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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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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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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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Leung, Donmienne Doen Mun. "Studies of serine and cysteine protease inhibitors /." St. Lucia, Qld, 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16491.pdf.
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Acquistapace, Bethany R. "Analysis of a trichomonas vaginalis cysteine protease." Scholarly Commons, 2007. https://scholarlycommons.pacific.edu/uop_etds/669.
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Trichomoniasis affects 170 million people worldwide, and 7.4 million in the USA. There is increasing focus on the role of cysteine proteases in Trichomonas vaginalis because of their role in virulence of other parasitic protozoa. Determining their location and function will provide insight about their role in the pathogenicity of T. vaginalis and their feasibility as a drug target. This study begins to characterize the first sequenced cysteine protease (CP1). E. coli and P. pastoris expression systems were developed to produce CP1 to generate antiserum, and to have enough active protein for biochemical characterization. Secondly, endogenous and epitope tagged CP1 were localized in T. vaginalis vesicles. These vesicles were confirmed to have alkaline phosphatase activity which is a characteristic of lysosomes. Lastly, deletion mutants of CP1 were created to determine the role of the prodomain in targeting CP1 to vesicles.
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Shabab, M. "Study on cysteine protease and their inhibitors." Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 2009. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/2736.
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Ovat, Asli. "Design, synthesis and evaluation of cysteine protease inhibitors." Diss., Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/33822.
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Cysteine proteases are important drug targets due to their involvement in many biological processes such as protein turnover, digestion, blood coagulation, apoptosis, cell differentiation, cell signaling, and the immune response. In this thesis, we have reported the design, synthesis and evaluation of clan CA and clan CD cysteine protease inhibitors.
Aza-peptidyl Michael acceptor and epoxide inhibitors for asparaginyl endopeptidases (legumains) from the bloodfluke, Schistosoma mansoni (SmAE) and the hard tick, Ixodes ricinus (IrAE) were designed and synthesized. SARs were similar, but with some notable exceptions. Both enzymes prefer disubstituted amides to monosubstituted amides in the P1' position and potency increased as we increased the hydrophobicity of the inhibitor in this position. Extending the inhibitor to P5 resulted in increased inhibitory potency, especially against IrAE, and both enzymes prefer small over large hydrophobic residues in the P2 position. Aza-peptide Michael acceptor inhibitors are more potent than aza-peptide epoxide inhibitors and, for some of these compounds, second order inhibition rate constants are the fastest yet discovered.
We have also synthesized aza-peptidyl Michael acceptor and epoxide inhibitors for the parasitic cysteine proteases; cruzain, rhodesain. We have found that monosubstituted amides were favored over disubstituted amides indicating the involvement of the amide hydrogen in a H-bond network. We have shown that aza-peptide epoxides were as potent as Michael acceptors and we have obtained compounds with IC50 values as low as 20 nM.
We have worked on the synthesis of heterocyclic peptidyl α-ketoamides, peptidyl ketones and aza-peptidyl ketones as calpain inhibitors. We have synthesized peptidyl α-ketoamides with nucleotide bases in the primed region to create compounds that can cross the blood-brain barrier. We have improved the potency by introducing a hydrophobic group on the adenine ring. We have obtained compounds with Ki values in the nanomolar range. We have designed peptidyl aminoketones as a new class of inhibitors for calpain. Peptidyl aminoketones were less potent than peptidyl α-ketoamides but still reasonable inhibitors of calpain that have the potential to cross the BBB.
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Campbell, Amy. "Design, synthesis, and evaluation of cysteine protease inhibitors." Diss., Available online, Georgia Institute of Technology, 2005, 2005. http://etd.gatech.edu/theses/available/etd-11222005-132114/.
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Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006.Murthy, Niren, Committee Member ; Doyle, Donald, Committee Member ; Fahrni, Christoph, Committee Member ; May, Sheldon, Committee Member ; Powers, James, Committee Chair.
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Bridges, Sylvia Shadinger. "Design, synthesis, and evaluation of cysteine protease inhibitors." Diss., Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/29738.
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Proteases are enzymes that cleave protein amide bonds. Proteases are involved in a myriad of biological processes and are considered good targets for drug design. The proteases described herein are cysteine proteases, which utilize a cysteine residue thiol to attack the amide carbonyl, leading to amide bond cleavage. Irreversible inhibitors of cysteine proteases react with the active site cysteine, forming a covalent bond and rendering the enzyme inactive.
The first project involved the design and synthesis of aza-peptide epoxide inhibitors for calpain, a clan CA, ubiquitous, calcium-activated human enzyme involved in neurodegeneration. These inhibitors proved to be poor inactivators of calpain, demonstrating that the aza-peptide epoxide is a warhead specific to clan CD cysteine proteases (caspases, gingipains). Subsequently, a known epoxide inhibitor of calpain was optimized to create a more potent inhibitor. Several of these inhibitors were more potent than the parent, and all were demonstrated to inhibit calpain in a breast cancer cell line which was treated with paclitaxel to spike calpain activity.
The second project involved the design and solid phase synthesis of aza-peptide Michael acceptor caspases inhibitors. The two goals of this project were to develop a solid phase method for synthesis of inhibitors that are tedious to synthesize in solution phase, and to use a variety of amino acid residues to determine the optimal interactions in the P3? position for various caspases. The synthesis was successful, and the optimal P3? residues were determined.
The third project involved the kinetic evaluation of aza-peptide epoxide and Michael acceptor inhibitor designed for the gingipains. Gingipains K and R are virulence factors in the pathology of Porphyromonas gingivalis involved in gingivitis and periodontal disease. These inhibitors proved to be extremely potent inactivators of gingipains, with some of the highest rates of inhibition measured in the Powers laboratory. Gingipain K preferred larger, aromatic moieties in the P1? position, while gingipain R preferred the Michael acceptor inhibitors, with the P1? substituent having less of an impact on potency.
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Håkansson, Katarina. "Cystatin C functions in vitro and in vivo studies on target enzyme inhibition by cystatin C variants and cystatin C deficient mice /." Lund : Dept. of Clinical Chemistry, University of Lund, University Hospital, 1998. http://catalog.hathitrust.org/api/volumes/oclc/40343026.html.
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Rapolu, Chaitanya. "Inhibition of Cysteine Protease by Platinum (II) Diamine Complexes." TopSCHOLAR®, 2011. http://digitalcommons.wku.edu/theses/1137.
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Chemotherapy is the first line of treatment used in cancer. Chemotherapy drugs such as cisplatin, carboplatin and oxaliplatin are used in treatment. Cisplatin enters the cell through copper transporter CTR1 by passive diffusion and bind to DNA and proteins. Cisplatin is found to inhibit several enzymes targeting cysteine, histidine and methionine residues, which are expected to be responsible for its anticancer activity. A better understanding of how the size and shape and leaving ligands of platinum complexes affect cysteine protease, papain enzyme are studied. This could give new ways to optimize anticancer activity. The activity of papain enzyme was measured on UV-Visible spectroscopy. The inhibition profile of papain with different platinum (II) complexes, and with different combinations was studied.
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Podivinsky, Ellen. "Molecular studies on actinidin, a cysteine protease from kiwifruit." Thesis, University of Auckland, 1991. http://hdl.handle.net/2292/2001.
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Research in this thesis describes the characterisation of mRNA sequences coding for actinidin, a cysteine protease found in abundance in the fruit of kiwifruit (Actinidia deliciosa). The first step in the characterisation required the isolation of mRNA from ripe kiwifruit tissue. The suitability of a number of RNA extraction procedures was investigated. The method finally adopted differed from that used for unripe fruit tissue, and was chosen as a result of the nature of the polysaccharide that contaminated nucleic acids prepared from extracts of kiwifruit fruit tissue. RNA extracted from ripe fruit was used to synthesis a partial cDNA library and clones for actinidin were isolated. A number of these cDNA clones were sequenced; three clones were almost full-length. The actinidin cDNA clones obtained fall into two broad sequence classes. The majority of them encode acidic proteins (pI˜4.7), with 97% homology to the published amino acid sequence of actinidin. The second class encode basic proteins (pI˜8.1), with 83% homology to the published amino acid sequence of actinidin. Both classes of actinidin cDNA sequence encode zymogens, which contain N- and C-terminal extensions not present in the mature form of the enzyme. The N-terminal extension of both sequence classes includes a putative signal peptide. Northern hybridization analysis was used to investigate the tissue specificity of actinidin mRNA expression, and the expression of mRNA for the two actinidin sequence classes during fruit ripening. Both actinidin sequence classes were expressed differentially during the latter stages of kiwifruit fruit development and through post-harvest fruit ripening. The expression of both sequence classes increased from just prior to fruit maturity through ripening and reached a maximum as fruit attained the stage of 'eating' ripeness. The level of expression of the sequences encoding acidic actinidin reached a plateau at this point, while the expression of the sequence encoding basic actinidin appeared to decrease slightly as fruit continued to ripen. The sequences encoding acidic actinidin were expressed during ripening at a much higher level than those encoding basic actinidin. No actinidin mRNA was detected in other tissues except for very low levels of the acidic form in kiwifruit leaf, and low levels of the basic form in senescing petals. A full-length, acidic, actinidin cDNA sequence was introduced into tobacco (Nicotiana tabacum) plants via Agrobacterium tumefaciens-mediated transformation. Using the binary vector pGA643, the sequence was introduced in both the sense and antisense orientation relative to the cauliflower mosaic virus 35S promoter and transgenic plants were obtained for both sequence orientations. The presence of the T-DNA cassette (containing the actinidin sequence) in the plant genomes was determined using PCR analysis, and confirmed by Southern hybridization. A number of the transgenic plants contained multiple insertions of the actinidin sequence, and most plants contained at least one intact copy of the T-DNA cassette. The transcription of the introduced actinidin sequence was investigated by Northern hybridization analysis. All of the plants containing actinidin in the sense orientation, and some of those incorporating the antisense construct, transcribed the actinidin sequence. Attempts to detect actinidin protein in the transgenic plants were unsuccessful. Acidic actinidin was identified as one of the most abundant bands in the total protein profile from ripe kiwifruit fruit tissue. The identity of the protein was confirmed by N-terminal sequence analysis. The electrophoretic mobility of actinidin, both in the total cell homogenate and when partially purified, suggested that the first step in post-translational processing of the zymogen may be the removal of the N-terminal extension. Actinidin was also partially purified and used to raise antibodies. Poor specificity of the antibody for actinidin led to preliminary evidence for the glycosylation of actinidin.
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Chen, Hongyuan. "Development of macrocyclic β-strand calpain cysteine protease inhibitors." Thesis, University of Canterbury. Chemistry, 2011. http://hdl.handle.net/10092/5582.
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The work in this thesis reports studies directed to developing a calpain cysteine protease
inhibitor that could be of value in slowing cataract development in humans. The work
focuses on the development of macrocyclic compounds which can have advantages over
acyclic compounds due to their resistance to proteolytic hydrolysis, improved selectivity,
bioavailability and membrane permeability. A review of X-ray crystal structures of
natural and synthetic calpain inhibitors complexed with the cysteine protease calpain
show the inhibitors generally bind in the enzyme active site in an extended β-strand
conformation.
The calpain inhibitor SJA-6017 has been identified as a suitable lead compound. The
importance of the para-fluoro group in SJA-6017 has been investigated. Modifications
have been made to constrain this basic structure within a macrocycle and restrict the
peptide chain as a β-strand conformation. Macrocycle CAT811 is a potent calpain 1 and
2 inhibitor and shows promise in slowing the progression of cortical cataract in trials with
sheep having a hereditary propensity towards the development of cataract.
In this thesis I report studies directed to improve the yield of the key RCM
macrocyclisation step in the synthesis of aldehyde CAT811 and of three ester analogues
(2.1, 2.3 and 2.4).
I also report the development of a more commercial route to CAT811 not involving
RCM but using intramolecular nucleophilic cyclisation.
This intramolecular nucleophilic cyclisation strategy was attempted for the preparation of
a histidine containing macrocyclic ester (4.1a) but was unsuccessful. An alternate
strategy involving intramolecular lactamization proved successful for the synthesis of
histidine-based macrocyclic esters (4.1a-4.3a). Reduction to the corresponding alcohols
(4.1b-4.3b) was successful and oxidation of (4.1b and 4.3b) afforded the corresponding
aldehydes (4.1c and 4.3c) for biological assay against ovine calpain 2.
Aldehyde 4.3c has an IC50 of 1 μM and the corresponding alcohol 4.3b shows no activity
(IC50 > 50 μM) consistent with the modelling which indicated that these two compounds
did not adopt a β-strand conformation in the docking studies. Aldehyde 4.1c, on the other
hand, shows significant inhibitory activity with an IC50 of 238 nM but as expected the
corresponding alcohol 4.1b shows little activity (IC50 = 29 μM). Modelling studies
showed that both the aldehyde 4.1c and the alcohol 4.1b on docking can form a β-strand
with appropriate H-bonding interactions. The aldehyde is more active than the alcohol
due to the reactivity of the aldehyde warhead allowing for the reversible formation of a
hemiacetal. A similar difference in reactivity is observed for CAT811 (30 nM) and its
alcohol analogue (700 nM).
These results demonstrate the value of molecular modelling as a screening mechanism
before unproductive synthetic work is considered.
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Chen, Yetian Tropsha Alexander. "Spatial motif discovery in papain-like cysteine protease family." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2008. http://dc.lib.unc.edu/u?/etd,1988.
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Thesis (M.S.)--University of North Carolina at Chapel Hill, 2008.Title from electronic title page (viewed Dec. 11, 2008). "... in partial fulfillment of the requirements for the degree of Master of Science in the Department of Biochemistry and Biophysics, School of Medicine." Discipline: Biochemistry and Biophysics; Department/School: Medicine.
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James, Allison Melissa. "Babesia microti cysteine protease-1 as a target for vaccine development." Thesis, Texas A&M University, 2005. http://hdl.handle.net/1969.1/4192.
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Babesia species have a worldwide distribution, affecting a wide range of mammalian
hosts. The major route of transmission is inoculation by an infected Ixodid tick. Babesia
species of major economic concern are those that cause bovine and equine babesiosis.
Historically, bovine Babesia species, Babesia bovis and Babesia bigemina caused
significant economic losses in the United States in the 1860âÂÂs, as thousands of cattle
died. Also, outbreaks of equine babesiosis, caused by Babesia equi or Babesia caballi,
have occurred in the United States resulting in the death of some horses and millions of
dollars in losses. A constant risk of reinfection with bovine and equine Babesia species
exists, as stray and smuggled animals from Mexico, where bovine babesiosis is endemic,
may carry infected ticks as they cross the border, and, thousands of horses from B. equiand
B. caballi-endemic regions are imported through Florida every year.
Vaccines have been developed for a number of Babesia species, none of which result
in sterile immunity. The live attenuated vaccine is the most commonly used vaccine
against Babesia species. However, the basis for the vaccine is to maintain a carrier state in order to prevent disease. Other vaccine designs have been developed to invoke
protection without a carrier state but have been unsuccessful.
It has been shown that the cysteine protease is important in the life cycle of a number
of parasitic organisms, making it a good target for vaccine development. The vaccine
design for this study incorporated the cysteine protease of Babesia microti. Babesia
microti naturally infects Peromyscus leucopus (white-footed mouse) and is the major
cause of human babesiosis in the United States. Using B. microti in the vaccine design
allowed for the use of a mouse model to determine whether the cysteine protease of
other economically important Babesia species may make a good vaccine target. The
vaccine design incorporated a prime-boost strategy, priming with DNA encoding the
cysteine protease and boosting two times with either DNA encoding the cysteine
protease or cysteine protease peptide, followed by parasite challenge. Analysis of daily
percent parasitemias, packed cell volume, and seroconversion of all groups revealed that
a protective immune response against B. microti was not elicited by this vaccine strategy.
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Rukamp, Karrie Eileen Adlington. "Design and synthesis of inhibitors for serine and cysteine proteases." Diss., Available online, Georgia Institute of Technology, 2004:, 2003. http://etd.gatech.edu/theses/available/etd-04082004-180343/unrestricted/rukamp%5Fkarrie%5Fe%5Fa%5F200312%5Fphd.pdf.
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Rukamp, Brian John. "Design, synthesis, and evaluation of novel thiobenzyl ester substrates and aza-peptide inhibitors for serine and cysteine proteases." Diss., Available online, Georgia Institute of Technology, 2004:, 2003. http://etd.gatech.edu/theses/available/etd-04072004-180202/unrestricted/rukamp%5Fbrian%5Fj%5F200312%5Fphd.pdf.
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Faucher, Ryan Michael John. "CHARACTERIZATION OF PHYTOCYSTATIN-LIKE CYSTEINE PROTEASE INHIBITORS OF TRICHOMONAS VAGINALIS." Scholarly Commons, 2017. https://scholarlycommons.pacific.edu/uop_etds/2970.
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Trichomoniasis is a common STD caused by the parasitic protozoan Trichomonas vaginalis. The parasite is estimated to have infected roughly 3.7 million Americans. Complications from trichomoniasis can lead to cervical cancer in women and prostate cancer in men. One of the mechanisms of the parasite employs is using cysteine proteases to break down the cellular matrix of its host. However, three endogenous phytocystatin-like protease inhibitors have been found within the parasite’s genome. By recombinantly expressing these cystatins we have been able to test their ability to inhibit cysteine proteases such as papain and those found in T. vaginalis to find their effectiveness. By characterizing these inhibitors, it appears that they are effective at reducing the ability of T. vaginalis cysteine proteases and thus could be useful against the pathogenicity of the parasite.
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Singh, J. P. "Studies on low molecular mass cysteine protease inhibitor from actinomycetes." Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 2009. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/2748.
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Mehrtens, (nee Nikkel) Janna Marie. "The Design, Synthesis and Biological Assay of Cysteine Protease Specific Inhibitors." Thesis, University of Canterbury. Chemistry, 2007. http://hdl.handle.net/10092/3271.
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This thesis investigates the design, synthesis and biological assay of cysteine protease inhibitors within the papain superfamily of cysteine proteases. This is achieved by examining the effect of inhibitor design, especially warheads, on IC₅₀ values and structureactivity relationships between cysteine protease inhibitors of the papain superfamily. The representative proteases used are m-calpain, μ-calpain, cathepsin B and papain. Chapter One is an introductory chapter; Chapters Two-Four describe the design and synthesis of cysteine protease inhibitors; Chapter Five discusses assay protocol; and Chapter Six contains the assay results and structure-activity relationships of the synthesised inhibitors. Chapter One introduces cysteine proteases of the papain family and examines the structure, physiology and role in disease of papain, cathepsin B, m-calpain and μ-calpain. The close structural homology that exists between these members of the papain superfamily is identified, as well characteristics unique to each protease. Covalent reversible, covalent irreversible and non-covalent warheads are defined. The generic inhibitor scaffold of address region, recognition and warhead, upon which the inhibitors synthesised in this thesis are based, is also introduced. Chapter Two introduces reversible cysteine protease inhibitors found in the literature and that little is known about the effect of inhibitor warhead on selectivity within the papain superfamily. Oxidation of the dipeptidyl alcohols 2.6, 2.26, 2.29, 2.30, 2.35 and 2.36 utilising the sulfur trioxide-pyridine complex gave the aldehydes 2.3, 2.27, 2.19, 2.2, 2.21 and 2.22. Semicarbazones 2.37-2.40 were synthesised by a condensation reaction between the alcohol 2.3 and four available semicarbazides. The amidoximes 2.48 and 2.49 separately underwent thermal intramolecular cyclodehydration to give the 3-methyl-1,2,4- oxadiazoles 2.41 and 2.50. The aldehydes 2.3 and 2.27 were reacted with potassium cyanide to give the cyanohydrins 2.51 and 2.52. The cyanohydrins 2.51 and 2.52 were separately reacted to give 1) the α-ketotetrazoles 2.43 and 2.55; 2) the α-ketooxazolines 2.42 and 2.58; 3) the esterified cyanohydrins 2.60 and 2.61. A two step SN2 displacement reaction of the alcohol 2.6 to give the azide 2.62, an example of a non-covalent cysteine protease inhibitor. Chapter Three introduces inhibitors with irreversible warheads. The well-known examples of epoxysuccinic acids 3.1 and 3.5 are discussed in detail, highlighting the lack of irreversible cysteine protease specific inhibitors. The aldehydes 2.3 and 2.27 were reacted under Wittig conditions to give the α,β-unsaturated carbonyls 3.14-3.18. Horner- Emmons-Wadsworth methodology was utilised for the synthesis of the vinyl sulfones 3.20- 3.23. The dipeptidyl acids 2.24 and 2.28 were separately reacted with diazomethane to give the diazoketones 3.25 and 3.26. The diazoketones 3.25 and 3.26 were separately reacted with hydrogen bromide in acetic acid (33%) to give the α-bromomethyl ketones 3.27 and 3.28, which were subsequently reduced to give the α-bromomethyl alcohols 3.29-3.32. Under basic conditions the α-bromomethyl alcohols 3.29-3.32 ring-closed to form the peptidyl epoxides 3.33-3.36. Chapter Four introduces the disadvantages of peptide-based inhibitors. A discussion is given on the benefits of constraining inhibitors into the extended bioactive conformation known as a β-strand. Ring closing metathesis is utilised in the synthesis of the macrocyclic aldehyde 4.4, macrocyclic semicarbazone 4.15, the macrocyclic cyanohydrin 4.16, the macrocyclic α-ketotetrazole 4.18 and the macrocyclic azide 4.19. Chapter Five introduces enzyme inhibition studies. The BODIPY-casein fluorogenic assay used for establishing inhibitor potency against m-calpain and μ-calpain is validated. Assay protocols are also established and validated for cathepsin B, papain, pepsin and α- chymotrypsin. A discussion of the effect of solvent on enzyme activity is also included as part of this study. Chapter Six presents the assay results for all the inhibitors synthesised throughout this thesis and an extensive structure-activity relationship study between inhibitors is included. The alcohols 2.26 and 2.30 are unprecedented examples of non-covalent, potent, cathepsin B inhibitors (IC₅₀ = 0.075 μM selectivity 80-fold and 1.1 μM, selectivity 18-fold). The macrocyclic semicarbazone 4.15 is an unprecedented example of a potent macrocyclic cysteine protease inhibitor (m-calpain: IC₅₀ = 0.16 μM, selectivity 8-fold). The cyanohydrin 2.51 contains an unprecedented cysteine protease warhead and is a potent and selective inhibitor of papain (IC₅₀ = 0.030 μM, selectivity 3-fold). The O-protected cyanohydrin 2.61 is a potent and selective inhibitor of pepsin (IC₅₀ = 1.6 μM, selectivity 1.5-fold). The top ten warheads for potent, selective cathepsin B inhibition are: carboxylic acid, methyl ester, diazoketone, esterified cyanohydrin, α-bromomethyl ketone, α,β- unsaturated aldehyde, vinyl sulfones, α-bromomethyl-C₃-S,R-alcohol, alcohol and α,β- unsaturated ethyl ester. The selectivity of these warheads was between 5- and 130-fold for cathepsin B. The best inhibitors for cathepsin B were the α-bromomethyl ketone 3.26 (IC₅₀ = 0.075 μM, selectivity 16-fold), the α,β-unsaturated aldehyde 3.18 (IC₅₀ = 0.13 μM, selectivity 13-fold) and the esterified cyanohydrin 3.59 (IC₅₀ = 0.35 μM, selectivity 22- fold). Chapter Seven outlines the experimental details and synthesis of the compounds prepared in this thesis.
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Krauser, Joel Anderson. "Design, synthesis and evaluation of novel inhibitors and fluorogenic substrates for cysteine proteases and metallo proteases." Diss., Georgia Institute of Technology, 2001. http://hdl.handle.net/1853/30003.
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Koot, Gretchen E. "Serine and cysteine protease inhibitors for blockade of cell mediated cytotoxicity /." abstract and full text PDF (UNR users only), 2002. http://0-gateway.proquest.com.innopac.library.unr.edu/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3121138.
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Mohan, Srinidi. "Functional role of recombinant cysteine protease on Spodoptera frugiperda peritrophic matrix." Diss., Mississippi State : Mississippi State University, 2006. http://library.msstate.edu/etd/show.asp?etd=etd-11072006-150055.
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Musonda, Chitalu Christopher. "Antimalarial and cysteine protease inhibitor pharmacophores as scaffolds for new antimalarial agents." Doctoral thesis, University of Cape Town, 2005. http://hdl.handle.net/11427/11811.
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Includes bibliographical references.The work in this thesis is threefold: (i) A new series of antiplasmodial agents were initially designed based on the β-amino alcohol bioactiphore, a subunit that is found in a number of antimalarial agents. (ii) Various thiosemicarbozones and semicarbozones were designed and synthesized as potential mechanism-based inhibiotrs of parasitic cysteine proteases. (iii) Multicomponenet reactions offer the advantage of introducing chemical diversity in fewer steps than conventional multi step organic synthesis. New chloroquine-type compounds were designed and synthesized using the Ugi 4 component condensation reaction and its variants. The synthesized compounds ranged from simple peptidic molecules to rigid heterocycles.
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Petzold, Herman Earl III. "Promoter Deletion Analysis of Xylem Cysteine Protease 2 (XCP2) in Arabidopsis thaliana." Thesis, Virginia Tech, 2007. http://hdl.handle.net/10919/32582.
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The process of xylem tracheary element differentiation involves the coordination of vascular cambium activity, cell fate determination, cell expansion/elongation, secondary wall synthesis, programmed cell death, and cellular autolysis. The end result of tracheary element differentiation is a cellular corpse lacking a protoplast and consisting of a thickened cell wall composed mostly of lignin and cellulose. Little is known about the genetic mechanisms regulating the process of tracheary element differentiation. XCP2 expression localizes to tracheary elements according to two independent methods of analysis: promoter reporter experiments and immunogold localization by electron microscopy. XCP2 may be involved in catalyzing the degeneration of the protoplast during the final autolytic stages of tracheary element differentiation. To this date XCP2 function has not been directly demonstrated. In principle, any tracheary element-specific markers can be linked to upstream regulatory genes with roles in tracheary element differentiation. To develop the XCP2 promoter as a tool for identification of transacting factors, a promoter deletion analysis was carried out. Utilizing information from 5â and 3â deletion constructs, a 70-bp region upstream of the XCP2 translational start site is both necessary and sufficient for TE-specific expression of the UidA reporter gene. Mutational analysis of the ACTTTA element at position -113-bp strongly suggests it is a cis element required for XCP2 expression. In silico analysis of an 18-bp promoter region located within 200-bp of the translation start site and including the ACTTTA element revealed high indentity shared between xylem-specific XCP2 homologs from Zinnia elegans, Populus trichocarpa, and XCP1 from Arabidopsis thaliana.Master of Science
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Gräsberg, Sofia. "Recombinant production of the Giardia intestinalis cysteine protease CP10217 in Pichia pastoris." Thesis, Uppsala universitet, Institutionen för cell- och molekylärbiologi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-385649.
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Giardia intestinalis is one of the leading causes of diarrheal diseases, affecting about 280 million people every year. By characterizing the virulence factors of G. intestinalis, new drug targets can be found to treat giardiasis. In addition to the adhesive disc and the variant surface proteins, cysteine proteases are some of the most interesting virulence factors in Giardia. In this project, one of the major secreted cysteine proteases, CP10217, was studied. The intent was to study the structure by modelling and to characterize CP10217 by expressing it in yeast cells and purifying the supernatant by using Immobilized Metal Affinity Chromatography (IMAC). The molecular modelling showed that the Giardia CP can be modelled on the structures of human and Trypanosoma brucei CPs. The process of expressing and purifying CP10217 in this manner proved difficult. The protease seemed to be very active when expressed, probably resulting in self-cleavage into its active form and later digestion of the whole protein, leading to a low protein yield from the purification. Two approaches were tested in order to increase the protein yield. First, expression at different pH ranges, and secondly, by re-cloning CP10217 with an extra 108 bp sequence at the 5’ end. While changes in pH did not seem to affect the yield, sequencing results of the new vector showed that the cloning worked. More work on this new vector is needed to further analyse, and possibly characterize CP10217.
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Kroon, Matthys Christoffel. "High-throughput modelling and structural investigation of cysteine protease complexes with protein inhibitors." Thesis, Rhodes University, 2013. http://hdl.handle.net/10962/d1001619.
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The papain-like cysteine protease family (C1 proteases) is highly important because of its involvement in research and industrial applications and its role in various human diseases. Protein inhibitors are an important aspect of C1 protease biology and are relevant to its clinical, industrial and research importance. To study the interaction between the proteases and the inhibitors it is very useful to have accurate structural models of the protease-inhibitor complexes. To this end, a high-throughput pipeline for modelling complexes of papain-like cysteine proteases and protein inhibitors was implemented and tested (Tastan Bishop & Kroon, 2011). The pipeline utilizes a novel technique for obtaining modelling templates by using superpositioning to combine coordinates from separate experimental structures. To test the pipeline, models of complexes with known structures (test set) were modelled using many different templates and the resultant models evaluated to compare the quality of the different templates. It was found that use of the new technique to obtain templates did not introduce significant errors, while allowing closer homologs to be used for modelling - leading to more accurate models. The test set models were also used to evaluate certain steps of the modelling protocol. The effect of Rosetta energy minimization on model accuracy and the use of Rosetta energy and DOPE Z-score values to identify accurate models were investigated. Several complexes were then modelled using the best available templates according to criteria informed by the previous results. A website was built that allows a user to download any of the metrics or models produced in the study. This website is accessible at http://rubi.ru.ac.za/cpmdb
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Vindebro, Reine. "Studies on secreted cysteine proteases of Streptococcus pyogenes : IdeS and SpeB." Doctoral thesis, Umeå universitet, Institutionen för molekylärbiologi (Medicinska fakulteten), 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-88223.
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The pathogen Streptococcus pyogenes is a significant cause of human morbidity and mortality. Most of the work in this thesis is focused on streptococcal virulence factor IdeS, but the thesis also features work on SpeB, another streptococcal virulence factor. Both IdeS and SpeB are secreted cysteine proteases and both have previously been shown to degrade human IgG. IgG is the only known substrate for IdeS while SpeB is a more promiscuous protease with a larger number of identified substrates. A significant part of the data presented in this thesis is the result of designing and optimizing methods to detect and accurately measure the proteolytic degradation of IgG. Methods aimed at measuring the binding interactions between enzyme and substrate have also been frequently utilized. I show that IdeS is a monomeric protease, as opposed to previously published data that suggested it to be dimeric. IdeS cleaves the two heavy chains of IgG in a two-step reaction and I demonstrate that the first cleavage is magnitudes faster than the second one. This means that IdeS is a more efficient enzyme than previously thought. The difference in rate cannot completely be explained by a loss of affinity between IdeS and IgG after the cleavage of the first heavy chain. The velocity of IdeS is further increased by the presence of human Cystatin C, via an unknown mechanism. Cystatin C is normally a protease inhibitor and it having an opposite effect is puzzling.The synthesis and evaluation of novel inhibitors are also described. Peptide analogues mimicking the sequence surrounding the scissile bond on IgG - with an amino acid replaced with a more rigid motif - act as specific, but low-affinity, inhibitors of IdeS. The peptide analogues’ inhibitory capacity for SpeB and papain was also assayed.When it comes to SpeB, I show that it does not have IgG as a substrate under physiological conditions, in contrast to what was previously thought. This thesis does not only present findings on the IgG degrading capacity of IdeS and SpeB but also include data on fundamental enzymatic properties for these proteases.
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SERVEAU, CAROLE, and Francis Gauthier. "Ciblage des proteases a cysteine par de nouveaux substrats et inhibiteurs peptidiques derives de leurs inhibiteurs naturels (cystatines). Application a la protease a cysteine majeure de trypanosoma cruzi." Tours, 1995. http://www.theses.fr/1995TOUR4011.
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Les cathepsines lysosomiales b, h et l de mammiferes, proteases a cysteine apparentees a la papaine, sont impliquees dans la degradation proteique intracellulaire mais aussi dans des processus pathologiques. Le protozoaire trypanosoma cruzi exprime une protease a cysteine homologue a la cathepsine l. Cette protease, ou cruzipaine, est impliquee dans la pathologie de la maladie de chagas transmise par t. Cruzi. C'est une cible potentielle pour developper de nouvelles drogues anti-parasitaires. Sa caracterisation enzymatique par comparaison aux cathepsines mammaliennes est necessaire pour definir un substrat ou un inhibiteur selectif de la protease parasitaire. Nous avons developpe des substrats fluorogeniques et des inhibiteurs irreversibles a partir de deux regions principales (lvg et qxvxg) tres conservees des cystatines (inhibiteurs naturels des proteases a cysteine), regions impliquees dans le mecanisme d'inhibition. Nous avons ainsi obtenu de nouveaux substrats et inhibiteurs sensibles et specifiques des enzymes apparentees a la papaine, adaptes pour la caracterisation comparee des proteases d'origine mammalienne et parasitaire. Contrairement aux cathepsines b et l, la cruzipaine est tres sensible a la nature des residus localises au niveau de l'extremite carboxy-terminale du site de clivage, plus particulierement une proline en p2'. Afin de developper des inhibiteurs peptidiques irreversibles, nous avons etudie la specificite de la region p du site de clivage pour les cathepsines b et l, et la cruzipaine. La combinaison des resultats obtenus nous a permis de selectionner une sequence peptidique (hpggp) selective de la cruzipaine. A partir de cette sequence, un substrat fluorogenique selectif de la cruzipaine est d'ores et deja synthetise et un inhibiteur irreversible est en cours de developpement pour une utilisation potentielle sur des cultures parasitaires in vitro
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Tomás, Ana Maria Luís Ramos. "Functional analysis of the major cysteine protease of `Trypanosoma cruzi' using genetic approaches." Thesis, London School of Hygiene and Tropical Medicine (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.590546.
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Liu, Yong Bo. "Characterization of major cysteine protease isoforms in embryos and larvae of Artemia franciscana." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0014/MQ30956.pdf.
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Schulz, Oliver. "The pro-allergic potential of the cysteine protease activity of DER P 1." Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262954.
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Yelpaala, Yuora. "The characterization of cysteine protease 4 and superoxide dismutase 6 in Trichomonas vaginalis." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/189.
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Pathogenesis attributable to infection with Trichomonas vaginalis , the causative agent of Trichomoniasis, is largely unknown although cysteine proteases have been implicated. This paper investigated the role of cysteine protease 4 (CP4) in T. vaginalis through characterization and expression of CP4. T. vaginalis strains showed differential protein and mRNA expression, although it was unclear which CP4 variants (other than TVAG_355480 and TVAG_467970) were recognized by the CP4 antibody for the protein studies. Iron did not regulate expression of TVAG_355480 and TVAG_467970 at the transcriptional but possibly at the post-transcriptional level. Our results also suggested that processing of recombinant TVAG_467970 occurred through cleavage by proteins rather than autocatalytic processing. Finally, endogenous CP4 was localized to vesicles though it was unclear which CP4 variant was recognized and was co-localized with HA-tagged VAMP1/2. Localization of HA-tagged TVAG_467970 proved problematic when co-staining with anti-HA and anti-CP4, so further localization studies need to be optimized. This paper also examined the role of iron superoxide dismutase 6 (TvFeSOD6) in resistance to metronidazole, the current drug used for Trichomoniasis infections. Of the two types of resistance, aerobic resistance may occur due to a high concentration of intracellular oxygen which can outcompete metronidazole for electrons or can re-oxidize reduced metronidazole to its inactive form. We determined that there was differential expression of TvFeSODs in T. vaginalis strains with varying levels of resistance although this may not correlate with the degree of resistance. Our results also showed that an increase in ectopic TvFeSOD6 in MSA1121 led to an augmentation in SOD activity and in resistance under aerobic conditions due to the possible role of TvFeSOD6 in also contributing to a higher intracellular oxygen concentration in MSA1121 (which is already sensitive to oxygen), leading to an increase in resistance.
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Musyoka, Thommas Mutemi. "Combined in silico approaches towards the identification of novel malarial cysteine protease inhibitors." Thesis, Rhodes University, 2017. http://hdl.handle.net/10962/4488.
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Malaria an infectious disease caused by a group of parasitic organisms of the Plasmodium genus remains a severe public health problem in Africa, South America and parts of Asia. The leading causes for the persistence of malaria are the emergence of drug resistance to common antimalarial drugs, lack of effective vaccines and the inadequate control of mosquito vectors. Worryingly, accumulating evidence shows that the parasite has developed resistant to the current first-line treatment based on artemisinin. Hence, the identification and characterization of novel drug targets and drugs with unique mode of action remains an urgent priority. The successful sequencing and assembly of genomes from several Plasmodium species has opened an opportune window for the identification of new drug targets. Cysteine proteases are one of the major drug targets to be identified so far. The use of cysteine protease inhibitors coupled with gene manipulation studies has defined specific and putative roles of cysteine proteases which include hemoglobin degradation, erythrocyte rupture, immune evasion and erythrocyte invasion, steps which are central for the completion of the Plasmodium parasite life cycle. In an aim to discover potential novel antimalarials, this thesis focussed on falcipains (FPs), a group of four papain-like cysteine proteases from Plasmodium falciparum. Two of these enzymes, FP-2 and FP-3 are the major hemoglobinases and have been validated as drug targets. For the successful elimination of malaria, drugs must be safe and target both human and wild Plasmodium infective forms. Thus, an incipient aim was to identify protein homologs of these two proteases from other Plasmodium species and the host (human). From BLASTP analysis, up to 16 FP-2 and FP-3 homologs were identified (13 plasmodial proteases and 3 human cathepsins). Using in silico characterization approaches, the intra and inter group sequence, structural, phylogenetic and physicochemical differences were determined. To extend previous work (MSc student) involving docking studies on the identified proteins using known FP-2 and FP-3 inhibitors, a South African natural compound and its ZINC analogs, molecular dynamics and binding free energy studies were performed to determine the stabilities and quantification of the strength of interactions between the different protein-ligand complexes. From the results, key structural elements that regulate the binding and selectivity of non-peptidic compounds onto the different proteins were deciphered. Interaction fingerprints and energy decomposition analysis identified key residues and energetic terms that are central for effective ligand binding. This research presents novel insight essential for the structure-based molecular drug design of more potent antimalarial drugs.
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Ntuli, Nelson Axe. "New aminoquinoline antimalarial cysteine protease inhibitors based on the isatin natural product scaffold." Master's thesis, University of Cape Town, 2005. http://hdl.handle.net/11427/6352.
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Includes bibliographical references (leaves 120-125).Many antimalarial drugs including chloroquine are no longer effective against the disease, as their efficacy has been decreased by the spread of drug resistant strains. This loss has been a major barrier to the effective treatment of malaria and has necessitated an urgent need to discover new antimalarial drugs. New 4-aminoquinoline isatin derivatives have been designed and synthesised. Isatin was used as a biologically validated starting point for the design of chemical libraries directed at the intended target due to its privileged nature. Included in the design of these isatin derivatives is the thiosemicarbazone moiety previously demonstrated to inhibit cysteine proteases from mUltiple protozoan parasites. Synthesized compounds were tested against the enzyme falcipain-2 (Rec-FP-2) as well as the parasite source of this protease, Plasmodium Jalciparum (P.f. W2). The results of structure activity relationship studies demonstrate the influence of substituents at position 5 of the isatin scaffold. With respect to the chain length, a two carbon methylene spacer between the aminoquinoline and isatin moieties, was found optimum. A 5-bromo isatin derivative with this spacer, compound 79b, was found to be the most active with an IC₅₀ value of 163.5 nM against the W2 parasite strain and second most active against the enzyme (lC₅₀ = 3.65 μM).
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Cruz, Wallace Teixeira da. "Proteomics analysis , purification and characterization of a cysteine peptidase oligomeric make latex Thevetia peruviana (Pers .) Schum." Universidade Federal do CearÃ, 2015. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=15721.
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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel SuperiorA great number of plant species produce latex, including Apocynacea, Sapotacea, Papaveracea and Euphorbiaceae. Thevetia peruviana (Pers.) Schum is a laticifer shrub belonging to Apocynaceae family popularly known as âchapÃu-de-napoleÃoâ. It is very limited the proteomic information about this specie. Thus, a proteomic analysis of protein fraction (TpLP) from T. peruviana latex was performed using two dimensional gel electrophoresis and mass spectrometry. A total of 33 proteins (86%) were identified, including storage proteins, peptidase inhibitor, cysteine peptidases, peroxidases and osmotins. This protein fraction showed strong proteolytic activity at pH 5.0 which was increased in the presence of low concentrations of the reducing agent DTT. The inhibition this activity in the presence of specific inhibitors E-64 and IAA and the high activity with BANA showed the predominance of cysteine peptidases in latex. A cysteine peptidase, termed peruvianin-I, was purified from the latex by a single chromatographic step involving gel filtration. The enzyme was inhibited by E-64 and iodoacetamide (IAA) and follows the Michaelis-Menten kinetics, showing high affinity for azocasein, with Km value of 17.6 uM, exhibiting an optimal pH and temperature of 5.0-6.0 and 25-37 ÂC, respectively. Two-dimensional gel electrophoresis and mass spectrometry revealed that peruvianin-I (100 kDa) possesses a pI of 4.0 and five subunits (20 kDa). The N-terminal amino acid sequence of peruvianin-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36) was similar to that of germin or germin-like proteins. High-resolution images from atomic force microscopy indicated the possible hexameric structure of peruvianin-I, which is organized as a trimer of dimers that form a central channel. TpLP and peruvianin-I exhibited no oxalate oxidase and superoxide dismutase activity or antifungal effects on the spore germination of Fusarium solani. This study showed that T. peruviana latex are a rich source of pathogenesis-related proteins, including cysteine peptidases. Interestingly, these peptidases exhibit different structural and biochemical characteristics that may be related to their specific physiological functions.
Um grande nÃmero de espÃcies vegetais produzem lÃtex, incluindo Apocynacea, Sapotacea, Papaveracea e Euphorbiaceae. Thevetia peruviana (Pers.) Schum à um arbusto laticÃfero pertencente à famÃlia Apocynaceae, popularmente conhecido como "chapÃu-de-NapoleÃo". SÃo bastante limitadas as informaÃÃes proteÃmicas sobre esta espÃcie. Por tanto, uma anÃlise proteÃmica da fraÃÃo proteica (TpLP) do lÃtex de T. peruviana foi realizada a partir de eletroforeses bidimensionais e espectrometria de massas. Um total de 33 proteÃnas (86%) foi identificado, incluindo proteÃnas de reserva, inibidor de peptidase, peptidases cisteÃnicas, peroxidases e osmotinas. As proteÃnas desta fraÃÃo apresentaram uma forte atividade proteolÃtica no pH 5,0, a qual foi aumentada na presenÃa de baixas concentraÃÃes do agente redutor DTT. A inibiÃÃo desta atividade na presenÃa dos inibidores especÃficos E-64 e IAA e a alta atividade com o substrato BANA evidenciou a predominÃncia de peptidases cisteÃnicas no lÃtex. Uma peptidase cisteÃnica denominada peruvianina-I, foi purificada a partir do lÃtex atravÃs de um Ãnico passo cromatogrÃfico envolvendo filtraÃÃo em gel. A enzima foi inibida por E-64 e iodoacetamida (IAA) e seguiu a cinÃtica de Michaelis-Menten, apresentando alta afinidade à azocaseÃna, com um valor de Km de 17,6 ÂM, exibindo pH e temperatura Ãtimos de 5,0-6,0 e 25-37 ÂC, respectivamente. A peruvianina-I nÃo foi reconhecida por anticorpos anti-papaÃna. As Eletroforeses bidimensionais e a espectrometria de massas revelaram que a peruvianina-I (100 kDa) possui um pI de 4,0 e cinco subunidades (20 kDa). A sequÃncia de aminoÃcidos N-terminal da peruvianina-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36) mostrou similaridade à germinas ou âgermin-like proteinsâ. Imagens de alta resoluÃÃo a partir da microscopia de forÃa atÃmica indicaram uma possÃvel estrutura hexamÃrica da peruvianina-I, que està organizada como um trÃmero de dÃmeros, formando um canal central. TpLP e Peruvianina-I nÃo exibiram atividade de oxalato oxidase e superÃxido dismutase ou efeitos antifÃngicos sobre a germinaÃÃo de esporos de Fusarium solani. Este estudo mostrou que o lÃtex de T. peruviana à uma fonte rica em proteÃnas relacionadas à patogÃnese, incluindo peptidases cisteÃnicas. Curiosamente, estas peptidases apresentam caracterÃsticas estruturais e bioquÃmicas diferentes, que podem estar relacionadas com as suas funÃÃes fisiolÃgicas especÃficas.
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Gheura, Iuliana L. "Design, synthesis and evaluation of AZA-peptide epoxides as inhibitors of cysteine proteases." Diss., Georgia Institute of Technology, 2002. http://hdl.handle.net/1853/30571.
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Maury, Frédéric. "Etude de l'activite synoviale des cysteine-proteases au cours de la polyarthrite rhumatoide." Lille 2, 1993. http://www.theses.fr/1993LIL2M235.
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Gotz, Marion Gabriele. "Design, synthesis, and evaluation of irreversible peptidyl inhibitors for clan CA and clan CD cysteine proteases." Diss., Georgia Institute of Technology, 2004. http://hdl.handle.net/1853/8072.
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Cysteine proteases are a class of proteolytic enzymes, which are involved in a series of metabolic and catabolic processes, such as protein turnover, digestion, blood coagulation, apoptosis, fertilization and cell differentiation, and the immune response system. The development of novel potent and selective inhibitors for cysteine proteases has therefore gained increasing attention among medicinal chemists. In this thesis we have reported the design, synthesis, and evaluation of several peptidyl inhibitors for clan CA and clan CD cysteine proteases.
We have continued the investigation of dipeptidyl vinyl sulfones as potent and selective inhibitors for dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease, which is involved in the processing of intracellular proteases, such as granzymes. We have found that DPPI tolerates negatively charged amino acid residues in the P2 position with inhibition rates of 7,600 M-1s-1. Dipeptidyl vinyl sulfones with positively charged amino acid residues at the P1 position, however, do not inhibit DPPI at all.
A second project focused on the epoxidation of the double bond of the vinyl sulfone moiety of the dipeptidyl vinyl sulfones. Instead of epoxidizing the double bond, we found that an isomerization had occurred. The newly formed compounds were determined to be allyl sulfones. We tested this new class of inhibitors with clan CA proteases and obtained inhibition rates of 560 M-1s-1 for Cbz-Leu-Phe-AS-Ph with calpain I.
Two new classes of compounds for the clan CD protease S. mansoni legumain were designed, synthesized, and evaluated. Aza-peptidyl epoxides were found to be potent and selective inhibitors of S. mansoni legumain with IC50’s as low as 45 nM. Aza-peptide Michael acceptors were derived from the aza-peptide epoxide design and synthesized in an analogous fashion. The aza-peptide Michael acceptors inhibited S. mansoni legumain with even lower IC50’s, as low as 10 nM. However, the aza-peptide Michael acceptors react with thioalkylating agents contained in the buffer, such as DTT. The rates of degradation were determined spectroscopically, and half-lives of 3 to 20 minutes were measured. This observation gave us insights into the enzymatic mechanism and allowed us to determine the point of attack for the legumain active site cysteine thiol.
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Singh, Shweta. "Mechanisms of cell death in cerebellar granule neurones." Thesis, University of Kent, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.344143.
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Gurjar, Purujit. "Design and Synthesis of Anti Cancer Agents that Inhibit Cysteine Proteases, Limit Oxidative Stress or Terminate Proliferation of BCR-ABL Expressing Cells." University of Cincinnati / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1535635261401718.
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Götz, Marion Gabriele. "Design, synthesis, and evaluation of irreversible peptidyl inhibitors for clan CA and clan CD cysteine proteases." Available online, Georgia Institute of Technology, 2004, 2004. http://etd.gatech.edu/theses/available/etd-01282004-095929/unrestricted/Gotz%5FMarionG%5F200405%5Fphd2.pdf.
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Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2004.Dr. Suzanne Shuker, Committee Member ; Dr. Niren Murthy, Committee Member ; Dr. Donald Doyle, Committee Member ; Dr. Nicholas Hud, Committee Member ; Dr. James C. Powers, Committee Chair. Includes bibliographical references.
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Pullumbi, Ervin. "Further characterization of the large subunit of the major embryonic Artemia franciscana cysteine protease." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0019/MQ52640.pdf.
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Chiyanzu, Idan. "Synthesis and biological evaluation of antiparasitic Cysteine protease inhibitors based on the Isatin Scaffold." Master's thesis, University of Cape Town, 2004. http://hdl.handle.net/11427/6300.
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Includes bibliographical references.Widespread drug resistance, loss of efficacy and toxicity has limited the full utilization of the current available drugs against malaria and other parasitic diseases. This necessitates the development of new drugs. Meanwhile, the cysteine protease family of enzymes has been identified as potential targets for new modes of chemotherapy due to the numerous critical roles they play in the disease-causing agents. In this project, a non-peptidic and low molecular weight isatin (indole-2, 3-dione) possessing a wide range of pharmacological properties was used as a scaffold to which different moeities were appended. Potential inhibitors of parasitic cysteine proteases and three strains of P. falciparum were identified from synthesized libraries of compounds. Various N-substituted isatin derivatives were synthesized by KF/Ah03-mediated reaction of isatins with an alkyl, acyl or sulfonyl halide. A series of isatin-3-thiosemicarbazones were prepared by condensation of isatin I substituted isatins with thiosemicarbazide, and also a series of isatin-based Schiff and Mannich bases were prepared by reacting selected isatin-3-thiosemicarbazones with formaldehyde and appropriate secondary amines. To compare the effects of replacing the Mannich bases, a similar series of aminoquinolineethylene isatin-based derivatives were then synthesized. The synthesis was accomplished by condensation of quinoline-ethylene ketone forms with thiosemicarbazide. All synthesized compound were obtained in reasonable to excellent yields and characterized by spectroscopic and analytical techniques.
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Faro, Aline Regis. "Estudos estruturais e funcionais da Xylellaína, uma cisteíno protease da bactéria Xylella fastidiosa." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-15122008-150833/.
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A Xylella fastidiosa é uma bactéria gram-negativa que infecta o xilema das plantas causando muitas vezes a maturação precoce e a diminuição dos frutos. Ela é responsável por importantes perdas na economia, no Brasil é a causadora de doenças de Citrus Variegated Chlorosis (CVC) e a da doença de Pierce nos Estados Unidos. As proteases desempenham funções vitais no ciclo de vida de muitos parasitas, muitas estão envolvidas em processos infecciosos, a Xylellaína é uma cisteíno protease que é diferentemente expressa em cepas patogênicas a não patogênica. A compreensão de seu mecanismo catalítico, através do estudo da sua estrutura e função, pode ajudar no planejamento de inibidores seletivos, potenciais agentes contra as doenças fitopatológicas ocasionadas pela X. fastidiosa. Sua estrutura molecular foi elucidada no Laboratório de Cristalografia de Proteínas e Biologia Estrutural do Instituto de Física de São Carlos (USP), estudos estruturais mostraram que a proteína se apresenta na forma de uma pró-proteína, pois está inativa devido a uma pró-região que bloqueia o sítio catalítico. Também foi verificada a presença de um nucleotídeo na estrutura da Xylellaína próximo a pró-região, como hipótese foi considerada a relação entre o nucleotídeo e o mecanismo de ativação da proteína. A influência do nucleotídeo na atividade funcional da enzima foi constatada através da comparação de ensaios enzimáticos entre a enzima nativa e mutantes. As mutações foram planejadas com a intenção de ocasionar a desestabilização do nucleotídeo, por isso foram mutados os resíduos da pró-região que interagem diretamente com o ele. As mutações foram Fenilalanina 45 (F45), Arginina 43 (R43) e F45/R43, todos os resíduos foram mutados para Alaninas (A). Os resultados mostraram que os valores de Km obtidos para a proteína nativa e suas mutantes apresentaram consideráveis alterações quando comparado entre eles, esse efeito não foi percebido para a eficiência catalítica. Conclui-se que as mutações pouco alteraram a capacidade da enzima converter o subsrato em produto, mas houve significantes alterações no reconhecimento do substrato. Esse resultado corrobora com a hipótese de que a existência do nucleotídeo está relacionada com o mecanismo de ativação da proteína.Xylella fastidiosa is a Gram-negative bacterium which infects the plant xylem system causing in many cases precocious maturation and diminution of fruits. It is responsible for economically important plant diseases, such as the Citrus Variegated Chlorosis (CVC). Proteases might be involved in the infection process by disrupting plant tissue. Xylellain is a cysteine protease which is differently expressed in strain pathogen and non-pathogen of X. fastidiosa. The 3D structure of xylellain was solved by our group and structural studies show that this protein has a proenzyme form and a ribonucleotideo close to the amine terminal region. Our hypothesis is that protein-nucleotide interactions are related to xylellains activation mechanism. To evaluate the influence of the nucleotide in the functional activity of enzyme, point mutations in aminoacids which interact directly with this ribonucleotide were carried out. The point mutations are phenylalanine 45 (F45) and arginine 43 (R43), individually mutated for alanine (A) residues. One way to quantify the changes caused by the alteration of a nucleotide is the direct comparison between the kinetic enzyme assays of native and mutant proteins. Greater variations between the values of Km than in the values of catalytic efficiency were observed. This suggests that the speed of production varied by enzyme-substrate. However the mutations caused little change on the ability of the protease to catalyze the reaction. This result is in agreement with the hypothesis that the nucleotide provides the structural support for the hinge formation on the N-terminal domain, thus directing the inhibitory peptide inside the active site of the enzyme. Therefore, the nucleotide may be exerting regulatory functions in vivo, possibly in the folding or activation of the protein and performance of catalytic function.
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Arispe, Angulo Wara Milenka Trawick Mary Lynn. "Inhibitors of human cathepsin L and cruzain as therapeutic agents." Waco, Tex. : Baylor University, 2008. http://hdl.handle.net/2104/5290.
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Ranjit, Najju. "Characterisation of proteases involved in proteolytic degradation of haemoglobin in the human hookworm Necator americanus." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/20651/1/Najju_Ranjit_Thesis.pdf.
Full textAbstract:
With over a billion people infected world wide, hookworms are considered as important human pathogens, particularly in developing countries which have the highest rates of infections. Hookworms reside in the gastrointestinal tract of the host where they continuously feed on blood, leading to conditions such as chronic irondeficiency anaemia. The majority of blood-feeding parasites rely on proteins found in blood to provide many of their nutritional requirements for growth, reproduction and survival. Of the numerous proteins found in blood, haemoglobin (Hb) is one of the most abundant. In order to acquire amino acids for protein synthesis, it is thought that haematophagous parasites degrade Hb using various classes of endo- and exoproteases, in a manner similar to that which occurs in catabolism of proteins in mammalian cellular lysosomes. This study identified and characterised proteases involved in the Hb degradation process in the human hookworm, Necator americanus, in order to identify potential candidate antigens for a vaccine that interrupts blood-feeding. Red blood cells ingested by hookworms are lysed to release Hb, which is cleaved by various proteases into dipeptides or free amino acids and these are taken up through the gut membrane by amino acid transporters. Proteases expressed in the intestinal tract of hookworms are thought to play a major role in this process and would therefore make good targets for vaccine candidates aimed at interrupting blood-feeding. To identify these proteases, adult hookworms (both N. americanus and Ancylostoma caninum) were sectioned and intestinal tissue was dissected via laser microdissection microscopy. RNA extracted from the dissected tissue was used to generate gut-specific cDNA, which then was used to create plasmid libraries. Each library was subjected to shotgun sequencing, and of the 480 expressed sequence tags (ESTs) sequenced from each species, 268 and 276 contigs were assembled from the N. americanus and A. caninum libraries, respectively. Nine percent of N. americanus and 6.5% of A. caninum contigs were considered novel as no homologues were identified in any published/accessible database. The gene ontology (GO) classification system was used to categorise the contigs to predicted biological functions. Only 17% and 38% of N. americanus and A. caninum contigs, respectively, were assigned GO categories, while the rest were classified as being of unknown function. The most highly represented GO categories were molecular functions such as protein binding and catalytic activity. The most abundant transcripts encoded fatty acid binding proteins, C-type lectins and activation associated secreted proteins, indicative of the diversity of functions that occur in this complex organ. Of particular interest to this study were the contigs that encoded for cysteine and metalloproteases, expanding the list of potential N. americanus haemoglobinases. In the N. americanus cDNA library, four contigs encoding for cathepsin B cysteine proteases were identified. Three contigs from the A. caninum and one contig from the N. americanus cDNA libraries encoded for metalloproteases, including astacin-like and O-sialoglycoprotein endopeptidases, neither of which had previously been reported from adult hookworms. Apart from haemoglobinases, other mRNAs encoding potential vaccine candidate molecules were identified, including anti-clotting factors, defensins and membrane proteins. This study confirmed that the gut of hookworms encodes a diverse range of proteases, some of which are likely to be involved in Hb digestion and have the potential to be hidden (cryptic) vaccine antigens. Four cysteine proteases (Na-CP-2, -3, -4 and -5) were identified from the gut cDNA library of N. americanus. All four proteases belong to the clan CA, family C1, share homology with human cathepsin B and possess a modified occluding loop. Real-time PCR indicated that all transcripts were up-regulated in the adult stage of the hookworm parasite with high levels of mRNA expression detected in gut cDNA. All four proteases were expressed in recombinant form, but only Na-CP-3 was successfully expressed in soluble form in the yeast Pichia pastoris. Proteolytic activity for Na-CP-3 was detected on a gelatin zymogen gel, however no catalytic activity was detected against the class-specific fluorogenic peptides Z-Phe-Arg-AMC and Z-Arg-Arg-AMC. Mass spectrometry analysis of the purified protein suggested that the pro-region had not been processed in trans when the protein was secreted by yeast. Incubation of Na-CP-3 in salt buffers containing dextran sulfate resulted in autoprocessing of the pro-region as detected by Western blot and catalytic activity was detected against Z-Phe-Arg-AMC. Activated Na-CP-3 did not digest intact tetrameric human Hb. The other three cysteine proteases (Na-CP-2, -4, and -5) were expressed in insoluble form in Escherichia coli. Antibodies to all four proteins (Na- CP-2 to 5) immunolocalised to the gut region of the adult worm, supporting mRNA amplification results and strongly indicated that they might play a role in nutrient acquisition. Hb digestion in blood feeding parasites such as schistosomes and Plasmodium spp. occurs via a semi-ordered cascade of proteolysis involving numerous enzymes. In Plasmodium falciparum, at least three distinct mechanistic classes of endopeptidases have been implicated in this process, and at least two classes have been implicated in schistosomes. A similar process is thought to occur in hookworms. An aspartic protease, Na-APR-1, was expressed in P. pastoris and purified protein was shown to cleave the class-specific fluorogenic peptide 7- Methoxycoumarin-4-Acetyl-GKPILFFRLK(DNP)-D-Arg-Amide. Recombinant Na- APR-1 was able to cleave intact human Hb and completely degrade the 16 kDa monomer and 32 kDa dimer within one hour. Recombinant Na-CP-3 was not able to cleave intact Hb, but was able to further digest globin fragments that had previously been digested with Na-APR-1. A clan MA metalloprotease, Na-MEP-1, was identified in gut tissue of N. americanus and was expressed in recombinant form in Hi5 insect cells using the baculovirus expression system. Recombinant Na-MEP-1 displayed proteolytic activity when assessed by gelatin zymography, but was incapable of cleaving intact Hb. However, Na-MEP-1 did cleave globin fragments which had previously been incubated with Na-APR-1 and Na-CP-3. Hb digested with all three proteases was subjected to reverse phase HPLC and peptides were analysed using Liquid Chromatography-Mass Spectrometry (LC-MS). A total of 74 cleavage sites were identified within Hb ƒ¿ and ƒÀ chains. Na-APR-1 was responsible for cleavage of Hb at the hinge region, probably unravelling the molecule so that Na- CP-3 and Na-MEP-1 could gain access to globin peptides. All three proteases were promiscuous in their subsite specificities, but the most common P1-P1�Œ residues were hydrophobic and/or bulky in nature, such as Phe, Leu and Ala. Antibodies to all three proteins (Na-APR-1, -CP-3, -MEP-1) immunolocalised to the gut region of the worm, further supporting their roles in Hb degradation. These results suggest that Hb degradation in N. americanus follows a similar pattern to that which has been described in Plasomdium falciparum. Studies conducted in this project have identified a number of potential haemoglobinases and have demonstrated that the gut region of the hookworm contains a multitude of proteases which could be targeted for production of new chemotherapies or as vaccine candidates. Results presented here also suggest that the Hb degradation process occurs in an ordered cascade, similar to those which have been reported in other haematophagous parasites. More importantly, it has been confirmed that Na-APR-1 plays a crucial role in the initiation of the Hb degradation process and therefore targeting this molecule as a vaccine candidate could provide high levels of protection against hookworm infection.
48
Ranjit, Najju. "Characterisation of proteases involved in proteolytic degradation of haemoglobin in the human hookworm Necator americanus." Queensland University of Technology, 2008. http://eprints.qut.edu.au/20651/.
Full textAbstract:
With over a billion people infected world wide, hookworms are considered as important human pathogens, particularly in developing countries which have the highest rates of infections. Hookworms reside in the gastrointestinal tract of the host where they continuously feed on blood, leading to conditions such as chronic irondeficiency anaemia. The majority of blood-feeding parasites rely on proteins found in blood to provide many of their nutritional requirements for growth, reproduction and survival. Of the numerous proteins found in blood, haemoglobin (Hb) is one of the most abundant. In order to acquire amino acids for protein synthesis, it is thought that haematophagous parasites degrade Hb using various classes of endo- and exoproteases, in a manner similar to that which occurs in catabolism of proteins in mammalian cellular lysosomes. This study identified and characterised proteases involved in the Hb degradation process in the human hookworm, Necator americanus, in order to identify potential candidate antigens for a vaccine that interrupts blood-feeding. Red blood cells ingested by hookworms are lysed to release Hb, which is cleaved by various proteases into dipeptides or free amino acids and these are taken up through the gut membrane by amino acid transporters. Proteases expressed in the intestinal tract of hookworms are thought to play a major role in this process and would therefore make good targets for vaccine candidates aimed at interrupting blood-feeding. To identify these proteases, adult hookworms (both N. americanus and Ancylostoma caninum) were sectioned and intestinal tissue was dissected via laser microdissection microscopy. RNA extracted from the dissected tissue was used to generate gut-specific cDNA, which then was used to create plasmid libraries. Each library was subjected to shotgun sequencing, and of the 480 expressed sequence tags (ESTs) sequenced from each species, 268 and 276 contigs were assembled from the N. americanus and A. caninum libraries, respectively. Nine percent of N. americanus and 6.5% of A. caninum contigs were considered novel as no homologues were identified in any published/accessible database. The gene ontology (GO) classification system was used to categorise the contigs to predicted biological functions. Only 17% and 38% of N. americanus and A. caninum contigs, respectively, were assigned GO categories, while the rest were classified as being of unknown function. The most highly represented GO categories were molecular functions such as protein binding and catalytic activity. The most abundant transcripts encoded fatty acid binding proteins, C-type lectins and activation associated secreted proteins, indicative of the diversity of functions that occur in this complex organ. Of particular interest to this study were the contigs that encoded for cysteine and metalloproteases, expanding the list of potential N. americanus haemoglobinases. In the N. americanus cDNA library, four contigs encoding for cathepsin B cysteine proteases were identified. Three contigs from the A. caninum and one contig from the N. americanus cDNA libraries encoded for metalloproteases, including astacin-like and O-sialoglycoprotein endopeptidases, neither of which had previously been reported from adult hookworms. Apart from haemoglobinases, other mRNAs encoding potential vaccine candidate molecules were identified, including anti-clotting factors, defensins and membrane proteins. This study confirmed that the gut of hookworms encodes a diverse range of proteases, some of which are likely to be involved in Hb digestion and have the potential to be hidden (cryptic) vaccine antigens. Four cysteine proteases (Na-CP-2, -3, -4 and -5) were identified from the gut cDNA library of N. americanus. All four proteases belong to the clan CA, family C1, share homology with human cathepsin B and possess a modified occluding loop. Real-time PCR indicated that all transcripts were up-regulated in the adult stage of the hookworm parasite with high levels of mRNA expression detected in gut cDNA. All four proteases were expressed in recombinant form, but only Na-CP-3 was successfully expressed in soluble form in the yeast Pichia pastoris. Proteolytic activity for Na-CP-3 was detected on a gelatin zymogen gel, however no catalytic activity was detected against the class-specific fluorogenic peptides Z-Phe-Arg-AMC and Z-Arg-Arg-AMC. Mass spectrometry analysis of the purified protein suggested that the pro-region had not been processed in trans when the protein was secreted by yeast. Incubation of Na-CP-3 in salt buffers containing dextran sulfate resulted in autoprocessing of the pro-region as detected by Western blot and catalytic activity was detected against Z-Phe-Arg-AMC. Activated Na-CP-3 did not digest intact tetrameric human Hb. The other three cysteine proteases (Na-CP-2, -4, and -5) were expressed in insoluble form in Escherichia coli. Antibodies to all four proteins (Na- CP-2 to 5) immunolocalised to the gut region of the adult worm, supporting mRNA amplification results and strongly indicated that they might play a role in nutrient acquisition. Hb digestion in blood feeding parasites such as schistosomes and Plasmodium spp. occurs via a semi-ordered cascade of proteolysis involving numerous enzymes. In Plasmodium falciparum, at least three distinct mechanistic classes of endopeptidases have been implicated in this process, and at least two classes have been implicated in schistosomes. A similar process is thought to occur in hookworms. An aspartic protease, Na-APR-1, was expressed in P. pastoris and purified protein was shown to cleave the class-specific fluorogenic peptide 7- Methoxycoumarin-4-Acetyl-GKPILFFRLK(DNP)-D-Arg-Amide. Recombinant Na- APR-1 was able to cleave intact human Hb and completely degrade the 16 kDa monomer and 32 kDa dimer within one hour. Recombinant Na-CP-3 was not able to cleave intact Hb, but was able to further digest globin fragments that had previously been digested with Na-APR-1. A clan MA metalloprotease, Na-MEP-1, was identified in gut tissue of N. americanus and was expressed in recombinant form in Hi5 insect cells using the baculovirus expression system. Recombinant Na-MEP-1 displayed proteolytic activity when assessed by gelatin zymography, but was incapable of cleaving intact Hb. However, Na-MEP-1 did cleave globin fragments which had previously been incubated with Na-APR-1 and Na-CP-3. Hb digested with all three proteases was subjected to reverse phase HPLC and peptides were analysed using Liquid Chromatography-Mass Spectrometry (LC-MS). A total of 74 cleavage sites were identified within Hb ƒ¿ and ƒÀ chains. Na-APR-1 was responsible for cleavage of Hb at the hinge region, probably unravelling the molecule so that Na- CP-3 and Na-MEP-1 could gain access to globin peptides. All three proteases were promiscuous in their subsite specificities, but the most common P1-P1Œ residues were hydrophobic and/or bulky in nature, such as Phe, Leu and Ala. Antibodies to all three proteins (Na-APR-1, -CP-3, -MEP-1) immunolocalised to the gut region of the worm, further supporting their roles in Hb degradation. These results suggest that Hb degradation in N. americanus follows a similar pattern to that which has been described in Plasomdium falciparum. Studies conducted in this project have identified a number of potential haemoglobinases and have demonstrated that the gut region of the hookworm contains a multitude of proteases which could be targeted for production of new chemotherapies or as vaccine candidates. Results presented here also suggest that the Hb degradation process occurs in an ordered cascade, similar to those which have been reported in other haematophagous parasites. More importantly, it has been confirmed that Na-APR-1 plays a crucial role in the initiation of the Hb degradation process and therefore targeting this molecule as a vaccine candidate could provide high levels of protection against hookworm infection.
49
Aiton, Andrea Lynn. "Further characterization of the major cysteine protease of Artemia franciscana cysts, and the isolation of a cDNA encoding the small subunit of the protease." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0022/MQ30860.pdf.
Full text
50
Massimi, Isabella. "SspB cysteine protease of Staphylococcus aureus promotes detachment of human keratinocytes and degrades fibronectin and vitronectin." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/MQ63102.pdf.
Full text