Les inhibiteurs des protéases à sérine (Serpins) constituent une classe d'enzymes très peu étudiée chez les bactéries. Dans ce travail de thèse nous nous sommes intéressés à l'étude des serpins provenant du microbiote intestinal et l'investigation de leur potentiel anti-inflammatoire pour le traitement des maladies inflammatoires chroniques de l'intestin (MICI) chez l'homme. Pour cela nous avons identifié les serpins provenant du microbiote intestinal humain et analysé leur diversité ainsi que leur distribution entre les individus malades et sains. Ces données nous ont permis d'isoler les serpins significativement associées aux MICI. La purification de quarte d'entre elles nous a amené à démontrer qu'elles inhibent les protéases humaines impliquées dans les MICI. L'analyse biochimique et cinétique approfondie de ces protéines a montré qu'elles possèdent des propriétés originales notamment leur efficacité d'inhibition élevée. L'étude de l'effet protecteur de trois serpins chez un modèle animal de colite a démontré pour la première fois l'efficacité des serpins in vivo démontrant ainsi leur potentiel thérapeutique. / Serine protease inhibitors (Serpins) are a class of proteins that reamin poorly studied in bacteria. In this thesis we are interested in the study of serpins originating from the intestinal microbiota and the investigation of their anti-inflammatory potential for the treatment of inflammatory bowel diseases (IBD) in humans. For this we have identified serpins from the human gut microbiota and analyzed their diversity as well as their distribution between healthy and IBD patients. These data allowed isolating serpins significantly associated with IBD. The purification of four of them led us to demonstrate that they inhibit human proteases involved in IBD. Biochemical and kinetic analysis of these proteins showed that they exhibit original properties, in particular their high inhibition efficiency. The study of the protective effect of three serpins in an animal model of colitis demonstrated for the first time the efficacy of serpins in vivo demonstrating thus their therapeutic potential.

Growing evidence indicates that serine proteases known as kallikreins are associated with malignancy and may have potential diagnostic/prognostic applications in cancer. Kallikreins are the largest group of serine proteases. Kallikrein enzymes are often involved in proteolytic cascades through their function in degradation of extracellular matrix proteins and promotion of angiogenesis. Kallikrein 6 (KLK6) is a member of the family of fifteen highly conserved secreted trypsin- or chemotrypsin-like serine proteases. Over-expression of KLK6 has been observed in different pathophysiological states such as neurodegenerative diseases, inflammation and various cancers, including colorectal cancer. In Chapter 3 we elucidated the miRNA-based mechanism of regulation of invasion in metastatic colorectal cancer over-expressing KLK6. We developed HCT116 colon stable isogenic cell lines with knockdown of KLK6 expression using short-hairpin interference RNA (shKLK6 clones). The shKLK6 clones had decreased expression and secretion of KLK6 protein with a minimal effect on cell growth and viability in cell culture. SCID mice injected with shKLK6-3 clone 3 cells exhibited a statistically significant increase in the survival rates (P=0.005), decrease in the incidence of distant metastases and a shift in the location of the metastatic foci closer to the cell's injection site. Levels of KLK6 protein secreted into the bloodstream were significantly lower in animals injected with shKLK6-3 clone 3 compared to HCT116 control clone 1 (P < 0.04). Through bioinformatics analyses we identified and validated three miRNAs, which are important in post-translational modification of bioactive proteins, proliferation, migration and p38 MAPK signaling pathway. In Chapter 4 we developed Caco-2 colon stable isogenic cell lines with expressing enzymatically active or mutant KLK6 protein (Caco-2 stable clones). We employed these cell lines to investigate the importance of KLK6 enzymatic activity of initiation of cell invasion using in vitro and in vivo models.

No description available.

Dorsal-ventral (DV) polarity in the Drosophila embryo is defined by spatially regulated activation of the transmembrane receptor Toll, which is uniformly distributed throughout the early embryo's plasma membrane. Ventral activation of Toll is accomplished through the local production of its activating ligand, a processed C-terminal fragment of the Spätzle protein, which is generated in the last step of a proteolytic cascade involving the sequentially-acting proteases Gastrulation Defective (GD), Snake and Easter. Pipe protein, a homologue of vertebrate glycosaminoglycan modifying enzymes, which is expressed during oogenesis in ventral follicle cells adjacent to the developing oocyte, is believed to control the ventrally restricted processing of Spätzle. pipe expression and the sulfation of its enzymatic target in the ventral follicle cells leads to the formation of a stable ventral cue, embedded in the eggshell. Recently the Pipe enzymatic target has been identified as several protein components of the vitelline membrane, the inner layer of the eggshell. Prior to this work, an important piece of information missing from our understanding of Drosophila DV patterning was the identity of the initial step in the protease cascade that requires Pipe activity. Here, I show that the processing of Snake is independent of Pipe activity, while the processing of Easter requires Pipe function, indicating that Easter processing by Snake is the key proteolytic step that is controlled by Pipe activity and presumably the first cleavage event that is spatially regulated. A second key gap in our understanding of Drosophila embryonic DV patterning concerned the role of GD in the protease cascade. While GD is the protease that cleaves and activates Snake, the existence of two distinct classes of complementing gd alleles has suggested that GD provides another, distinct function. Investigations described here indicate that the second function of GD is to promote the ability of activated Snake to process Easter, independent of its Snake-processing function. Finally, I provide evidence for the formation of protein complexes containing various components of the serine protease cascade, which suggest that conformational changes in the complexes, which act to promote productive interactions between the proteins, are an important aspect of their activation. / text

<p>Mast cells are key effector cells in allergic and inflammatory diseases. However, their primary role is most likely in host defence against parasitic and bacterial infections. Mast cells are a particularly rich source of serine proteases. These proteases belong to the chymase or the tryptase family, which are encoded from the mast cell chymase and the multigene tryptase loci, respectively. To better understand the biological functions and the molecular evolution of these enzymes we have studied the organisation of these two loci in species ranging from fish to human. We show that the mast cell chymase locus has evolved from a single founder gene to a complex locus during the past 200 Myr of mammalian evolution. Forty-five fish candidate genes for hematopoietic serine proteases were also identified. However, in phylogenetic analyses none of them grouped with individual branches holding mammalian mast cell chymase locus genes, indicating an independent parallel evolution in fish. </p><p>Studies of the evolution of the multigene tryptase locus showed that this locus has been highly conserved between marsupials and eutherians. However, no genes belonging to the individual subfamilies identified in eutherians could be identified in fish, amphibians or in birds, which also here indicates parallel evolution.</p><p>To study the evolution of specific cleavage specificities associated with these proteases, the extended cleavage specificity of opossum α-chymase was determined and found to be nearly identical to human mast cell chymase and the major mouse mast cell chymase mMCP-4. This indicates a strong pressure to maintain this specificity during mammalian evolution.</p><p>Basophils are rare blood cells with functions similar to mast cells that when mature almost completely lack mRNA. To study the proteome and to primarily characterize the granule protein content of basophils, an <i>in vitro</i> purification protocol was developed to obtain transcriptionally active umbilical cord blood-derived basophil precursors.</p>

Background: Receptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown. Methods and results: We demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1(-/-)mice and Pim1(-/-)2(-/-)3(-/-) triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1 (-/-) and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit S-35 methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level. Conclusions: Our study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.

The serpin antiplasmin (APL) is the primary inhibitor of plasmin, a proteinase that digests fibrin, the main component of blood clots. Most serpins are serine protease inhibitors, which undergo dramatic conformational change in forming a tight covalent complex with the target protease. Plasmin has been shown to be angiogenic through its protease activity, but it is also angiostatic, being the source of angiostatin, which inhibits angiogenesis. The main objective of our study is to obtain antiplasmin in large amounts, for crystallization and structure determination of APL and of its complex with plasmin, and for solution studies of the complex. Bacterially expressed APL will not be glycosylated, an advantage in crystallization trials.Bacterial expression of rAPL has been problematic. We have found that it can be greatly enhanced through the use of host E.coli cells that carry extra copies of genes for tRNAs coding for rarely used codons in E.coli that occur in high frequency in eukaryotic genes. Several vectors were screened for rAPL expression (pET19b, pET20b and pET28b). rAPL is expressed in high yield from a pET28b construct in host BL-21 RIPL codon plus cells. rAPL thus expressed accumulates as inclusion bodies, but can be solubilized using N-lauroyl sarcosine at pH11. Refolding and purification of rAPL is achieved by using a sizing column followed by a Nickel His-tag affinity column with an imidazole gradient. rAPL fractions thus obtained are stable at 4°C in the presence of EDTA. However, no inhibitor activity of this rAPL towards trypsin was observed, nor did it form inhibition complex with trypsin. The presence of trace protease and/or failure to fold correctly may be preventing recovery of inhibitory activity. A screen of various refolding buffers failed to yield soluble, stable APL.

The work in this thesis is divided into two aims. The first aim is to provide a detailed analysis of water molecules at protein-protein interfaces as well as quantifying their contributions with respect to different residue types. To achieve this aim a data set of 4741 water molecules abstracted from 179 high-resolution (≤ 2.30 Å) X-ray crystal structures of protein-protein complexes was analyzed with a suite of modeling tools based on HINT. The second aim is to observe the effect of adding interfacial water molecules in developing a model for the protein-protein interaction between pyridoxal kinase and serine hydroxymethyltransferase. This model was created to explore the possibility of the formation of a channel between the two proteins upon interaction providing a safe way to transport the substrate pyridoxal 5’-phosphate (active form of vitamin B6). This work demonstrates a substantial progress in the understanding of the role of water molecules in protein-protein binding.

Serine racemase (SR) is a pyridoxal-5'-phosphate-dependent enzyme responsible for biosynthesis of D-serine, a recognized neurotransmitter acting as a co-activator of N-methyl- D-aspartate (NMDA) type of glutamate receptors in the mammalian central nervous system. The hyperfunction of the mentioned receptors have been shown to be implicated in many neuropathological conditions including Alzheimer's disease, amyotrophic lateral sclerosis and epilepsy. To alleviate the symptoms of these diseases, several artificial blockers of NMDA receptors have been introduced into the clinical practice. However, many of these compounds cause undesirable side effects and it is thus necessary to search for either less harmful blockers or regulators of other targets of pharmaceutical intervention that are involved in NMDA receptor activation. In this context, specific inhibition of serine racemase seems to be a promising strategy for regulation of NMDA receptor overstimulation. Mouse serine racemase shares 89% identity with its human ortholog and it was also shown that both enzymes possess similar kinetic parameters and inhibitor specificity. Therefore, the mouse models can be used to search for a potent human serine racemase inhibitor. Although many different compounds for their inhibitory potency towards serine...

Small chiral molecules are very important building blocks in the synthesis of biologically active compounds. These building blocks include nitrogen and oxygen-containing heterocycles such as 2-oxazolidinones, 1,3-oxazinan-2-ones, 2-oxazolines, oxazines, morpholine and morpholinones. Because of their interesting properties, chiral heterocycles have stirred great interest in the synthetic chemist community to develop useful and efficient strategies to these molecules. In this dissertation, the design and syntheses of various heterocyclic building blocks are presented, as well as the testing of their biological activities as antibacterial. Another very interesting family of heterocycle-containing molecules are the Aeruginosins. They are a family of marine natural products isolated from a blue-green algae, which display inhibitory activity against serine proteases such as thrombin, trypsin, and factor VIIa. Most aeruginosins contain an heterocyclic moiety called the 2-carboxy-6-hydroxyoctahydroindole (Choi) ring; this Choi moiety is a rigid bicyclic unnatural amino acid and is the core structure in the aeruginosins, indispensable to their biological activity. A synthesis of a ring-oxygenated variant of the Choi from D-mannose is reported in this dissertation. The ring-oxygenated variant of 2-carboxy-6-hydroxyoctahydroindole can potentially be used as a surrogate of Choi in the design and synthesis of aeruginosin-based thrombin inhibitors.