretion of other relevant peptides. Our results show that a negatively charged residue (preferably Glu) at cleavage site is important for LicP efficacy. Some mutations of the lichenicidin hexapeptide such as Val-4Ala, Asp-5Ala, Asn-6Ser, and the alteration of GG-motif to GA resulted in higher processing rates, indicating the possibility of improved lichenicidin production in Escherichia coli. More importantly, insulin A, amylin (non-lanthipeptides), and epidermin were produced and secreted to E. coli supernatant, when fused to the LicA2 leader peptide. This work aids in clarifying the activity of lantibiotic-related transporters and proteases and to evaluate their possible application in industrial processes of relevant compounds, taking advantage of the potential of microorganisms as biofactories. KEY POINTS: • LicM2 correct activity implies a negatively charged residue at position -1. • Hexapeptide mutations can increase the amount of fully processed Bliß. • LicA2 leader peptide directs LicTP cleavage and secretion of other peptides.

Spatial Venomics─Cobra Venom System Reveals Spatial Differentiation of Snake Toxins by Mass Spectrometry Imaging.

Among venomous animals, toxic secretions have evolved as biochemical weapons associated with various highly specialized delivery systems on many occasions. Despite extensive research, there is still limited knowledge of the functional biology of most animal toxins, including their venom production and storage, as well as the morphological structures within sophisticated venom producing tissues that might underpin venom modulation. Here, we report on the spatial exploration of a snake venom gland system by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), in combination with standard proteotranscriptomic approaches, to enable in situ toxin mapping in spatial intensity maps across a venom gland sourced from the Egyptian cobra (Naja haje). MALDI-MSI toxin visualization on the elapid venom gland reveals a high spatial heterogeneity of different toxin classes at the proteoform level, which may be the result of physiological constraints on venom production and/or storage that reflects the potential for venom modulation under diverse stimuli.

New Approach Methods for Hazard Identification: A Case Study with Azole Fungicides Affecting Molecular Targets Associated with the Adverse Outcome Pathway for Cholestasis.

Triazole fungicides such as propiconazole (Pi) or tebuconazole (Te) show hepatotoxicity in vivo, e.g., hypertrophy and vacuolization of liver cells following interaction with nuclear receptors such as PXR (pregnane-X-receptor) and CAR (constitutive androstane receptor). Accordingly, azoles affect gene expression associated with these adverse outcomes in vivo but also in human liver cells in vitro. Additionally, genes indicative of liver cholestasis are affected in vivo and in vitro. We therefore analyzed the capability of Pi and Te to cause cholestasis in an adverse outcome pathway (AOP)-driven approach in hepatic cells of human origin in vitro, considering also previous in vivo studies. Bile salt export pump (BSEP) activity assays confirmed that both azoles are weak inhibitors of BSEP. They alternate the expression of various cholestasis-associated target genes and proteins as well as the mitochondrial membrane function. Published in vivo data, however, demonstrate that neither Pi nor Te cause cholestasis in rodent bioassays. This discrepancy can be explained by the in vivo concentrations of both azoles being well below their EC50 for BSEP inhibition. From a regulatory perspective, this illustrates that toxicogenomics and human in vitro models are valuable tools to detect the potential of a substance to cause a specific type of toxicity. To come to a sound regulatory conclusion on the in vivo relevance of such a finding, results will have to be considered in a broader context also including toxicokinetics in a weight-of-evidence approach.

The occurrence of ansamers in the synthesis of cyclic peptides.

α-Amanitin is a bicyclic octapeptide composed of a macrolactam with a tryptathionine cross-link forming a handle. Previously, the occurrence of isomers of amanitin, termed atropisomers has been postulated. Although the total synthesis of α-amanitin has been accomplished this aspect still remains unsolved. We perform the synthesis of amanitin analogs, accompanied by in-depth spectroscopic, crystallographic and molecular dynamics studies. The data unambiguously confirms the synthesis of two amatoxin-type isomers, for which we propose the term ansamers. The natural structure of the P-ansamer can be ansa-selectively synthesized using an optimized synthetic strategy. We believe that the here described terminology does also have implications for many other peptide structures, e.g. norbornapeptides, lasso peptides, tryptorubins and others, and helps to unambiguously describe conformational isomerism of cyclic peptides.

One-Pot Chemoenzymatic Synthesis of Microviridin Analogs Containing Functional Tags.

Microviridins are a prominent family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) featuring characteristic lactone and lactam rings. Their unusual cage-like architecture renders them highly potent serine protease inhibitors of which individual variants specifically inhibit different types of proteases of pharmacological interest. While posttranslational modifications are key for the stability and bioactivity of RiPPs, additional attractive properties can be introduced by functional tags. To date - although highly desirable - no method has been reported to incorporate functional tags in microviridin scaffolds or the overarching class of graspetides. In this study, a chemoenzymatic in vitro platform is used to introduce functional tags in various microviridin variants yielding biotinylated, dansylated or propargylated congeners. This straightforward approach paves the way for customized protease inhibitors with built-in functionalities that can help to unravel the still elusive ecological roles and targets of this remarkable class of compounds and to foster applications based on protease inhibition.

DFT-Guided Discovery of Ethynyl-Triazolyl-Phosphinates as Modular Electrophiles for Chemoselective Cysteine Bioconjugation and Profiling.

We report the density functional theory (DFT) guided discovery of ethynyl-triazolyl-phosphinates (ETPs) as a new class of electrophilic warheads for cysteine selective bioconjugation. By using CuI -catalysed azide alkyne cycloaddition (CuAAC) in aqueous buffer, we were able to access a variety of functional electrophilic building blocks, including proteins, from diethynyl-phosphinate. ETP-reagents were used to obtain fluorescent peptide-conjugates for receptor labelling on live cells and a stable and a biologically active antibody-drug-conjugate. Moreover, we were able to incorporate ETP-electrophiles into an azide-containing ubiquitin under native conditions and demonstrate their potential in protein-protein conjugation. Finally, we showcase the excellent cysteine-selectivity of this new class of electrophile in mass spectrometry based, proteome-wide cysteine profiling, underscoring the applicability in homogeneous bioconjugation strategies to connect two complex biomolecules.

Molecular basis of antibiotic self-resistance in a bee larvae pathogen.

Paenibacillus larvae, the causative agent of the devastating honey-bee disease American Foulbrood, produces the cationic polyketide-peptide hybrid paenilamicin that displays antibacterial and antifungal activity. Its biosynthetic gene cluster contains a gene coding for the N-acetyltransferase PamZ. We show that PamZ acts as self-resistance factor in Paenibacillus larvae by deactivation of paenilamicin. Using tandem mass spectrometry, nuclear magnetic resonance spectroscopy and synthetic diastereomers, we identified the N-terminal amino group of the agmatinamic acid as the N-acetylation site. These findings highlight the pharmacophore region of paenilamicin, which we very recently identified as a ribosome inhibitor. Here, we further determined the crystal structure of PamZ:acetyl-CoA complex at 1.34 Å resolution. An unusual tandem-domain architecture provides a well-defined substrate-binding groove decorated with negatively-charged residues to specifically attract the cationic paenilamicin. Our results will help to understand the mode of action of paenilamicin and its role in pathogenicity of Paenibacillus larvae to fight American Foulbrood.

Total Synthesis and Biosynthesis of Cyclodepsipeptide Cochinmicin I.

Phenylglycines are building blocks of many non-ribosomally synthesized peptides. The dihydroxyphenylglycine-containing cyclodepsipeptide cochinmicin I exhibits endothelin receptor antagonist activity. Therefore, it represents an interesting and synthetically challenging molecule because of the racemization-prone nature of dihydroxyphenylglycine. We present the total synthesis of cochinmicin I and the non-natural derivative cochinmicin VI and describe the identification and assignment of the cochinmicin (cmn) biosynthesis gene cluster, encoding a five-module non-ribosomal peptide synthetase for cochinmicin assembly.

Quantum Chemistry-based Molecular Dynamics Simulations as a Tool for the Assignment of ESI-MS/MS Spectra of Drug Molecules.

In organic mass spectrometry, fragment ions provide important information on the analyte as a central part of its structure elucidation. With increasing molecular size and possible protonation sites, the potential energy surface (PES) of the analyte can become very complex, which results in a large number of possible fragmentation patterns. Quantum chemical (QC) calculations can help here, enabling the fast calculation of the PES and thus enhancing the mass spectrometry-based structure elucidation processes. In this work, the previously unknown fragmentation pathways of the two drug molecules Nateglinide (45 atoms) and Zopiclone (51 atoms) were investigated using a combination of generic formalisms and calculations conducted with the Quantum Chemical Mass Spectrometry (QCxMS) program. The computations of the de novo fragment spectra were conducted with the semi-empirical GFNn-xTB (n=1, 2) methods and compared against Orbitrap measured electrospray ionization (ESI) spectra in positive ion mode. It was found that the unbiased QC calculations are particularly suitable to predict non-evident fragment ion structures, sometimes contrasting the accepted generic formulation of fragment ion structures from electron migration rules, where the "true" ion fragment structures are approximated. For the first time, all fragment and intermediate structures of these large-sized molecules could be elucidated completely and routinely using this merger of methods, finding new undocumented mechanisms, that are not considered in common rules published so far. Given the importance of ESI for medicinal chemistry, pharmacokinetics, and metabolomics, this approach can significantly enhance the mass spectrometry-based structure elucidation processes and contribute to the understanding of previously unknown fragmentation pathways.

Aurora Kinase A Is Involved in Controlling the Localization of Aquaporin-2 in Renal Principal Cells.

The cAMP-dependent aquaporin-2 (AQP2) redistribution from intracellular vesicles into the plasma membrane of renal collecting duct principal cells induces water reabsorption and fine-tunes body water homeostasis. However, the mechanisms controlling the localization of AQP2 are not understood in detail. Using immortalized mouse medullary collecting duct (MCD4) and primary rat inner medullary collecting duct (IMCD) cells as model systems, we here discovered a key regulatory role of Aurora kinase A (AURKA) in the control of AQP2. The AURKA-selective inhibitor Aurora-A inhibitor I and novel derivatives as well as a structurally different inhibitor, Alisertib, prevented the cAMP-induced redistribution of AQP2. Aurora-A inhibitor I led to a depolymerization of actin stress fibers, which serve as tracks for the translocation of AQP2-bearing vesicles to the plasma membrane. The phosphorylation of cofilin-1 (CFL1) inactivates the actin-depolymerizing function of CFL1. Aurora-A inhibitor I decreased the CFL1 phosphorylation, accounting for the removal of the actin stress fibers and the inhibition of the redistribution of AQP2. Surprisingly, Alisertib caused an increase in actin stress fibers and did not affect CFL1 phosphorylation, indicating that AURKA exerts its control over AQP2 through different mechanisms. An involvement of AURKA and CFL1 in the control of the localization of AQP2 was hitherto unknown.