Protein modification and phospholipid oxidation

Phospholipid oxidation can generate reactive and electrophilic products that are capable of modifying proteins, especially at cysteine, lysine and histidine residues. Such lipoxidation reactions are known to alter protein structure and function, both with gain of function and loss of activity effects. As well as potential importance in the redox regulation of cell behaviour, lipoxidation products in plasma could also be useful biomarkers for stress conditions. Although studies with antibodies suggested the occurrence of lipoxidation adducts on ApoB-100, these products had not previously been characterized at a molecular level. We have developed new mass spectrometry-based approaches to detect and locate adducts of oxidized phospholipids in plasma proteins, as well as direct oxidation modifications of proteins, which avoid some of the problems typically encountered with database search engines leading to erroneous identifications of oxidative PTMs. This approach uses accurate mass extracted ion chromatograms (XICs) of fragment ions from peptides containing oxPTMs, and allows multiple modifications to be examined regardless of the protein that contains them. For example, a reporter ion at 184.074 Da/e corresponding to phosphocholine indicated the presence of oxidized phosphatidylcholine adducts, while 2 reporter ions at 100.078 and 82.025 Da/e were selective for allysine. ApoB-100-oxidized phospholipid adducts were detected even in healthy human samples, as well as LDL from patients with inflammatory disease. Lipidomic studies showed that more than 350 different species of lipid were present in LDL, and were altered in disease conditions. LDL clearly represents a very complex carrier system and one that offers a rich source of information about systemic conditions, with potential as indicators of oxidative damage in ageing or inflammatory diseases.

Residue-specific investigation of the relationship between oxidation and activity of a dual specificity phosphatase by mass spectrometry

Redox regulation of signalling pathways is critical in proliferation and apoptosis; redox imbalance can lead to pathologies such as inflammation and cancer. Vaccinia H1-related protein (VHR; DUSP3) is a dual-specificity phosphatase important in controlling MAP kinase activity during cell cycle. the active-site motif contains a cysteine that acts as a nucleophile during catalysis. We used VHR to investigate the effect of oxidation in vitro on phosphatase activity, with the aim of determining how the profile of site-specific modification related to catalytic activity. Recombinant human VHR was expressed in E. coli and purified using a GST-tag. Protein was subjected to oxidation with various concentrations of SIN-1 or tetranitromethane (TNM) as nitrating agents, or HOCl. the activity was assayed using either 3-O-methylfluorescein phosphate with fluorescence detection or PIP3 by phosphate release with malachite green. the sites of oxidation were mapped using HPLC coupled to tandem mass spectrometry on an ABSciex 5600TripleTOF following in-gel digestion. More than 25 different concentration-dependent oxidative modifications to the protein were detected, including oxidations of methionine, cysteine, histidine, lysine, proline and tyrosine, and the % oxidized peptide (versus unmodified peptide) was determined from the extracted ion chromatograms. Unsurprisingly, methionine residues were very susceptible to oxidation, but there was a significant different in the extent of their oxidation. Similarly, tyrosine residues varied greatly in their modifications: Y85 and Y138 were readily nitrated, whereas Y38, Y78 and Y101 showed little modification. Y138 must be phosphorylated for MAPK phosphatase activity, so this susceptibility impacts on signalling pathways. Di- and tri- oxidations of cysteine residues were observed, but did not correlate directly with loss of activity. Overall, the catalytic activity did not correlate with redox state of any individual residue, but the total oxidative load correlated with treatment concentration and activity. This study provides the first comprehensive analysis of oxidation modifications of VHR, and demonstrates both heterogenous oxidant effects and differential residue susceptibility in a signalling phosphatase.

Targeted mass spectrometry methods for detecting oxidative post-translational modifications

Oxidative post-translational modifications (oxPTMs) can alter the function of proteins, and are important in the redox regulation of cell behaviour. The most informative technique to detect and locate oxPTMs within proteins is mass spectrometry (MS). However, proteomic MS data are usually searched against theoretical databases using statistical search engines, and the occurrence of unspecified or multiple modifications, or other unexpected features, can lead to failure to detect the modifications and erroneous identifications of oxPTMs. We have developed a new approach for mining data from accurate mass instruments that allows multiple modifications to be examined. Accurate mass extracted ion chromatograms (XIC) for specific reporter ions from peptides containing oxPTMs were generated from standard LC-MSMS data acquired on a rapid-scanning high-resolution mass spectrometer (ABSciex 5600 Triple TOF). The method was tested using proteins from human plasma or isolated LDL. A variety of modifications including chlorotyrosine, nitrotyrosine, kynurenine, oxidation of lysine, and oxidized phospholipid adducts were detected. For example, the use of a reporter ion at 184.074 Da/e, corresponding to phosphocholine, was used to identify for the first time intact oxidized phosphatidylcholine adducts on LDL. In all cases the modifications were confirmed by manual sequencing. ApoB-100 containing oxidized lipid adducts was detected even in healthy human samples, as well as LDL from patients with chronic kidney disease. The accurate mass XIC method gave a lower false positive rate than normal database searching using statistical search engines, and identified more oxidatively modified peptides. A major advantage was that additional modifications could be searched after data collection, and multiple modifications on a single peptide identified. The oxPTMs present on albumin and ApoB-100 have potential as indicators of oxidative damage in ageing or inflammatory diseases.

Importance of tissue transglutaminasenitrosylation in fibroblasts migration and MMPregulation

As an extracellular second messenger, nitric oxide (NO) mediates the modification of proteins through nitrosylation of cysteine andtyrosine residues. Tissue Transglutaminase (TG2) is a Ca2+ activated, sulfhydryl rich protein with 18 free cysteine residues, which catalyzes ε-(γ glutamyl)lysine crosslink between extracellular and intracellular proteins. NO can nitrosylate up to 15 of the cysteine residues in TG2, leading to the irreversible inactivation of the enzyme activity. The interplay between these two agents was revealed for the first time by our study showing that NO inhibited the TG2-induced transcriptional activation of TGFb1and extracellular matrix (ECM) protein synthesis by nitrosylating TG2 in an inactive confirmation with inert catalytic activity. However, nitrosylated TG2 was still able to serve as a novel cell adhesion protein. In the light of our previous findings, in this study we aim to elucidate the NO modified function of TG2 in cell migration using an in vitro model mimicking the tissue matrix remodeling phases of wound healing. Using transfected fibroblasts expressing TG2 under the control of the tetracycline-off promoter, we demonstrate that upregulation of TG2 expression and activity inhibited the cell migration through the activation of TGFβ1. Increased TG2 activity led to arise in the biosynthesis and activity of the gelatinases, MMP-2 andMMP-9, while decreasing the biosynthesis and activity of the col-lagenases MMP-1a and MMP-13. NO donor S-Nitroso-N-acetyl-penicillamine (SNAP) treatment relieved the TG2 obstructed-cellmigration by blocking the TG2 enzyme activity. In addition,decrease in TG2 activity due to nitrosylation led to an inhibition of TGFβ1, which in turn affected the pattern of MMP activation. Recent evidence suggests that, once in complex with fibronectin in the ECM, TG2 can interact with syndecan-4 or integrinβ-1and regulate the cell adhesion. In the other part of this study, the possible role of nitrosylated TG2 on the regulation of cell migration during wound healing was investigated with respect to its interactions with integrin β1 (ITGβ1) and syndecan-4 (SDC4). Treatment with TG2 inhibitor Z-DON resulted in a 50% decrease in the TG2 interaction with ITGB1 and SDC4, while increasing concentrations of SNAP firstly led to a substantial decrease and then completely abolished the TG2/ITGβ1 and TG2/SDC4 complex formation on the cell surface. Taken together, data obtained from this study suggests that nitrosylation of TG2 leads to a change not only in the binding partners of TG2 on cell surface but also in TGFβ1-dependent MMP activation, which give rise to an increase in the migration potential of fibroblasts.

Tailoring heme -thiolate proteins into efficient biocatalysts with high specificity and selectivity

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

Covalent protein adduction of nitrogen mustards and related compounds

Chemical warfare agents continue to pose a global threat despite the efforts of the international community to prohibit their use in warfare. For this reason, improvement in the detection of these compounds remains of forensic interest. Protein adducts formed by the covalent modification of an electrophilic xenobiotic and a nucleophilic amino acid may provide a biomarker of exposure that is stable and specific to compounds of interest (such as chemical warfare agents), and have the capability to extend the window of detection further than the parent compound or circulating metabolites. This research investigated the formation of protein adducts of the nitrogen mustard chemical warfare agents mechlorethamine (HN-2) and tris(2-chloroethyl)amine (HN-3) to lysine and histidine residues found on the blood proteins hemoglobin and human serum albumin. Identified adducts were assessed for reproducibility and stability both in model peptide and whole protein assays. Specificity of these identified adducts was assessed using in vitro assays to metabolize common therapeutic drugs containing nitrogen mustard moieties. Results of the model peptide assays demonstrated that HN-2 and HN-3 were able to form stable adducts with lysine and histidine residues under physiological conditions. Results for whole protein assays identified three histidine adducts on hemoglobin, and three adducts (two lysine residues and one histidine residue) on human serum albumin that were previously unknown. These protein adducts were determined to be reproducible and stable at physiological conditions over a three-week analysis period. Results from the in vitro metabolic assays revealed that adducts formed by HN-2 and HN-3 are specific to these agents, as metabolized therapeutic drugs (chlorambucil, cyclophosphamide, and melphalan) did not form the same adducts on lysine or histidine residues as the previously identified adducts formed by HN-2 and HN-3. Results obtained from the model peptide and full protein work were enhanced by comparing experimental data to theoretical calculations for adduct formation, providing further confirmatory data. This project was successful in identifying and characterizing biomarkers of exposure to HN-2 and HN-3 that are specific and stable and which have the potential to be used for the forensic determination of exposure to these dangerous agents.

Tailoring Heme-Thiolate Proteins into Efficient Biocatalysts with High Specificity and Selectivity

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

Exoenzimas em rações de frangos de corte

The study aimed to evaluate performance, relative weight of the small intestine, digestibility and deposition of minerals in the bone of broilers supplemented with enzymatic complex (carbohydrases and phytase) in sorghum-based diets or sorghum and millet. In the experiments based of sorghum and sorghum and millet were used 912 day-old male and female Hubbard Flex chicks (50:50) were distributed in a completely randomized design in a 2x2 factorial arrangement (Feed Control base Sorghum (Contcs); FeedReducedbase Sorghum (RedS); Feed Control base sorghum + Enzymatic Complex (Contcs + Enz);. Feed reduced base sorghum + Enzymatic Complex (RedS + Enz) in the study of feed basis of sorghum and millet the design was similar (Feed Control base sorghum + millet ( ContSM); Reduced feed based on Millet + Sorghum (RedSM); Feed Control based Sorghum + Millet + EnzimaticComplex (ContSM + Enz); Reduced Feed base Sorghum+ Millet + Enzimatic Complex (RedSM + Enz). At 35 and 42 days of age were determined performance data: feed intake (CR), body weight (BW), feed conversion (FC), viability (VIAB), relative weight of the small intestine and deposition of minerals in the bone. The digestibility was evaluated sorghum grain size (crushed and whole) with and without exoenzimatico complex. They used 32 birds, eight birds per treatment, in periods from 17 to 21 (initial) and 31 to 35 days of age (fattening). The exoenzimático complex used in feed favored the weight gain results, feed conversion and bone mineralization when compared to a control diet not added to enzymes, demonstrating its effect on non-starch polysaccharides and phosphorus phytic present as anti-nutritional factors in these diets, increasing the digestibility and supply of metabolizable energy, essential amino acids, methionine and lysine and calcium and phosphorus for bone formation. It is concluded that a safe strategy for inclusion in feed is based on the reduction of energy levels, essential amino acids, methionine and lysine and calcium and phosphorus in the expected result of the constant activity of the enzymes of this exoenzimático complex.

Caractérisation de l’activité Nur77 et de ses complexes en essais BRET par complémentation

Dans le système nerveux central, la dopamine joue un rôle crucial dans de nombreuses fonctions physiologiques telles que : l’apprentissage, le mouvement volontaire, la motivation, la cognition et la production hormonale. Il a été aussi démontré que le système de signalisation dopaminergique est altéré dans plusieurs maladies neurologiques et psychiatriques comme la maladie de Parkinson et la schizophrénie. Des études, effectuées dans le laboratoire du Dr.Daniel Lévesque (laboratoire d’accueil), ont montré que les récepteurs nucléaires Nur77 (NR4A1, NGFI-B) et RXRγ (retinoid X receptors γ) sont impliqués dans la régulation des effets de la dopamine dans le système nerveux central. De plus, ces données suggèrent que le complexe Nur77 et RXR joueraient un rôle crucial dans l’effet des médicaments antipsychotiques et antiparkinsoniens. Toutefois, très peu de médicaments ciblant Nur77 ont été identifiés à ce jour et les médicaments agissant sur RXRγ restent mal caractérisés. En outre, les analyses actuellement disponibles ne peuvent pas résumer la complexité des activités des NRs et génèrent des mesures indirectes des activités des drogues. Afin de mieux comprendre comment est régulée l’interaction Nur77/RXRγ dans ces processus, mon projet a été de mettre au point un essai BRET (Bioluminescence Resonance Energy Transfer) et PCA-BRET (Protein Complementation Assay-BRET) basé sur le recrutement d'un motif mimant un co-activateur fusionné avec la YFP. Nos différents essais ont été validés par courbes dose-réponse en utilisant différents composés RXR . Les EC50 (concentration efficace médiane, qui permet de mesurer l'efficacité d'un composé) obtenues étaient très semblables aux valeurs précédemment rapportées dans la littérature. Nous avons aussi pu identifier un composé le SR11237 (BMS649) qui semble posséder une sélectivité pour le complexe Nur77/RXRγ par rapport aux complexes Nurr1/RXRγ et RXRγ /RXRγ. Nos résultats indiquent que ces essais de BRET peuvent être utilisés pour évaluer la sélectivité de nouveaux composés pour les complexes Nur77/RXRγ, Nurr1/RXRγ et RXRγ /RXRγ. Un autre aspect de mon projet de doctorat a été de mettre en évidence par BRET l’importance de la SUMOylation dans la régulation de l'activité de Nur77 dans sa forme monomèrique, homodimèrique et hétérodimèrique. Nous avons ainsi identifié que Nur77 recrute principalement SUMO2 sur sa lysine 577. Il est intéressant de noté que le recrutement de la SUMO2 à Nur77 est potentialisé en présence de la SUMO E3 Ligase PIASγ. Aussi, la perte de la SUMOylation sur la lysine 577 entraîne l'incapacité de Nur77 de recruter divers motifs de co-activation mais pas pour ses formes homo- et hétérodimèrique. Cependant, la présence de PIASγ ne potentialise pas le recrutement du co-activateur, suggérant que cette SUMO E3 Ligase est seulement impliqué dans le processus de recrutement de la SUMO mais pas dans celui du co-activateur. Nous avons ainsi déterminé une nouvelle modification post-traductionnelle sur Nur77 régulant spécifiquement son activité monomérique Ces projets pourraient donc apporter de nouvelles données cruciales pour l’amélioration du traitement de la maladie de Parkinson ou de la schizophrénie, ainsi que d'obtenir une meilleure compréhension sur les mécanismes permettant la régulation de la fonction de Nur77

Pre-formulation and systematic evaluation of amino acid assisted permeability of insulin across in vitro buccal cell layers

The aim of this work was to investigate alternative safe and effective permeation enhancers for buccal peptide delivery. Basic amino acids improved insulin solubility in water while 200 and 400 µg/mL lysine significantly increased insulin solubility in HBSS. Permeability data showed a significant improvement in insulin permeation especially for 10 µg/mL of lysine (p < 0.05) and 10 µg/mL histidine (p < 0.001), 100 µg/mL of glutamic acid (p < 0.05) and 200 µg/mL of glutamic acid and aspartic acid (p < 0.001) without affecting cell integrity; in contrast to sodium deoxycholate which enhanced insulin permeability but was toxic to the cells. It was hypothesized that both amino acids and insulin were ionised at buccal cavity pH and able to form stable ion pairs which penetrated the cells as one entity; while possibly triggering amino acid nutrient transporters on cell surfaces. Evidence of these transport mechanisms was seen with reduction of insulin transport at suboptimal temperatures as well as with basal-to-apical vectoral transport, and confocal imaging of transcellular insulin transport. These results obtained for insulin is the first indication of a possible amino acid mediated transport of insulin via formation of insulin-amino acid neutral complexes by the ion pairing mechanism.

Sortase as a Tool in Biotechnology and Medicine

We have harnessed two reactions catalyzed by the enzyme sortase A and applied them to generate new methods for the purification and site-selective modification of recombinant protein therapeutics.

We utilized native peptide ligation —a well-known function of sortase A— to attach a small molecule drug specifically to the carboxy-terminus of a recombinant protein. By combining this reaction with the unique phase behavior of elastin-like polypeptides, we developed a protocol that produces homogenously-labeled protein-small molecule conjugates using only centrifugation. The same reaction can be used to produce unmodified therapeutic proteins simply by substituting a single reactant. The isolated proteins or protein-small molecule conjugates do not have any exogenous purification tags, eliminating the potential influence of these tags on bioactivity. Because both unmodified and modified proteins are produced by a general process that is the same for any protein of interest and does not require any chromatography, the time, effort, and cost associated with protein purification and modification is greatly reduced.

We also developed an innovative and unique method that attaches a tunable number of drug molecules to any recombinant protein of interest in a site-specific manner. Although the ability of sortase A to carry out native peptide ligation is widely used, we demonstrated that Sortase A is also capable of attaching small molecules to proteins through an isopeptide bond at lysine side chains within a unique amino acid sequence. This reaction —isopeptide ligation— is a new site-specific conjugation method that is orthogonal to all available protein-small conjugation technologies and is the first site-specific conjugation method that attaches the payload to lysine residues. We show that isopeptide ligation can be applied broadly to peptides, proteins, and antibodies using a variety of small molecule cargoes to efficiently generate stable conjugates. We thoroughly assessed the site-selectivity of this reaction using a variety of analytical methods and showed that in many cases the reaction is site-specific for lysines in flexible, disordered regions of the substrate proteins. Finally, we showed that isopeptide ligation can be used to create clinically-relevant antibody-drug conjugates that have potent cytotoxicity towards cancerous cells