Comparative study of clan CA cysteine proteases: an insight into the protozoan parasites
- Authors: Moyo, Sipho Dugunye
- Date: 2015
- Subjects: Cysteine proteinases , Proteolytic enzymes , Protozoan diseases , Parasites , Protozoan diseases -- Chemotherapy , Bioinformatics , Plasmodium , Antiprotozoal agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4165 , http://hdl.handle.net/10962/d1020309
- Description: Protozoan infections such as Malaria, Leishmaniasis, Toxoplasmosis, Chaga’s disease and African trypanosomiasis caused by the Plasmodium, Leishmania, Toxoplasma and Trypanosoma genuses respectively; inflict a huge economic, health and social impact in endemic regions particularly tropical and sub-tropical regions. The combined infections are estimated at over a billion annually and approximately 1.1 million deaths annually. The global burden of the protozoan infections is worsened by the increased drug resistance, toxicity and the relatively high cost of treatment and prophylaxis. Therefore there has been a high demand for new drugs and drug targets that play a role in parasite virulence. Cysteine proteases have been validated as viable drug targets due to their role in the infectivity stage of the parasites within the human host. There is a variety of cysteine proteases hence they are subdivided into families and in this study we focus on the clan CA, papain family C1 proteases. The current inhibitors for the protozoan cysteine proteases lack selectivity and specificity which contributes to drug toxicity. Therefore there is a need to identify the differences and similarities between the host, vector and protozoan proteases. This study uses a variety of bioinformatics tools to assess these differences and similarities. The Plasmodium cysteine protease FP-2 is the most characterized protease hence it was used as a reference to all the other proteases and its homologs were retrieved, aligned and the evolutionary relationships established. The homologs were also analysed for common motifs and the physicochemical properties determined which were validated using the Kruskal-Wallis test. These analyses revealed that the host and vector cathepsins share similar properties while the parasite cathepsins differ. At sub-site level sub-site 2 showed greater variations suggesting diverse ligand specificity within the proteases, a revelation that is vital in the design of antiprotozoan inhibitors.
- Full Text:
- Date Issued: 2015
Malarial drug targets cysteine proteases as hemoglobinases
- Authors: Mokoena, Fortunate
- Date: 2012
- Subjects: Malaria -- Chemotherapy , Antimalarials , Hemoglobin , Proteolytic enzymes , Cysteine proteinases , Plasmodium falciparum , Plasmodium vivax , Papain
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4005 , http://hdl.handle.net/10962/d1004065 , Malaria -- Chemotherapy , Antimalarials , Hemoglobin , Proteolytic enzymes , Cysteine proteinases , Plasmodium falciparum , Plasmodium vivax , Papain
- Description: Malaria has consistently been rated as the worst parasitic disease in the world. This disease affects an estimated 5 billion households annually. Malaria has a high mortality rate leading to distorted socio-economic development of the world at large. The major challenge pertaining to malaria is its continuous and rapid spread together with the emergence of drug resistance in Plasmodium species (vector agent of the disease). For this reason, researchers throughout the world are following new leads for possible drug targets and therefore, investigating ways of curbing the spread of the disease. Cysteine proteases have emerged as potential antimalarial chemotherapeutic targets. These particular proteases are found in all living organisms, Plasmodium cysteine proteases are known to degrade host hemoglobin during the life cycle of the parasite within the human host. The main objective of this study was to use various in silico methods to analyze the hemoglobinase function of cysteine proteases in P. falciparum and P. vivax. Falcipain-2 (FP2) of P. falciparum is the best characterized of these enzymes, it is a validated drug target. Both the three-dimensional structures of FP2 and its close homologue falcipain-3 (FP3) have been solved by the experimental technique X-ray crystallography. However, the homologue falcipain-2 (FP2’)’ and orthologues from P.vivax vivapain-2 (VP2) and vivapain-3 (VP3) have yet to be elucidated by experimental techniques. In an effort to achieve the principal goal of the study, homology models of the protein structures not already elucidated by experimental methods (FP2’, VP2 and VP3) were calculated using the well known spatial restraint program MODELLER. The derived models, FP2 and FP3 were docked to hemoglobin (their natural substrate). The protein-protein docking was done using the unbound docking program ZDOCK. The substrate-enzyme interactions were analyzed and amino acids involved in binding were observed. It is anticipated that the results obtained from the study will help focus inhibitor design for potential drugs against malaria. The residues found in both the P. falciparum and P. vivax cysteine proteases involved in hemoglobin binding have been identified and some of these are proposed to be the main focus for the design of a peptidomimetric inhibitor.
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- Date Issued: 2012
Purification and partial characterisation of cathepsin D from ostrich skeletal muscle, and its activity during meat maturation
- Authors: Krause, Jason
- Date: 2009
- Subjects: Proteolytic enzymes , Ostrich products industry
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10313 , http://hdl.handle.net/10948/1461 , Proteolytic enzymes , Ostrich products industry
- Description: Cathepsin D, a muscle proteinase, participates in lysosomally mediated protein degradation in vivo. This enzyme has been proposed to play a significant role in the postmortem proteolysis process apparently associated with tenderisation. The lack of data on the postmortem characteristics of ostrich meat, especially on the ageing process and its influence on meat tenderness, called for an investigation into this process. There is no data available for purified ostrich cathepsin D, and the aim of this study was, therefore, to isolate, purify and characterise cathepsin D from ostrich skeletal muscle and subsequently investigate the possible role that it may have in the tenderisation process of meat. Cathepsin D was successfully isolated and purified from ostrich skeletal muscle using pepstatin A-agarose chromatography. The purified enzyme was composed of two subunits (14 and 29kDa). The amino acid composition as well as the N-terminal amino acid sequence of both subunits were determined. Kinetic parameters (Km and Vm), thermodynamic parameters (Ea, ∆H, ∆S and ∆G) and functional characteristics (effect of pH, temperature and various inhibitors on cathepsin D activity) were determined and are reported in this study. Ostrich muscle cathepsin D showed a pH optimum of 4 and a temperature optimum of 45°C. The activity of cathepsin D was strongly inhibited by pepstatin A and DTT. Purified ostrich cathepsin D displayed kinetic and functional properties similar to previously reported values from various species. The effect of storage on the activity of cathepsin D was investigated over a 30 day period. It was established that substantial postmortem cathepsin D activity remained throughout the storage period, to implicate cathepsin D, fulfilling a possible role in meat maturation.
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- Date Issued: 2009
Purification and partial characterization of a Myofibril-Bound Serine Protease and its endogenous inhibitor from skeletal muscle of the ostrich
- Authors: Tshidino, Shonisani Cathphonia
- Date: 2008
- Subjects: Proteolytic enzymes , Ostrich products industry
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10330 , http://hdl.handle.net/10948/703 , Proteolytic enzymes , Ostrich products industry
- Description: The ostrich is becoming an important source of meat for humans in developed and developing countries. This study was conducted to purify and characterize myofibrilbound serine protease (MBSP) and its endogenous inhibitor (MBSPI) from skeletal muscle of the ostrich. It is well documented that MBSP is tightly bound to myofibrils and its endogenous inhibitor has been purified from the same tissue of other studied mammalian species. Literature supports an association of MBSP and its endogenous inhibitor with the degradation of myofribrillar proteins, resulting in the softening of muscle that lead to the conversion of muscle into meat with the control of the inhibitor. MBSP was successfully dissociated from washed myofibrils by 40 percent ethylene glycol at pH 8.5. Following centrifugation, MBSP was partially purified in two chromatographic steps, namely Toyopearl Super Q 650S and p-aminobenzamidine-Agarose. On the other hand, MBSPI was fractionated from the sarcoplasmic fraction using 75 percent ammonium sulfate saturation, followed by centrifugation and partially purified by three chromatographic steps, namely Toyopearl Super Q 650S, Superdex 200 and HiTrap SP HR. Ostrich MBSP was physicochemically and kinetically characterized, while MBSPI was only physicochemically characterized. Ostrich MBSP revealed an Mr of 21 kDa, cleaving synthetic fluorogenic substrates specifically at the carboxyl side of arginine residues. Optimum pH and temperature of ostrich MBSP were 8.0 and 40˚C, respectively. Kinetic parameters (Km and Vmax values) were calculated from Lineweaver-Burk plots. The characteristics of ostrich MBSP were compared to the values obtained for commercial bovine trypsin in this study, as well as that obtained for MBSP from various fish species and mouse. The results suggest that ostrich MBSP is a trypsin-like serine protease, thereby confirming the existence of MBSP in ostrich skeletal muscle. Partially purified ostrich MBSPI (Mr 17 kDa) (one form) shares 100 percent identity to myoglobin from the same species, while 2 other forms of MBSPI (Mr values of 35 and 36 kDa) exhibited high sequence identity to glyceraldehyde 3- phosphate dehydrogenase (GAPDH) (76 percent) from human and rat.
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- Date Issued: 2008
SphereZyme (TM) technology for enhanced enzyme immobilisation application in biosensors
- Authors: Molawa, Letshego Gloria
- Date: 2011
- Subjects: Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3989 , http://hdl.handle.net/10962/d1004048 , Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Description: Self-immobilisation enzyme technologies, such as SphereZyme™, suffer from the lack of applicability to hydrolyse large substrates. Solid support immobilisation is usually a method of choice, to produce a stable biocatalyst for large substrates hydrolysis in the industry. In order to investigate this limitation, a commercial protease called Alcalase® was chosen as a model enzyme due to its natural activity (hydrolysis of large substrates-proteins). Prior to immobilising through the SphereZyme™ technology, Alcalase® was partially purified through dialysis followed by CM Sepharose™ FF cation exchanger. Sample contaminants, such as salts and stabilisers can inhibit protein crosslinking by reacting with glutaraldehyde. Alcalase® was successfully separated into 3 proteases with the major peak correlating to a positive control run on native PAGE, indicating that it was likely subtilisin Carlsberg. A 16% alkaline protease activity for azo-casein hydrolysis was retained when 5% v/v PEI: 25% v/v glutaraldehyde solution was used as a crosslinking agent in Alcalase® SphereZyme™ production. An increase in activity was also observed for monomeric substrates (PNPA) where the highest was 55%. The highest % activities maintained when 0.33 M EDA: 25% v/v glutaraldehyde solution was initially used as crosslinking agent were 4.5% and 1.6% for monomeric and polymeric substrates, respectively. PEI is a hydrophilic branched polymer with an abundance of amine groups compared to EDA. A comparison study of immobilisation efficiencies of SphereZyme™, Eupergit® and Dendrispheres was also performed for large substrate biocatalysis. The two latter technologies are solid-support immobilisation methods. Dendrispheres reached its maximum loading capacity in the first 5 minute of the one hour binding time. Twenty minutes was chosen as a maximum binding time since there was constant protein maintained on the solid support and no enzyme loss was observed during the 1 hour binding time. PEI at pH 11.5, its native pH, gave the highest immobilisation yield and specific activity over the PEI pH range of 11.5 to 7. SphereZyme™ had the highest ratio for azocasein hydrolysis followed by Dendrispheres and Eupergit®. The SphereZyme™ was also shown to be applicable to biosensors for phenol detection. Different modifications of glassy carbon electrode (GCE) were evaluated as a benchmark for the fabrication of SphereZyme™ modified phenol biosensor. GCE modified with laccase SphereZyme™ entrapped in cellulose membrane was the best modification due to the broad catechol range (<0.950 mM), high correlation coefficient (R2, 0.995) and relative high sensitivity factor (0.305 μA.mM-1). This type of biosensor was also shown to be electroactive at pH 7.0 for which its control, free laccase, lacked electroactivity. From the catalytic constants calculated, GCE modified with laccase SphereZyme™ entrapped in cellulose membrane also gave the highest effectiveness factor (Imax/Km app) of 1.84 μA.mM-1. The modified GCE with Alcalase® SphereZyme™ was relatively more sensitive than GCE modified with free Alcalase®.
- Full Text:
- Date Issued: 2011
Structural analysis of prodomain inhibition of cysteine proteases in plasmodium species
- Authors: Njuguna, Joyce Njoki
- Date: 2012
- Subjects: Plasmodium , Cysteine proteinases , Proteolytic enzymes , Malaria -- Chemotherapy , Antimalarials , Plasmodium falciparum
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4021 , http://hdl.handle.net/10962/d1004081 , Plasmodium , Cysteine proteinases , Proteolytic enzymes , Malaria -- Chemotherapy , Antimalarials , Plasmodium falciparum
- Description: Plasmodium is a genus of parasites causing malaria, a virulent protozoan infection in humans resulting in over a million deaths annually. Treatment of malaria is increasingly limited by parasite resistance to available drugs. Hence, there is a need to identify new drug targets and authenticate antimalarial compounds that act on these targets. A relatively new therapeutic approach targets proteolytic enzymes responsible for parasite‟s invasion, rupture and hemoglobin degradation at the erythrocytic stage of infection. Cysteine proteases (CPs) are essential for these crucial roles in the intraerythrocytic parasite. CPs are a diverse group of enzymes subdivided into clans and further subdivided into families. Our interest is in Clan CA, papain family C1 proteases, whose members play numerous roles in human and parasitic metabolism. These proteases are produced as zymogens having an N-terminal extension known as the prodomain which regulates the protease activity by selectively inhibiting its active site, preventing substrate access. A Clan CA protease Falcipain-2 (FP-2) of Plasmodium falciparum is a validated drug target but little is known of its orthologs in other malarial Plasmodium species. This study uses various structural bioinformatics approaches to characterise the prodomain‟s regulatory effect in FP-2 and its orthologs in Plasmodium species (P. vivax, P. berghei, P. knowlesi, P. ovale, P. chabaudi and P. yoelii). This was in an effort to discover short peptides with essential residues to mimic the prodomain‟s inhibition of these proteases, as potential peptidomimetic therapeutic agents. Residues in the prodomain region that spans over the active site are most likely to interact with the subsite residues inhibiting the protease. Sequence analysis revealed conservation of residues in this region of Plasmodium proteases that differed significantly in human proteases. Further prediction of the 3D structure of these proteases by homology modelling allowed visualisation of these interactions revealing differences between parasite and human proteases which will lead to significant contribution in structure based malarial inhibitor design.
- Full Text:
- Date Issued: 2012