Identification of a neuropeptide precursor protein that gives rise to a "cocktail" of peptides that bind Cu(II) and generate metal-linked dimers

The Transparency document associated with this article can be found,in online version.This work was supported by a Leverhulme Trust grant (RPG-2013-351) awarded to MRE and National Science Foundation (USA) grant awards DEB 1036416, 1036358, 1036366, and 1036368

Novel highly emissive non proteinogenic amino acids : synthesis of 1,3,4-thiadiazolyl asparagines and evaluation as fluorimetric chemosensors for biologically relevant transition metal cations

Highly emissive heterocyclic asparagine derivatives bearing a 1,3,4-thiadiazolyl unit at the side chain, functionalised with electron donor or acceptor groups, were synthesised and evaluated as amino acid based fluorimetric chemosensors for metal cations such as Cu2+, Zn2+, Co2+ and Ni2+. The results suggest that there is a strong interaction through the donor heteroatoms at the side chain of the various asparagine derivatives, with high sensitivity towards Cu2+ in a ligand-metal complex with 1:2 stoichiometry. Association constants and detection limits for Cu2+ were calculated. The photophysical and metal ion sensing properties of these asparagine derivatives confirm their potential as fluorimetric chemosensors and suggest that they can be suitable for incorporation into chemosensory peptidic frameworks.Fundação para a Ciência e a Tecnologia (FCT) - PTDC/QUI/66250/2006 (FCOMP-01-0124-FEDER-007428

Inreasing the Stability and Applications of Highly Active Naturally Occurring Antimicrobial Peptides

Antimicrobial Resistance is a fundamental threat to global health and safety” (WHO, 2016). Nisin, a 34 amino acid lanthipeptide, is currently used as a food preservative worldwide and has been used for decades without significant bacterial resistance having developed. It is an extremely active molecule which kills a range of different bacterial species. However nisin, along with some other similar peptides, suffers from low stability and solubility at physiological pH, which severely restricts its possible use in human and veterinary medicine. This research is concerned with increasing the stability of nisin in physiological pH systems; the stability of the dehydroalanine (Dha) at position 33 was examined as one of the project aims, along with the generation of novel analogues of the nisin tail and also the incorporation of a cyclopropyl amino acid as a Dha bioisostere moiety conserving the native conformation. This derivatisation of the tail region of nisin may increase its stability while preserving its bioactivity

Fragmentation of Lanthanide (III) Cationized Small Peptides: Generation of Peptide Radical Cations and Dipositive a and b ions.

This research work examines the dissociation chemistry of tripositive complexes formed by trivalent lanthanide ions and small peptides with tandem mass spectrometry under low-energy collision-induced dissociation (CID). By fragmentation of the tripositive lanthanide(III) cationized small peptide, a new route to generate peptide radical cations has been discovered. The dipositive b ions are also observed and the mechanisms by which they fragment are investigated by MSn. Tripositive complexes of lanthanide(III)/peptide have similar fragmentation chemistries in the gas phase when lanthanide = yttrium, lanthanum, cerium, samarium, gadolinium and terbium; [a3+H]2+ ions are formed and there are no peptide radical cations observed. When the lanthanide is europium(III), radical cations of tryptophan-, tyrosine-, phenylalanine-, methionine-containing peptides and of aliphatic peptides have been generated. Fragmentations of tripositive Ce(III)/peptide and Eu(III)/peptide complexes show very different behaviours. Abundant CO loss is only observed for dissociation of Ce(III)/peptide complexes, whereas CO2 loss is the predominant channel for Eu(III)/peptide complexes. Similarly, CO loss and CO2 loss are the predominant channels for the dissociations of [Ce(peptide-H)]2+ and [Eu(peptide-H)]2+, respectively. Peptide radical cations are only generated by the fragmentation of Eu(III)/peptide complexes, while protonated a and b ions are only observed when Ce(III)/peptide complexes dissociate. The dissociations of aliphatic [peptide]+ions generate [b3-H]+/ [b2-H]+ions for most peptides. In the dissociation of [a3+H]+ions, [b2-H]+ions are formed from most peptides. [a3+H]2+ ions usually cleave at the C-terminal amide bonds, creating two singly charged ions, a [b2]+ ion and an iminium ion derived from the C-terminal residue. Some [a3+H]2+ ions also lose small neutral molecules. The composition of the peptides dictates the preferred mode of the fragmentation of [b3+H]2+ ions, either loss of CO to form [a3+H]2+, or loss of CO plus H2O. Fragmentations of [Ce(peptide-H)]2+ ions show CO loss, and CO2 losses are observed for peptides with aromatic side chains or a methionine residue at C-terminus. For [Ce(peptide-H)(peptide)]2+ complexes, neutral losses are also observed but formation of two singly charged ions is dominant. The dissociation behaviour of [Ce(peptide-H)(CH3CN)]2+ and [Eu(peptide-H)(CH3CN)]2+ complexes are quite different. The former loses only CH3CN whereas the latter loses only CO2

Ruthenium-catalyzed azide alkyne cycloaddition reaction: scope, mechanism and applications

The ruthenium-catalyzed azide alkyne cycloaddition (RuAAC) affords 1,5-disubstituted 1,2,3-triazoles in one step and complements the more established copper-catalyzed reaction providing the 1,4-isomer. The RuAAC reaction has quickly found its way into the organic chemistry toolbox and found applications in many different areas, such as medicinal chemistry, polymer synthesis, organocatalysis, supramolecular chemistry, and the construction of electronic devices. This Review discusses the mechanism, scope, and applications of the RuAAC reaction, covering the literature from the last 10 years

Development of a Sel-tag for Multimodality Imaging and Studies of Mammalian Thioredoxin Reductase 1

Selenocysteine (Sec; U in one-letter code), the 21st amino acid existing in all selenoproteins, has unique biochemical properties due to its selenium atom, including a low pKa and a high reactivity with many electrophilic agents. When a selenoprotein is translated, the insertion of Sec occurs at a UGA codon, normally yielding translational termination. Here UGA can be recoded as Sec through the presence of a species-specific downstream mRNA sequence called Sec insertion sequence (SECIS) element. As one of about 25 mammalian selenoproteins, thioredoxin reductase 1 (TrxR1) contains one Sec residue within its C-terminal tetrapeptide motif, -Gly-Cys- Sec-Gly-COOH. By utilizing an engineered bacterial SECIS element in the rat TrxR1 cDNA, we are able to produce recombinant rat TrxR1 with both high yields and specific activities, which gives us an international leading edge, as selenoprotein production is a common bottleneck for researchers in the field of selenoprotein biology. Based on the studies on TrxR1, we have developed a Sel-tag for recombinant proteins using the C-terminal tetrapeptide motif of TrxR1. We have subsequently demonstrated that the selenolate of a reduced Sel-tag provides a handle which can be utilized for a wide range of selenolate-targeted applications, including a one-step protein purification procedure, residue-specific fluorescent labeling and radiolabeling with either gamma emitters (75Se) or positron emitting radionuclides (11C). We first utilized Sel-tag to investigate the mechanism of antigen presentation through carbohydrate based particles (CBP) by tracking the selenium-75 labeled Sel-tagged Fel d 1, the major cat allergen, in a mouse model. We further applied this technique for studies of apoptosis with a particular interest in utilizing Sel-tagged annexin A5 to accomplish multimodality imaging. We demonstrate that Seltagged annexin A5 labeled either with fluorescent 5-iodoacetamidofluorescein (5-IAF) or the positron emitter carbon11 remains functional and specifically binds apoptotic cells. This proved to be useful for (i) utilizing fluorescently labeled annexin A5 in either the conventional annexin A5 affinity assay with flow cytometry or in fluorescent microscopy of cells undergoing cell death, (ii) PET imaging of apoptotic liver of health BALB/c mice treated with anti-FAS antibody, and (iii) PET imaging of xenograft tumors developed from FaDu cells in SCID mice. We have solved the crystal structure of wild type rat TrxR1 and probed its catalytic mechanism. We have shown that the oxidized protein presents a stabilized selenenylsulfide motif in cis-configuration, with the Sec residue coordinated to the planar surface of a tyrosine side chain (Tyr116), thus located far from the redox active moieties of the enzyme proposed to be involved in electron transport to the C-terminal motif during catalysis. We believe that Tyr116 plays an important role for catalysis of TrxR1, possibly by involvement in electron transfer during the reductive half reaction, or by stabilizing the selenenylsulfide configuration of oxidized TrxR1. It has been shown that TrxR1 has pro-oxidant roles especially upon redox cycling with certain low molecular weight substrates. Thus we have investigated the potential of ROS generation by TrxR1 using wild type enzyme as well as several TrxR1 mutants. Utilizing Electron Spin Resonance (ESR) spin trapping with 5-Diethoxyphos-phoryl-5-methyl-1pyrroline-N-oxide (DEPMPO), we found that TrxR1 could generate free radicals including both hydroxyl radicals (HO.) and superoxide (O2.-) upon NADPH reduction in the absence of other substrates, and the HO. generation was largely dependent upon prior enzyme-catalyzed O2.-generation, indicating an inherent peroxidase activity of TrxR1. Closer analyses revealed that the the DEPMPO/HOO. adduct was a direct substrate of TrxR that could be reduced to DEPMPO/HO. in a Sec-dependent manner

Structural and functional characterisation of LdcB LD-carboxypeptidases from Streptococcus pneumoniae and Bacillus subtilis

PhD ThesisThe bacterial cell wall surrounds the cytoplasmic membrane and protects the cell against osmolysis in addition to providing shape. The cell wall is comprised of peptidoglycan, repeating units of N-acetly glucosamine and N-acetyl muramic acid form glycan strands and are crosslinked by short peptides that contain both L- and D-amino acids. Owing to the unique nature of peptidoglycan, and its absence in eukaryotic organisms, the cell wall has become an important target for many antibiotics, including the β-lactams and glycopeptides. Newly synthesised peptidoglycan contains pentapeptides, which extend from the lactyl moiety of the MurNAc sugar. These chains consist of L-alanine-D-γ- glutamate/glutamine-L-lysine/meso-diaminopimelic acid-D-alanine-D-alanine. The terminal D-alanine is often lost during cell wall maturation, either as a result of the crosslinking reaction, in which the penultimate D-alanine is attached to the side-chain of a neighbouring L-lysine or meso-diaminopimelic acid by an isopeptide bond, or as a consequence of the activities of DD-carboxypeptidases, and results in a tetrapeptide. The tetrapeptide can then be trimmed further to form a tripeptide by the action of LD-carboxypeptidases. Although many DD-carboxypeptidases have been well characterised, the majority of LD-carboxypeptidases that have been studied are active only against peptidoglycan fragments and so cannot be responsible for producing the tripeptides found in the cell wall. Of the LD-carboxypeptidases active against the mature cell wall, DacB (Streptococcus pneumoniae), Csd6 (Helicobacter pylori) and Pgp2 (Campylobacter jejuni), each has been shown to be essential in maintaining cell morphology. It should be noted, however, that neither Csd6 nor Pgp2 share any sequence similarity with DacB and belong to different peptidase families. This thesis concerns the structural and biochemical characterisation of DacB, herein renamed to LdcB (LD-carboxypeptidase B). The crystal structures of the apo form of LdcB from both S. pneumoniae and Bacillus subtilis were solved, revealing a single domain, globular protein with 2 sub-domains forming a V-shaped cleft in which the active site is located. LdcB binds one zinc ion per monomer, located at the bottom of the active site, and is a member of the LAS (lysostaphin, D-Ala-D-Ala peptidases, sonic hedgehog) family of metalloproteins. Additionally, the activity of LdcB as an LDcarboxypeptidase was confirmed and the crystal structure of LdcB from S. pneumoniae ii was solved in complex with a product mimic, M-Tri-Lys(D-Asn), revealing the molecular basis for peptidoglycan recognition in this family of enzymes. Finally, the affinity of LdcB for zinc and copper has been determined and it has been shown that catalysis is not inhibited by the substitution of zinc by copper or cobalt

New peptidic Cu(I) chelators as potential candidates for the treatment of Wilson’s disease

Le cuivre est un micronutriment essentiel qui participe à de nombreux processus biologiques. Cependant, le cuivre libre est toxique pour l’organisme parce qu’il catalyse une réaction de type Fenton formant des espèces réactives de l’oxygène. Par conséquent la concentration en cuivre est finement régulée dans tous les organismes vivants. Les maladies de Menkes et de Wilson sont dues à des dérèglements de l’homéostasie du cuivre qui se manifestent respectivement par une déficience ou une accumulation de cuivre dans l’organisme. La maladie de Wilson est traitée avec des chélateurs du cuivre, qui provoquent des effets secondaires importants chez certains patients.Mon projet de doctorat consiste en l’élaboration de trois familles de peptides qui contiennent des acides aminés cystéines et en l’étude de leurs complexes de Cu(I) pour déterminer s’ils sont des candidats adaptés pour le traitement de la maladie de Wilson. L'interaction de certains peptides avec les ions Hg(II) ou Zn(II) a également été étudiée. En effet, le Hg(II) est un cation métallique possédant des propriétés similaires au Cu(I) et donc souvent utilisé pour modéliser le Cu(I) qui est sensible à l'oxygène et se dismute dans l’eau. Le Zn(II) est quant à lui omniprésent dans les cellules et un compétiteur intracellulaire potentiel du Cu().Les séquences des peptides ont été choisies selon trois stratégies différentes. Dans la première, des séquences inspirées de la boucle de liaison du cuivre de la protéine bactérienne CueR (copper efflux regulator), contenant deux cystéines, ont été étudiées afin de bénéficier de la sélectivité et de la sensibilité de ce régulateur. Dans une deuxième approche, des peptides contenant trois cystéines dans les motifs CxCxxC et CxCxC ont été étudiés pour combiner les avantage des peptides (bonne internalisation dans les cellules hépatiques quand ils sont judicieusement fonctionnalisés) et des tripodes (très forte affinité pour le Cu(I)) de l’équipe CIBEST. Finalement, la pré-organisation a été exploitée dans un tétrapeptide rigide où les deux cysteines sont liées dans un coude β préformé.Les trois peptides modèles du régulateur CueR miment la capacité de la protéine à accueillir exclusivement un ion Cu(I) dans des conditions d'excès de ligand et une forte affinité et sélectivité par rapport au Zn(II). Ces caractéristiques sont avantageuses dans la perspective du développement de nouveaux chélateurs du Cu(I).Les peptides contenant trois cystéines s’avèrent trop flexibles pour contrôler la spéciation des complexes du Cu(I). Par ailleur, ces peptides sont bien adaptés pour une coordination efficace du Hg(II) par trois groupes thiolates. Les différences structurales n’ont qu’une influence modeste sur les stabilités des complexes. La différence dans la coordination des peptides vis-à-vis des deux ions mous Hg(II) et Cu(I) démontre que l'utilisation du Hg(II) comme ion modèle pour la coordination du Cu(I) avec des peptides ou des protéines riches en soufre dans des conditions physiologiques n’est pas toujours appropriée.La pré-organisation de la structure peptidique est un élément clé du contrôle de la spéciation du complexe Cu(I) et de l’affinité des ligands pour le Cu(I). Le peptide CDPPC forme uniquement le cluster Cu4P3 avec une grande stabilité et une bonne sélectivité Cu(I)/Zn(II). Au contraire, les données expérimentales avec le tétrapeptide plus flexible CPGC montrent la formation d’un mélange de complexes polymétalliques de Cu(I). Il est intéressant de noter que le peptide simple CDPPC est capable d’imiter la formation des clusters Cu(I)-thiolates identifiés dans de nombreuses protéines impliquées dans l’homéostasie du cuivre, comme Cox17 ou Ctr1. CDPPC est intéressant pour mettre au point un chélateur intracellulaire du Cu(I), et sa fonctionnalisation afin de pouvoir cibler les cellules hépatiques pour le traitement de maladie Wilson sera donc pertinente dans le futur.The essential micronutrient copper participates in several biological processes, like respiration, iron homeostasis, antioxidant defense or pigment formation. However, excess of copper can promote ROS formation and thus induce oxidative damages. Therefore, intracellular copper concentration is under strict control. Menkes and Wilson’s diseases are genetic disorders causing impairment in copper homeostasis leading to copper deficiency or overload, respectively. Wilson’s disease is treated by chelation therapy, but the presently used drugs have several adverse side effects.The aim of my Ph.D. work consisted of the design of three groups of cysteine containing peptides and the characterization of their Cu(I) complexes to determine whether they are appropriate candidates for the treatment of Wilson’s disease.The peptides were designed following three different approaches. In a first strategy, we attempted to take advantage of the outstanding selectivity and sensitivity of the bacterial copper efflux regulator protein CueR by studying oligopeptides based on the metal binding motif of CueR involving two cysteine residues. Second, three-cysteine containing linear and cyclic peptides were designed with the aim of merging the better internalization of peptides by hepatocytes and the high Cu(I) affinity of tripods previously studied in the Delangle’s lab. Finally, the advantages of a highly preorganized peptide structure were exploited in a short, rigid tetrapeptide where two cysteines were linked by a turn motif (CDPPC). For comparative purposes studies were also performed with another, less rigid tetrapeptide ligand containing the PG unit as a turn inducing motif.The three CueR model peptides resemble the ability of the protein to exclusively accommodate one metal ion under ligand excess conditions. This, combined with the large affinity and high selectivity vs. Zn(II), are the features that are advantageous in the view of the development of new Cu(I) chelators.The three-cysteine-containing peptides proved to be too flexible to control the speciation and hereby leading to the formation of several species. On the other hand, they are well adapted for an efficient trithiolate coordination of the thiophilic cation Hg(II). Structural differences in the three-cysteine containing peptides have minor effect on the affinity of the ligands towards Cu(I) and Hg(II) ions. The striking difference in the behavior of the peptides towards the two soft metal ions demonstrate that the use of Hg(II) as a probe for Cu(I) coordination with sulfur-rich peptides or proteins in physiological conditions may not always be fully appropriate.Preorganization of the peptide structure is a key element in the control of Cu(I) complex speciation and in the affinity of the ligands for Cu(I).CdPPC forms a single Cu4P3 cluster with high stability and displays large selectivity for Cu(I) with respect to the ubiquitous Zn(II). In contrast, The CPGC-Cu(I) system is characterized by a more complicated complex formation. It is worth to note, that the simple CdPPC peptide is able to mimic the Cu(I)-thiolate cluster formation that are typical in proteins like Ctr1 or Cox17. CDPPC is an interesting simple peptide candidate to be targeted to the liver cells for the localized treatment of Cu overload in Wilson’s disease