Dissertations / Theses on the topic 'Peptide substrates'

Protein tyrosine kinases (PTKs) mediate important signaling events associated with cellular growth, differentiation, and mitogenesis. The p60c-src protein is the first described cellular protein tyrosine kinase. Human p60c-src PTK has been implicated in the development of colon and breast cancer, and leukemia. However, the exact physiological role of p60c-src PTK or its physiological target proteins are not well known, and the mechanism by which the p60c-src PTK activity is regulated is not completely understood. Peptide substrates can be used to determine the substrate specificity and kinetic parameters, and therefore to provide important information for understanding of the physiological role and mechanism of action of this enzyme. Peptide substrates can also be used to develop pseudosubstrate-based peptide inhibitors for p60c-src PTK. Combinatorial peptide library methods have proven to be very powerful in identifying ligands for receptors and in discovering peptide substrates for protein kinases. In this dissertation, a "one-bead one-compound" combinatorial peptide library method was applied to identify peptide substrates for p60c-src PTK, the structure-activity relationship of the identified peptide substrates was studied, and the pseudosubstrate-based peptide inhibitors for p60c-src PTK were developed. Using the "one-bead one-compound" combinatorial peptide library method, a novel peptide, YIYGSFK, was identified as an efficient substrate for p60c-src PTK. The structure-activity relationship study was performed on over 70 analogs of YIYGSFK. It was determined that -Ile-Tyr- were the two critical residues required for activity. Based on this dipeptide motif a secondary library was synthesized (XIYXXXX, wherein X = all 19 eukaryotic amino acids except cysteine, I = isoleucine, Y = tyrosine) and screened with p60c-src PTK. One of the identified peptides, GIYWHHY, was found to be more efficient for p60c-src PTK than the parental compound, YIYGSFK. Several potent psedosubstrate based inhibitors were developed using GIYWHHY as a template. Some of the more potent inhibitors have branched structure indicating the enzyme active site can accommodate more than a linear motif. These data demonstrate that the "one-bead one-compound" combinatorial library method is a powerful tool to discover novel peptide substrates, and to develop pseudosubstrate-based peptide inhibitors for PTKs.

Ribosomal synthesized and post translational modified peptide natural products have attracted a lot of interest in the past decade. Backbone cyclization of the translated linear peptides is generally catalysed by specific enzymes giving them peptidase resistance, thermodynamic stability and various other physiological activities. These features have made backbone cyclic peptide to become an attractive resource for drug discovery. Here, we described the synthesis of linear peptides containing natural and unnatural residues and its biosynthetic mechanism to generate man-made cyclic peptides. In this thesis we used SPPS to make short and medium linear peptide chains, we purified them using HPLC, and analysed them using MS. We incorporated unnatural residues such as homocysteine, homoserine, aminoalanine, propargyl glycine and the substrates were subjected to different enzymatic reaction such as prenylation, heterocyclization and macrocyclization modification reactions to generate small macrocycles (4-6 residues), prenylated linear peptides, and patellamime analogues. The final products were analysed using LC-MS. In our results, we verified that kawaguchipeptin (kgp) gene cluster is responsible for the production of kawaguchipeptins through heterologous expression of the kgp gene cluster in Escherichia coli. The KgpF prenyltransferase was overexpressed and was shown to prenylate C-3 of Trp residues in both linear and cyclic peptides in vitro. We also found out that PatGmac can macrocyclise short peptides (4-6 residues) to generate small macrocyclic peptides. We also tested the flexibility of OscGmac using unnatural amino acid residues such as pseudoprolines and pipecolic acid that can mimic the heterocyle incorporated as the final residue in the natural product. Our results show that OscGmac recognises pseudoprolines before AYD(G) to process a linear peptide.

ABH1 and Ofd2 are human and Schizosaccharomyces pombe members of the 2-oxyglutarate-iron(II)-dependent dioxygenase family respectively. Little is known about these proteins, and no substrates for them have been identified. Using positionally addressable synthesis of peptides on a cellulose membrane (SPOT synthesis) random peptide libraries were generated. These were screened with ABH1 and Ofd2 using a 2-oxoglutarate turnover assay, to generate preferred sequence motifs. The motifs were searched against protein databases to create a list of potential substrates. The potential substrates were tested both for binding and activity. Several highly active peptides were systematically mutated in an attempt to further define the preferred motifs. These sequence preferences were used to conduct another search of the protein databases. Ultimately none of the sequences studied could be confirmed as substrates, however, refinement of the technique used could produce a viable method of identifying unknown substrates.

Lysine methylation modulates diverse biological processes and is catalyzed by SET domain methyltransferases such as the SMYDs (SMYD1-5), which possess a SET domain split by a MYND motif. Through association with NCoR, the H4 Lys20 methyltransferase activity of SMYD5 represses inflammation by restricting TLR-4 mediated expression in macrophages, yet biochemical and structural features required for SMYD5 methylation activity remain elusive. To determine how SMYD5 catalyses methylation, crystallization screens were conducted with SMYD5 in complex with the co-factor AdoMet and histone H4. Screens yielded lead conditions but no crystals. To determine the motif recognized by SMYD5 and decipher its methylome, peptide arrays were conducted to produce a methylation motif used to identify putative substrates. Surprisingly, arrays revealed that substitution of Lys16, not Lys20, is detrimental to SMYD5 activity. Further enzymatic assays are required to determine if SMYD5 methylates residues other than Lys20 on the H4 tail, or if structural determinants or interacting partners restrict methylation of target lysines.

Les cyclodipeptides constituent, avec leurs dérivés plus complexes les dicétopiperazines (DKP), une importante famille de produits naturels, synthétisés essentiellement par des micro-organismes. Une approche intéressante pour synthétiser une grande diversité de DKP consiste à étudier et manipuler les voies de biosynthèse de ces molécules. Les synthases de cyclodipeptides (CDPS) constituent une famille d’enzymes dédiés à la production de cyclodipeptides, qui ont la particularité d’utiliser les ARNt aminoacylés (AA-ARNt) comme substrats. Afin d’exploiter complètement le potentiel de ces enzymes, il est nécessaire de mieux comprendre leur spécificité, notamment vis-à-vis de substrats non naturels. Dans cette thèse, nous avons tout d’abord démontré que les CDPS sont capables d’incorporer des acides aminés non naturels, en utilisant la promiscuité des aminoacyl-ARNt synthétases (AARS) d’Escherichia coli. Puis nous avons amélioré notre compréhension de la reconnaissance de la partie ARNt des susbtrats par les CDPS. En utilisant les flexizymes, des ribozymes à activité AARS, nous avons généré des analogues d’AA-ARNt avec des parties ARNt tronquées. Nous avons pu montrer que des « mini AA-ARNt » reproduisant les 7 paires de bases du bras accepteur des ARNt sont d’aussi bons substrats que les AA-ARNt complets, ce qui suggère que les CDPS interagissent principalement avec les bras accepteurs de leurs substrats ARNt
Cyclodipeptides and their complex derivatives, the diketopiperazines (DKPs), constitute a large class of natural products with diverse and noteworthy pharmacological activities observed for many naturally occurring DKPs. A promising approach to generate diverse DKPs is to study and manipulate DKP biosynthetic pathways. Cyclodipeptide synthases (CDPSs) constitute an enzyme family dedicated to the synthesis of cyclodipeptides, with the particularity to use aminoacylated-tRNAs (AA-tRNAs) as substrates. In order to unlock the biosynthetic potential of these enzymes, better understanding their specificity, in particular towards non-natural substrates, is required.In this thesis, we first significantly expanded the diversity of cyclodipeptides accessible with CDPSs by showing that CDPSs could incorporate non-canonical amino acids, through the use of the promiscuity of E. coli aminoacyl-tRNA synthetases. Then, we gave new insights into the recognition by CDPSs of the tRNA moieties of their substrates. By using an innovative RNA acylation strategy based on a class of ribozymes called flexizymes, we generated analogues of AA-tRNAs with truncated RNA moieties. Among these “AA-minitRNAs”, we showed that those mimicking the entire 7 bp stems of tRNAs are as good substrates as AA-tRNAs, which suggests that CDPSs interact mainly with the acceptor arms of tRNAs and paves the way for promising biophysical and structural studies

Transglutaminases (protein-glutamine:amine y-glutamyl- transferase, EC 2.3.2.13) are a family of calcium-dependent enzymes which catalyze an acyl transfer between glutamine residues and a wide variety of primary amines. When lysine acts as the acyl-acceptor substrate, α-glutamyl lysine isopeptide bond is formed. Isopeptide catalyzation results in protein cross-linkage which is prevalent throughout biological processes. Microbial transglutaminase (mTG) is a bacterial variant of the transglutaminase family, distinct by virtue of its calcium-independent catalysis of the isopeptidic bond. Furthermore, mTGs promiscuity in donor substrate preference highlights its biocatalytic potential. To realize the potential of the enzyme, a high-reactivity tag was necessary for protein labelling. To address this, an enzyme-coupled assay was developed to characterize peptides in the hopes of developing orthogonal substrates to facilitate mTG-mediated labelling and biocatalysis. The discovery of high-reactivity peptide tags allowed the realization of in vitro protein labelling- facilitated by mTG. The 7M48 peptide was fused to a test protein, where it was subsequently propargylated with propargyl amine to fluorescently label or immobilize a test protein. Although there are endless possibilities for in vitro bio-conjugation through mTG, proteolytic activation limits any in-cell labelling strategies with this enzyme. To circumvent this issue, development of an alternative bacterial enzyme, Bacillus subtilis transglutaminase (bTG), was chosen to replace mTG. bTG maintains the advantages associated with mTG but is expressed in its active form. Unlike mTG, there is limited preliminary research associated with the enzyme or its substrate scope. To better understanding substrate reactivity, a FRET-based assay was developed allows for the discovery of new high-reactivity peptides for bTG. These peptides were then used in labelling strategies to demonstrate the potential bTG-mediated bioconjugation. This strategy includes the added advantage of potential for in-cellulo labelling.

Alzheimer Disease (AD) is the most common neurodegenerative disorder in the world. One of the neuropathological hallmarks of AD is the senile plaques in the brain. The plaques are mainly composed of the amyloid β (Aβ) peptide. Aβ is generated from the amyloid precursor protein, APP, when it is first cleaved by the β-secretase and subsequently the γ-secretase complex. The γ-secretase complex cleaves at different sites, called γ and ε, where the γ-cleavage site generates Aβ peptides of different lengths and ε-cleavage generates the APP intracellular domain (AICD). The two major forms of Aβ is 40 and 42 amino acids long peptides, where the latter is more prone to aggregate and is the main component in senile plaques. The γ-secretase complex is composed of four proteins; Pen-2, Aph-1, nicastrin and presenilin (PS). The PS protein harbours the catalytic site of the complex, where two aspartate residues in position 257 and 385 (Presenilin 1 numbering) are situated. Most Familial AD (FAD) mutations in the PS gene cause a change in the γ-cleavage site, leading to a shift from producing Aβ40 to the longer more toxic variant Aβ42. Frequently, this often leads to impairments of the AICD production. Another substrate for the γ-secretase complex is Notch. It is important to maintain the Notch signaling since an intracellular domain (NICD) is formed after cleavage by the γ-secretase complex in the membrane (S3-site) and this domain is involved in transcription of genes important for cell fate decisions.

It has been reported that certain APP luminal juxtamembrane mutations could drastically alter Aβ secretion, however their effect on AICD production remains unknown. In this study we want to analyse wether the juxtamembrane region is important for the AICD production. To gain more insight into the luminal juxtamembrane function for γ-secretase-dependent proteolysis, we have made a juxtamembrane chimeric construct. A four-residue sequence preceding the transmembrane domain (TMD) of APP (GSNK), was replaced by its topological counterpart from the human Notch1 receptor (PPAQ). The resulting chimeric vector C99GVP-PPAQ and the wildtype counterpart were expressed in cells lacking PS1 and PS2 (BD8) together with PS1wt. We observed that the chimeric construct did not alter production of AICD when using a cell based luciferase reporter gene assay monitoring AICD production. We also introduced a PS1 variant lacking a big portion of the large hydrophilic loop, PS1∆exon10, since our group has previously observed that this region affect Aβ production¹⁴³. We found that the absence of the large hydrophilic loop in PS1 gave a 2-fold decrease in AICD-GVP formation from C99GVPwt compared to PS1wt.  The activity of PS1wt and PS1Δexon10 using C99GVP-PPAQ as a substrate gave similar result as the C99GVPwt substrate, i.e. a 2-fold decrease in AICD-GVP formation when comparing PS1Δexon10 with PS1wt. From this data we therefore suggest that the four residues in the juxtramembrane domain (JMD) (GSNK) is not altering ε-cleavage of APP when changed to Notch1 counterpart, PPAQ. Furthermore, we also show that the 2-fold decrease in AICD-production by the PS1Δexon10 molecule is not changed between the two substrates C99GVPwt and C99GVP-PPAQ. This indicates that the luminal region of APP is not directly involved in the ε-site processing. If the luminal region is affecting processing in the γ-cleavage sites, remains however to be investigated.

Arguably the causative agent of Alzheimer's disease (AD), amyloid beta (Aβ)- peptides, are generated by sequential proteolysis of the amyloid precursor protein (APP) by β- and γ-secretase activities. Alternatively, APP can be processed nonamyloidogenically by an α-secretase activity that cleaves within the Aβ-domain, precluding the formation of intact Aβ-peptides and liberating a neuroprotective fragment, sAPPα. This latter proteolytic event is catalysed by a disintegrin and metalloproteinase (ADAM) 10. Recent research has suggested that glucagon-like peptide-1 (GLP-1) analogues; Lixisenatide, Dulaglutide, Liraglutide, and Exenatide (Exendin-4), currently used to treat diabetes, may also be beneficial in the treatment of neurodegenerative conditions such as AD, as they have been shown to exert neuroprotective properties. Research has also suggested that one of these compounds, exendin-4, can enhance the amount of membrane-associated ADAM10. The current study, therefore, aims to determine firstly; whether GLP-1 analogues can protect neuroblastoma, SH-SY5Y cells, against chemical stressors of relevance to AD and, secondly, whether these compounds alter ADAM10 maturation/activity and the proteolysis of the enzyme's substrates, APP, prion protein (PrP) and neuroligin1 (NLGN-1). Cells treated with hydrogen peroxide (as an oxidative stressor), cobalt chloride (as a hypoxic mimic), methylglyoxal (MG) or thapsigargin (TG) (as Endoplasmic Reticulum stressors) were co-treated with or without GLP-1 analogues and cell viability was subsequently monitored along with the expression and proteolysis of ADAM10 and its substrates. The results of the study showed that the GLP-1 analogue, liraglutide, had no major effect in terms of protecting SH-SY5Y cells against any of the afore-mentioned chemical stressors. However, a second compound, exendin-4, was protective against TG-induced cytotoxicity. Thapsigargin impaired the proteolytic maturation of ADAM10 suggesting a decrease in the activity of the enzyme but exendin-4 was unable to reverse this effect. Nonetheless, exendin-4 was able to partly reverse the inhibitory effect of TG on the expression of endogenous APP, and shedding of over-expressed NLGN1, and sAPPα in cells over-expressing APP695. Interestingly, TG caused an increase in expression of APP695, and an intracellular accumulation of PrP and, subsequently, did not alter the cell surface ADAM-mediated shedding of this protein. Collectively, these data indicate that liraglutide was ineffective in the protection of SH-SY5Y cells against the chemical stressors employed. However, exendin-4, is mildly protective against thapsigargin-mediated oxidative stress and is able to partly restore the TG-induced decrease in expression of endogenous APP, and shedding of APP695 and NLGN1. Unusually though, this latter effect was not associated with restored proteolytic maturation of ADAM10.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009.
Committee Chair: Collard, David M.; Committee Co-Chair: Garcia, Andres J.; Committee Member: France, Stefan; Committee Member: Ragauskas, Arthur; Committee Member: Temenoff, Johnna. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Two highly homologous synthetic peptides MLC(3-13) (K-R-A-K-A-K-T-TK-K-R-G) and MLC(5-13) (A-K-A-K-T-T-K-K-R-G) corresponding to the amino terminal amino acid sequence of smooth muscle myosin light chain were utilized as substrates for protein kinase C purified from murine lymphosarcoma tumors to determine the role of the primary amino acid sequence of protein kinase C substrates in defining the lipid (phosphatidyl serine and diacylglycerol) requirements for the activation of the enzyme. Removal of the basic residues lysine and arginine from the amino terminus of MLC(3-13) did not have a significant effect on the Ka value of diacylglycerol. The binding of effector to calcium-protein kinase C appears to be random since binding of one effector did not block the binding of the other.

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Aspártico (E.C. 3.4.23), cisteíno (E.C. 3.4.22) e serino peptidases (E.C. 3.4.21) são endopeptidases, cujos modos de ação são dependentes de resíduos de ácido aspártico, cisteína e serina presentes no sítio catalítico, respectivamente. Atualmente, vários estudos são realizados na busca por novas enzimas com relevantes propriedades bioquímicas para aplicação industrial. Neste contexto, nós propomos a produção de enzimas em bioprocesso submerso, purificação, estudo das propriedades bioquímicas e determinação da especificidade catalítica das peptidases secretadas pelos fungos filamentosos Rhizomucor miehei, Phanerochaete chrysosporium e Leptosphaeria sp. Inicialmente, após produção por bioprocesso submerso, estas enzimas foram purificadas utilizando cromatografias de exclusão molecular e troca iônica. Em ensaios de inibidores na atividade enzimática, notamos inibição das peptidases por pepstatina A (R. miehei), ácido iodoacético/N-Etilmaleimida (P. chrysosporium) e fluoreto de fenil metil sulfonila (Leptosphaeria sp), sendo então definidas como aspártico, cisteíno e serino peptidases, respectivamente. Por SDS-PAGE (12%), as massas moleculares foram estimadas em 37 kDa (aspártico), 23 kDa (cisteíno) e 35 kDa (serino). O máximo de atividade proteolítica foi alcançado em pH 5,5 e 55 ºC para peptidase aspártica secretada por R. miehei; pH 7 e faixa de temperatura 45-55 ºC para cisteíno peptidase secretada por P. chrysosporium, e pH 7 e 45 ºC para serino peptidase secretada por Leptosphaeria sp. Sob efeito de incubação a diferentes pH, a peptidase aspártica mostrou-se estável em condições ácidas (pH 3-5); cisteíno peptidase foi estável em ampla faixa de pH (pH 4-9), e serino peptidase mostrou-se mais estável em condições com tendências alcalinas e pH ligeiramente ácido (pH 5-9). Em todas estas faixas de pH citadas, as peptidases apresentaram atividade proteolítica acima de 80% por 1 hora de incubação. Quanto à estabilidade térmica, a cisteíno peptidase mostrou-se mais termoestável dentre as três enzimas e serino peptidase descreveu a menor tolerância à temperatura. Em incubação com agentes desnaturantes, observamos redução na atividade proteolítica sob efeito de surfactantes iônicos (0,02-1%): dodecil sulfato de sódio (SDS) e brometo de cetil-trimetil amônio (CTAB); íon cobre II (5 mM); Ditiotreitol (DTT) e guanidina (ambos na faixa de 10-200 mM) para todas as peptidases. Por último, em estudo de especificidade catalítica destas enzimas, observamos a preferência por aminoácidos aromáticos (F e W), básicos (K e R) e apolares (em particular, resíduo de metionina) para peptidase aspártica. Alta especificidade descrita por cisteíno peptidase, cuja preferência catalítica é notória por aminoácidos básicos (K, H e R), especialmente na posição P3 e lisina-dependência para catálise na posição P'3. Em serino peptidase, notamos maior aceitação por aminoácidos apolares (G, I, L, M e V), básicos (H e R) e polares neutros (N e Q) para as diferentes posições avaliadas no substrato.
Aspartic (EC 3.4.23), cysteine (EC 3.4.22) and serine peptidases (EC 3.4.21) are endopeptidases whose modes of action are dependent on aspartic acid, cysteine and serine residues present in the catalytic site, respectively. Currently, several studies are conducted in the search for new enzymes with relevant biochemical properties for industrial application. In this context, we propose the production of enzymes in submerged bioprocess, purification, the study of biochemical properties and determining the catalytic specificity peptidases secreted by the filamentous fungus Rhizomucor miehei, Phanerochaete chrysosporium and Leptosphaeria sp. Initially, after production submerged bioprocess, these enzymes have been purified using size-exclusion and ion exchange chromatographies. In the effect of inhibitors on enzyme activity, we note peptidase inhibition by pepstatin A (R. miehei), iodoacetic acid/ N-Ethylmaleimide (P. chrysosporium) and phenyl methyl sulfonyl fluoride (Leptosphaeria sp), suggesting that these enzymes are aspartic, cysteine and serine peptidases, respectively. For SDS-PAGE (12%), molecular weights were estimated at 37 kDa (aspartic), 23 kDa (cysteine) and 35 kDa (serine). Maximum proteolytic activity was achieved at pH 5.5 and 55 °C for aspartic peptidase secreted by R. miehei; pH 7 and temperature range 45-55 °C for cysteine peptidase secreted by P. chrysosporium and pH 7 and 45 °C for serine peptidase secreted by Leptosphaeria sp. Under incubation at different pH effect, aspartic peptidase was stable under acidic conditions (pH 3-5); cysteine peptidase was stable in wide pH range (pH 4-9), and serine peptidase was more stable under alkaline conditions and pH slightly acidic (pH 5-9). In all these pH ranges mentioned, peptidases showed proteolytic activity above 80% by 1 hour incubation. As regards the thermal stability, cysteine peptidase was more thermostable enzyme and serine peptidase described the lowest temperature tolerance. In incubation with denaturing agents, we observed a decrease in proteolytic activity under the effect of ionic surfactant (0.02-1%) sodium dodecyl sulfate (SDS) bromide and cetyl-trimethyl ammonium bromide (CTAB); copper (II) ion (5 mM); Dithiothreitol (DTT) and guanidine (both in the range of 10-200 mM) for all peptidases. Finally, the study of catalytic specificity of these enzymes, we found a preference for aromatic amino acids (F and W), basic (K and R) and nonpolar (in particular, methionine residue) to aspartic peptidase. High specificity described by cysteine peptidase, which a catalytic preference is notorious for basic amino acids (K, R and H), especially in position P3 and lysine-dependence for catalysis at position P'3. In serine peptidase, for different evaluated positions, we noticed greater acceptance by nonpolar amino acids (G, I, L, M and V), basic (M and R) and neutral polar (N and Q).

Il est de plus en plus évident que la matrice extracellulaire (MEC), au-delà de sa fonction d’échafaudage cellulaire, génère des signaux de nature biochimique et biophysique jouant un rôle primordial au cours du processus de différenciation des cellules souches. A l’heure actuelle, plus de 15 différents facteurs extrinsèques (environnementaux), incluant l’organisation spatiale de la MEC, sa topographie, rigidité, porosité, biodégradabilité et chimie ont été identifiés comme modulateurs potentiels de la différenciation des cellules souches en lignées cellulaires spécialisées. Ainsi, il est plausible que l’intégration d’un biomatériau au sein de l’organisme dépendra largement de sa capacité à mimer les propriétés de la MEC du tissu à remplacer. Récemment, les techniques de micro-ingénierie ont émergé comme outil innovant pour découpler les différentes propriétés de la MEC et étudier l’impact individuel ou combiné de ces facteurs sur le comportement des cellules souches. De plus, ces techniques de microfabrication ont un intérêt particulier dans une perspective de reconstruction de la MEC dans tous ses aspects, in vitro. Dans ce projet de thèse, le concept de déconstruction/reconstruction de la complexité de la MEC a été appliqué pour récapituler, in vitro, plusieurs aspects inhérents à la MEC osseuse et explorer leurs effets individuels ou combinés sur la différenciation ostéoblastique des cellules souches mésenchymateuses (CSMs) humaines. Trois principales composantes ont été utilisées tout au long du projet : un matériau modèle (verre borosilicate), des séquences peptidiques mimétiques dérivées de la MEC naturelle, favorisant à la fois l’adhérence cellulaire (peptide RGD) et la différenciation ostéoblastique (peptide BMP-2) des CSMs prélevées de la moelle osseuse des patients. La première étude du projet consiste à greffer, d’une manière aléatoire, les peptides RGD et/ou BMP-2 sur la surface du matériau. Brièvement, nous avons développé trois types de matériaux bioactifs : matériaux fonctionnalisés avec le peptide RGD, matériaux fonctionnalisés avec le peptide BMP-2 et matériaux bi-fonctionnalisés avec les peptides RGD/BMP-2. La caractérisation physicochimique de ces matériaux a été réalisée en utilisant la spectrométrie photoélectrique à rayons X (XPS) pour évaluer la composition chimique de la surface, la microscopie à force atomique (AFM) pour évaluer la topographie de la surface et la microscopie à fluorescence pour confirmer la présence des peptides sur la surface et évaluer leur densité. L’objectif de cette étude est d’évaluer le potentiel individuel et synergétique de ces peptides à induire et contrôler la différentiation ostéoblastique des CSMs. Dans un premier temps, la caractérisation physicochimique nous a permis de confirmer l’immobilisation covalente des peptides sur la surface et de mesurer leur densité. En effet, la densité des peptides, mesurée sur les surfaces greffées uniquement avec le peptide RGD ou BMP-2, était de 1.8 ± 0.2 pmol/mm² et 2.2 ± 0.3 pmol/mm², respectivement. Cependant, sur les surfaces bifonctionnalisées, la densité de chaque peptide a diminué de presque la moitié, atteignant 0.7 ± 0.1 pmol/mm² pour le peptide RGD et 1 ± 0.1 pmol/mm² pour le peptide BMP-2. Ensuite, l’évaluation biologique des différents matériaux fonctionnalisés a clairement révélé que contrairement au peptide RGD, le peptide BMP-2 induit la différenciation ostéoblastique des CSMs. Cependant, le greffage simultané des peptides RGD/BMP-2 améliore significativement la différenciation des CSMs en ostéoblastes et cela malgré la diminution significative de la densité de chaque peptide sur les surfaces bi-fonctionnalisées, comparativement aux surfaces contenant qu’un seul peptide. Ces résultats montrent que les peptides RGD et BMP-2 peuvent engendrer un effet synergétique pour améliorer la différenciation ostéoblastique des CSMs. Le second chapitre de thèse vise à déterminer si la microstructuration de la surface des matériaux avec des ligands bioactifs améliore la différenciation ostéoblastique des CSMs. En effet, les peptides RGD et BMP-2 ont été greffés séparément sur la surface du matériau sous forme de micro-motifs de différentes formes mais de taille similaire. En se basant sur des précédents travaux de littérature – discutés dans le chapitre II – nous avons sélectionné trois différentes formes de motifs peptidiques (triangle, carré et rectangle) dont la surface est de 50 μm². Ces micromotifs ont été créées grâce à une technique assez répondue et facile à utiliser qui est la photolithographie. Les surfaces microstructurées ont été caractérisées avec l’interférométrie optique et la microscopie à fluorescence. Les résultats montrent que les micromotifs peptidiques ont à la fois la forme et les dimensions prédéfinies. In vitro, les résultats de différenciation cellulaire ont révélé que la distribution spatiale des ligands à l’échelle micrométrique joue un rôle très important dans l’engagement et la différenciation des CSMs en ostéoblastes. En effet, contrairement aux micromotifs peptidiques en forme de rectangles, les micromotifs triangulaires et carrés améliorent significativement l’expression des marqueurs ostéogéniques (Runx-2 et Ostéopontine) comparativement à la distribution aléatoire des peptides. Il est important de noter que ce profile d’expression des marqueurs biologiques a été observé que sur les matériaux fonctionnalisés avec le peptide BMP-2, tant dis que les matériaux fonctionnalisés avec le peptide RGD n’ont induit aucun effet spécifique sur la différenciation des CSMs et cela peu importe la forme des micromotifs peptidiques. En conclusion, cette étude a permis d’identifier un nouveau facteur extracellulaire capable de contrôler la différenciation des CSMs. De plus, nous avons démontré que la distribution spatiale des ligands à l’échelle micrométrique affecte le devenir des CSMs, dépendamment de la nature du principe actif. Finalement, la troisième étude de ce projet de thèse est une suite logique de l’étude 1 et 2, puisqu’elle consiste à greffer simultanément les peptides RGD et BMP-2 sous forme de micromotifs. En effet, ces surfaces ont été développées afin de bénéficier à la fois de l’effet synergétique des peptides RGD/BMP-2, observé dans l’étude 1 (facteur 1), et de l’effet de la distribution spatiale contrôlée des ligands, observé dans l’étude 2 (facteur 2). Les différents types de matériaux ont été caractérisés avec les mêmes techniques de caractérisation de surface mentionnées dans l’étude 2. Les résultats montrent clairement que les surfaces microstructurées sont très bien définies et correspondent à un damier de micromotifs RGD, intercalé par un damier de micromotifs BMP-2. L’évaluation de la différenciation des CSMs sur ces matériaux a révélé que la combinaison des facteurs 1 et 2 améliore significativement la différenciation des CSMs vers le lignage ostéoblastique, comparativement à l’exposition des CSMs à un seul facteur extracellulaire (1 ou 2). De plus, cette étude confirme les résultats obtenus dans l’étude 2, puisque les micromotifs triangulaires et carrés ont permis une meilleure différenciation cellulaire, comparativement aux micromotifs rectangulaires. Il est important de noter également que l’évaluation biologique des différentes surfaces biomimétiques a été réalisée dans un milieu de culture basal qui ne contient pas de facteurs ostéogéniques solubles, afin d’étudier d’une manière assez précise et fiable les interactions des CSMs avec les différents microenvironnements in vitro développés dans ce projet. En conclusion générale, les travaux effectués jusqu’à présent ont permis d’identifier deux aspects de la MEC qui influencent considérablement la différenciation ostéoblastique des CSMs. De plus, nous avons démontré que ces deux facteurs peuvent coopérer pour induire une meilleure différenciation cellulaire. Cela révèle clairement l’intérêt des techniques de micro-ingénierie pour une meilleure et plus profonde compréhension des mécanismes d’interactions des cellules souches avec leurs niches, ce qui permettra éventuellement de concevoir des produits d’ingénierie tissulaire sur-mesure. Mots clés : Microstructuration de la surface des matériaux, matrice extracellulaire biomimétique, peptides mimétiques, BMP-2, cellules souches, ostéogenèse.
It is becoming increasingly appreciated that the role of extracellular matrix (ECM) extends beyond acting as scaffolds to providing biochemical and biophysical cues, which are critically important in regulating stem cell self-renewal and differentiation. To date, more than 15 different extrinsic (environmental) factors, including the matrix spatial organization, topography, stiffness, porosity, biodegradability and chemistry have been identified as potent regulators of stem cells specification into lineage-specific progenies. Thus, it is plausible that the behavior of biomaterials inside the human body will depend to a large extent on their ability to mimic ECM properties of the tissue to be replaced. Recently, nano- and microengineering methods have emerged as an innovative tool to dissect the individual role of ECM features and understand how each element regulates stem cell fate. In addition, such tools are believed to be useful in reconstructing complex tissue-like structures resembling the native ECM to better predict and control cellular functions. In the thesis project presented here, the concept of deconstructing and reconstructing the ECM complexity was applied to reproduce several aspects inherent to the bone ECM and harness their individual or combinatorial effect on directing human mesenchymal stem cells (hMSCs) differentiation towards the osteoblastic lineage. Three main components were used throughout this project: a model material (borosilicate glass), ECM derived peptides (adhesive RGD and osteoinductive BMP-2 mimetic peptides) and bone marrow derived hMSCs. All cell differentiation experiments were performed in the absence of soluble osteogenic factors in the medium in order to precisely assess the interplay between hMSCs and the different artificial matrices developed in the current study. First, RGD and/or BMP-2 peptides were covalently immobilized and randomly distributed on glass surfaces. The objective here was to investigate the effect of each peptide as well as their combination on regulating hMSCs osteogenic differentiation. The most important funding was that RGD and BMP-2 peptides can act synergistically to enhance hMSCs osteogenesis. Then, micropatterning technique (photolithography) was introduced to control the spatial distribution of RGD and BMP-2 at the micrometer scale. The peptides were grafted individually onto glass substrates, as specific micropatterns of varied shapes (triangular, square and rectangle geometries) but constant size (50 μm² per pattern). In this second part of the project, the focus was made on investigating the role of ligands presentation in a spatially controlled manner in directing hMSCs differentiation into osteoblasts. Herein, we demonstrated that the effect of microscale geometric cues on stem cell differentiation is peptide dependent. Finally, glass surfaces modified with combined and spatially distributed peptides were used as in vitro cell culture models to evaluate the interplay between RGD/BMP-2 crosstalk and microscale geometric cues in regulating stem cell fate. In this study, we revealed that the combination of several ECM cues (ligand crosstalk and geometric cues), instead of the action of individual cues further enhances hMSCs osteogenesis. Overall, our findings provide new insights into the role of single ECM features as well their cooperation in regulating hMSCs fate. Such studies would allow the reconstruction of stem cell microenvironment in all the aspects ex vivo, which may pave the way towards the development of clinically relevant tissue-engineered constructs. Keywords: Chemical micropatterning, bioactive surfaces, mimetic peptides, BMP-2, mesenchymal stem cells, stem-cell differentiation, stem-cell niche, osteogenesis.

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xiv, 110 p.; also includes graphics. Includes bibliographical references (p. 100-110). Available online via OhioLINK's ETD Center

Dans le but de developper un dosage specifique, sensible et selectif de l'elastase de pseudomonas aeruginosa, nous avons synthetise par voie chimique ou enzymatique des derives peptidiques. Ceux-ci ont ensuite ete evalues comme substrats de l'elastase en utilisant la methode conductimetrique. Nous avons aussi evalue trois inhibiteurs potentiels de l'enzyme. Enfin, nous avons effectue des modifications chimiques de l'enzyme et evalue sa stabilisation vis-a-vis de la temperature

La preparation de peptides fluores incorporant des residus 3-fluorophenylalanine, 3,3-difluorophenylalanine et 3-fluoroalanine dans leur structure est decrite. Ces residus aminoacides fluores ont ete obtenus sous forme d'esters par ouverture d'aziridines et d'azirines au moyen du reactif d'olah (hf/pyridine). La preparation des peptides fluores suit une strategie adaptee a la presence de l'atome de fluor en 3. Le choix du groupement protecteur de l'aminoester s'est porte sur le 9-fluorenylmethyloxycarbonyle (fmoc) qui est stable dans les conditions de deprotection des esters fluores (milieu acide). Les reactions de couplage ont ete effectuees avec le chloroformate d'isobutyle. L'obtention des dipeptides amines libres a partir des derives n-fmoc correspondants a necessite l'utilisation originale de csf/18cr6 dans le dmf. Afin d'evaluer dans une hydrolyse enzymatique, l'influence de l'atome de fluor situe a proximite de la liaison peptidique devant etre hydrolysee, nous avons synthetise deux series de peptides fluores, analogues de ala-ala-phe-pro-val-val, substrat de la protease du vih, et des tripeptides fluores analogues de bz-phe-ala-pro-oh, substrat de l'enzyme de conversion ace. Ces composes ont fait l'objet d'evaluations biologiques dans les deux systemes concernes. Les hexapeptides fluores n'ont montre qu'une activite inhibitrice faible dans les tests d'inhibition de la protease du vih. Les tripeptides fluores, analogues du substrat de l'ace ont montre une activite equivalente au compose non fluore

The antibody directed enzyme pro-drug therapy (ADEPT) is a new anticancer treatment, where pre-clinical experiments concluded that an intermediate step involving the inhibition of carboxypeptidase G2 (CPG2) from the circulatory system prior to pro-drug administration is crucial to prevent systemic toxicity (Bagshawe et al. 1991). The research described in this thesis sought to better understand the mode of binding of these inhibitors to CPG2 using solution state nuclear magnetic resonance (NMR) spectroscopy. A high-yield expression of active and soluble mature CPG2 (in the absence of the leader peptide) in E. coli suitable for NMR studies and co-crystallisation screening is reported. We have used this method to routinely produce milligrams quantities of 1H/13C/15N isotopically-labelled protein suitable for NMR studies. The second aim of this thesis is interactions; interactions between CPG2 and selected inhibitors provided by our industrial partner Mologic Ltd. (Bedford, UK). Different structural parts of the inhibitors were identified by NMR to directly interact with CPG2: the naphthalene and the glutamate groups. Chemical shift perturbations studies show different patterns for CP06 and CP67 inhibitors suggesting that they have different binding mechanisms. Site-directed mutagenesis of residues in P1 pocket of CPG2 reveal no activity against methotrexate (MTX), suggesting that they are key players in substrate recognition, while H285A and E200A mutant proteins display similar activity to wild type CPG2 protein. Although the NMR data described here for CPG2 were incomplete and thus did not yield resonance assignment, we show attempts at a "divide-and-conquer" approach. The CPG2CAT construct shows great promise for downstream NMR studies as it has favourable solution properties and retains key properties of the parent protein, namely enzymatic activity and the ability to self associate.

Les métalloprotéases de la matrice extracellulaire (MMPs) sont une famille d’enzymes responsables de la dégradation de la matrice extracellulaire. Elles jouent un rôle important à tous les stades du développement tumoral, de la prolifération des cellules cancéreuses à l’émission de métastases. Elles sont donc une cible biologique de choix pour contrôler ou imager les processus tumoraux. Dans le travail présenté, nous avons développé de nouveaux substrats fluorescents sélectifs et spécifiques des MMPs, qui ont permis de suivre l’activité protéolytique de ces enzymes. Ces substrats sont stables en présence de plasma. Nous avons entrepris des études de complexation afin d’intégrer ces substrats d’un nouveau genre dans des systèmes supramoléculaire de libération contrôlée de principe actif
Abstract

Les protéine arginine méthyltransférases (PRMT) sont impliquées dans de nombreux processus cellulaires, incluant la régulation de l’expression des gènes, le contrôle de l’épissage, le maintien de l’intégrité du génome et la transduction du signal. De nombreuses études montrent que la dérégulation de l’activité des PRMT est associée au développement de pathologies, et en particulier de cancers. Les PRMT constituent ainsi une des nouvelles cibles potentielles en chimiothérapie. Les travaux présentés dans ce manuscrit portent sur trois cibles : PRMT2, PRMT3 et PRMT4/CARM1. Combinant des approches biochimiques, biophysiques et structurales (cristallographie et cryo- microscopie électronique), ces travaux comportent deux aspects : (I) comprendre au niveau atomique la régulation de la réaction de méthylation des protéines (reconnaissance protéines-protéines et interactions entre modifications post-traductionnelles) ; (II) découvrir des inhibiteurs spécifiques et puissants de plusieurs PRMT cibles
Protein arginine methyltransferases (PRMT) are involved in many cellular processes, including gene expression regulation, splicing control, maintenance of genome integrity and signal transduction.Since deregulation of those biological processes appears to be implicated in the pathogenesis of different diseases, PRMTs have emerged as potential new targets for the development of novel therapeutic modulators. Despite the large amount of biological and structural data on PRMTs, two challenges remain to be solved by structural biology ; (I) understanding how PRMTs recognize and bind their full-length substrates ; (II) revealing how PRMTs achieve specific arginine methylation on different target sites. The works presented here focused on 3 targets: PRMT2, PRMT3 and PRMT4/ CARM1. We used biochemical, biophysical and structural methods (bio-crystallography and cryo- electron microscopy) to decipher structural clues that drive PRMT-substrate recognition. We developed new chemical probes that can be used in early drug discovery for the conception of PRMT inhibitors

Proteins and protein interactions are involved in virtually all processes of life. Here we study interactions between globular domains and short linear motifs, which form a small interface ideal for transient interactions. Despite the small number of contacts involved, these domain-motif interactions (DMIs) are known to be highly specific in vivo. We have identified hundreds of instances of DMIs in high-resolution 3-dimensional (3D) structures to analyze the molecular basis of their high specificity. Furthermore, we have derived structural parameters to identify DMIs in 3D structures in a more general, motif-independent way. An important class of DMIs are kinase-substrate interactions. By combining the phosphorylation motif with different kinds of contextual information, we could predict substrates of the human kinase Aurora A. Lastly, we have incorporated DMIs into our database of 3D interacting domains (3did) to disseminate our results to the scientific community for future research.

Los procesos moleculares subyacentes a la mayoría de funciones biológicas implican la participación directa de una infinidad de proteínas y múltiples interacciones entre ellas. En esta tesis estudiamos un tipo particular de estas interacciones, de carácter transitorio y altamente específicas, dónde un dominio globular en una proteína reconoce un corto péptido lineal en otra (DMIs). En concreto, identificamos múltiples casos de DMIs en estructuras tridimensionales (3D) de alta resolución y analizamos las bases moleculares de su especificidad. Además, derivamos parámetros estructurales globales que nos permiten identificar nuevos casos de DMIs. Así mismo, y como caso práctico, combinamos el motivo de fosforilación propio de la quinasa humana Aurora A con diversas clases de información contextual para predecir y validar 90 nuevos substratos. Por último, incorporamos las caracterizadas DMIs en nuestra base de datos de interacciones en 3D (3did) con el fin de diseminar nuestros resultados entre la comunidad científica.

Thesis(Ph.D.)--Case Western Reserve University, 2009
Title from PDF (viewed on 2009-12-30) Department of Chemistry Includes abstract Includes bibliographical references and appendices Available online via the OhioLINK ETD Center

Ces dernières années, les nanotubes de carbone fonctionnalisés sont apparus comme un nouveau matériau prometteur pour de nombreuses applications biomédicales et biotechnologiques. Les études toxicologiques et sur leur biodistribution ont montré des effets toxiques réduits sur les cellules, les tissus et les organes comparés aux nanotubes non fonctionnalisés du fait de leur meilleure solubilité. La fonctionnalisation des nanotubes avec des peptides peut être envisagée pour de nombreuses applications comme le développement des outils de vectorisation de médicaments, comme agent ciblant les cellules tumorales, ou comme nouveaux substrats pour la croissance cellulaire. Durant ma thèse, j’ai développé une stratégie pour la conjugaison de nanotubes de carbone avec des peptides bioactifs comme substrats pour les neurones, ou pour cibler les cellules B autoréactives impliquées dans le lupus érythémateux disséminé. Dans le premier cas, la cytotoxicité et l’impact sur les fonctions des neurones ont été étudiés ainsi que l’immunogénicité des conjugués CNT-peptides. Pour les applications de ciblage cellulaire, nous avons utilisé différentes stratégies pour fonctionnaliser les nanotubes avec les peptides et étudié la reconnaissance spécifique de ces conjugués par des anticorps. Ces recherches représentent une avancée pour l’utilisation de nanotubes fonctionnalisés comme substrats cellulaires ou comme nouveau système de vectorisation de médicaments
In the recent years functionalised carbon nanotubes (f-CNTs) have emerged as new and promising materials for biomedical and biotechnological applications. The toxicological and biodistribution studies have shown functionalised nanotubes have reduced toxic effects on cell, tissues and organs compared to pristine, non functionalized CNTs because of their higher solubility. Functionalisation of carbon nanotubes with peptides can be envisaged for various applications like development of drug delivery tools, tumour cell targeting carriers, or cell growth substrates. In this thesis, I have developed a strategy for the conjugation of the carbon nanotubes with bioactive peptides as neuronal substrates and for the targeting of abnormal cells B involved in systemic lupus erythematosus. In the first case, the cytotoxicity and the impact on neuron functions were studied as well as the immunogenicity of the conjugate CNT-peptides. For the applications in cell targeting, we have used different kind of strategies to functionalise CNTs with peptide, and we have studied the specific binding of those conjugates to antibodies. This work show great promise for the use of functionalised CNTs as substrates for growth cells, and as new drug vectors

M42 aminopeptidases are dinuclear enzymes widely found in prokaryotes but completely absent from eukaryotes. They have been proposed to hydrolyze peptides downstream the proteasome or other related proteolytic complexes. Their description relies mainly on the pioneering work on four M42 aminopeptidases from Pyrococcus horikoshii. Their quaternary structure consists of twelve subunits adopting a tetrahedral-shaped structure. Such a spatial organization allows the compartmentalization of the active sites which are only accessible to unfolded peptides. The dodecamer assembly results from the self-association of dimers under the control of the metal ion cofactors. Both oligomers have been shown to co-exist in vivo and heterododecamers with broadened substrate specificity may even occur. Yet, the molecular determinants behind the dodecamer assembly remain unknown due the lack of a high-resolution structure of a stable dimer. In addition, the bacterial M42 aminopeptidases are still ill-described due to the paucity of structural studies. This work focuses mainly on the characterization of TmPep1050, an M42 aminopeptidase from Thermotoga maritima. As expected, TmPep1050 adopts the genuine tetrahedral-shaped structure with twelve subunits. It also displays a leucyl-aminopeptidase activity requiring Co2+ as a cofactor. In addition to its catalytic function, Co2+ has a role in the enzyme thermostability and oligomerization. The absence of Co2+ provokes the disassembly of active TmPep1050 dodecamers into inactive dimers. The process, however, is reversible since Co2+ triggers the self-association of dimers into dodecamers, as shown by native MS. The main achievement of this work is the determination of the first high-resolution structure of a dimer, allowing to better understand the dimer-dodecamer transition. Several structural motifs involved in oligomerization are displaced or highly flexible in the TmPep1050 dimer structure. Furthermore, a loop bringing two catalytic relevant residues is displaced outside the catalytic site. These residues are the catalytic base and a ligand involved in the Co2+ binding at the M1 site. The metal ion binding sites have been further investigated to define how they influence the oligomerization of TmPep1050. A mutational study shows that the M1 site strictly controls the dodecamer formation while the M2 site contributes only partly to it. A strictly conserved aspartate residue of the M2 site second shell also plays an important structural role in maintaining the active site integrity. Indeed, its substitution prevents the formation of dodecamer probably due to the lack of stabilization of the active site loop. The characterization of TmPep1050 supports that bacterial M42 aminopeptidases probably share the quaternary structures and dodecamer assembly with their archaeal counterparts. The dimer structure highlights several structural modifications occurring in the dimer-dodecamer transition. Yet, based on current knowledge, no general rules can be drawn for the role of the M1 and M2 sites in oligomerization. Besides, the physiological function of the M42 aminopeptidases is under-examined albeit the proposed link to the proteasome. In this work, this has been investigated using the Escherichia coli M42 aminopeptidases as a model. Yet, no phenotype has been associated to the deletion of their coding genes. Preliminary results have shown that the three enzymes (i) display a redundant substrate specificity, (ii) could be localized partly to the membrane, and (iii) form heterocomplexes. Further experiments are still required to crack the function of these M42 aminopeptidases.
Option Biologie moléculaire du Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Many intricate and highly conserved mechanisms have evolved to safeguard organisms against errors in gene expression. The nonsense-mediated mRNA decay pathway (NMD) exemplifies one such mechanism, specifically by eliminating mRNAs containing premature translation termination codons within their protein coding regions, thereby limiting the synthesis of potentially deleterious truncated polypeptides. Studies in Saccharomyces Cerevisiae have found that the activity of at least three trans-acting factors, known as UPF1, UPF2/NMD2, and UPF3is necessary for the proper function of the NMD pathway. Further research conducted in yeast indicates that the degradation of substrates of the NMD pathway is dependent on their translation, and that the sub-cellular site of their degradation in the cytoplasm. Although most evidence in yeast suggests that substrates of the NMD pathway are degraded in the cytoplasm while in association with the translation apparatus, some mammalian studies have found several mRNAs whose decay appears to occur within the nucleus or before their transport to the cytoplasm has been completed. In addition, study of the mammalian TPI mRNA found that this transcript was unavailable as a substrate for the NMD pathway once it had been successfully exported to the cytoplasm, further supporting the notion that the degradation of mammalian substrates of the NMD pathway occurs in association with the nucleus, or during export from the nucleus to the cytoplasm. To determine if yeast cytoplasmic nonsense-containing mRNA can become immune to the NMD pathway we examined the decay kinetics of two NMDS substrate mRNAs in response to repressing or activating the NMD pathway. Both the ade2-1 and pgk1-UAG-2nonsense-containing mRNAs were stabilized by repressing this pathway, while activation of NMD resulted in the rapid and immediate degradation of each transcripts. These findings demonstrate that nonsense-containing mRNAs residing in the nucleus are potentially susceptible to NMD at each round of translation. The remainder of this thesis utilizes protein overexpression studies to gain understanding into the function of factors related to the processes of nonsense-mediated mRNA decay and translation in Saccharomyces cerevisiae. Overexpression of a C-terminal truncated form of Nmd3p was found to be dominant-negative for cell viability, translation and the normal course of rRNA biogenesis. Overexpression studies conducted with mutant forms of the nonsense-mediated mRNA decay protein Upf1p, found that overexpression of mutants in the ATP binding and ATP hydrolysis region ofUpflp were dominant-negative for growth in an otherwise wild-type yeast strain. Furthermore, overexpression of the ATP hydrolysis mutant of Upf1p (DE572AA), resulted in the partial inhibition of NMD and a general perturbation of the translation apparatus. These results support previous studies suggesting a general role for Upf1p function in translation.

Pour exercer ses effets biologiques, l'insuline se lie sur un récepteur transmembranaire appartenant à la famille des récepteurs à activité tyrosine kinase (RTKs). Ces récepteurs permettent la transduction du signal au sein de la cellule en initiant une cascade d'interactions protéine-protéine, qui conduit à la réponse de la cellule cible. Ces dix dernières années de nombreux travaux ont permis d'identifier une nouvelle catégorie de protéines impliquées dans la signalisation : les adaptateurs moléculaires. Ces effecteurs sont caractérisés par une succession de domaines d'interaction protéine­protéine ou protéine-lipide, et sont dépourvus d'activité catalytique. Les protéines de la famille Grb7 constituent une nouvelle famille d'adaptateurs impliqués dans la signalisation. Cette famille comprend trois membres, les protéines Grb7, GrblO et Grb14, qui se caractérisent par une succession de domaines protéiques : une région N-terminale présentant un motif riche en pralines (PP), une région centrale homologue à la protéine MiglO de C. Elegans comprenant un domaine "Ras-Associated-like", appelée GM pour «Grb and Mig»; un domaine PH (Plextrin Homology), un domaine PIR (Phosphotyrosine Interacting Region) ainsi qu'un domaine SH2 (Src Homology 2) C-terminal. Au cours d'un crible double-hybride réalisé au laboratoire;la protéine Grb7 avait été identifiée comme partenaire du récepteur de l'insuline (IR). Des expériences d'interaction in vitro et de coimmunoprécipitation in vivo dans le foie de rat nous ont permis de confirmer cette interaction. Au cours de cette étude, nous avons montré que Grb7 semble interagir préférentiellement avec le IR, comparé à d'autres RTKs. Grb7 n'est pas un substrat du IR. De façon similaire à Grb14 et GrblO, la protéine Grb7 se lie au domaine catalytique du IR activé, par le biais de ses deux domaines PIR et SH2. Ces domaines sont également impliqués dans l'interaction des protéines Grbs avec d'autres RTKs mais leur importance relative dépend du récepteur et de la protéine Grb considérée. Les domaines PIR et SH2 semblent donc jouer un rôle important dans la spécificité de liaison des protéines Grb et leur implication dans la signalisation des récepteurs des facteurs de croissance. Dans la seconde partie de ma thèse, nous nous sommes intéressés au mécanisme d'action des protéines de la famille Grb7 dans la signalisation de l'insuline. Ce travail nous a permis de mettre en évidence que Grbl4 inhibe in vitro l'activité tyrosine kinase du IR. Cependant, la présence de Grbl4 n'altère pas l'autophosphorylation du récepteur et ne bloque pas l'accès des sites de liaison de l'ATP et des substrats. Des expériences similaires réalisées avec les autres membres de la famille ou le récepteur de l'IGF-1 sont en faveur d'une inhibition spécifique de l'activité catalytique du IR exercée par Grbl4. En effet, Grb7 n'inhibe que faiblement l'activité tyrosine kinase du IR. Le domaine PIR, qui est le domaine majeur impliqué dans la liaison de Grb14 au IR, est responsable de cet effet inhibiteur. Dans des cellules CHO-IR/Grb14, Grb14 se lie très rapidement au IR, en réponse à l'insuline. Cette association maintient le récepteur dans un état phosphorylé, Grb14 le protégeant de l'action des tyrosine phosphatases. Cependant, la présence de Grb14 sur le IR induit un décalage dans l'activation des protéines Akt et ERKl/2, ainsi que la phosphorylation des ERKs. L'ensemble de ces données montre que Grbl4 est un inhibiteur spécifique direct de l'activité catalytique des IR et peut être considéré comme un régulateur négatif de la signalisation de l'insuline.

La peptidyl-ARNt hydrolase est une enzyme qui hydrolyse les peptidyl-ARNt issus d'une terminaison prématurée de la traduction. Cette protéine est essentielle à la viabilité des bactéries, mais pas à celle des eucaryotes, ce qui fait d'elle une cible potentielle pour l'action d'anti-bactériens. Cela justifie également qu'on cherche à cartographier l'interaction de cette protéine avec son substrat, pour faciliter la conception d'inhibiteur. Les tentatives d'obtention de cristaux de complexes enzyme:analogue de substrat étant restées vaines, nous avons choisi d'étudier de tels complexes en solution, par RMN. Grâce à un double marquage 15N/13C, nous avons tout d'abord attribué les fréquences de résonance des atomes du squelette de la protéine et d'une grande partie des chaînes latérale. Nous avons ensuite étudié l'interaction entre la PTH d'E. coli et un analogue de son substrat synthétisé chimiquement, la diacétyl-Lys-(3'NH)-adénosine. Cette étude nous a permis de caractériser le rôle de nombreux résidus du site actif, notamment celui d'une phénylalanine (F66) interagissant via son cycle aromatique avec l'adénine 3'-terminale du substrat, celui d'une asparagine (N114) stabilisant une molécule d'eau responsable de l'hydrolyse du substrat et celui d'une autre asparagine (N10) permettant à la PTH de discriminer positivement les peptidyl-ARNt par rapport aux aminoacyl-ARNt. Nous avons aussi étudié l'interaction entre la protéine et des mini-ARNt mimant la tige acceptrice et le bras TΨC d'un ARNt. Ce travail a permis de cartographier la surface de la PTH où l'ARN s'ancre à la protéine. Il a confirmé l'importance de deux résidus basiques, la lysine K105 et l'arginine R133, pour la reconnaissance du phosphate en 5' de l'ARNt. Il a également révélé une interaction entre l'hélice C-terminale de la protéine et le bras TΨC de l'ARNt, à 30 Å du site actif. La pertinence fonctionnelle de ce dernier contact a pu être établie par mutagenèse dirigée. L'ensemble de ces résultats permet de proposer un modèle complet de l'interaction entre la PTH et un peptidyl-ARNt.

The insulin-like growth factor-1 receptor (IGF-1R) and many of its downstream signaling components have long been implicated in tumor progression and resistance to therapy. The insulin receptor substrate-1 (IRS-1) and IRS-2 adaptor proteins are two of the major downstream signaling intermediates of the IGF-1R. Despite their considerable homology, previous work in our lab and others has shown that IRS-1 and IRS-2 play divergent roles in breast cancer cells. Signaling through IRS-1 promotes cell proliferation, whereas signaling through IRS-2 promotes cell motility and invasion, as well as glycolysis. Moreover, using a mouse model of mammary tumorigenesis, our lab demonstrated that IRS-2 acts as a positive regulator of metastasis, while IRS-1 cannot compensate for this function. The focus of my thesis research is to understand how IRS-2, but not IRS-1, promotes breast carcinoma cell invasion and metabolism to support metastasis. In preliminary studies, I have found that IRS-1 and IRS-2 exhibit different expression patterns in both cell lines and human tumors with correlations to patient survival, which provides a potential mechanism for their distinct functions. The localization of IRS-1 and IRS-2 within separate intracellular compartments would determine their access to downstream effectors and substrates, and this would result in unique cellular outcomes. Specifically, I have observed that IRS-2, but not IRS-1, co-localizes with microtubules in breast carcinoma cell lines with implications for signaling through AKT and mTORC2. The goal of this research is to determine how the localization of IRS-2 contributes to its regulation of breast cancer progression and response to therapy and how this information could be used to better predict patient outcomes.

Thesis (Ph.D. in Cell and Developmental Biology) -- University of Colorado at Denver and Health Sciences Center, 2005.
Typescript. Includes bibliographical references (leaves 123-141). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Ce travail concerne l'étude de l'activité de la chymosine sur des substrats immobilisés (cytochrome C, chaine B de l'insuline, caséine K et peptides synthétisés au laboratoire). Une première partie a porté sur la recherche d'une méthode optimale d'immobilisation sur 4 résines commerciales (CNBR-, AH-, CH-, et CH-sépharose-activée). Les résines AH et CH-sépharose activées par l'hexafluorophosphate de N, N benzotriazolyl-tetramethyl uronium (H. B. T. U. ) et par la carbodiimide donnent de bons rendements d'immobilisation du cytochrome C. Un meilleur rendement est obtenu avec la CH-sépharose-activée. En deuxième partie l'étude du modèle cytochrome C/chymosine a permis de mettre au point le réacteur enzymatique. La séparation par C. L. H. P. Des produits de dégradation du cytochrome C immobilisé sur deux résines différentes a mis en évidence l'intérêt de l'utilisation d'une résine à bras espaceur. Une troisième partie est consacrée à l'étude de la spécificité de coupure sur la chaîne B de l'insuline immobilisée. L'analyse chromatographique des produits de la réaction montre une hydrolyse rapide dès les premières minutes de la réaction. Ainsi, 8 sites de coupure ont pu être identifiés après séparation par C. L. H. P. Et identification. Enfin la dernière partie de ce travail a porté sur l'hydrolyse de la caséine K, substrat naturel de l'enzyme. La séparation des produits d'hydrolyse par C. L. H. P. Montre 1 pic non aromatique qui pourrait correspondre au caséino-macropeptide. Contrairement aux autres substrats l'heptapeptide synthétisé s'est montré résistant à l'action de la chymosine dans ces conditions réactionnelles

Abstract Collagen prolyl 4-hydroxylase is the key enzyme in the biosynthesis of collagens, a family of extracellular matrix proteins. Vertebrate collagen prolyl 4-hydroxylases are α2β2 tetramers, the β subunit being identical to the multifunctional protein disulphide isomerase (PDI). Several isoforms of the catalytic α subunit have been identified in various organisms. Prolyl 4-hydroxylases have also been isolated from plants, where they hydroxylate proline-rich structural glycoproteins of the cell walls. The structural and functional properties of the peptide-substrate-binding domain of human collagen prolyl 4-hydroxylase are characterized here. Data obtained from NMR studies indicate that the domain consists of five α helices and one short β strand, this structure being quite different from those of other proline-rich peptide-binding modules. Several residues involved in the binding of a short synthetic peptide were also identified by NMR. Kd values for the binding of several synthetic peptides to the α(I) and α(II) domains were determined by surface plasmon resonance and isothermal calorimetry, and the results indicated that the binding properties of the type I and type II collagen prolyl 4-hydroxylase tetramers can mainly be explained by the binding of peptides to this domain rather than to the catalytic domain. The peptide-substrate-binding domain of human type I collagen prolyl 4-hydroxylase was also crystallized. The crystals were well ordered and diffracted to at least 3 Å, the asymmetric unit most probably containing a domain dimer. The genome of Arabidopsis thaliana was found to encode at least six putative prolyl 4-hydroxylase polypeptides, one of which was cloned and characterized here as a recombinant protein. All the catalytically critical residues identified in animal prolyl 4-hydroxylases were also conserved in this plant prolyl 4-hydroxylase, and their mutagenesis led to inactivation of the enzyme. The recombinant plant enzyme was effective in hydroxylating poly(L-proline) and several synthetic proline-rich peptides. Surprisingly, contrary to previous reports on plant prolyl 4-hydroxylases, the collagen-like peptides were found to be good substrates, the enzyme preferentially hydroxylating prolines in the Y positions of the -X-Y-Gly- triplets, thus resembling the vertebrate collagen prolyl 4-hydroxylases even in this respect. The recombinant plant prolyl 4-hydroxylase also hydroxylated peptides representing the N and C-terminal hydroxylation sites present in the hypoxia-inducible transcription factor α. The fact that these peptides contain only one proline residue indicated that a poly(L-proline) type II conformation was not required for hydroxylation.