Emerging Roles of ADAM and ADAMTS Metalloproteinases in Cancer

A disintegrin and metalloproteinases (ADAMs) are a recently discovered family of proteins that share the metalloproteinase domain with matrix metalloproteinases (MMPs). Among this family, structural features distinguish the membrane-anchored ADAMs and the secreted ADAMs with thrombospondin motifs referred to as ADAMTSs. By acting on a large panel of membrane-associated and extracellular substrates, they control several cell functions such as adhesion, fusion, migration and proliferation. The current review addresses the contribution of these proteinases in the positive and negative regulation of cancer progression as mainly mediated by the regulation of growth factor activities and integrin functions

Apport de la protéomique à l'étude de la physiopathologie de l'asthme

L’asthme bronchique est une affection chronique des voies respiratoires. Cette pathologie est à la fois complexe et multifactorielle. En effet, son développement implique différents types de cellules inflammatoires et structurelles mais également des médiateurs de nature peptidique et lipidique. Dans ce contexte, les études protéomiques représentent un intérêt considérable car des techniques émergentes ont rendu possible l’analyse d’échantillons biologiques complexes d’origines différentes, provenant notamment de patients ou d’animaux modélisant des maladies inflammatoires chroniques telles, entre autres, l’asthme. Ces études basées sur la protéomique ont pour but d'approfondir les connaissances sur le mécanisme physiopathologique de la maladie ou encore de découvrir de nouveaux biomarqueurs voire de nouvelles cibles thérapeutiques.Au cours de ce travail, deux études protéomiques ont été réalisées sur des échantillons provenant de souris soumises à deux types de protocoles d’asthme expérimental. Ces protocoles, comportant des durées d’exposition aux allergènes variables, reproduisent de nombreuses caractéristiques de l’asthme allergique humain. Les médiateurs peptidiques connus pour jouer un rôle prépondérant dans les pathologies inflammatoires dont l’asthme sont le plus souvent des cytokines, des chimiokines, des facteurs de croissance et des fragments protéolytiques. Ces protéines de faibles poids moléculaires sont difficilement accessibles par la technique du SDS-PAGE. Une approche protéomique adaptée à la détection de changements d’intensité de peptides et de protéines de petites tailles (<20-30 kDa), le SELDI-TOF-MS, a donc été nécessaire pour atteindre nos objectifs. L’analyse des profils protéiques détectés par SELDI-TOF-MS révèle différents pics discriminatoires entre nos conditions contrôles et pathologiques. Suite à ces analyses, plusieurs pics (m/z) sont considérés comme discriminatoires. Parmi ces pics, certains ont été identifiés par MS/MS. Il s’agit de Found Inflammatory Zone 1(Fizz-1), Calcyclin (S100A6), Clara Cell secretory protein 10 (CC10), Ubiquitine and Histone H4. Plus précisément, nos données expérimentales ont validé une forte différence d’intensité des pics correspondant à Fizz-1, S100A6, CC10 et Ubiquitine dans les souris exposées à l’allergène comparativement aux souris contrôles correspondantes. Au contraire, le pic correspondant à l’Histone H4 est significativement diminué suite à l'exposition à l'allergène. Les résultats ont été validés par western blot, immunohistochimie et RT-PCR semi-quantitative. Le deuxième versant de ce travail de doctorat porte sur l’utilisation de l’électrophorèse sur gel de polyacrylamide à deux dimensions. Parmi les protéines identifiées, 7 d’entre elles ont fait l’objet d’une étude plus approfondie. Plus spécifiquement, les protéines GRP78 (Glucose Related Protein 78), PDIA6 (Protein Disulfide Isomérase 6), Annexin A6, hnRNPA3 (Heterogeneous Nuclear Ribonucleoprotein A3) et Enolase montrent une production accrue après exposition aigue à l’allergène, alors qu’Apolipoprotéine A1 est modulée de manière négative suite à la même exposition. La protéine Calreticulin est modulée négativement suite à une exposition de longue durée à l’allergène. Les résultats ont été validés par western blot et immunohistochimie. Au terme de ce travail, différents biomarqueurs et médiateurs potentiellement impliqués dans la pathologie asthmatique ont pu être mis en évidence. Ceci confirme l’intérêt d’étudier le rôle des protéines les plus diverses dans la mise en place du processus asthmatique

Comparison of serum fractionation methods by data independent label-free proteomics

Off-line sample prefractionations applied prior to biomarker discovery proteomics are options to enable more protein identifications and detect low-abundance proteins. This work compared five commercial methods efficiency to raw serum analysis using label-free proteomics. The variability of the protein quantities determined for each process was similar to the unprefractionated serum. A 49% increase in protein identifications and 12.2% of reliable quantification were obtained. A 61 times lower limit of protein quantitation was reached compared to protein concentrations observed in raw serum. The concentrations of detected proteins were confronted to estimated reference values

Role of A Disintegrin and Metalloprotease-12 in Neutrophil Recruitment Induced by Airway Epithelium.

Among proteases, metalloproteases are implicated in tissue remodeling, as shown in numerous diseases including allergy. ADAMs (A Disintegrin And Metalloprotease) metalloproteases are implicated in physiologic processes such as cytokine and growth factor shedding, cell migration, adhesion, or repulsion. Our aim was to measure ADAM-12 expression in airway epithelium and to define its role during the allergic response. To raise this question, we analyzed the ADAM-12 expression ex vivo after allergen exposure in patients with allergic rhinitis and in vitro in cultured primary human airway epithelial cells (AEC). Clones of BEAS-2B cells transfected with the full-length form of ADAM-12 were generated to study the consequences of ADAM-12 up-regulation on AEC function. After allergen challenge, a strong increase of ADAM-12 expression was observed in airway epithelium from patients with allergic rhinitis but not from control subjects. In contrast with the other HB-epidermal growth factor sheddases, ADAM-10 and -17, TNF-alpha in vitro increased the expression of ADAM-12 by AEC, an effect amplified by IL-4 and IL-13. Up-regulation of ADAM-12 in AEC increased the expression of alpha3 and alpha4 integrins and to the modulation of cell migration on fibronectin but not on collagen. Moreover, overexpression of ADAM-12 in BEAS-2B enhanced the secretion of CXCL1 and CXCL8 and their capacity to recruit neutrophils. CD47 was strongly decreased by ADAM-12 overexpression, a process associated with a reduced adhesion of neutrophils. These effects were mainly dependent on epidermal growth factor receptor activation. In summary, ADAM-12 is produced during allergic reaction by AEC and might increase neutrophil recruitment within airway mucosa

Potential Proteomic Biomarkers Associated To Mucosal Healing In Crohn’s Disease

Introduction and objectives: In Crohn's disease (CD), there is a discrepancy between clinical activity of the disease (symptoms) and intestinal healing. However absence of tissue healing is associated with the risk of relapse and tissue damage progression. Endoscopy is costly and invasive. Hence biomarkers correlating with intestinal healing could improve disease management. We aimed to identify potential biomarkers associated to CD mucosal healing by a shotgun proteomics label free study. Methods: We used the STORI clinical trial cohort (n=103) aiming at identifying markers associated to relapse prediction after Infliximab treatment withdrawals. We used serum samples of patients in clinical remission, grouped according to the degree of intestinal healing seen at endoscopy. We performed depletion of the 20 most abundant plasma proteins on each serum pools and ran a proteomics label free differential analysis using 2D-nanoUPLC-MSE HDMS Synapt G1 for data acquisition and Protein Lynks Global Server vs 2.4 for data analysis (Waters, Corp., Milford, USA). Results and Discussion: We obtained potential biomarkers and designed a multiplexed -selected reaction monitoring (SRM) method for validation of these candidates in each individual patient. The method may also be tested in an independent set of IBD patients with and without mucosal healing. Conclusions: This research strategy and results of SRM validation of potential biomarkers associated to mucosal healing in this cohort of CD patients as well as the tests done on other CD patients, may provide new opportunities for CD follow-up tests development

MMP-19 Deficiency Promotes Tenascin-C Accumulation and Allergen-induced Airway Inflammation.

Matrix metalloproteinases (MMPs) recently appeared as key regulators of inflammation, allowing recruitment and clearance of inflammatory cells and modifying the biological activity of many peptidic mediators by cleavage. MMP-19 is a newly described MMP and preferentially cleaves matrix proteins such as collagens and tenascin-C. The role of MMP-19 in asthma has not been described to date. The purpose of the present study was to assess MMP-19 expression in a murine asthma model and to address biological effects of MMP-19 deficiency in mice. Allergenexposed wild-type (WT) mice displayed an increased expression of MMP-19 mRNA and an increased number of MMP-19-positive cells in the lungs detected by immunohistochemistry. After allergen challenge of MMP-19 knockout (MMP-19-/-) mice, an exacerbated eosinophilic inflammation was detected in bronchoalveolar lavage fluid and bronchial tissue along with an increased airway responsiveness to methacholine. A shift towards increased Th2-driven inflammation in MMP-19-/- mice was demonstrated by 1) increased numbers of cells expressing the IL-33 receptor T1/ST2 in lung parenchyma, 2) increased IgG1 levels in serum and 3) higher levels of IL-13 and CCL11 in lung extracts. Tenascin-C was found accumulated in peribronchial areas of MMP-19-/- after allergen challenges as assessed by Western blot and immunohistochemistry analysis. We conclude that MMP-19 is a new mediator in asthma, preventing tenascin-C accumulation and directly or indirectly controlling Th2-driven airway eosinophilia and airway hyperreactivity. Our data suggest that MMP-19 might act on Th2 inflammation homeostasis through preventing tenascin protein accumulation