Serine protease inhibitors (serpins) are a large superfamily of proteins that regulate critical proteolytic pathways by inhibiting serine proteases. Tick-encoded serpins are thought to play a vital role in the feeding process. To determine the relationship of Amblyomma americanum serpin 6 (S6) to tick feeding regulation, this study attempted to define the biological significance of this molecule through transcription and protein expression profiles, biochemical characterization of recombinant s6 (rS6), and the effects of in vivo post-transcriptional gene silencing on blood meal acquisition and fecundity. Transcriptional analysis revealed that S6 mRNA is ubiquitously expressed in unfed and partially fed ticks through the initial 5 days of the feeding period. S6 mRNA abundance in dissected tick organs showed a 3.7, 3.4, and 1.7- fold upregulation from 24 h to 96 h in the salivary gland (SG), midgut (MG) and the carcass (CA) remnant after removal of SG, MG respectively before downregulating at 120 h. Native S6 protein is downregulated in response to tick feeding, with correlation between transcription and protein expression profiles only consistent from the unfed to 48 h. Similarly, S6 protein expression in dissected female tick tissues is reduced as feeding progresses, with S6 being identified in SG, MG, ovary (OV), and CA from 24 h until 72 h. Biochemical characterization of S6 was not achieved, as rS6 did not form an irreversible complex when incubated with chymotrypsin or trypsin. Although complete silencing of S6 and S6/S17 mRNA was achieved, post-transcriptional gene knockdown had no effect on tick feeding efficiency or fecundity. These findings have been discussed in regards to the development of a vaccine against A. americanum and necessary future studies have been suggested for further characterization and assessment of biological significance.

Master of Science / Division of Biology / Kristin Michel / To date malaria is the most important tropical disease, which is caused by Plasmodium sp. and vectored by anopheline mosquitoes. The mosquito’s immune system is one of the limiting factors of malaria transmission. Immune reactions, such as the prophenoloxidase (PPO) pathway result in the melanization of pathogens, and are effective at limiting parasite numbers. Novel strategies for malaria control aim to exploit the immune system to interrupt parasite transmission by boosting the immune responses in the mosquito vector. Serpins play a crucial role in regulating protease cascades involved in immunity of arthropods. In Anopheles gambiae, the major malaria vector in Sub-Saharan Africa, 18 SRPN genes encoding 23 distinct proteins have been identified. So far, two are identified as active inhibitors, and both affect parasite survival. This research aims to identify additional inhibitory serpins in An. gambiae and elucidate their potential function. Identification of such serpins will enhance our understanding of the immune system of this important vector species and may identify immunoregulators to be used in malaria control. SRPN7, 9, and 18 were tested for their ability to inhibit commercial proteases in vitro. Recombinant SRPN18 had no inhibitory activity, while SRPN7 and 9 inhibited several serine proteases. SRPN7, 9 and 18 were tested against two recombinant An. gambiae clip serine proteases (CLIPBs) that are required for activation of phenoloxidase and thus regulate melanization. Only SRPN9 strongly inhibited CLIPB9 in vitro, suggesting that this serpin is a potential negative regulator of melanization. This hypothesis is further supported by the finding that SRPN9 can inhibit PO activity in insect hemolymph, ex vivo. Taken together, this research identifies SRPN18 as the first non-inhibitory serpin described in mosquitoes. Additionally, this study describes the larval-specific SRPN7 as a functional inhibitor. Future studies on these proteins will elucidate their precise physiological functions. Finally, this thesis provides strong evidence that SRPN9 is a negative regulator of melanization in An. gambiae and may therefore affect pathogen survival within this important vector species.

Chronic kidney disease (CKD), defined as a decrease in renal function, is a global issue. The treatment of CKD and its comorbidities imparts a costly burden on the American healthcare system, therefore the need for therapeutics that prevent the progression of chronic kidney disease is urgent. Microarray studies have shown that the serine protease inhibitor clade A member 3 (SERPINA3) is transcriptionally upregulated in kidney injury. SERPINA3 is an extracellular protease inhibitor that maintains the homeostasis of extracellular matrix proteins. Our lab hypothesizes that SERPINA3 might not only be a transcriptional biomarker for kidney injury, but the SERPINA3 protein might act as a key upstream regulator in the advancement of renal inflammation and fibrosis. Our research characterizes the expression patterns of SERPINA3 in models of acute and chronic kidney injury through immunoblotting and immunohistochemistry. Our unilateral ureteral obstruction (UUO) model of chronic renal injury displays significant glomerular localization of SERPINA3. The adenine diet model of chronic kidney injury and the renal ischemic reperfusion injury (RIRI) model of acute kidney injury both display tubular upregulation of SERPINA3. The DOCA-salt hypertension model of chronic kidney injury was imposed on two strains of mice, C57BL/6 and 129/sv, both of which display tubular and glomerular upregulation of SERPINA3. However, the C57BL/6 strain, which is known for its resistance to glomerular sclerosis, displays higher renal localization of SERPINA3 when exposed to DOCA-salt hypertension, than does the 129/sv strain. In conclusion, our data suggests that SERPINA3 protein is upregulated in both acute and chronic kidney injury. The role of SERPINA3 in these models remains unknown, however, our lab theorizes that SERPINA3 protein may be renoprotective in certain instances of kidney injury. Functional assays must be performed to elucidate the role of SERPINA3 in these models of kidney injury. Characterizing the function of SERPINA3 in chronic and acute kidney injury might aid in the development of novel therapeutics to prevent the advancement of CKD.

Orientador: Prof. Dr. Sergio Daishi Sasaki / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017. / O Feijão Caupi (Vigna unguiculata (L.) Walp) é uma leguminosa com importante representatividade econômica e nutricional, especialmente no Brasil. Inibidores de serino proteases, como a tripsina, já foram descritos na espécie, assim como em outras plantas. No entanto, nesta espécie, ainda não foram identificados inibidores que apresentem atividade sobre a elastase de neutrófilos humana (HNE), protease envolvida em muitos processos patológicos, como na instalação e progressão da doença pulmonar obstrutiva crônica (DPOC). Nesse estudo, purificamos um inibidor a partir do extrato protéico de Vigna unguiculata que apresenta atividade sobre HNE. Inicialmente, foi realizado o processo de extração alcalina de proteínas, seguido de três passos cromatográficos distintos, utilizando as colunas Hitrap-Q (Troca-iônica), Source15RPC (Fase-Reversa) e ACE18 (Fase-Reversa). Essas etapas foram acompanhadas por testes de atividade inibitória, utilizando os substratos fluorogênicos Meo-Suc-Ala-Ala-Pro-Val-MCA (Elastase) e Z-Phe-Arg-MCA (Tripsina), além de ensaios da quantificação de concentração total de proteínas. Para determinar a massa do inibidor, foram utilizadas as técnicas de espectrometria de massa por MALDI-TOF e SDS-PAGE, o inibidor apresenta massa molecular de 10,99 KDa. O Ki para HNE foi determinado no valor de 9 pM. O inibidor não apresentou atividade inibitória sobre tripsina e trombina, porém foi observada atividade sobre subtilisina e quimotripsina. Estes dados indicam que o inibidor purificado trata-se de uma molécula ainda não caracterizada, devido às suas atividades inibitórias o nomeamos de Vigna unguiculata Elastase Inhibitor (VuEI). / The cowpea (Vigna unguiculata (L.) Walp) is a legume of important economic and nutritional representativeness, especially in Brazil. Serine protease inhibitors, such as trypsin, have been described in many species, as well as in other plants. In this specie an inhibitor with activity on human neutrophil elastase (HNE) has not yet been identified. This protease is involved in many pathological processes, such as the onset and progression of chronic obstructive pulmonary disease (COPD). We purified and characterized an inhibitor from the protein extract of Vigna unguiculata presenting activity towards HNE. Firstly, we performed the alkaline extraction procedure for proteins followed by three different chromatographic steps using Hitrap Q (ion exchange), Source15RPC (Reversed-Phase) and ACE18 (Reversed Phase) columns. These steps were followed by the inhibitory activity tests using fluorogenic substrates, MeO-Suc-Ala-Ala-Pro-Val-MCA (elastase) and Z-Phe-Arg-MCA (trypsin), and quantitation assays of protein concentration. To determinate the size of the molecule, we used MALDI-TOF mass spectrometry and SDS-PAGE. The molecular mass of the inhibitor was 10,99 kDa. The dissociation constant (Ki) toward HNE was 9 pM. HNE inhibitor showed no inhibitory activities toward trypsin and thrombin. However, the inhibitor presented activity toward subtilisin and chymotrypsin. These datas indicate that this molecule is a novel inhibitor to HNE and we named it Vigna unguiculata Elastase Inhibitor (VuEI).

Ixodes ricinus est l’espèce de tique la plus abondante et ayant la plus vaste répartition géographique en Europe. Elle est le vecteur de nombreux agents pathogènes d’importance en santé publique et vétérinaire. Le remplacement des acaricides générant pollution environnementale et apparition croissante de résistances requiert le développement urgent de nouvelles stratégies de lutte efficaces contre les tiques et les agents pathogènes qu’elles transmettent. La découverte de telles stratégies passe nécessairement par une meilleure connaissance des interactions entre les tiques, leurs hôtes et les agents pathogènes transmis. La salive de tique, à l’interface de ces interactions, est un fluide essentiel pour ces arthropodes et possède notamment des propriétés protéolytiques, anticoagulantes, immunomodulatrices, analgésique, et anti-inflammatoires qui permettent à la tique de réaliser ses repas sanguins extrêmement longs. Afin de comprendre les mécanismes moléculaires impliqués dans la transmission des agents pathogènes et pour identifier de possibles candidats vaccinaux contre I. ricinus, une étude transcriptomique comparative entre des glandes salivaires infectées et non infectées par la bactérie Bartonella henselae a été antérieurement réalisée. Le transcrit le plus surexprimé suite à cette infection était IrSPI, un inhibiteur de sérine protéase de la famille des Kunitz. Les analyses fonctionnelles par ARN interférence ont montré l’implication de ce gène dans le gorgement et de l’infection des glandes salivaires par B. henselae. Ainsi, les travaux de thèse présentés ici ont concerné l’analyse structurelle, biochimique et fonctionnelle de IrSPI en tant que molécule impliquée dans les interactions tick-hôte-pathogène. Le premier objectif était de définir la structure et la séquence du gène IrSPI mais, malheureusement, bien que nos résultats aient permit des avancés sur cette question, nous n'avons pu obtenir la totalité de sa séquence. Dans un second temps, la dynamique d’expression d’IrSPI a été évaluée au cours du gorgement et de l’infection des tiques par différents agents pathogènes, montrant que son expression est induite par le repas sanguin, par des agents transmis par la tique mais pas par Escherichia coli, bactérie non transmise. De plus, nos résultats ont montré l’expression de IrSPI dans plusieurs organes de la tique, suggérant son implication dans diverses fonctions au sein de ce vecteur. Parmi elles, la mise en évidence d'une injection, par la salive, de la protéine à l'hôte vertébré nous a permis d'envisager un rôle sur les réponses de l'hôte à la piqûre de tique. Nos résultats n’ont montré aucune implication dans la voie extrinsèque de la coagulation ni dans la fibrinolyse, ni dans l’angiogenèse. En revanche, ils ont démontré que IrSPI inhibe la prolifération des lymphocytes TCD4+ sous stimulation monogénique quand chez des lymphocytes B non stimulés IRSPI, il induit une hausse de la prolifération. De plus IrSPI a montré une action négative significative sur la production de la majorité des cytokines et chimiokines pro-inflammatoires produites par les macrophages et les splénocytes. Ainsi, IrSPI, correspond à un des composants salivaires d’I. ricinus lui permettant de moduler la réponse immune de l’hôte pour lui permettre de prélever son repas sanguin tout en favorisant la transmission des agents pathogènes. Enfin, des résultats préliminaires dans l'identification des interactants de IrSPI à la fois chez la tique et l’hôte vertébré ouvre de nombreuses voies de recherche quant à la compréhension de ses fonctions. / Ixodes ricinus tick species, the most abundant and widespread tick in Europe, is an important vector of pathogens affecting both animal and human health. To replace the use of acaricides that generate environmental contamination and resistances, new environmentally sustainable approaches providing broad protection against ticks and tick-borne pathogens (TBP) are urgently needed. Such development requires improved understanding of the biology of ticks and more particularly of their interactions with vertebrate hosts and TBP. Tick saliva is an essential biofluid for ticks, as its proteolytic, anticoagulant, immunomodulatory, analgesic and anti-inflammatory activities allow ticks to acquire their blood meal under optimal conditions. Moreover, injection of saliva during blood feeding represents the principal route by which TBP are transmitted to the host. To understand the molecular mechanisms involved in TBP transmission, as well as to identify putative vaccine candidates against I. ricinus, salivary glands from bacteria infected and uninfected ticks were previously compared by high throughput transcriptomics. The most up-regulated transcript following infection was IrSPI, which belongs to the Kunitz/BPTI inhibitor family. Functional analyses via RNAi knockdown experiments revealed that IrSPI enhances both blood feeding and bacterial burden in the salivary glands. This present PhD work concerns then the structural, biochemical and functional characterization of IrSPI as a molecule involved in tick-host-pathogen interactions. Our aim was first to define the structure of IrSPI gene but, unfortunately, while our results have led to progress on this issue, we have not been able to get the full sequence. Then, the dynamic of IrSPI expression was evaluated during both tick feeding and colonization of ticks by pathogens, showing that its expression is induced by blood feeding and TBP but not by Escherichia coli that is not transmitted by I. ricinus. In addition, our results shown the expression of IrSPI in several tick organs, suggesting its implication in several functions in tick physiology. Among them, the discovery of the injection of IrSPI, through the saliva, to the vertebrate host allowed us to consider a role in host responses to tick bite. Evaluation of IrSPI effect on host showed no impact on coagulation through extrinsic pathway, as determined by analysis of thrombin generation time and by fibrinolysis, or in angiogenesis. However, it inhibited the proliferation of mitogen-stimulated CD4+ lymphocytes and increased unstimulated-B cell proliferation. In addition, IrSPI also modulated cytokine production from macrophages and splenocytes, repressing significantly most of proinflammatory cytokines and chemokines. Thus, we demonstrated that IrSPI plays a role in modulating the host immune response during blood feeding. Finally, preliminary results in the identification of the protein’s interactants open many research perspectives for understanding how IrSPI acts in tick physiology and counteracts host responses to tick injury and pathogen transmission.

Proteins that recognise the sugar surface structures on cells have an enormous potential to be used as tools in the characterisation of these structures. A group of proteins, called lectins, have been identified that can bind to carbohydrate complexes on the receptors of cells. The crude extract from Grevillea robusta seeds was found to contain lectin-like proteins that were different from most other lectins, as they would specifically target the receptors of white blood cells and not those found on red blood cells. Therefore, the lectin isolated from G.robusta could be used as a tool to identify the specific surface structures on white blood cells. The lectin was isolated using affinity chromatography where a complex (oligosaccharide) matrix was used. Agglutination, binding and sugar inhibition assays confirmed the isolated protein was a lectin. The lectin was found in low amounts (up to 5% of the total protein content) within the seeds of G.robusta. As a result of this low yield, the identification of the lectin by PAGE was difficult because the levels of protein were beyond the detection limit of the commercial staining reagents. The lectin was called the GR2 protein and was characterised as a monocot mannose binding lectin based on its sugar specificity for only mannose. A serine protease inhibitor was isolated from the seeds of G.robusta using two different chromatography methods, reverse phase HPLC (GR1.HPLC) and gel filtration chromatography (GR1.GF). Ion exchange chromatography was used to initially separate the proteins in the crude extract and the fraction containing the GR1 protein was further purified using reverse phase HPLC (GR1.HPLC). N-terminal sequencing results of the GR1.HPLC protein, showed evidence of proteolytic cleavage during the extraction process, which lead to the second purification method being established. Protease inhibitors were added to the buffers prior to being purified by gel filtration chromatography, which resulted in the GR1 protein being isolated from the crude extract without the presence of the contaminating protein. Mass spectroscopy identified the molecular weight of the GR1 protein to be 6669Da and the full amino acid sequence was derived by cDNA techniques. Sequence alignment studies of the GR1 protein showed significant similarities with the Bowman-Birk inhibitor. The positioning of the cysteine residues were conserved throughout the Bowman-Birk superfamily, however these residues were not conserved within the GR1 protein. Competitive inhibition assays on the GR1 protein revealed the protein could inhibit both trypsin and chymotrypsin at similar levels to that seen for the Bowman-Birk inhibitor. Therefore, the GR1 protein was characterised as a member of the Bowman-Birk superfamily of serine protease inhibitors. The three-dimensional structure of the GR1 protein was determined using two-dimensional NMR spectroscopy. Computer programs such as XEASY, DYANA and SYBYL® were used to tabulate the information taken from the 2D experiments, generate structures and minimise these structures respectively. The solution structure of the GR1 protein was found to contain a region of antiparallel β-sheet structure that corresponded to the trypsin binding site and the remainder of the protein consisted of loops and turns that were held together by disulfide bridges (the chymotrypsin-binding region). Structural similarities between the GR1 protein and the Bowman-Birk inhibitor existed only in the trypsin-binding site of the Bowman-Birk inhibitor. The GR1 protein is the first member of the Proteaceae family to be characterised as a Bowman-Birk inhibitor. This thesis outlines the isolation and biochemical characterisation of the two proteins found within Grevillea robusta and also describes the steps involved and results obtained in determining the three-dimensional structure of the GR1 protein.

科学研究費補助金 研究種目:一般研究(B)(2) 課題番号:07457240 研究代表者:宮田 敏男 研究期間:1995-1996年度

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

Die Erkrankung Huntington s Chorea ist eine autosomal dominant vererbte Erkrankung, die gewöhnlich im mittleren Lebensabschnitt beginnt und unausweichlich zum Tode führt. In unserem Bestreben, Proteine zu identifizieren, welche an Prozessen "Upstream" oder "Downstream" des krankheitsverursachenden Proteins Huntingtin beteiligt sind, wurde das Proteom eines sehr gut etablierten Mausmodells mit Hilfe der Großgel 2D-Elektrophorese untersucht. Es konnte zum ersten Mal auf Proteinebene nachweisen werden, dass die Expression von zwei Serinproteasehemmern, alpha1-Antitrypsin und Contraspin und darüber hinaus eines Chaperons, alphaB-Kristallin, im Verlauf der Erkrankung abnimmt. Reduzierte Expression von alpha1-Antitrypsin und Contraspin konnte in Gehirn, Leber, Herz und Testes nahe dem Endstadium der Erkrankung nachgewiesen werden. Hier ist es wichtig festzustellen, dass die Expressionsabnahme von alpha1-Antitrypsin im Gehirn der Abnahme in der Leber im Herzen und in den Testes vorangeht. Eine verminderte Expression des Chaperons alphaB-Kristallin wurde nur im Gehirn gefunden. Für ein weiteres Protein, das Major Urinary Protein, wurde eine verminderte Expression in der Leber und im Urin von betroffenen Mäusen festgestellt. Damit konnte demonstriert werden, dass die Erkrankung auf Proteinebene auch ein Protein, das im Gehirn von transgenen Mäusen nicht vorkommt, beeinflusst. Bei Untersuchungen am Menschen wurde in drei Gehirnregionen von Postmortem-Gehirnen von Huntington s Chorea Patienten eine veränderte Expression von alpha1-Antitrypsin festgestellt. Wenn gewährleistet werden kann, dass die Konzentration von alpha1-Antitrypsin und alphaB-Kristallin während Huntington s Chorea im Gewebe nicht absinkt, könnte dies vielleicht neuronalen Zelltod verhindern und somit bei der Verzögerung des Krankheitsverlaufs nutzbringend eingesetzt werden. / Huntington disease is an autosomal dominantly inherited disease that usually starts in midlife and inevitably leads to death. In an effort to identify proteins involved in processes upstream or downstream of the disease causing huntingtin, the proteome of a well-established mouse model was studied by large-gel 2D electrophoresis. It could be demonstrated for the first time at the protein level that two serin protease inhibitors, alpha1-antitrypsin and contraspin and the chaperone alphaB-crystallin decrease in expression over the course of disease. Importantly, the alpha1-antitrypsin decrease in the brain precedes that in liver, heart and testes in mice. Reduced expression of alpha1-antitrypsin and contraspin could be detected in the brain, liver heart and testes close to terminal disease. Decreased expression of the chaperone alphaB-crystallin was found exclusively in the brain. Reduced expression of the liver specific major urinary proteins not found in the brain, was seen in affected mice, demonstrating that the disease exerts its influence on a protein not present in the brain of transgenic mice at the protein level. When investigating three human brain regions obtained post-mortem from Huntington s disease patients, alpha1-antitrypsin expression was also altered. Maintaining alpha1-antitrypsin and alphaB-crystallin availability during the course of Huntington s disease might prevent neuronal cell death and therefore could be useful in delaying the disease progression.