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A linhagem queratinolítica Chryseobacterium sp. Kr6 mostrou-se com possibilidade de aplicação em processos envolvendo queratinólise, principalmente na hidrólise de penas de frango e depilação de couro bovino. No presente trabalho avaliou-se o efeito da composição do meio sobre o crescimento e atividade proteolítica deste isolado e uma protease queratinolítica (queratinase) foi purificada e caracterizada. O microrganismo mostrou-se adaptado à utilização de queratina como substrato durante o crescimento, produziu diferentes proteases dependendo do meio utilizado e a maior atividade proteolítica foi atingida quando utilizado meio de cultivo com penas como única fonte de carbono e nitrogênio. A adição de fonte extra de nutrientes resultou em uma parcial repressão catabólica. Uma protease extracelular (Q1) foi purificada cerca de 14 vezes utilizando cromatografia de interação hidrofóbica em Phenyl-Sepharose CL 4B e gel filtração em Superose H12R. Q1 mostrou ser uma proteína monomérica com peso molecular de 64 KDa determinado por SDS-PAGE e pH e temperatura ótimos de 8,5 e 50°C respectivamente. O perfil de inibição indica tratar-se uma metaloprotease e as seqüências internas dos peptídeos resultantes de digestão tríptica mostraram homologia ao sítio ativo e de ligação ao Zn da família M14 (Carboxipeptidase). A atividade proteolítica foi estimulada pela presença de íons Ca2+ e Mg2+ e inibida por Cu2+, Zn2+, Al2+, Hg 2+ e agentes redutores. Q1 apresentou atividade queratinolítica sobre o substrato keratin azure, mas não foi capaz de hidrolisar penas de frango sugerindo a necessidade de outras enzimas durante o processo de degradação de penas. Utilizando os iniciadores degenerados desenhados com base na seqüência dos peptídeos, foi amplificado um fragmento de 470 pb correspondente a uma região do possível gene desta metaloproteína utilizando DNA e cDNA como molde. A seqüência do fragmento pode estar sendo expressa, mas não apresentou similaridade e homologia a proteínas conhecidas e portando, indicativa de uma nova metaloprotease. / The strain Chryseobacterium sp. kr6 shown to be useful for biotechnological purposes such as hydrolysis of poultry feathers and de-hairing of bovine pelts. The effect of media composition on the protease production and growth by this strain was studied and a keratinolytic protease (keratinase) was purified and characterized. The strain was adapted to use keratin as substrate to growth, produced different proteases in different media composition and the higher proteolytic activity was reached when used feather as only source of carbon and nitrogen. The addition of sources of nutrients has resulted in partially repressed catabolism. An extracellular protease Q1) was purified 14-fold by chromatography using the hydrophobic interaction Phenyl-Sepharose CL 4B column and gel filtração in Superose 12HR. SDS-PAGE indicated that the Q1 is a monomeric protein with molecular mass of 64 KDa. and optima pH and temperature were 8,5 e 50°C, respectively. The inhibition profile indicates to be a Zn-metalloprotease and analysis of tryptic peptides sequence revealed sequence homology to the conserved active site and Zn binding site, which may characterize keratinase Q1 as a member of M14 metalloprotease family (Carboxipeptidase). The activity was stimulated by of Ca2+ and Mg2+ and inhibited by Cu2+, Zn2+, Al2+, Hg 2+ and reducing agents. Q1 presented keratinolytic activity under substrate keratin azure, but was unable to hydrolyze poultry feather, suggesting the requirement by other enzymes in the feather hydrolysis mechanism. Degenerate primers amplified a 470 bp, corresponding to a probable gene region of this metalloprotein, with DNA and cDNA. The sequence is being expressed but do not showed similarity and homology to known proteins, thus indicating a new metalloprotease.

A linhagem queratinolítica Chryseobacterium sp. Kr6 mostrou-se com possibilidade de aplicação em processos envolvendo queratinólise, principalmente na hidrólise de penas de frango e depilação de couro bovino. No presente trabalho avaliou-se o efeito da composição do meio sobre o crescimento e atividade proteolítica deste isolado e uma protease queratinolítica (queratinase) foi purificada e caracterizada. O microrganismo mostrou-se adaptado à utilização de queratina como substrato durante o crescimento, produziu diferentes proteases dependendo do meio utilizado e a maior atividade proteolítica foi atingida quando utilizado meio de cultivo com penas como única fonte de carbono e nitrogênio. A adição de fonte extra de nutrientes resultou em uma parcial repressão catabólica. Uma protease extracelular (Q1) foi purificada cerca de 14 vezes utilizando cromatografia de interação hidrofóbica em Phenyl-Sepharose CL 4B e gel filtração em Superose H12R. Q1 mostrou ser uma proteína monomérica com peso molecular de 64 KDa determinado por SDS-PAGE e pH e temperatura ótimos de 8,5 e 50°C respectivamente. O perfil de inibição indica tratar-se uma metaloprotease e as seqüências internas dos peptídeos resultantes de digestão tríptica mostraram homologia ao sítio ativo e de ligação ao Zn da família M14 (Carboxipeptidase). A atividade proteolítica foi estimulada pela presença de íons Ca2+ e Mg2+ e inibida por Cu2+, Zn2+, Al2+, Hg 2+ e agentes redutores. Q1 apresentou atividade queratinolítica sobre o substrato keratin azure, mas não foi capaz de hidrolisar penas de frango sugerindo a necessidade de outras enzimas durante o processo de degradação de penas. Utilizando os iniciadores degenerados desenhados com base na seqüência dos peptídeos, foi amplificado um fragmento de 470 pb correspondente a uma região do possível gene desta metaloproteína utilizando DNA e cDNA como molde. A seqüência do fragmento pode estar sendo expressa, mas não apresentou similaridade e homologia a proteínas conhecidas e portando, indicativa de uma nova metaloprotease. / The strain Chryseobacterium sp. kr6 shown to be useful for biotechnological purposes such as hydrolysis of poultry feathers and de-hairing of bovine pelts. The effect of media composition on the protease production and growth by this strain was studied and a keratinolytic protease (keratinase) was purified and characterized. The strain was adapted to use keratin as substrate to growth, produced different proteases in different media composition and the higher proteolytic activity was reached when used feather as only source of carbon and nitrogen. The addition of sources of nutrients has resulted in partially repressed catabolism. An extracellular protease Q1) was purified 14-fold by chromatography using the hydrophobic interaction Phenyl-Sepharose CL 4B column and gel filtração in Superose 12HR. SDS-PAGE indicated that the Q1 is a monomeric protein with molecular mass of 64 KDa. and optima pH and temperature were 8,5 e 50°C, respectively. The inhibition profile indicates to be a Zn-metalloprotease and analysis of tryptic peptides sequence revealed sequence homology to the conserved active site and Zn binding site, which may characterize keratinase Q1 as a member of M14 metalloprotease family (Carboxipeptidase). The activity was stimulated by of Ca2+ and Mg2+ and inhibited by Cu2+, Zn2+, Al2+, Hg 2+ and reducing agents. Q1 presented keratinolytic activity under substrate keratin azure, but was unable to hydrolyze poultry feather, suggesting the requirement by other enzymes in the feather hydrolysis mechanism. Degenerate primers amplified a 470 bp, corresponding to a probable gene region of this metalloprotein, with DNA and cDNA. The sequence is being expressed but do not showed similarity and homology to known proteins, thus indicating a new metalloprotease.

Les ADAMs forment une sous-famille d’enzymes appelées “métalloprotéases”. Elles sont impliquées dans de nombreux processus aussi bien physiologiques que pathologiques de par leur capacité à cliver un certain nombre de substrats tels que des facteurs de croissance, des cytokines ou des protéines d’adhérence. Malgré de nombreuses études sur l’activité des ADAMs, on ne connaît que très peu d’éléments de leur régulation.Les tétraspanines constituent une super-famille de protéines membranaires ayant en commun une structure particulière. Elles sont impliquées dans un grand nombre de processus biologiques fondamentaux tels que la migration, les interactions intercellulaires, la réponse immunitaire, la fusion des gamètes… Les tétraspanines interagissent non seulement entre elles mais aussi avec un certain nombre de partenaires protéiques à la membrane plasmique, formant ainsi un réseau multi-moléculaire appelé « réseau de tétraspanines » ou « tetraspanin web ». Les travaux précédents de notre laboratoire ont montré que la métalloprotéase ADAM10 est associée au réseau de tétraspanines. Cependant, la tétraspanine en association directe avec ADAM10 permettant à cette dernière d’être incluse dans le réseau n’avait jusqu’ici pas été identifiée.Tout d’abord, afin d’établir un modèle permettant une mesure de la modulation de l’activité d’ADAM10 par les tétraspanines, nous avons démontré que l’engagement des tétraspanines par des anticorps monoclonaux augmente le clivage d’E-cadhérine par ADAM10. De plus, l’activation d’un récepteur muscarinique à l’acétylcholine permet aussi une augmentation du clivage d’E-cadhérine mais de manière ADAM17-dépendante cette fois. La transactivation de l’EGFR n’est pas impliquée dans la régulation muscarinique du clivage de l’E-cadhérine alors que l’activation directe de l’EGFR par un de ses ligands conduit, elle, à une stimulation de ce clivage.Revenant à notre quête initiale des conséquences de l’interaction entre ADAM10 et les tétraspanines, nous avons démontré que la métalloprotéase ADAM10 interagit avec l’ensemble des membres de la sous-famille de tétraspanines à 8 cystéines « TspanC8 » regroupant Tspan5, Tspan17, Tspan14, Tspan15, Tspan10 et Tspan33. Ces interactions ainsi que l’expression relative de chacun des membres des TspanC8s influent sur la sortie d’ADAM10 du réticulum endoplasmique. ADAM10 et son interaction avec les TspanC8s sont conservées dans l’Evolution et jouent un rôle dans la régulation de la voie de signalisation Notch. Lorsque nous avons examiné plus en détail l’interaction entre la tétraspanine Tspan5 et ADAM10, nous avons découvert qu’elle avait un effet négatif sur les expressions membranaires et totales d’ADAM10. De plus, cette interaction semble impliquée dans la prolifération de la lignée cellulaire PC3 dérivée de cancer de la prostate. En effet, la surexpression de Tspan5 cause une croissance diminuée de cette lignée. Cette inhibition semble due à un ou plusieurs facteurs solubles qui pourraient être sécrétés moins efficacement par les cellules surexprimant Tspan5 que par leurs homologues sauvages. Egalement, de manière inattendue, les cellules PC3 surexprimant Tspan5 sont totalement insensibles aux drogues ciblant le récepteur à tyrosine-kinase EGFR alors que la croissance des PC3 sauvages est très réduite après de tels traitements. Ceci impliquant donc que la croissance de ces dernières est au moins partiellement dépendante de la signalisation EGFR. Enfin, nous montrons qu’un autre récepteur à tyrosine-kinase appelé EphA2 pourrait avoir un rôle important dans la régulation de la dépendance à la signalisation EGFR des cellules PC3. / ADAMs are a sub-family of enzymes called “metalloproteases” which are implicated in a variety of physiological as well as pathological processes through their ability to cleave a number of substrates including growth factors, cytokines or adhesion proteins. Despite numerous studies on ADAM activity, very little is known about their regulation.Tetraspanins form a super-family of membrane proteins with a common conserved structure. They are implicated in numerous biological processes including migration, intercellular interactions, immune response, gamete fusion… Tetraspanins are known to interact with one another and with a restricted number of protein partners at the cell surface, thus forming a multi-molecular network referred to as « the tetraspanin web ». Previous studies in our laboratory have shown that the metalloprotease ADAM10 is associated to the tetraspanin web. Nevertheless, the tetraspanin in direct interaction with ADAM10 enabling it to be part of the web was not identified at the time. To begin with, in order to establish a model providing a read-out for a modulation of ADAM10 activity by tetraspanins, we demonstrate that tetraspanin engagement by monoclonal antibodies enhances E-cadherin shedding by ADAM10. Furthermore, muscarinic receptor activation also augments E-cadherin shedding but this time in an ADAM17-dependent manner. This occurs without the intervention of EGFR transactivation whereas a direct EGFR activation is able to stimulate E-cadherin shedding. Refocusing on the initial subject of the consequences of an interaction between ADAM10 and the tetraspanins, we conclusively show that the metalloprotease ADAM10 interacts with members of the conserved TspanC8 subfamily consisting of tetraspanins Tspan5, Tspan17, Tspan14, Tspan15, Tspan10 and Tspan33. These interactions and the relative expression of each of the TspanC8 members play a role in ADAM10 trafficking. ADAM10 and TspanC8 interactions are conserved throughout the Evolution and play a role in Notch signaling pathway regulation. When we examined in more details the particular interaction between the tetraspanin Tspan5 and ADAM10, we discovered that it had a negative effect on ADAM10 membrane as well as total expression. Moreover, this interaction seems to have implications on prostate cancer PC3 cell proliferation as Tspan5 overexpression causes a diminished growth rate. This inhibition could be caused by one or more soluble factors which could be less secreted by cells overexpressing Tspan5 than wild-type counterparts. Furthermore, oddly enough, PC3 cells overexpressing Tspan5 were completely unaffected by drugs targeted against the tyrosine-kinase receptor EGFR whereas this type of treatment impaired PC3 WT cell growth which therefore seems at least partly dependent on EGFR signalling. Finally, we reveal that another tyrosine-kinase receptor called EphA2 could play the proeminent role of regulating EGFR signalling-dependence in PC3 cells.

A bothropasina é uma proteína hemorrágica de 48 kDa, pertencente à classe P-III das metaloproteases, isolada a partir do veneno bruto da serpente brasileira Bothrops jararaca, e que possui os domínios adesivos desintegrina (D) e rico em cisteína (C). Neste trabalho, nós apresentamos a estrutura cristalográfica da bothropasina complexada ao inibidor POL647. O domínio catalítico , metaloprotease (M), pode ser dividido em dois subdomínios, dispostos de maneira muito similar aos descritos para essa família de metaloproteases de venenos de serpentes (em inglês \"SVMPs\"), que inclui os sítios de ligação ao zinco e ao cálcio. A cisteína livre, resíduo Cys189, está localizado em um núcleo hidrofóbico e, sendo assim, impossibilitado de fazer pontes dissulfeto ou qualquer outra interação. O domínio D não apresenta estruturas secundárias bem definidas, sendo constituído, majoritariamente, por estruturas desordenadas como \"loops\", porém estabilizados por 7 pontes dissulfeto e por dois íons cálcio. A região do motivo ECD está localizada em um \"loop\" e é estruturalmente relacionado à região RGD das desintegrinas-RGD, derivadas de SVMPs da classe P-II. O motivo ECD é estabilizado pela ponte dissulfeto Cys277-Cys310 (entre os domínios D e C), além de um íon cálcio. A cadeia lateral do Glu276 do motivo ECD está exposta ao solvente. Na bothropasina, a região hiper variada (em inglês HVR), descrita para outras P-III de SVMPs, presente no domínio C, de fato, é bastante conservada quando comparada a outros membros da classe P-III de diversas espécies. Nós propomos que esse subgrupo deva ser referido como PIII-HCR (região altamente conservada) SVMPs. Ainda é proposto que as diferenças estruturais dos domínios D, C ou DC possam estar envolvidas em uma melhor adaptação da estrutura na interação com diferentes alvos, além do reconhecimento e especificidade a um substrato para o domínio M. / Bothropasin is a 48kDa hemorrhagic P-III metalloprotease isolated from the venom of the Brazilian snake Bothrops jararaca, which has the disintegrin (D) and cysteine-rich (C) adhesive domains. We present the crystal structure of the bothropasin complexed with the inhibitor POL647. The catalytic domain, metalloprotease (M), consists of two subdomains in a very similar scaffold to the ones described for other snake venom metalloproteases (SVMPs) including the zinc and calcium binding sites. The free cysteine, residue Cys189, is in a hydrophobic core and it is not available for disulfide bonding or other interactions. The D domain does not have a defined secondary structure, but instead is composed by mostly loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and it is structurally related to the RGD region of RGD-disintegrins, which are derived from P-II SVMPs. The ECD motif is stabilized by the Cys277-Cys310 disulfide bond (between D and C domains) and by one calcium ion. The side chain of the Glu276 of the ECD motif is solvent exposed. In bothropasi, the HVR (hyper-variable region) described for other P-III SVMPs in the C domain in fact presents a well conserved sequence with respect to several other P-III members from different species. We propose that this subset be referred to as PIII-HCR (highly-conserved region) SVMPs. We further propose that the structural differences in the D, C or DC domains may be involved in selecting target binding which in turn could generate substrate diversity or specificity for the M domain.

With over a billion people infected world wide, hookworms are considered as important human pathogens, particularly in developing countries which have the highest rates of infections. Hookworms reside in the gastrointestinal tract of the host where they continuously feed on blood, leading to conditions such as chronic irondeficiency anaemia. The majority of blood-feeding parasites rely on proteins found in blood to provide many of their nutritional requirements for growth, reproduction and survival. Of the numerous proteins found in blood, haemoglobin (Hb) is one of the most abundant. In order to acquire amino acids for protein synthesis, it is thought that haematophagous parasites degrade Hb using various classes of endo- and exoproteases, in a manner similar to that which occurs in catabolism of proteins in mammalian cellular lysosomes. This study identified and characterised proteases involved in the Hb degradation process in the human hookworm, Necator americanus, in order to identify potential candidate antigens for a vaccine that interrupts blood-feeding. Red blood cells ingested by hookworms are lysed to release Hb, which is cleaved by various proteases into dipeptides or free amino acids and these are taken up through the gut membrane by amino acid transporters. Proteases expressed in the intestinal tract of hookworms are thought to play a major role in this process and would therefore make good targets for vaccine candidates aimed at interrupting blood-feeding. To identify these proteases, adult hookworms (both N. americanus and Ancylostoma caninum) were sectioned and intestinal tissue was dissected via laser microdissection microscopy. RNA extracted from the dissected tissue was used to generate gut-specific cDNA, which then was used to create plasmid libraries. Each library was subjected to shotgun sequencing, and of the 480 expressed sequence tags (ESTs) sequenced from each species, 268 and 276 contigs were assembled from the N. americanus and A. caninum libraries, respectively. Nine percent of N. americanus and 6.5% of A. caninum contigs were considered novel as no homologues were identified in any published/accessible database. The gene ontology (GO) classification system was used to categorise the contigs to predicted biological functions. Only 17% and 38% of N. americanus and A. caninum contigs, respectively, were assigned GO categories, while the rest were classified as being of unknown function. The most highly represented GO categories were molecular functions such as protein binding and catalytic activity. The most abundant transcripts encoded fatty acid binding proteins, C-type lectins and activation associated secreted proteins, indicative of the diversity of functions that occur in this complex organ. Of particular interest to this study were the contigs that encoded for cysteine and metalloproteases, expanding the list of potential N. americanus haemoglobinases. In the N. americanus cDNA library, four contigs encoding for cathepsin B cysteine proteases were identified. Three contigs from the A. caninum and one contig from the N. americanus cDNA libraries encoded for metalloproteases, including astacin-like and O-sialoglycoprotein endopeptidases, neither of which had previously been reported from adult hookworms. Apart from haemoglobinases, other mRNAs encoding potential vaccine candidate molecules were identified, including anti-clotting factors, defensins and membrane proteins. This study confirmed that the gut of hookworms encodes a diverse range of proteases, some of which are likely to be involved in Hb digestion and have the potential to be hidden (cryptic) vaccine antigens. Four cysteine proteases (Na-CP-2, -3, -4 and -5) were identified from the gut cDNA library of N. americanus. All four proteases belong to the clan CA, family C1, share homology with human cathepsin B and possess a modified occluding loop. Real-time PCR indicated that all transcripts were up-regulated in the adult stage of the hookworm parasite with high levels of mRNA expression detected in gut cDNA. All four proteases were expressed in recombinant form, but only Na-CP-3 was successfully expressed in soluble form in the yeast Pichia pastoris. Proteolytic activity for Na-CP-3 was detected on a gelatin zymogen gel, however no catalytic activity was detected against the class-specific fluorogenic peptides Z-Phe-Arg-AMC and Z-Arg-Arg-AMC. Mass spectrometry analysis of the purified protein suggested that the pro-region had not been processed in trans when the protein was secreted by yeast. Incubation of Na-CP-3 in salt buffers containing dextran sulfate resulted in autoprocessing of the pro-region as detected by Western blot and catalytic activity was detected against Z-Phe-Arg-AMC. Activated Na-CP-3 did not digest intact tetrameric human Hb. The other three cysteine proteases (Na-CP-2, -4, and -5) were expressed in insoluble form in Escherichia coli. Antibodies to all four proteins (Na- CP-2 to 5) immunolocalised to the gut region of the adult worm, supporting mRNA amplification results and strongly indicated that they might play a role in nutrient acquisition. Hb digestion in blood feeding parasites such as schistosomes and Plasmodium spp. occurs via a semi-ordered cascade of proteolysis involving numerous enzymes. In Plasmodium falciparum, at least three distinct mechanistic classes of endopeptidases have been implicated in this process, and at least two classes have been implicated in schistosomes. A similar process is thought to occur in hookworms. An aspartic protease, Na-APR-1, was expressed in P. pastoris and purified protein was shown to cleave the class-specific fluorogenic peptide 7- Methoxycoumarin-4-Acetyl-GKPILFFRLK(DNP)-D-Arg-Amide. Recombinant Na- APR-1 was able to cleave intact human Hb and completely degrade the 16 kDa monomer and 32 kDa dimer within one hour. Recombinant Na-CP-3 was not able to cleave intact Hb, but was able to further digest globin fragments that had previously been digested with Na-APR-1. A clan MA metalloprotease, Na-MEP-1, was identified in gut tissue of N. americanus and was expressed in recombinant form in Hi5 insect cells using the baculovirus expression system. Recombinant Na-MEP-1 displayed proteolytic activity when assessed by gelatin zymography, but was incapable of cleaving intact Hb. However, Na-MEP-1 did cleave globin fragments which had previously been incubated with Na-APR-1 and Na-CP-3. Hb digested with all three proteases was subjected to reverse phase HPLC and peptides were analysed using Liquid Chromatography-Mass Spectrometry (LC-MS). A total of 74 cleavage sites were identified within Hb ƒ¿ and ƒÀ chains. Na-APR-1 was responsible for cleavage of Hb at the hinge region, probably unravelling the molecule so that Na- CP-3 and Na-MEP-1 could gain access to globin peptides. All three proteases were promiscuous in their subsite specificities, but the most common P1-P1Œ residues were hydrophobic and/or bulky in nature, such as Phe, Leu and Ala. Antibodies to all three proteins (Na-APR-1, -CP-3, -MEP-1) immunolocalised to the gut region of the worm, further supporting their roles in Hb degradation. These results suggest that Hb degradation in N. americanus follows a similar pattern to that which has been described in Plasomdium falciparum. Studies conducted in this project have identified a number of potential haemoglobinases and have demonstrated that the gut region of the hookworm contains a multitude of proteases which could be targeted for production of new chemotherapies or as vaccine candidates. Results presented here also suggest that the Hb degradation process occurs in an ordered cascade, similar to those which have been reported in other haematophagous parasites. More importantly, it has been confirmed that Na-APR-1 plays a crucial role in the initiation of the Hb degradation process and therefore targeting this molecule as a vaccine candidate could provide high levels of protection against hookworm infection.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Anna Zolkiewska / Cancer cells exploit key signaling pathways in order to survive, proliferate, and metastasize. Understanding the intricacies of the aberrant signaling in cancer may provide new insight into how to therapeutically target tumor cells. The goal of my research was to explore the role of two modulators of transmembrane signaling, the secretory pathway and cell surface proteolysis, in the aggressiveness of breast cancer cells. To study the role of the secretory pathway, I focused on the family of endoplasmic reticulum (ER) chaperones. I found that several ER chaperones were upregulated in breast cancer cells grown under anchorage-independent conditions as mammospheres versus those grown under adherent conditions. Furthermore, certain members of the protein disulfide isomerase (PDI) family were consistently upregulated in two different cell lines at both the mRNA and protein levels. Knocking down these PDIs decreased the ability of the cells to form mammospheres. I demonstrated that the requirement for PDI chaperones in mammosphere growth is likely due to an increased flux of extracellular matrix (ECM) components through the ER. Next, I examined the role of cell surface proteolysis in modulating the aggressiveness of breast cancer cells. Cell-surface metalloproteases release soluble growth factors from cells and activate the corresponding growth factor receptors. I determined that specific metalloproteases (ADAM9 or ADAM12), modulate the activation of Epidermal Growth Factor Receptor (EGFR). I demonstrated that EGFR activation enhances the CD44⁺/CD24⁻ cell surface marker profile, which is a measure of cancer cell aggressiveness. I found that the MEK/ERK pathway, which is a downstream effector of EGFR activation, modulates the CD44⁺/CD24⁻ phenotype. When DUSP4, a negative regulator of the MEK/ERK pathway, is lost, activation of EGFR by metalloproteases no longer plays a significant role in cancer cell aggressiveness. This indicates that the ligand dependent activation of the EGFR/MEK/ERK pathway is a critical step in DUSP4-positive aggressive breast cancer. Finally, I examined the importance of metalloproteases in the regulation of Programmed-death ligand 1 (PD-L1), a transmembrane protein expressed by some cancer cells that plays a major role in suppressing the immune system. I demonstrated that cell-surface metalloproteases have the ability to cleave PD-L1 and release its receptor-binding domain to the extracellular environment. Collectively, these data indicate that (a) ER chaperones support anchorage-independent cell growth, (b) metalloproteases are important in regulation of an aggressive phenotype through the EGFR/MEK/ERK pathway, and (c) metalloproteases cleave PD-L1, a key component of immunosuppression in cancer.

A bothropasina é uma proteína hemorrágica de 48 kDa, pertencente à classe P-III das metaloproteases, isolada a partir do veneno bruto da serpente brasileira Bothrops jararaca, e que possui os domínios adesivos desintegrina (D) e rico em cisteína (C). Neste trabalho, nós apresentamos a estrutura cristalográfica da bothropasina complexada ao inibidor POL647. O domínio catalítico , metaloprotease (M), pode ser dividido em dois subdomínios, dispostos de maneira muito similar aos descritos para essa família de metaloproteases de venenos de serpentes (em inglês \"SVMPs\"), que inclui os sítios de ligação ao zinco e ao cálcio. A cisteína livre, resíduo Cys189, está localizado em um núcleo hidrofóbico e, sendo assim, impossibilitado de fazer pontes dissulfeto ou qualquer outra interação. O domínio D não apresenta estruturas secundárias bem definidas, sendo constituído, majoritariamente, por estruturas desordenadas como \"loops\", porém estabilizados por 7 pontes dissulfeto e por dois íons cálcio. A região do motivo ECD está localizada em um \"loop\" e é estruturalmente relacionado à região RGD das desintegrinas-RGD, derivadas de SVMPs da classe P-II. O motivo ECD é estabilizado pela ponte dissulfeto Cys277-Cys310 (entre os domínios D e C), além de um íon cálcio. A cadeia lateral do Glu276 do motivo ECD está exposta ao solvente. Na bothropasina, a região hiper variada (em inglês HVR), descrita para outras P-III de SVMPs, presente no domínio C, de fato, é bastante conservada quando comparada a outros membros da classe P-III de diversas espécies. Nós propomos que esse subgrupo deva ser referido como PIII-HCR (região altamente conservada) SVMPs. Ainda é proposto que as diferenças estruturais dos domínios D, C ou DC possam estar envolvidas em uma melhor adaptação da estrutura na interação com diferentes alvos, além do reconhecimento e especificidade a um substrato para o domínio M. / Bothropasin is a 48kDa hemorrhagic P-III metalloprotease isolated from the venom of the Brazilian snake Bothrops jararaca, which has the disintegrin (D) and cysteine-rich (C) adhesive domains. We present the crystal structure of the bothropasin complexed with the inhibitor POL647. The catalytic domain, metalloprotease (M), consists of two subdomains in a very similar scaffold to the ones described for other snake venom metalloproteases (SVMPs) including the zinc and calcium binding sites. The free cysteine, residue Cys189, is in a hydrophobic core and it is not available for disulfide bonding or other interactions. The D domain does not have a defined secondary structure, but instead is composed by mostly loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and it is structurally related to the RGD region of RGD-disintegrins, which are derived from P-II SVMPs. The ECD motif is stabilized by the Cys277-Cys310 disulfide bond (between D and C domains) and by one calcium ion. The side chain of the Glu276 of the ECD motif is solvent exposed. In bothropasi, the HVR (hyper-variable region) described for other P-III SVMPs in the C domain in fact presents a well conserved sequence with respect to several other P-III members from different species. We propose that this subset be referred to as PIII-HCR (highly-conserved region) SVMPs. We further propose that the structural differences in the D, C or DC domains may be involved in selecting target binding which in turn could generate substrate diversity or specificity for the M domain.

Im Menschen regulieren extrazelluläre Metalloproteasen eine Vielzahl von physiologischen Prozessen, wobei deren exakte Funktionen bei der Ausbildung humaner Krankheiten wie beispielsweise Krebs, der Alzheimerschen Krankheit oder Störungen des Herz-Kreislaufsystems vielfach noch unbekannt sind. Insbesondere die Proteinfamilie der Neprilysine wird seit einigen Jahren vermehrt in Bezug auf eine mögliche Anwendung als Therapeutikum gegen die genannten Erkrankungen hin diskutiert. In dieser Arbeit wurde erstmals die M13-Metalloprotease Neprilysin 4 (Nep4) aus dem Modellorganismus Drosophila melanogaster charakterisiert. Zusätzlich wurde zu Beginn dieser Arbeit das ADAM (A Disintegrin And Metalloprotease)-Protein Meltrin analysiert. Innerhalb der Neprilysin-Familie kommt Nep4 eine Sonderrolle zu, da es im Gegensatz zu den meisten Neprilysinen nicht ausschließlich als membrangebundenes Protein, sondern isoformspezifisch auch in löslicher Form exprimiert wird. In diesem Zusammenhang deuten RT-PCRs, in situ Hybridisierungen und Antikörperfärbungen auf ein breites Funktionsspektrum beider Isoformen hin, das in den zahlreichen Geweben, in denen Nep4 exprimiert wird, hauptsächlich der Etablierung und Aufrechterhaltung der Homöostase verschiedener bioaktiver Peptide dienen dürfte. Über den gesamten Lebenszyklus der Fruchtfliege kann Nep4 in Gliazellen des ZNS und in den männlichen Geschlechtszellen nachgewiesen werden, während es im Verlauf der Embryogenese zusätzlich in Herz- und Muskelzellen exprimiert wird. Die regulatorischen Elemente zur Steuerung der neuronalen (ZNS) und mesodermalen (Herz und Muskel) Nep4 Expression konnten in dieser Arbeit identifiziert und für die Erzeugung transgener Fliegenlinien genutzt werden. Mittels semi-quantitativer PCR und durch Untersuchungen von Fliegen, die GFP unter der Kontrolle des mesodermalen Enhancers exprimieren, wurde die endogene Expression von Nep4 im Muskel von Larven des dritten Stadiums nachgewiesen. Da alle nachfolgenden Stadien Reporteraktivität im Herzen und Muskel zeigen, wird die Peptidase vermutlich durchgängig in diesen Geweben benötigt. Die katalytische Aktivität von Nep4 konnte anhand der Peptide Substanz P und Angiotensin I demonstriert werden. Dabei ist die Enzymaktivität, wie für die Neutralen Endopeptidasen (Nep) typisch, von einem neutralen pH-Wert abhängig und wird durch bekannte Inhibitoren der humanen Neprilysine, Nep und Nep2 reduziert. Bei einer künstlich erhöhten Expression von Nep4 in der Muskulatur der Fruchtfliege ist, entgegen der Erwartung, nicht die katalytische Aktivität, sondern ausschließlich die nicht katalytische, intrazelluläre Domäne ursächlich für eine nekrotische Gewebedegeneration. Um eine mögliche Funktion der intrazellulären Domäne des Nep4 Proteins im Muskel genauer zu erforschen, wurden Proteininteraktionsstudien durchgeführt, erste Interaktionspartner identifiziert und deren Interaktion auf Proteinebene nachgewiesen.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.