Letteratura scientifica selezionata sul tema "Proteins – Affinity labeling"

Subsequent to swelling, the Amphiuma red blood cells lose K+, Cl-, and water until normal cell volume is restored. Net solute loss is the result of K+/H+ and Cl-/HCO3- exchangers functionally coupled through changes in pH and therefore HCO3-. Whereas the Cl-/HCO3- exchanger is constitutively active, K+/H+ actively is induced by cell swelling. The constitutive Cl-/HCO3- exchanger is inhibited by low concentrations (< 1 microM) of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or H2DIDS, yet the concentration of H2DIDS > 25 microM irreversibly modifies the K+/H+ exchanger in swollen cells. We exploited the volume-dependent irreversible low-affinity reaction between H2DIDS and the K+/H+ to identify the protein(s) associated with K+/H+ exchange activity. Labeling of the membrane proteins of intact cells with 3H2DIDS results in high-affinity labeling of a broad 100-kDa band, thought to be the anion exchanger. Additional swelling-dependent low-affinity labeling at 110 kDa suggests the possibility of a volume-induced population of anion exchangers. Finally, the correlation between volume-sensitive K+/H+ modification and low-affinity labeling suggests that transport activity is associated with a protein of approximately 85 kDa. Although a 55-kDa protein is also labeled, it is a less likely candidate, since label incorporation and transport modification are less well correlated than that of the 85- and 110-kDa proteins.

Solid-phase affinity labeling of target proteins with the specifically designed chemical tools selectively and effectively furnished the labeled target proteins without the need for tedious manipulations.

There is increasing evidence that hydrogen peroxide (H2O2) can act as a signalling molecule capable of modulating a variety of biochemical and genetic systems. Using Jurkat T-lymphocytes, this study initially investigated the involvement of H2O2 in the activation of a specific signalling protein extracellular signal-regulated protein kinase (ERK). It was found that as a result of H2O2 treatment, mitochondrial complex activities decreased which led to subsequent increase of mitochondrial reactive oxygen species (ROS) production. The increase of ROS resulted in higher cellular H2O2 as well as increased ERK activation. This study demonstrated that in an oxidative stress setting, H2O2 production from the mitochondria was an essential component in maintaining the activation of a signalling protein. One way in which H2O2 could influence protein function is by the oxidation of susceptible thiol groups of cysteine residues. To further understand the variety of signalling pathways that H2O2 may be involved in, an improved proteomics technique was developed to globally identify proteins with susceptible thiol groups. The

KCN-1 is known to reduce tumor growth 6-fold in mice implanted with LN229 glioma cells. Although this inhibitor is effective, the mechanism of action for KCN-1 is not well understood. Based on preliminary studies, KCN-1 reduces tumor growth by disrupting the HIF 1 (hypoxia-induced factor-1) pathway. The binding target of KCN-1 needs to be investigated in order to develop KCN-1 or its analogs for therapeutic applications. In this research, a molecule was designed and synthesized for the identification of the binding target of KCN-1. Specifically, this molecule contains the inhibitor (KCN-1), a photocleavable linker, beads, and the affinity label (L DOPA). When UV light shines on the linker, the trans-alkene isomerizes to cis-alkene and undergoes intramolecular ring-closing reaction, which helps cleave the immobilized bead from the linker. The immobilized bead is used to separate the binding fragment attached to the photocleavable linker from the solution after enzyme digestion. The affinity label (L-DOPA) reacts with a nucleophile from the binding target and creates a covalent bond. If the design is successful, this method is able to analyze the mass of the peptide sequence and determine the binding target of KCN-1.

Les Métallo Protéases Matricielles (MMP) en tant qu'endopeptidases à zinc ont une large gamme de fonctions biologiques allant du remodelage tissulaire à la modulation de la réponse cellulaire. Une modification de leur activité protéolytique est souvent associée à de nombreux désordres biologiques. In vivo, ces protéases sont soumises à de nombreuses modifications post-traductionnelles. Elles sont sécrétées sous formes latentes à l'extérieur des cellules pour être ensuite transformées en forme fonctionnelles. Ces dernières sont ensuite inhibées par des inhibiteurs endogènes. En raison de leur sécrétion dans l’espace extra cellulaire, les MMP sous formes actives ont longtemps été considérées comme de simples ciseaux moléculaires capable de dégrader uniquement la matrice extracellulaire. Cependant, le remodelage tissulaire ne constitue pas la fonction unique et encore moins la fonction principale de ces enzymes. Elles peuvent en effet cliver une grande variété de substrats non matriciels et à ce titre sont impliquées dans la progression tumorale, l'immunité et l'inflammation. Pour ajouter une complexité supplémentaire à la biologie des MMP, il a été récemment montré que certaines MMP ont une localisation intracellulaire associée à des fonctions non protéolytiques. Ces observations, mais aussi celles montrant que ces protease participent à la progression de la maladie alors que d'autres ont une fonction protectrice, soulignent la nécessité de mieux documenter leur activation spatiale et temporelle dans divers contextes biologiques.Le profilage protéique basé sur l'activité vise à analyser l'état fonctionnel des protéines dans des échantillons biologiques complexes. À cette fin, des sondes basées sur l'activité (ABP), qui réagissent avec les enzymes en s’appuyant sur leur mécanisme catalytique, ont été développées pour la détection d’enzymes sous formes actives, notamment dans le cas des protéases à sérine et à cystéine. Une sonde basée sur l’activité (ABP) est classiquement composée : i) d’un groupement réactif conduisant à la modification covalente de résidus au sein du site actif de l’enzyme, ii) d’un motif de liaison imposant la sélectivité au groupement réactif et iii) d’un groupement rapporteur permettant la détection des enzymes ciblées. Cette approche ne s’applique toutefois pas aux MMP, pour lesquelles il n’existe pas de résidus nucléophiles conservés au sein du site actif. À cet égard, tous les ABP ciblant les MMP comportent un groupement photo activable qui, sous irradiation UV, favorise la formation du complexe covalent. De telles sondes photo sensibles ont permis de détecter les MMP sous leurs formes actives dans des tissus et des fluides, mais pas chez les animaux vivants au sein desquels l’étape de photo-activation ne peut être réalisé.Dans ce contexte, en nous appuyant sur un contexte structural favorable et en exploitant la chimie de l'acyl imidazole (LDAI) dirigée par un ligand, nous avons identifié une nouvelle série de sondes capables de modifier de manière covalente les MMP sans recourir à la photo-activation. Nous avons ainsi validé la capacité de ces sondes à marquer de manière sélective et efficace la MMP12 humaine in vitro et dans des protéomes complexes. Dans ce dernier cas, jusqu’à 50ng de hMMP12 correspondant à 0,05% du protéome total peuvent être détectés. Nous avons également déterminé l'identité de l’unique résidu modifié de façon covalente au sein du site actif de la hMMP-12 et vérifié que cette modification avait peu d'impact sur l’activité protéolytique de cette dernière. Nous avons démontré que cette approche permettait de détecter des MMP endogènes. Enfin, nous avons étendu cette stratégie de marquage à un panel plus large de MMP.En développant la première stratégie de marquage des formes actives de MMP «sans photo-activation», il semble maintenant possible d’envisager la détection de ces enzymes à la fois dans les protéomes complexes et in vivo
Matrix MetalloProteases (MMPs) as zinc endopeptidases have a wide range of biological functions, and changes in their proteolytic activity underlie many biological disorders. Since their proteolytic activity has to be tightly controlled to prevent tissue destruction, theses proteases are subjected to numerous posttranslational modifications in vivo. They are secreted under latent forms outside of the cells, and are subsequently processed into their functional form that can be further inhibited by endogenous inhibitors. Due to their delineated area of activation, MMP active forms have long been considered for their unique ability to degrade extracellular substrates. However, turnover and breakdown of the extracellular matrix are neither the sole nor the main function of MMPs. These enzymes can indeed process a wide variety of non-matrix substrates and are involved in the regulation of multiple aspects of tumor progression, immunity and inflammation. To add further complexity to MMPs biology, some members within the family were recently reported to have intracellular localization associated to non-proteolytic functions. These observations but also those evidencing that some MMPs participate in disease progression while others have a protective function, stress the need to better document their spatial and temporal activation in various biological contexts.Activity-based protein profiling (ABPP) aims to analyze the functional state of proteins within complex biological samples. To this purpose, activity-based probes (ABPs) that react with enzymes in a mechanism-based manner have been successfully developed for the profiling of several enzymes including serine and cysteine proteases. A typical Activity-Based probe (ABP) is composed of i) a reactive warhead, which reacts in a covalent manner with enzyme active site residues, ii) a targeting moiety that imposes selectivity upon the reactive group and iii) a detectable group for subsequent analyses. This approach is not applicable to MMPs, which lack a targetable nucleophile involved in the catalysis. In this respect, all ABPs directed to MMPs are affinity-based probes (AfBPs) containing within their structure a photo cross-linking group that promotes the formation of a covalent complex upon UV-irradiation. Such photoactivatable probes have been successfully developed for the detection of MMPs under their active forms in fluids and tissue extracts, but not in living animals where the photo-activation step is not feasible.By relying on a favorable structural context and by exploiting the ligand-directed acyl imidazole (LDAI) chemistry, we have identified a novel series of AfBPs capable of covalently modifying matrix metalloproteases without making use of photo-activation. These active-site-directed probes whose structure was derived from that of a MMP12 selective inhibitor harbored a reactive acyl imidazole in their P3' position. They demonstrated their labelling specificity in vitro by covalently modifying a single Lysine residue within the MMP-12 S3' region. We also showed that these probes only targeted functional states of hMMP-12 and spared forms whose active site was occluded either by a synthetic or a natural inhibitor. We have validated the ability of these chemical probes to efficiently label human MMP12 in complex proteomes. In this case, down to 50 ng of hMMP12 corresponding to 0.05% of the whole proteome can be labelled and detected by in-gel fluorescence analysis. We demonstrated that this approach also allowed detecting endogenous MMPs secreted by stimulated-macrophages. In addition, by modifying the nature of the targeting moiety, we have extended this affinity-labeling approach to six other MMPs.By developing the first “photo activation-free” strategy to covalently modify active forms of MMPs, the unresolved proteomic profiling of native MMPs should be now accessible both in complex proteomes and in preclinical model in which MMPs are potential relevant targets

Indiana University-Purdue University Indianapolis (IUPUI)
The discovery and isolation of naturally occurring fluorescent proteins, FPs, have provided much needed tools for molecular and cellular level studies. Specifically the cloning of green fluorescent protein, GFP, revolutionized the field of biotechnology and biochemical research. Recently, a red fluorescent protein, DsRed, isolated from the Discosoma coral has further expanded the pallet of available fluorescent tools. DsRed shares only 23 % amino acid sequence homology with GFP, however the X-ray crystal structures of the two proteins are nearly identical. DsRed has been subjected to a number of mutagenesis studies, which have been found to offer improved physical and spectral characteristics. One such mutant, DsRed-Monomer, with a total of 45 amino acid substitutions in native DsRed, has shown improved fluorescence characteristics without the toxic oligomerization seen for the native protein. In our laboratory, we have demonstrated that DsRed proteins have a unique and selective copper-binding affinity, which results in fluorescence quenching. This copper-binding property was utilized in the purification of DsRed proteins using copper-bound affinity columns. The work presented here has explored the mechanism of copper-binding by DsRed-Monomer using binding studies, molecular biology, and other biochemical techniques. Another focus of this thesis work was to demonstrate the applications of DsRed-Monomer in biochemical studies based on the copper-binding affinity and fluorescence properties of the protein. To achieve this, we have focused on genetic fusions of DsRed-Monomer with peptides and proteins. The work with these fusions have demonstrated the feasibility of using DsRed-Monomer as a dual functional tag, as both an affinity tag and as a label in the development of a fluorescence assay to detect a ligand of interest. Further, a complex between DsRed-Monomer-bait peptide/protein fusion and an interacting protein has been isolated taking advantage of the copper-binding affinity of DsRed-Monomer. We have also demonstrated the use of non-natural amino acid analogues, incorporated into the fluorophore of DsRed-Monomer, as a tool for varying the spectral properties of the protein. These mutations demonstrated not only shifted fluorescence emission compared to the native protein, but also improved extinction coefficients and quantum yields.

Plasminogen activator (PAs) are enzymes that convert the zymogen plasminogen into the trypsin-like protease plasmin, which degrades extracellular matrix proteins and fibrin in the course of fibrinolysis, embryogenesis, tissue remodeling and in tumor metastasis. Plasminogen activator inhibitors (PAIs) are important modulators of PA activity. Several proteins have been identified which inhibit at fast rates urokinase (u-PA) and tissue-type PA (t-PA). In the order of inhibition rate constants these are: a) PAI-1, present in human plasma and platelet extracts and purified from human endothelial cell, fibrosarcoma cell and melanoma cell conditioned media; b) PAI-2, first identified in extracts of human placenta and later also in extracts and conditioned media of human granulocytes and monocytes; and c) protease nexin, a broad specificity protease inhibitor that was first identified and purified from human fibroblasts. We have chosen to use phorbol myristate acetate (30 ng/ml) stimulated histiocytic lymphoma cells (U-937) for the purification of PAI-2. The concentration of PAI-2 in the conditioned media after three days culture in the absence of fetal calf serum is 5 mg/1 and PAI-2 represents 3% of total protein. PAI-2 was purified by a two step procedure consisting of isoelectric focusing and affinity chromatography on Cibacron-Blue agarose. Two forms of PAI-2 were identified: a 47 kDa, nonglycosylated, pi 5.0 form and a 60 kDa glycosylated, pi 4.4 form. Immunctolot analysis and in vivo protein labeling studies under culture conditions that assure 100% viability of the cells showed that the glycosylated Torn is secreted, whereas the 47 kDa, nonglycosylated form remains intracellular. The glycosylation does not affect the activity of the inhibitors since both forms of PAI-2 react with the same rate with u-PA. PAI-2 is a fast inhibitor of u-PA (kl=9×l05M−1s−1) and two-chain t-PA (kl=2×l05) and a rather slow inhibitor of one chain t-PA (kl=l×l02) and of plasmin (kl×l02), but does not inhibit glandular and plasma kallikrein or thrombin. The inhibition spectrum and the kinetics of inhibition clearly distinguish PAI-2 from PAI-1 (kl of reaction with u-PA and two and one chain t-PA above 107) and from protease nexin, that is an efficient inhibitor also of thrombin and plasmin.We have cloned a 1880 Ip fragment of PAI-2 cDNA and determined its nucleotide sequence. The derived acid sequence reveals that PAI-2 is like PAI-1 and protease nexin a member of the serpin family of proteins and contains arginine at its putative active site. In an attenpt to identify parts of the inhibitor proteins that are responsible for conferring PA specificity to PAI-1 and PAI-2 we have compared the primary structures of PAI-1 and PAI-2 with each other and with antithrombin III (AT III). Surprisingly, PAI-2 exhibits no homology with PAI-1 in the region close to the active site except for the active site arginine, whereas, in that region, AT III showed three and seven conserved aminoacids when compared to PAI-1 and PAI-2, respectively. This finding suggests that other regions than those close to the active site contribute to the specificity of PAIs.Plasma concentrations of PAI-2 were measured by a specific radioimmunoassay in over 50 healthy individuals, PAI-2 levels were below detection limit (15 ng/ml) in half of the saitples. Maximal concentrations encountered were in the 30 ng/ml range. PAI-2 measurements in over 300 hospitalized patients demonstrated significantly elevated PAI-2 concentrations only in pregnant women. Measurements in various stages of pregnancy showed a steady increase of PAI-2 from below detection limit in nonpregnant women to values of 250 ng/ml at term and of PAI-1 frcm 25 ng/ml to 150 ng/ml. Unlike to PAI-1 concentrations that normalize rapidly after delivery, PAI-2 concentrations remain significantly elevated for several days.