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1
Wartchow, Charles Aaron. "Carbohydrate protease conjugates (CPC) : stabilized proteases for peptide synthesis /". The Ohio State University, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487847309050511.
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De, Veer Simon J. "Development of novel protease inhibitors for epidermal kallikrein proteases". Thesis, Queensland University of Technology, 2014. https://eprints.qut.edu.au/114508/1/Simon_de%20Veer_Thesis.pdf.
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Preserving the integrity of the skin's outermost layer (the epidermis) is vital for humans to thrive in hostile surroundings. Covering the entire body, the epidermis forms a thin but impenetrable cellular cordon that repels external assaults and blocks the escape of water and electrolytes from within. This structure exists in a perpetual state of repair and regeneration where the production of new cellular subunits (keratinocytes) at the base of the epidermis is offset by the gradual release of terminally differentiated corneocytes from the surface. It is increasingly clear that proteinases (hereafter termed proteases) are essential for assembling and maintaining the epidermal barrier. More than thirty proteases are expressed by keratinocytes or immune cells that infiltrate the skin, and the activity of each must be maintained within narrow limits and confined to the correct time and place. Accordingly, dysregulated proteolytic activity is a common factor in a multitude of skin disorders that range in severity from relatively mild to life-threatening.
Serine proteases from the kallikrein-related peptidase (KLK) family are widely recognised as key modulators of epidermal barrier function. For several decades, KLK proteases have been regarded as major contributors to the natural shedding of corneocytes from the skin's surface by degrading (corneo)desmosomes in the stratum corneum. Additionally, controlled KLK proteolytic activity influences the step-wise processing of key molecules involved in hydration and acidification (pro-filaggrin), and anti-microbial defence (cathelicidins).
Further, KLK proteases have prominent roles in the response to barrier disruption that involve stimulating inflammation and alerting the immune system. Thus, failure to properly control KLK proteolytic activity represents a significant threat to epidermal barrier function, as seen in a spectrum of skin disorders, including Netherton syndrome and atopic dermatitis.
The focus of this study was to develop novel inhibitors for three KLK proteases with the strongest links to epidermal (patho)physiology (KLK5, KLK7 and KLK14) by engineering the naturally occurring cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1). Initially, each target protease was screened against a dedicated library of individually synthesised peptide substrates (sparse matrix library) to identify sequence combinations that bound to the active site with high affinity. This approach yielded a series of efficient tetrapeptide substrates for each protease that outperformed existing substrates from conventional screening methods, such as positional scanning and phage display. Strikingly, the optimal substrates for KLK5, KLK7 and KLK14 each displayed a unique physicochemical signature, indicating that the active site of each protease was configured to recognise distinct cleavage motifs. This phenomenon was explored in more detail by analysing structures of diverse serine proteases from the S1A (chymo)trypsin fold, which identified several points of high sequence variation in the active site cleft that likely contribute to divergent cleavage site specificities.
Subsequently, favoured cleavage sequences for KLK5, KLK7 and KLK14 were substituted into the contact β-strand of SFTI-1 to generate inhibitor variants with improved affinity and selectivity. Whereas most inhibitor design strategies based on Laskowski inhibitors (including SFTI-1) focus mainly on optimising the interaction between protease and inhibitor, this study paired binding loop modifications with a second step that aimed to refine the intramolecular hydrogen bond network, as shown recently for engineered SFTI variants targeting KLK4. Like the previous study, improving the tendency for an inhibitor to form intramolecular hydrogen bonds generally led to improvements in inhibitory activity.
However, it was also evident that certain hydrogen bonds were detrimental, revealing that the quantity of hydrogen bonds should not be the only criteria during this screening process.
Additionally, the iterative optimisation of inhibitors for KLK5 highlighted the need to consider both sides of the reactive site when engineering Laskowski inhibitors as generating a selective KLK5 variant was dependent on substituting the P2′ residue. Using this design process, it was possible to successfully re-direct the inhibitory activity of SFTI-1 to three separate protease targets that showed varying affinity for the wild-type inhibitor.
To delve further into the Laskowski mechanism, SFTI-1 was used as a model system to explore the molecular basis for Laskowski inhibitor potency and specificity. Here, inhibitor association and dissociation kinetics were characterised for a series of variants with different binding sequences and hydrogen bond tendencies. These analyses revealed that the primary determinant for rapid association was the pre-organised conformation of the inhibitor binding loop rather than its sequence, whereas coordinated inter- and intramolecular interactions promoted efficient religation and slow dissociation. As the conformation of the binding loop is conserved, inhibitor selectivity dictated by the binding sequence was found to arise from modulating the off-rate. Performing additional analyses on eight fold-divergent inhibitor families revealed that these concepts were generally applicable to Laskowski inhibitors, providing broad new insights on protease inhibitor function and design. These findings were subsequently applied to engineer an additional series of potent inhibitors with broad-range activity.
Finally, the substrates and inhibitors developed for KLK5, KLK7 and KLK14 were used in biological assays to investigate the activity and significance of each target protease in healthy and diseased skin. KLK peptide substrates were applied to profile KLK hyperactivity in skin extracts from transgenic KLK5 mice and Spink5-/- mice. Additionally, KLK inhibitors were used to sequentially block different KLKs in gel-based and in situ zymography experiments. Selective and broad-range inhibitors were also evaluated in ex vivo desquamation assays to examine the relative importance of KLK5, KLK7 and KLK14 during corneocyte shedding, which revealed a major role for KLK7. Collectively, these findings shed light on the individual contributions of KLK proteases to maintaining the epidermal barrier and identify a series of therapeutic leads for further development as novel treatments for skin disorders associated with dysregulated KLK proteolytic activity.
3
Josh, R. S. "Tailoring potent plant protease inhibitor against helicoverpa armigera proteases". Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 2014. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/1969.
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Hamill, J. A. "Involvement of proteases and protease inhibitors in potato late blight". Thesis, Queen's University Belfast, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.426731.
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James, Karen Amanda Ellis. "Design, synthesis, and evaluation of novel cysteine protease inhibitors". Diss., Georgia Institute of Technology, 2002. http://hdl.handle.net/1853/30283.
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Lourbakos, Afrodite 1972. "Activation of human protease-activated receptors by proteases from a periodontal pathogen". Monash University, Dept. of Biochemistry and Molecular Biology, 2001. http://arrow.monash.edu.au/hdl/1959.1/8876.
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Deo, Shivdeep. "DETECTION OF SECRETED PROTEASES AND A MEMBRANE PROTEASE IN PATHOGENIC ACANTHAMOEBA CULBERTSONI". VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/256.
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Acanthamoeba culbertsoni (A. culbertsoni) is an amphizoic amoeba that is the causative agent of Granulomatous Amoebic Encephalitis (GAE), an often fatal central nervous system infection that is seen most frequently in severely immunocompromised patients and is characterized by hemorrhagic and necrotic lesions of the brain as well as varying degrees of granuloma formation. A.culbertsoni isolates have also been identified in a few cases of Amoebic Keratitis, a painful, sight-threatening corneal infection that disproportionately affects contact lens users irrespective of immune status. Common features of both infections include amoebic interaction with host extracellular matrix (ECM) components as requisites for both attachment to, and subsequent invasion of, host tissues to facilitate disease establishment. Previous studies have demonstrated that pathogenic species of Acanthamoeba , such as A.culbertsoni, bind to the ECM proteins Laminin-1 and Collagen I to a greater extent than non-pathogenic species. It has also been documented in the literature that secreted Acanthamoeba proteases have the ability to degrade components of the extracellular matrix. The role of amoebic proteases in mediating the attachment and invasion processes is not entirely understood. Initial experiments conducted in the present study revealed secretion of approximately 150 and 55-kDa serine proteases during attachment as well as invasion of the ECM by A. culbertsoni. However, inhibition of these serine proteases using phenylmethylsulfonyl fluoride (PMSF) did not diminish the ability of amoebae to attach or invade. It was demonstrated that secretion of the observed proteases occurred in a constitutive rather than substrate-induced manner and that amoebae secrete these proteases under a number of different conditions. Additionally, a 140-kDa membrane-associated serine protease was identified which may prove to play a role in focal proteolytic degradation. Collectively, our results suggest that attachment to extracellular matrix components is mediated through non-protease-dependent mechanisms. We also suggest that ECM invasion by A.culbertsoni is predominately a mechanical process that may be supplemented or enhanced by focal proteolytic degradation of extracellular matrix components by membrane-associated proteases.
8
Groll, Michael. "Strukturelle und funktionelle Zusammenhänge und Unterschiede archaebakterieller und eukaryontischer 20S-Proteasome". Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2005. http://dx.doi.org/10.18452/13957.
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In eukaryotes protein degradation is performed by the ubiquitin-proteasome system. The 26S proteasome, a 2.5MDa large multimeric molecular machine, consists of more than 30 subunits and represents the core component of this proteolytic pathway. The complex is assembled from a proteolytically active 20S proteasome and two 19S regulator cap complexes. So far crystal structure, topology and enzymatic mechanism have only been elucidated for the 20S proteasome core particle (CP). CPs are assembled from four stacked rings of seven subunits each, following an alpha7beta7beta7alpha7-stochiometry. The strict established order of the proteasomal assembly and maturation is essential to prevent uncontrolled and premature protein degradation in the cell. CPs belong to the class of Ntn-hydrolases. Peptide hydrolysis is performed inside a central cavity at the active sites of the beta-type subunits, with Ogam of the hydroxyl group of the N-terminal threonine acting as the nucleophile. Release of the proteolytically active threonine through N-O-Acetyl rearrangement is the last step of the proteasomal assembly. Compartmentalisation of CPs is an important way to regulate substrate access to the central cavity as well as release of the generated oligopeptides. The activity of eukaryotic CPs are controlled by an unique mechanism: docking of regulatory complexes, like Blm3, PA28 or 19S, causes a conformational change of the N-terminal residues of the latent alpha-subunits, resulting in an activation of the proteolytically active sites. Archaebacterial CPs lack such regulatory gating mechanism. The controlled degradation of proteins by the proteasome dominates a variety of biological essential processes, like metabolic adaptation, apoptosis, inflammation, immune and stress response, as well as cell proliferation and cell differentiation. Selective and specific natural and synthetic inhibitors of CPs might find their practical application in treatment of cancer or inflammatory diseases.
9
Fishovitz, Jennifer. "A Chemical Approach to Distinguish ATP-dependent Proteases". Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1291142553.
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Tiew, Kok-Chuan. "Dengue virus protease inhibitors". Thesis, Wichita State University, 2011. http://hdl.handle.net/10057/6117.
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Dengue virus (DENV) is a major health threat that affects 2.5 billion people, or 40% of the world’s population. However, there are no approved antiviral drugs or vaccines to treat Dengue infection. This thesis describes the design, synthesis and discovery of a new class of inhibitors of DENV NS3 protease. Structure-activity relationship studies have been carried out in order to delineate the structural elements responsible for the activity of this series of compounds. A lead compound suitable for further development has been identified.Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry.
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Mariani, Victoria L. "Understanding HTLV-I protease". Diss., Georgia Institute of Technology, 2003. http://hdl.handle.net/1853/30374.
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Donnelly, P. K. "Protease and human immunity". Thesis, University of Newcastle Upon Tyne, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.371254.
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Ozdemir, Fatma Inci. "Purification And Characterization Of Cytoplasmic And Proteasome Associated Chymotrypsin-like Proteases From Thermoplasma Volcanium". Phd thesis, METU, 2003. http://etd.lib.metu.edu.tr/upload/3/1137629/index.pdf.
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ABSTRACT
PURIFICATION AND CHARACTERIZATION OF CYTOPLASMIC AND PROTEASOME ASSOCIATED CHYMOTRYPSIN-LIKE PROTEASES
FROM THERMOPLASMA VOLCANIUM
Özdemir, F.inci Ph.D., Department of Biology Supervisor: Prof. Dr. Semra Kocabiyik September, 147 pages In this study, two novel cytoplasmic serine proteases were isolated and characterized from thermophilic archaea Thermoplasma volcanium. The first protease was purified by ion exchange and affinity chromatographies and identified as a chymotrypsin-like serine protease mainly based on its substrate profile and inhibition pattern. The presence of protease activity was analyzed by gelatin zymography which was detected as a single band (35 kDa). Optimum temperature was found to be 60oC for azocasein hydrolysis and 50oC for N-Suc-Phe-pNA hydrolysis. Optimum activity was observed in the pH range of 6.0-8.0 with a maximum value at pH 7.0. The Km and Vmax values for the purified protease were calculated to be 2.2 mM and 40 µ
moles of p-nitroanilide released min-1.ml-1, respectively, for N-Suc-Phe-PNA as substrate. Ca2+ and Mg2+ at 4 mM concentrations were the most effective divalent cations in activating the enzyme. In the second stage of this study, 20S proteasome of Tp. volcanium with substantial chymotrypsin-like activity was purified and characterized. This enzyme complex was purified with 19.1 U/mg specific activities from cell free extract by a four-step procedure. SDS-PAGE analysis revealed two strong bands with relative molecular masses of 26 kDa (&
#945
-subunit) and 21.9 kDa (&
#946
-subunit). Tp. volcanium 20S proteasome predominantly catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residue Phe (chymotrypsin-like activity) in short chromogenic peptides. Low-level hydrolyzing activity was also detected carboxyl to basic residue Arg (trypsin-like activity). Chymotrypsin-like activity of Tp. volcanium 20S proteasome was significantly inhibited by chymotrypsin specific serine protease inhibitor chymostatin. When N-CBZ-Arg was used which is a substrate for trypsin, 20S proteasome was strongly inhibited by TLCK. The optimum temperature for Ala-Ala-Phe-pNA hydrolysis by the Tp. volcanium 20S proteasome was 55oC and the optimum pH was 7.5. The chymotryptic activity was significantly enhanced by divalent cations such as Ca+2 and Mg2+ at high concentrations, i.e. 125-250 mM. Keywords:Serine protease, 20S proteasome, archaea, thermophilic protease, Thermoplasma volcanium, chymotrypsin-like serine protease.
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Johansson, Per-Ola. "Design and synthesis of inhibitors that target the serine protease thrombin, the malarial aspartyl proteases plasmepsin I and II, and the hepatitis C virus NS3 serine protease /". Linköping : Linköpings universitet, 2005. http://www.bibl.liu.se/liupubl/disp/disp2005/tek981s.pdf.
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Cameron, Angus. "Proteolytic enzymes from the hepatopancreas of the Kamchatkan King crab". Thesis, London South Bank University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264822.
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Mahajan, N. "Efficacy of diverse Capsicum annuum protease inhibitors against the adaptive plasticity of Helicoverpa armigera proteases". Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 2014. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/1966.
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Dyllick-Brenzinger, Melanie. "The relationship between alpha-synuclein and the proteasome and its role in Parkinson's pathology". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://dx.doi.org/10.18452/16707.
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Morbus Parkinson (PD) ist eine Bewegungsstörung die durch intrazelluläre Einschlüsse, sogenannte Lewy Bodies, charakterisiert ist. Das synaptische Protein alpha-Synuclein (alpha-Syn) und Polyubiquitin sind Hauptkomponenten von Lewy Bodies. Das Ziel dieser Arbeit war es erstens herauszufinden ob alpha-Syn die proteolytische Aktivität des Proteasoms beeinflusst und zweitens die Auswirkungen der Proteasominhibition auf die Löslichkeit und Lokalisation von alpha-Syn zu prüfen. alpha-Syn inhibierte in vitro reversibel die 20S Proteasomenaktivität während in vivo alpha-Syn Überexpression weder in Neuroblastoma- (SH-SY5Y und SK-N-BE), HEK293 Zellen, noch in alpha-Syn transgenen Mäusen die Aktivität beeinflusste. Umgekehrt wurde der Einfluss pharmakologischer Proteasominhibition (mit MG132 und Epoxomicin) auf die Löslichkeit von alpha-Syn in SH-SY5Y Zellen untersucht. Diese Behandlung führte nicht zur Aggregation von alpha-Syn, wie sie in PD beobachtet wird, jedoch zu einer Verschiebung von mehr löslichem zu mehr membrangebundenem alpha-Syn in Zellen mit endogenem (mock) und mit WT alpha-Syn transfizierten Zellen. Diese Behandlung führte auch zur Aussparung von alpha-Syn und Polyubiquitin im Zellkern und zu cytoplasmatischen alpha-Syn-Einschlüssen ohne Polyubiquitin in einem geringen Anteil der Zellen. Die Kombination aus Proteasominhibition und Serum-Depletion, welches in den Zellen zu oxidativem Stress führen kann, verursachte die Bildung von höhermolekularem alpha-Syn im Western Blot, vergleichbar mit jenem, was in Lewy Bodies gefunden wird. Diese Daten zeigen, dass hohe Konzentrationen an alpha-Syn in vitro die Proteasomfunktion beeinflussen kann, dass dies in unseren Modellsystemen aber nicht in vivo geschieht. Außerdem führt Proteasominhibition zu strukturellen Veränderungen von alpha-Syn. Jedoch muss mehr als eine krankhafte Bedingung (z.B. oxidativer Stress und Proteasominhibition) vorhanden sein, um pathologische Veränderungen zu induzieren.Parkinson’s disease (PD) is a movement disorder characterized by the appearance of inclusions known as Lewy bodies. The synaptic protein alpha-synuclein (alpha-syn) and polyubiquitin are major components of Lewy bodies. The objective of this study was, firstly, to evaluate whether alpha-syn inhibits proteolytic function of the proteasome and, secondly, to determine the effects of proteasome inhibition on alpha-syn solubility and localization. alpha-Syn inhibited 20S proteasome activity reversibly in vitro, while alpha-syn overexpression did not affect activity in neuroblastoma (SH-SY5Y and SK-N-BE) or HEK293 cells nor in alpha-syn transgenic mice in vivo. A reciprocal approach was used to analyze the effects of pharmacological proteasome inhibition (with MG132 and epoxomicin) on alpha-syn solubility in SH-SY5Y cells. This treatment did not lead to alpha-syn aggregation, as seen in PD. However, it induced a shift from more soluble alpha-syn toward more membrane bound alpha-syn in endogenous (mock) and WT alpha-syn transfected cells. This treatment also led to the clearing of nuclei of alpha-syn and ubiquitin, as well as to cytoplasmic alpha-syn inclusions devoid of polyubiquitin in a small percentage of the cells. The combination of proteasome inhibition with serum deprivation, which is known to produce oxidative dysfunction, caused the appearance of high molecular weight alpha-syn species, such as those found in Lewy bodies. So far these data suggest that, although not observed in our in vivo models, high concentrations of alpha-syn can interfere with proteasome function under certain conditions, while proteasome inhibition leads to structural changes in alpha-syn that may precede neurodegeneration. However, more than one condition (e.g. oxidative stress and proteasome inhibition) needs to be met to induce pathological changes.
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Omnus, Deike J. "Regulation of a Transcription Factor Activating Protease". Licentiate thesis, Stockholms universitet, Avdelningen för cellbiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-57814.
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Fagundes, José Américo Gonçalves. "Avaliação da expressão do receptor tipo 2 ativado por protease (PAR2) e da atividade proteolítica presente no fluido crevicular de pacientes com periodontite crônica". Universidade de Taubaté, 2010. http://www.bdtd.unitau.br/tedesimplificado/tde_busca/arquivo.php?codArquivo=429.
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O receptor tipo 2 ativado por protease (PAR2) é um receptor pro-inflamatório que pode ser ativado por tripsina, triptase, protease 3 produzida pelo neutrófilo e pela gingipaína (produzida por Porphyromonas gingivalis). Objetivo: o objetivo do presente estudo foi investigar a expressão do PAR2 na periodontite crônica em humanos, e avaliar se esta expressão está relacionada com a presença de atividade proteolítica no fluido crevicular. Metodologia: foram coletadas amostras de fluido crevicular gengival de indivíduos do grupo controle (sítios saudáveis com profundidade de sondagem ≤ 3mm, ausência de sangramento à sondagem; n=40), grupo periodontite crônica com destruição de leve a moderada (n=40) e grupo periodontite crônica com destruição avançada (n=40). A expressão do PAR2 foi determinada por RT-PCR e a atividade proteolítica no fluido crevicular foi analisada utilizando-se o substrato BApNA. Resultados: observou-se um aumento significativo (p<0.001) da expressão do PAR2 no grupo periodontite em relação ao grupo controle, independentemente da severidade da doença. Além disso, no grupo periodontite houve um aumento significativo na atividade proteolítica (p<0.001) presente no fluido crevicular comparado com o grupo controle. Conclusões: concluiu-se que na periodontite crônica, há um aumento da expressão do PAR2 e um aumento da atividade proteolítica do fluido crevicular. Desta forma, sugere-se que o PAR2 pode estar envolvido com a inflamação periodontal em humanos.Proteinase-activated receptor-2 (PAR2) is a pro-inflammatory receptor that can be activated by trypsin, tryptase, neutrophil-serine protease-3 (P3), and gingipain (produced by Porphyromonas gingivalis). Objectives: the objective of the present study was to investigate the expression of PAR2 in chronic periodontitis, and to evaluate whether its expression is related to the presence of proteolytic activity at the crevicular fluid. Methodology: gingival crevicular fluid (CF) samples were collected from subjects from control group (health sites with probing depth ≤ 3mm, absence of bleeding on probing; n=40), chronic periodontitis group with slight to moderate destruction (n=40), and chronic periodontitis group with advanced destruction (n=40). PAR2 expression was determined by reverse transcriptase-PCR, and the proteolytic activity (PA) at the CF was quantified using the specific substrate BApNA. Results: PAR2 expression was significantly higher (p<0.001) in periodontitis compared to healthy individuals, independently of the diseases severity. In addition, in periodontitis group, there was a significantly increased proteolytic activity (p<0.001) compared with controls. Conclusions: we conclude that in chronic periodontitis there is an increased expression of PAR2 and an increased proteolytic activity at the crevicular fluid. Thus, it is suggested that PAR2 might be involved in human periodontal inflammation.
20
Mendieta, Martínez Laura. "Protease inhibitors as therapeutic agents". Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/279388.
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Proteases are involved in a high number of diseases, and thus, are relevant targets. For that reason our main goal was the discovery of protease inhibitors as therapeutic agents.
We focused our study in four proteases: dipeptidyl peptidase IV (diabetes mellitus type 2), prolyl oligopeptidase (cognitive disorders) and cathepsins L and B (cancer).For the discovery of inhibitors, three strategies were selected: medicinal plant screening, high throughput screening and the characterization of a combinatorial chemistry library.
Once accomplished the DPP IV recombinant expression optimization, the protein was studied by means of nuclear magnetic resonance (NMR) in order to obtain information of its dynamism. Since DPP IV is a large protein, a strategy combining selective labeling and the use of TROSY-HSQC experiments was used. From the 14 methionine residues of the protease, 11 of them were detected in the NMR spectra.
Then, a study of the inhibitor effect on the NMR spectra of DPP IV was done. Interestingly, the corresponding spectra of DPP IV / inactivator afforded an extra signal.
We believe that it is a consequence of a small structural change that the protease suffers after inhibition.
Afterwards, we planned to find DPP IV from botanical sources.
First, we selected plants that were already reported to have antidiabetic action.
Common antidiabetic plants were chosen, as well as Brazilian plants and others from the Traditional Chinese Medicine. Besides, a library of Mediterranean plants was also selected.
After, extraction and testing of DPP IV inhibitory activity was done.
From our tailored collection, the plant AP-3 was selected for further analysis. After fractionation and purification, two molecules were found to be DPP IV inhibitors. Kinetic experiments of the best one, AP-3-a, demonstrated that it was inhibiting DPP IV in a parabolic manner.
Then, AP-3-a inhibition of DPP IV was analysed by NMR. The extra signal that was observed with competitive inhibitors was not present.
We hypothesized that the lack of appearance of this signal is a result of the parabolic inhibition of AP-3-a.
Then, we planned the identification of POP inhibitors by HTS.
Our strategy was based in the use of libraries containing non-toxic compounds and lead-like properties. The assay we selected was FP, which allowed the identification of protein binders by competition with a fluorophore-labelled probe.
First, the peptide probes were validated as useful probes for the FP assay. Then, the HTS was carried out. Over the 4,500 tested compounds, 73 hits were found to be POP binders. Later, 37 hits were selected by means of clustering, docking data and FP results in order to be validated as POP inhibitors. The subset of molecules was evaluated by enzymatic assays. Six compounds presenting the highest POP inhibition ration were selected for further study. Finally, two POP inhibitors have been described. HTS-43 is a competitive POP inhibitor and HTS-75 displays a parabolic behaviour.
It was the first time that parabolic inhibition is reported for POP. We believe that the existence of a non-competitive site would help in the understanding of the relationship between POP and mental diseases.
Finally, a novel peptidyl aryl vinyl sulfone library was tested for its inhibitory activity against cathepsins L and B.
Among all the 20 molecules of the library, a potent covalent irreversible cathepsin L inhibitor has been found, PAVS-20. The progress-curve of the pre-incubation time representation allowed the calculation of its inhibition constants.
Furthermore, evaluation of subsite preferences was done by docking analysis. This allowed understanding the experimental differences in inhibition constants obtained for similar compounds.
Since cathepsins L and B are targets for cancer, molecules of the PAVS library are promising candidates for the development of new anticancer drugs.Las proteasas están involucradas en un alto número de enfermedades y por lo tanto, son dianas terapéuticas relevantes. Por este motivo, nuestra principal meta era el descubrimiento de inhibidores de proteasas cómo agentes terapéuticos. Para ello nuestro estudio en cuatro proteasas: la dipeptidil peptidasa IV, la prolil oligopeptidasa y las catepsinas L y B. Para la búsqueda de inhibidores, se seleccionaron tres estrategias: cribado de plantas medicinales, cribado de alto rendimiento y caracterización de una biblioteca proveniente de la química combinatoria. Una vez se llevó a cabo la expresión recombinante de la DPPIV, la proteína se estudió mediante resonancia magnética nuclear (NMR) para obtener información acerca de su dinamismo. Dado que es una proteína grande, se utilizó una estrategia en la que se combinó el marcaje selectivo y el uso de experimentos TROSY-HSQC. Posteriormente, se realizó el estudio de la DPP IV en presencia de sus inhibidores, para observar como estos afectan a la estructura proteica. Después, se realizó la búsqueda de inhibidores de la DPP IV a partir de extractos de plantas medicinales. De nuestra colección, se seleccionó el extracto de la planta AP-3 para un análisis en profundidad. Se detectaron dos inhibidores de la proteasa. El más potente, AP-3-a, se caracterizó cómo un inhibidor parabólico. Después se llevó a cabo el estudio del complejo DPP IV/AP-3-a por NMR. En cuanto a la POP, se realizó la búsqueda de inhibidores mediante cribado de alto rendimiento (HTS). De los 4,500 compuestos testados se obtuvo un total de 73 hits en el ensayo de polarización de la fluorescencia (FP). La validación de estas moléculas mediante docking, clustering y ensayos enzimáticos, permitió identificar seis potentes inhibidores de POP. Uno de ellos, HTS-75, se caracterizó como un inhibidor parabólico. Esta es la primera vez que se describe un inactivador de este tipo para POP. Finalmente, en cuanto a las catepsinas L y B, se cribó una librería de peptidil aril vinil sulfonas. Entre las 20 moléculas testadas se encontró un potente inhibidor irreversible de la catepsina L, el PAVS-20. Además, se realizó un estudio de docking que permitió evaluar las preferencias de los subsitios de las dos proteasas.
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Dou, Dengfeng. "Mammalian and viral protease inhibitors". Diss., Wichita State University, 2010. http://hdl.handle.net/10057/3281.
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Chronic Obstructive Pulmonary Disease (COPD) is currently the fourth leading cause of death in the US. COPD is a multi-factorial disorder characterized by an oxidant/antioxidant imbalance, inflammation, a protease/antiprotease imbalance and apoptosis. This dissertation describes a general strategy for the design, synthesis and biochemical evaluation of dual function inhibitors which could potentially interrupt the above disorder, thereby enhancing the treatment of COPD. An example of this type inhibitor based on the 1,2,5-thiadiazolidin-3-one scaffold has been proven effective against both human neutrophil elastase (HNE) and caspase-1, two key enzymes responsible for elastin degradation and inflammation, respectively. In addition, an X-ray crystal structure and a high resolution mass spectrum of inhibitor bonded HNE have proven the proposed mechanism of HNE inactivation. Furhtermore, simple reversible competitive inhibitors of COPD-related enzymes (HNE and proteinase 3) have also been designed, synthesized and evaluated biochemically.
West Nile virus and Dengue virus are recognized as a major health threat that affects millions of people worldwide. However, there is currently no treatment or vaccine available for the virus infection. This dissertation describes the design, synthesis and biochemical evaluation of reversible competitive inhibitors of both West Nile virus and Dengue virus NS2B-NS3 protease. Combinatorial chemistry and click chemistry methods have been used in the design of the protease inhibitor and the identified hit was optimized using computational programs (AutoDock4 and SYBYL). Several more hits were identified during the optimization and further development could potentially lead to very potent inhibitors of NS2B-NS3 protease with good pharmacokinetics and oral bioavailability.Thesis (Ph.D.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry
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Allendorf, Sarah. "Site-1 protease in chrondrogenesis". Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66959.
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During mammalian skeletal development, endochondral ossification is preceded by the establishment of a cartilage template through a process termed chondrogenesis. Disruptions in chondrogenesis result in chondrodysplasia, a disorder of the appendicular skeleton which is characterized by disproportionately shortened limbs. A severe chondrodysplasia phenotype is observed when site-1 protease (S1P) is disrupted in both the zebrafish and the mouse, indicating that S1P is essential for normal skeletal development. To investigate the role of S1P during chondrogenesis, ATDC5 cells were used as an in vitro model. Inhibition of S1P activity in these cells decreases the expression of collagens type II and X, while type I collagen and aggrecan remain unaffected. The substrates of S1P are membrane-bound proteins which, when cleaved, release transcription factors promoting cholesterol biosynthesis and the unfolded protein response. Real time-PCR analysis determined that the expression of two substrates, ATF6 and CREB3/Luman, were significantly up-regulated during chondrogenesis. However, silencing of CREB3/Luman expression revealed no cartilage-related phenotype.Pendant le dévelopment du squelette des mammifères, l'ossification endochondrale est précédée par l'établissement d'une matrice cartilagineuse par un processus appelé chondrogenesis. Toute altération dans la chondrogenèse produisant une telle matrice résulte en une chondrodysplasie, maladie affectant le squelette appendiculaire et caractérisée par un raccourcissement disproportionnel des membres. Chez le poisson zèbre et la souris, l'ablation du gène encodant S1P (site-1-protease) engendre une chondrodysplasie sévère, démontrant ainsi que S1P est une protéine essentielle au développement normal du squelette. Ainsi, afin d'étudier le rôle de S1P dans l'ossification endochondrale, les cellules ATDC5 on été utilisées comme modèle in vitro de chondrogenèse. L'inhibition de l'activité de S1P dans ces cellules a eu pour effet de diminuer l'expression des collagènes de type II et X, alors que l'expression du collagène de type I et de l'aggrecane est restée inchangée. Les substrats de S1P sont des protéines membranaires qui, une fois clivées, libèrent des facteurs de transcription actifs lesquels, après translocation nucléaire, vont promouvoir la synthèse du cholestérol, ainsi que la voie UPR (unfolded protein response). L'analyse par PCR en temps réel a permis de démontrer que l'expression génique de deux substrats de S1P, ATF6 et CREB3/Luman, est augmentée de façon significative lors de la chondrogenèse. Cependant, les expériences de suppression génique de CREB3/Luman n'ont pas permis de révéler de phénotype spécifique du cartilage.
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Lee, Victor. "Synthesis of HIV protease inhibitors". Thesis, University of Oxford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.291434.
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Fox, M. T. "Characterisation of protease activated receptors". Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599152.
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In order to characterise the thrombin receptor three mouse monoclonal antibodies were isolated by using a recombinant fusion protein incorporating the extracellular domain of the human thrombin receptor as an antigen. An affinity matrix prepared from one monoclonal antibody was used in the successful purification of the native receptor from human platelet membranes. Two of the monoclonal antibodies were capable of blocking thrombin-induced platelet aggregation, and this may lead to their use as possible platelet thrombin receptor antagonists. To characterise further the monoclonals, their epitopes were mapped. In addition the cDNA encoding the antibodies variable regions were cloned and sequenced. By using this procedure, the amino acid sequence of the antibody complementarity determining regions (CDRs) involved in binding to the target epitope were determined. The recently discovered human protease activated-2 receptor (hPAR-2) was studied using rabbit polyclonal antibodies generated with a multiple antigenic peptide comprising a region around the protease cleavage site. Immunocytochemistry and flow cytometry using affinity purified antibodies detected expression of hPAR-2 on human umbilical vein endothelial cells, keratinocytes and granulocytes. The expression of both PAR-2 and thrombin receptor were analysed using Northern analysis in a wide variety of murine haematopoietic cell lines. This analysis revealed the widespread distribution of the thrombin receptor. In contrast expression of PAR-2 was not observed in haematopoietic cells, suggesting that it is a marker of terminal differentiation in granulocytes. To identify potential activators of PAR-2, a variety of serine proteases were tested using a chloromethylketone inhibitor based on the peptide sequence of the cleavage site. These experiments revealed three potential trypsin-like enzymes that may activate PAR-2 in vivo namely, pancreatic trypsin, mast-cell tryptase and acrosin.
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Peng, Kah Whye. "Protease-activatable targeted retroviral vectors". Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624668.
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Gordon, Nathaniel Charles. "Protease engineering for therapeutic applications". Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648185.
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Barattini, Valeria. ""Protease-catalysed dynamic peptide libraries"". Thesis, University of Manchester, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.517733.
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Udukala, Dinusha Nishani. "Protease assays for cancer diagnostics". Diss., Kansas State University, 2014. http://hdl.handle.net/2097/17387.
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Doctor of PhilosophyDepartment of Chemistry
Stefan H. Bossmann
Numerous proteases are known to be necessary for cancer development and progression including Matrix Metalloproteinases (MMPs), Tissue Serine Proteases, and Cathepsins. The goal of this research is to develop a Fe/Fe₃O₄ nanoparticle-based system for clinical diagnostics, which has the potential to measure the activity of cancer-associated proteases in biospecimens. Our nanoparticle-based “light switches” for measuring protease activity, consist of fluorescent cyanine dyes which are directly attached to Fe/Fe₃O₄ nanoparticles and porphyrins that are attached to Fe/Fe₃O₄ nanoparticles via consensus sequences. The consensus (cleavage) sequences can be cleaved in the presence of the correct protease, thus releasing a fluorescent dye from the Fe/Fe₃O₄ nanoparticle resulting in highly sensitive (down to 1 x 10⁻¹⁶ mol L⁻¹ for 12 proteases), selective, and fast nanoplatforms (required time: 60 min.). Upon escape, the emission intensity of the organic dye will significantly increase, which can be detected using fluorescence spectroscopy. In order to demonstrate the potential of this new technology of early recognition of various cancers several analysis types have been used. Blood and urine samples from human cancer patients and healthy volunteers, tissue and blood serum samples from human cancer patients, and canine urine and blood serum samples are some of those types. Blood samples from human cancer patients and healthy volunteers were used to demonstrate the potential of this new technology for the early recognition of breast and lung cancers. We were able to establish several proteases with diagnostic potential for breast cancer and non-small cell lung cancer. It is very likely that different cancers will feature different “protease signatures”, meaning that different proteases will be activated, depending on the origin of cancer. This permits the diagnosis of various solid tumors at different stages. Tissue samples were collected from normal tissues, from the boundary of the tumor and from the tumor of the same person. Performed fluorescence experiments clearly indicate that tissue samples from the tumor show the highest fluorescence indicating the highest concentration of the protease. Results can be used excellently in a diagnostic system for breast cancer. Based on our results measuring protease signatures offers an inexpensive and fast approach towards early cancer diagnostics.
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Faria, Raquel Cristina Laranjeira. "Lichenicidin biosynthesis: LicP protease specificity". Master's thesis, Universidade de Aveiro, 2011. http://hdl.handle.net/10773/7460.
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Mestrado em MicrobiologiaThe lantibiotics are ribosomally synthesized peptides produced by Gram-positive cells that have antibiotic or morphogenic activity. After translation, the lantibiotics undergo chemical changes that result in the formation of unusual amino acids (lanthionine and methylanthionine), as well as the establishment of chemical bonds that confer a polycyclic structure to the peptide chain. After modifications occur, the leader sequence of the prelantibiotic is removed to yield the active lantibiotic, a process that may involve a single proteolitical reaction or two consecutive reactions. The lantibiotics are divided into classes according to their mechanism of biosynthesis and its biological activity. A particular sub-class of such compounds consists of two-component lantibiotics, in which each lantibiotic consists of two peptides, α and β, which act in synergy to confer it full activity. Lichenicidin is a two component lantibiotic produced by a microorganism commonly found in soil, Bacillus licheniformis. The leader sequence of one of the lichenicidin peptides (α-peptide) is totally removed through a single proteolytic reaction while the formation of the second peptide (β-peptide) implies the occurrence of two reactions of proteolysis, presumably made by the successive action of two enzymes, LicT and LicP, encoded in B. licheniformis genome. In this work, we evaluated the specificity of the LicP protease. For this purpose, we produced β-peptide mutants, in which some amino acids were replaced by Ala. The results indicated that LicP functionality is not impaired by single amino acid replacements. It was also shown that the Bliβ maturation process should, indeed, include the proteolysis of the leader sequence before the activity of LicP since an introduced mutation in the LicT cleavage site most probably inhibited the Bliβ full maturation.
Os lantibióticos são péptidos com actividade antibiótica ou morfogénica, sintetizados nos ribossomas de células Grampositivas. Após tradução, os lantibióticos sofrem modificações que resultam na formação de aminoácidos pouco comuns (lantionina e metil-lantionina) e na formação de ligações químicas que lhes conferem uma estrutura policíclica. Após estas modificações químicas, a sequência líder do pré-lantibiótico é removida tornando o péptido activo. Este processo pode envolver uma única reacção de proteólise ou duas reacções sucessivas. Os lantibióticos são divididos em diferentes classes consoante o seu mecanismo de biosíntese e a sua actividade biológica. Uma destas sub-classes inclui os lantibióticos de dois componentes, que são formados por dois péptidos, α e β, que, em conjunto, lhes conferem total actividade. A lichenicidina pertence a esta classe de lantibióticos. É produzida por Bacillus licheniformis, um microrganismo vulgarmente encontrado no solo. A remoção da sequência líder de um dos péptidos deste lantibiótico (péptido α) é removida numa única reacção proteolítica enquanto que, a formação do segundo péptido (o péptido β) implica a ocorrência de duas reacções de proteólise, presumivelmente efectuadas por acção sucessiva de duas enzimas, LicT e LicP, codificadas no genoma de B. licheniformis. Constituiu o objectivo do presente trabalho, avaliar a especificidade da protease LicP. Para tal, foram produzidos mutantes do péptido β nos quais, os aminoácidos da sequência líder foram substituídos por alanina. Os resultados mostraram que a funcionalidade da protease LicP não foi afectada pelas substituições de um único aminoácido. Os resultados mostraram, ainda, que a protease LicT deve actuar antes da protease LicP uma vez que, a introdução de uma mutação no local de corte da enzima LicT impediu a produção do péptido β.
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Webster, Ailsa. "Characterisation of the adenovirus protease". Thesis, University of St Andrews, 1992. http://hdl.handle.net/10023/14300.
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The substrate specificity, classification, expression and control of the adenovirus encoded protease, the activity of which is required for the production of virions, is described. The use of synthetic peptides has shown that the protease cleaves sites of the form (M,L,I)XGG-X or (M,L,I)XGX-G and these consensus sequences have been used to identify potential cleavage sites in all the known substrates of the protease. Putative cleavage sites have also been found in a number of other adenovirus proteins including the major coat proteins, the hexon and the penton, that had not previously been considered as substrates of the protease. The octapeptide, MSGGAFSW, has been used to develop a specific assay for the protease where digestion is monitored by HPLC reverse phase chromatography. Purification of the protease from adenovirus particles and the preparation of antipeptide sera against the L3 23-kDa protein have been used to confirm that the latter is the protease and that it is not proteolytically activated. Inhibitor studies reveal that the enzyme is inhibited by the thiol directed reagents iodoacetate, PCMB and DTDP, and activated by DTT or cysteine suggesting that it is a member of the cysteine class of proteases. Analysis of the sequence of the enzyme, however, shows that it is not a papain-like enzyme and it is suggested that it might be a member of the recently identified subclass of cysteine proteases that are related to trypsin. The 23-kDa protease has been expressed using E.coli and baculovirus expression systems and a purification schedule for the protein was developed using anion exchange and hydrophobic interaction chromatography. The purified enzyme, however, was not able to cleave the peptide substrate MSGGAFSW and the conclusion was drawn that the protein is probably synthesised as an apoenzyme. One of the substrates of the protease, the pre-terminal (pTP) protein was expressed in insect cells using a recombinant baculovirus. Coinfections of cells with recombinant 23-kDa and pTP baculoviruses resulted in efficient processing of the pTP to the intermediate terminal protein (iTP) in situ. The partially purified baculovirus expressed 23-kDa protein was not, however, found to digest the pTP in vitro, supplying further evidence that the adenovirus protease requires a cofactor.
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Song, Jing. "Functional characterization of extracellular protease inhibitors of phytophthora SPP and their targets tomato proteases". Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1194563833.
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Ismail, Ihab. "Function and Regulation of Xylem Cysteine Protease 1 and Xylem Cysteine Protease 2 in Arabidopsis". Diss., Virginia Tech, 2003. http://hdl.handle.net/10919/11243.
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A functional water-conducting system, the tracheary elements of the xylem, is required to sustain plant growth and development. Tracheary element formation is dependent on many biological processes terminated by programmed cell death and cellular autolysis. The final two processes are probably dependent on the activity of hydrolytic enzymes such as XCP1 and XCP2 known to be expressed in tracheary elements during these final two processes. Thus, the transcriptional regulation of XCP1 and the function of XCP2 were investigated. Qualitative and quantitative assessments of GUS activity as directed by various fragments of the XCP1 promoter showed that a 237-bp internal region was able to drive GUS expression in a tracheary element-specific manner in Arabidopsis. A 25-bp deletion at the 3' end of this region abolished GUS expression. The 237-bp region served as bait in a yeast one-hybrid analysis. Screening of yeast colonies retrieved 109 putative positive interactions, which included a potential transcriptional regulator, indole acetic acid-induced protein 8 (IAA8). An auxin responsive element that potentially binds auxin responsive transcription factors was found within the 25-bp deletion. Cis-elements were predicted by Genomatix and Athamap computer programs. The cis-elements form pyrimidine and gibberellic acid responsive elements that can potentially bind Dof and Myb transcription factors, respectively. In an independent effort, attempts to develop a mapping population to isolate upstream regulators of XCP1 expression did not succeed. Functionally, tracheary element-specific expression of XCP2 in Arabidopsis suggested a specialized role for XCP2 in final phases of tracheary element differentiation. The function of XCP2 was assessed using T-DNA insertional mutants, post-transcriptional gene silencing, and through tracheary element-specific expression of the cysteine protease inhibitor, soyacystatin N in Arabidopsis. Our findings revealed that the absence of XCP2 expression due to T-DNA insertional mutagenesis did not affect plant growth and development in the laboratory. Soyacystatin N was an effective in vitro inhibitor of cysteine proteases. Plants expressing 35S-driven cytosolic form of soyacystatin exhibited stunting and reduced apical dominance. Plants expressing pXCP1-driven cytosolic soyacystatin did not differ from wild type plants. Additionally, transgenic plants expressing pXCP1- and 35S-directed XCP2-double-stranded RNA for the silencing of XCP2 showed no unusual phenotypes compared to their wild type counterpartsPh. D.
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Larocque, Angela M. "Interaction between protease inhibitors and proteases of European corn borer larvae Ostrinia nubilalis Hubner (Lepidoptera: Pyralidae)". Thesis, University of Ottawa (Canada), 1989. http://hdl.handle.net/10393/5698.
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Pol, Ewa. "Mechanism of interaction of the mammalian cysteine protease inhibitors, cystatin A and B, with target proteases /". Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 2001. http://epsilon.slu.se/avh/2001/91-576-5927-3.pdf.
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Oetari, A. "Purification and characterisation of an extra cellular protease inhibitor and extracellular proteases of Stroptomyces lividans '66'". Thesis, Swansea University, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.638358.
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A proteinaceous protease inhibitor was identified and characterised from Streptomyces livdans '66' culture supernatants. The protein has a molecular weight of 11-kD. Comparison of the amino acid residues in a conserved region (two β-strands near the N-terminus involved in dimerization) indicated that this protein is related to other previously characterized Streptomyces proteinaceous protease inhibitors. The inhibitor obtained in this study has specificity for chymotrypsin-like serine proteases. A biological function of the inhibitor could be to control the activity of a native serine protease. Observations on the pH stability of the extracellular serine proteases of Streptomycs lividans '66' indicates that there are at least two types, one of which is stable in acidic pH and the other in neutral/alkaline pH. Their activities, however, are negligible in a liquid medium. Studies on the effect of carbon and nitrogen sources in complex and minimal solid media underline the importance of these sources for the production of proteases and protease inhibitor, and for morphological differentiation. Enhanced production of the serine protease and protease inhibitor occurred in the presence of glucose. The present study implies that the protease inhibitor of Streptomycs livdans '66' may regulate proteases during mycelial growth, aerial mycelium formation and sporulation. The neutral/alkaline serine protease may play a role in the proteolysis of substrate mycelia for aerial mycelium formation, while the acidic protease may play a role in the proteolysis of mycelia during sporulation.
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Conseil, Valérie. "Etude des proteolyses en culture et in vitro de la proteine p126 de plasmodium falciparum". Lille 2, 1996. http://www.theses.fr/1996LIL2T007.
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Lewinska, Marzena. "Verlängerung des N-Terminus von Proteinen mithilfe von IgA-Protease eine neue Methode zur posttranslationalen In-vitro-Modifikation von Proteinen /". [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=967122260.
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Dunlevy, Fiona Kathleen Carol. "Protease-antiprotease imbalance in cystic fibrosis". Thesis, Queen's University Belfast, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491992.
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Affects over 8000 patients In the UK. Persistent neutrophilic inflammation is associated with high levels of airway NE. DX-890 is a smallprotein inhibitor ofNE developed by Dyax, USA for use in CF. This project investigated the ex-vivo effects ofDX-890 on human sputum, neutrophils and epithelial cells, to help determine the potential ofDX-890 as a drug for CF. The Ki ofDX-890 was measured to be 11.12 pM. ICso values measured in pure NE and CF sol were similar, demonstrating that DX-890 retained activity in CF sol. Thickened dehydrated CF mucus may prevent access ofDX-890 to NE and reduce efficacy ill vivo. It was hypothesised that, by reducing stickiness of sputum with DNase or surfactant, DX-890 activity would be enhanced. DX-890 inhibited significantly more NE when sputum was pre-treated with surfactant ill vitro. DX-890 reduced release of active NE from fMLP-activated neutrophils and it was found that DX-890 also inhibits NE inside the neutrophil. DX-890 significantly reduced transmigration of neutrophils across a monolayer ofepithelial cells in response to fMLP, implicating NE activity in the process of transmigration. Nasal epithelial cells from CF and non-CF participants were grown in a monolayer and release of the proinflammatory mediator IL-8 was measured. DX-890 prevented NE induced IL-8 release and also IL-8 release induced by CF sol. In conclusion, inhibition ofNE with DX-890 may reduce airway inflammation by minimising production of IL-8 from epithelial cells, and release of active NE from neutrophils. DX-890 efficacy in whole sputum may be enhanced by co-treatment with surfactant. Used in conjunction with current CF therapies such as antibiotics and physiotherapy, DX-890 may help prevent lung damage and prolong life by reducing airway
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Pratt, Stephanie Ann. "Immunoglobulin A1 protease of Streptococcus pneumoniae". Thesis, University of Leicester, 1988. http://hdl.handle.net/2381/35410.
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The aim of this project was to examine the Streptococcal IgA1 proteases, with particular interest on the Streptococcus pneumoniae enzyme. IgA1 protease of S. pneumoniae was identified and characterised. A non-reducing polyacrylamide gel system was employed to screen clinical isolates for IgA1 protease activity. Of 187 isolates tested 18% were found to be IgA1 protease negative, there was no correlation with the site of isolation of the organism and its ability to produce the enzyme. Attempts were made to clone the pneumococcal IgA1 protease gene using the cosmid pEMBLcos4, the plasmid vector pLG339 and the ? replacement vector ?EMB4. Libraries were screened for pneumolysin and IgA1 protease activity. Clones that expressed pneumolysin were identified by overlaying with sheep red blood cells. One haemolytic clone was not inhibited in the presence of cholesterol. Screening for IgA1 protease activity identified clones with IgA1 protease-ike activity but this activity was not stably expressed. Closer analysis of the libraries suggested that pneumococcal DNA was highly unstable when cloned into E. coil plasmid and cosmid vectors.
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Gariani, Talal. "Design of serine protease inhibitor peptides". Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267244.
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Chong, Sannie Siaw Foong. "Anisotropic potential HIV-1 protease inhibitors". Thesis, University of Hull, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327289.
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Hattersley, Neil. "Characterisation of the SUMO protease SenP6". Thesis, University of Dundee, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.521677.
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Eagling, Victoria Anne. "HIV protease inhibitors and drug disposition". Thesis, University of Liverpool, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.484290.
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Orr, D. C. "Studies on the rhinovirus 3C protease". Thesis, University of Hertfordshire, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382223.
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Nager, Andrew R. (Andrew Ross). "Mechanistic studies of a AAA+ protease". Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/79189.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, February 2013.Cataloged from PDF version of thesis. "December 2012."
Includes bibliographical references.
AAA+ proteases are present in all branches of life and responsible for the energy-dependent degradation of most cytosolic proteins. Substrates for AAA+ proteases are unfolded and translocated into a compartmental peptidase. The requirement for protein unfolding raises several questions. How easily are proteins unfolded within the native environment of a cell? Are some proteins more difficult to unfold than others, and, if so, why? How do AAA+ ATPases convert the chemical energy of ATP binding and hydrolysis into mechanical unfolding and translocation? ClpXP is a AAA+ protease that consists of the hexameric ClpX unfoldase and polypeptide translocase and the ClpP compartmental peptidase. ClpX binds a substrate by an unstructured degradation tag and then, by multiple rounds of ATP-binding and hydrolysis, unfolds and translocates the substrate into the proteolytic chamber of ClpP. To study the features that allow a protein to resist unfolding, I investigate the degradation of degron-tagged Green Fluorescent Protein (GFP; Chapter 2). By engineering GFP substrates, I determine the steps of GFP unfolding and how structure local to the degron can hinder ClpX-mediated unfolding. In later chapters, my collaborators and I use ensemble and single-molecule fluorescent assays to study the mechanochemical cycle of ClpX6 . By these assays, we observe that subunits adopt unique classes which differ in structure and nucleotide binding and hydrolysis, subunit classes switch in a thermally-driven probabilistic fashion that is decoupled from the chemical cycle, and ClpX 6 form a staircase architecture similar to AAA+ helicases.
by Andrew R. Nager.
Ph.D.
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Farbman, Mary E. "Mechanistic studies of the ClpAP protease/". Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/46048.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.Vita.
Includes bibliographical references.
CIpA, a member of the Hspl00/Clp subset of the AAA+ superfamily, is an energy-dependent chaperone, disassembling and remodeling its protein substrates. CIpA also serves as the ATPase component of the ClpAP protease, where it is resonsible for binding specific substrates and unfolding and translocating them into its partner ClpP, the proteolytic component of the complex. We have used single molecule and traditional biochemical experiments to probe the mechanism of the unfolding and translocation steps of ClpA's enzymatic activity. We have identified two states of varying substrate affinities and shown that the conformational switch between these states is responsible for protein translocation. We have also investigated ClpA's autounfolding activity and have demonstrated that though CIpA can disaggregate some substrates in vivo, it does not act as an autodisaggregase.
by Mary E. Farbman.
Ph.D.
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Thorne, Christopher Mark Cornelius. "Characterisation of ubiquitin specific protease 33". Thesis, University of Liverpool, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548811.
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FALLER, BERNARD. "Interactions protease-inhibiteur. Influence de l'heparine". Université Louis Pasteur (Strasbourg) (1971-2008), 1991. http://www.theses.fr/1991STR13125.
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Nous avons evalue la capacite de l'eglin c a inhiber l'elastase pancreatique humaine, une enzyme soupconnee d'exacerber une affection grave du pancreas: la pancreatite aigue hemorragique. Nous avons mesure les constantes cinetiques d'association et de dissociation, parametres necessaires pour predire l'efficacite in vivo de cette molecule en vue d'une utilisation a des fins therapeutiques. La presence d'intermediaires reactionnels dans le processus d'inhibition est soupconnee lorsque la constante cinetique d'association est inferieure a la limite imposee par la diffusion. L'etude de l'interaction entre l'eglin c et la chymotrypsine par cinetique rapide montre que les premiers contacts entre ces deux partenaires font intervenir un equilibre de faible affinite bien que le complexe ei final soit tres stable. Nous nous sommes egalement interesses a l'etude de l'influence d'un glycosaminoglycane polysulfate, l'heparine sur l'inhibition de l'elastase leucocytaire par deux inhibiteurs proteiques humains: l'alpha#1-inhibiteur de protease et l'inhibiteur bronchique. Nous avons montre qu'en presence d'heparine la vitesse d'inhibition de l'elastase par l'alpha#1-inhibiteur de protease etait fortement diminuee. L'etude plus detaillee de la modification des parametres cinetiques en presence d'heparine a montre que ce compose agissait en provoquant l'accumulation d'un intermediaire reactionnel de forte affinite, partiellement actif sur le substrat, et s'isomerisant lentement pour donner naissance au complexe irreversible final ei. Par contre, l'effet de l'heparine sur l'inhibiteur du mucus bronchique s'apparente davantage a celui de ce compose sur le systeme thrombine/antithrombine iii puisqu'un effet accelerateur de l'inhibition de l'elastase correle a un changement de conformation de la proteine a ete observe en presence d'heparine. Ces resultats pourraient avoir des repercussions physiopathologiques importantes
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Swedberg, Joakim Erik. "Rational design of serine protease inhibitors". Thesis, Queensland University of Technology, 2011. https://eprints.qut.edu.au/48131/1/Joakim_Swedberg_Thesis.pdf.
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Proteases regulate a spectrum of diverse physiological processes, and dysregulation of proteolytic activity drives a plethora of pathological conditions. Understanding protease function is essential to appreciating many aspects of normal physiology and progression of disease. Consequently, development of potent and specific inhibitors of proteolytic enzymes is vital to provide tools for the dissection of protease function in biological systems and for the treatment of diseases linked to aberrant proteolytic activity. The studies in this thesis describe the rational design of potent inhibitors of three proteases that are implicated in disease development. Additionally, key features of the interaction of proteases and their cognate inhibitors or substrates are analysed and a series of rational inhibitor design principles are expounded and tested. Rational design of protease inhibitors relies on a comprehensive understanding of protease structure and biochemistry. Analysis of known protease cleavage sites in proteins and peptides is a commonly used source of such information. However, model peptide substrate and protein sequences have widely differing levels of backbone constraint and hence can adopt highly divergent structures when binding to a protease’s active site. This may result in identical sequences in peptides and proteins having different conformations and diverse spatial distribution of amino acid functionalities. Regardless of this, protein and peptide cleavage sites are often regarded as being equivalent. One of the key findings in the following studies is a definitive demonstration of the lack of equivalence between these two classes of substrate and invalidation of the common practice of using the sequences of model peptide substrates to predict cleavage of proteins in vivo. Another important feature for protease substrate recognition is subsite cooperativity. This type of cooperativity is commonly referred to as protease or substrate binding subsite cooperativity and is distinct from allosteric cooperativity, where binding of a molecule distant from the protease active site affects the binding affinity of a substrate. Subsite cooperativity may be intramolecular where neighbouring residues in substrates are interacting, affecting the scissile bond’s susceptibility to protease cleavage. Subsite cooperativity can also be intermolecular where a particular residue’s contribution to binding affinity changes depending on the identity of neighbouring amino acids. Although numerous studies have identified subsite cooperativity effects, these findings are frequently ignored in investigations probing subsite selectivity by screening against diverse combinatorial libraries of peptides (positional scanning synthetic combinatorial library; PS-SCL). This strategy for determining cleavage specificity relies on the averaged rates of hydrolysis for an uncharacterised ensemble of peptide sequences, as opposed to the defined rate of hydrolysis of a known specific substrate. Further, since PS-SCL screens probe the preference of the various protease subsites independently, this method is inherently unable to detect subsite cooperativity. However, mean hydrolysis rates from PS-SCL screens are often interpreted as being comparable to those produced by single peptide cleavages. Before this study no large systematic evaluation had been made to determine the level of correlation between protease selectivity as predicted by screening against a library of combinatorial peptides and cleavage of individual peptides. This subject is specifically explored in the studies described here. In order to establish whether PS-SCL screens could accurately determine the substrate preferences of proteases, a systematic comparison of data from PS-SCLs with libraries containing individually synthesised peptides (sparse matrix library; SML) was carried out. These SML libraries were designed to include all possible sequence combinations of the residues that were suggested to be preferred by a protease using the PS-SCL method. SML screening against the three serine proteases kallikrein 4 (KLK4), kallikrein 14 (KLK14) and plasmin revealed highly preferred peptide substrates that could not have been deduced by PS-SCL screening alone. Comparing protease subsite preference profiles from screens of the two types of peptide libraries showed that the most preferred substrates were not detected by PS SCL screening as a consequence of intermolecular cooperativity being negated by the very nature of PS SCL screening. Sequences that are highly favoured as result of intermolecular cooperativity achieve optimal protease subsite occupancy, and thereby interact with very specific determinants of the protease. Identifying these substrate sequences is important since they may be used to produce potent and selective inhibitors of protolytic enzymes. This study found that highly favoured substrate sequences that relied on intermolecular cooperativity allowed for the production of potent inhibitors of KLK4, KLK14 and plasmin. Peptide aldehydes based on preferred plasmin sequences produced high affinity transition state analogue inhibitors for this protease. The most potent of these maintained specificity over plasma kallikrein (known to have a very similar substrate preference to plasmin). Furthermore, the efficiency of this inhibitor in blocking fibrinolysis in vitro was comparable to aprotinin, which previously saw clinical use to reduce perioperative bleeding. One substrate sequence particularly favoured by KLK4 was substituted into the 14 amino acid, circular sunflower trypsin inhibitor (SFTI). This resulted in a highly potent and selective inhibitor (SFTI-FCQR) which attenuated protease activated receptor signalling by KLK4 in vitro. Moreover, SFTI-FCQR and paclitaxel synergistically reduced growth of ovarian cancer cells in vitro, making this inhibitor a lead compound for further therapeutic development. Similar incorporation of a preferred KLK14 amino acid sequence into the SFTI scaffold produced a potent inhibitor for this protease. However, the conformationally constrained SFTI backbone enforced a different intramolecular cooperativity, which masked a KLK14 specific determinant. As a consequence, the level of selectivity achievable was lower than that found for the KLK4 inhibitor. Standard mechanism inhibitors such as SFTI rely on a stable acyl-enzyme intermediate for high affinity binding. This is achieved by a conformationally constrained canonical binding loop that allows for reformation of the scissile peptide bond after cleavage. Amino acid substitutions within the inhibitor to target a particular protease may compromise structural determinants that support the rigidity of the binding loop and thereby prevent the engineered inhibitor reaching its full potential. An in silico analysis was carried out to examine the potential for further improvements to the potency and selectivity of the SFTI-based KLK4 and KLK14 inhibitors. Molecular dynamics simulations suggested that the substitutions within SFTI required to target KLK4 and KLK14 had compromised the intramolecular hydrogen bond network of the inhibitor and caused a concomitant loss of binding loop stability. Furthermore in silico amino acid substitution revealed a consistent correlation between a higher frequency of formation and the number of internal hydrogen bonds of SFTI-variants and lower inhibition constants. These predictions allowed for the production of second generation inhibitors with enhanced binding affinity toward both targets and highlight the importance of considering intramolecular cooperativity effects when engineering proteins or circular peptides to target proteases. The findings from this study show that although PS-SCLs are a useful tool for high throughput screening of approximate protease preference, later refinement by SML screening is needed to reveal optimal subsite occupancy due to cooperativity in substrate recognition. This investigation has also demonstrated the importance of maintaining structural determinants of backbone constraint and conformation when engineering standard mechanism inhibitors for new targets. Combined these results show that backbone conformation and amino acid cooperativity have more prominent roles than previously appreciated in determining substrate/inhibitor specificity and binding affinity. The three key inhibitors designed during this investigation are now being developed as lead compounds for cancer chemotherapy, control of fibrinolysis and cosmeceutical applications. These compounds form the basis of a portfolio of intellectual property which will be further developed in the coming years.
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Patrnogic, Jelena. "Serine proteases and serine protease homologs : genetic analysis of their involvement in immune response activation in Drosophila". Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAJ091/document.
Texto completoResumen
Lors de la réponse immunitaire de la drosophile, la voie Toll est activée lors d'un challenge immunitaire par des bactéries à Gram positif ou des champignons. Ce mécanisme est initié soit par la reconnaissance de motifs moléculaires associés aux pathogènes (PAMPs) qui activent la voie de reconnaissance, soit par des facteurs de virulence et des protéases produits par les agents pathogènes qui activent la voie des signaux de danger. Le travail que j'ai effectué a pour but de caractériser les différentes molécules impliquées dans ces cascades protéolytiques en amont de Toll. Cela permettra de reconstituer ces cascades in vitro et de comprendre comment elles sont organisées, comment et où des complexes peuvent être formés. La première partie concerne les approches génétiques utilisées pour générer des mutants des gènes pouvant être impliqués dans l'activation de la voie Toll par la voie des PAMPs. La deuxième partie se concentre sur un homologue inactif de protéase à sérine appelé spheroide et sur son implication dans la voie de reconnaissance des signaux de danger. Pour la première fois, nous avons pu démontrer qu'une protéase inactive est requise dans la cascade protéolytique, et plus particulièrement dans la détection des signaux de danger après un challenge immunitaire par des bactéries pathogènes à Gram positifThe Toll pathway in Drosophila immune response is activated upon immune challenge with Gram positive bacteria and fungi. This can be achieved either through recognition of Pathogen Associated Molecular Patterns (PAMPs), which triggers the recognition cascade; or by virulence factors and proteases produced by the pathogens, which triggers the danger signal cascade. The work I have done aimed to characterize the various molecules involved in proteolytic cascade supstream of Toll. This will help to reconstitute these cascades in vitro and understand how they are organized, how and where complexes could be formed. The first part focuses on genetic approaches used to generate mutants for genes suggested to be involved in the activation of Toll pathway via the recognition cascade. The second part focuses on an inactive serine protease, aserine protease homolog spheroide and its involvement in the danger signal cascade. For the first time, we could demonstrate that an inactive protease is required in the proteolytic cascade,involved in the sensing of danger signais upon immune challenge with pathogenic Gram-positive bacteria