Molecular Cloning, Expression, And Characterization Of Glutathione-Stransferase As A Novel Target In Antimalarial Drug Design And Discovery

Glutation-S-transferase (GSTs) adalah sekumpulan enzim detoksifikasi. Plasmodium falciparum mempunyai isoform tunggal GST (PfGST) yang terlibat dalam bagi detoksifikasi heme. The Glutathione-S-transferases (GSTs) are group of detoxification enzymes. Plasmodium falciparum has a single isoform of GST (PfGST) that involves in heme detoxification. While other GSTs isoforms from human (hGSTP1) and mouse (mGSTM1) are involved in apoptotic stress kinase pathway and mediate cancer cell resistance to chemotherapy

The role of a conserved interdomain salt bridge on the structure, function and stability of the Y-GSTs

Domain interfaces are important to the folding, stability, structure and function of multidomain proteins. In the case of human glutathione S-transferase A1-1 (hGSTA1-1) site-directed mutagenesis studies have previously implicated the interdomain Arg13 residue of the protein in maintaining the proper catalytic function of the GST though its exact role was never determined (Stenberg et al., 1991). In this study it was shown by structural and sequence alignment of many representatives of the GST family and other thioredoxin-fold containing proteins that Arg13 is also highly conserved throughout the Alpha, Mu, Pi, Plasmodium falciparum and Sigma classes, all of which are Y-GSTs, and that it forms an interdomain salt bridge. This study therefore chose to evaluate the contribution of Arg13 towards the structure, stability and function of hGSTA1-1 by mutating the Arg residue to an Ala and performing comparative studies between wild-type and R13A hGSTA1-1. The spectral properties of R13A hGSTA1-1 monitored using far-ultraviolet circular dichroism and fluorescence indicated no significant changes in the secondary structure as compared to the native protein though fluorescence did indicate local tertiary structural changes around Trp21. Additionally, the catalytic activity of the R13A variant was reduced by 70% as compared to that of the wild-type enzyme further indicating local tertiary structural changes at and possibly near the active site which is located near the Trp21 residue. Conformational stability studies were performed by monitoring both thermal- and chemical-induced protein unfolding. The stability of the R13A variant was lower than that of the wild-type protein as revealed by a thermal-induced unfolding study which indicated that the melting point (Tm) of the R13A variant was 6 °C lower than that of the wild-type. Thermal-induced unfolding was shown not to be reversible however and the thermodynamic parameters of unfolding could not be determined. Urea-induced equilibrium unfolding studies on the other hand were reversible and displayed a variant-induced destabilisation of the conformation of the protein with a ΔΔG(H2O) of 16.7 kJ.mol−1 between the mutant and native protein. Additionally urea-induced equilibrium unfolding studies in the presence of ANS indicated that the equilibrium unfolding of both wild-type and R13A hGSTA1-1 was three-state. In summary the Arg13 residue is more important to the function of the protein than it is for its global stability or structure. Also since the Arg13 residue was found to be highly conserved in all the Y-GSTs and that it forms an interdomain interaction, the residue most likely performs a similar role in each of the Y-GSTs as well

Drug Discovery

Natural products are a constant source of potentially active compounds for the treatment of various disorders. The Middle East and tropical regions are believed to have the richest supplies of natural products in the world. Plant derived secondary metabolites have been used by humans to treat acute infections, health disorders and chronic illness for tens of thousands of years. Only during the last 100 years have natural products been largely replaced by synthetic drugs. Estimates of 200 000 natural products in plant species have been revised upward as mass spectrometry techniques have developed. For developing countries the identification and use of endogenous medicinal plants as cures against cancers has become attractive. Books on drug discovery will play vital role in the new era of disease treatment using natural products

The Identification of Small Molecule Inhibitors to Candida albicans Phosphatidylserine Synthase

Candida albicans phosphatidylserine (PS) synthase, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis due to its importance in virulence, absence in the host and conservation among fungal pathogens. This dissertation is focused on the identification of inhibitors for this membrane enzyme. Cho1 has two substrates: cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine. Previous studies identified a conserved CDP-alcohol phosphotransferase (CAPT) binding motif present within Cho1, and here we revealed that mutations in all but one conserved amino acid within the CAPT motif resulted in decreased Cho1. For serine, we have predicted a serine-binding site based on sequence alignment and found that some of the residues in this putative serine-binding site are required for Cho1 function. One residue, R189, is particularly interesting because it was suggested to be involved in serine binding. Then, we attempted to perform a small molecule screening on C .albicans Cho1, which will be facilitated by purified Cho1 protein. Due to the transmembrane nature, several solubilizing reagents were used to solubilize Cho1 protein. Digitonin was determined to be the best detergent as it retained the most PS synthase activity. Pull-downs of HA-tagged Cho1 in the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Biochemical and electron microscopy analysis suggest that the hexamer is composed of a trimer of dimers. Cho1 protein was then purified to near-homogeneity as a hexamer and was optimized for high activity to be used in the small drug screening. For the screening, we developed a nucleotidase-coupled malachite green-based screen against purified Cho1. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform, and seven compounds were identified to inhibit purified Cho1. Among all, compound CBR-5884 disrupted in vivo Cho1 function by inducing phenotypes consistent with the cho1∆∆ mutant, including a reduction of cellular PS levels. Kinetic curves and computational docking suggest that CBR-5884 competes with serine for binding of Cho1 with a Ki of 1,550 ± 245.6 nM, thus this compound has the potential for further drug design

The N-subdomain of the thioredoxin fold of glutathione transferase is stabilised by topologically conserved leucine residue

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2012The thioredoxin-like (Trx-like) fold is preserved in various protein families with diverse functions despite their low sequence identity. Glutathione transferases (GSTs) are characterised by a conserved N-terminal domain with a thioredoxin–like βαβαββα secondary structure topology and an all alpha-helical domain. GSTs are the principal phase II enzymes involved in protecting cellular macromolecules from a wide variety of reactive electrophilic compounds. It catalyses the conjugation of reduced glutathione (GSH) to an electrophilic substrate to form a hydrophilic and non-toxic compound. The binding site for GSH (G-site) is located in the N-terminal domain of GSTs. The sequence identity within members of the Trx-like superfamily is low; however, the members of this family fold into a conserved βαβαββα topology. It, therefore, seems reasonable that there are topologically conserved residues within this fold whose main role is to drive folding and/or maintain the structural integrity of the Trx-like fold. Structural alignments of the N-subdomain (βαβ motif) of the GST family shows that Leu7 in β1 and Leu23 in α1 are topologically conserved residues. The Leu7 side chain is involved in the packing of α1β1α2 and α3, whilst Leu23 is mainly involved in van der Waals interactions with residues in α1 and the loop region connecting α1 and β2. Taking into account the types of interaction that both Leu7 and Leu23 are involved in, as well their location in close proximity to the G-site, it was postulated that both these residues may play a role in the structure, function and stability of the GST family of proteins. Leu7 and Leu23 are not directly involved in the binding of GSH but they could be important in maintaining the G-site in a functional conformation via correct packing of the Nsubdomain. The homodimeric human class Alpha of GST (hGSTA1-1) was used as the representative of the GST family to test this hypothesis. The bulky side chains of Leu7 and Leu23 were replaced with a less bulky alanine residue to prevent altering the hydrophobicity of the βαβ motif. The effect of the mutation on the structure, function and stability of hGSTA1-1 was, therefore, studied in comparison with the wild-type using spectroscopic tools, X-ray crystallography, functional assays and conformational stability studies. The impact of the mutations on the structure of the enzyme was determined using spectroscopic tools and X-ray crystallography. The X-ray structures of the L7A and L23A mutants were resolved at 1.79 Å and 2.2 Å, respectively. Analysis of both X-ray structures shows that the mutation did not significantly perturb the global structure of the protein, which correlates with far-UV CD and intrinsic fluorescence spectroscopic data. In addition, structural alignments using the C-alpha gave root mean square deviation (r.m.s.d) values of 0.63 Å (L7A) and 0.67 Å (L23A) between the wild-type and mutant structures. However, both the L7A and L23A structures showed the presence of a cavity within the local environment of each mutation. The functional properties of the mutants were also similar to those of the wild-type as determined by specific activity and 8-anilino-1-naphthalene sulfonate (ANS)-binding, indicating that Leu7 and Leu23 are not involved in the function of hGSTA1- 1. The conformational stability of L7A and L23A proteins was probed using thermal-induced unfolding, pulse proteolysis and urea-induced equilibrium unfolding studies. The thermal stability of L7A and L23A hGSTA1-1 was reduced in comparison to the wild-type protein. This was consistent with proteolytic susceptibility of L7A and L23A proteins which indicates that both mutants are more prone to thermolysin digestion when compared to wild-type hGSTA1-1. This also correlates with urea-induced equilibrium studies. The ΔG(H2O) value (23.88 kcal.mol-1) for the wild-type protein was reduced to 12.6 and 10.49 kcal.mol-1 in L7A and L23A hGSTA1-l, respectively. Furthermore, the m-values obtained for the L7A and L23A proteins were 1.46 and 1.06 kcal.mol-1.M-1 urea, respectively; these were much lower than that obtained for the wild-type protein (4.06 kcal.mol-1.M-1 urea). The low m-values obtained for the mutant proteins indicated that the cooperativity of hGSTA1-1 unfolding was significantly diminished in both mutations. The results obtained in this study indicate that the topologically conserved Leu7 and Leu23 in the N-subdomain of hGSTA1-1 play a crucial role in maintaining the structural stability of the thioredoxin-like domain and are not involved in the function of the enzyme

Functional Analysis of Protein S-Palmitoylation Enzymes.

Protein S-palmitoylation is a dynamic, hydrophobic, post-translational modification of cysteine residues that is required for the spatiotemporal organization of hundreds of proteins. In turn, protein palmitoylation contributes to the composition of cellular membrane environments and plays fundamental roles in cancer, neurological disorders and many other human diseases. Despite its central function in human pathology, still, little is known about the enzymes that catalyze the addition (protein S-acyl transferases) and removal (S-acyl protein thioesterases) of this modification. The two enzyme families, DHHCs and LYPLAs, are thought to make up the so-called dynamic palmitoylation machinery in which dual action of acyltransferases and thioesterases promote proper membrane targeting for an expanding list of dynamically palmitoylated proteins. Indeed, a steady growth in studies of these enzymes is beginning to shed light on their biological functions, revealing that the interplay between these opposing catalysts may be more complex than previously thought. More specific tools for these enzymes can therefore provide a more complete molecular description of dynamic palmitoylation events and its regulation in various biological settings. The work presented in this thesis explores the chemical mechanisms and physiological roles of DHHCs and LYPLAs by developing, characterizing and employing novel tools for their study in the context of cancer biology. In the second chapter, the cellular targets of a widely-used, mechanism-based protein acyltransferase inhibitor are profiled and analyzed. In the third chapter, a mechanistic description of divergent thioesterase active-site ligand specificities is presented using both a structural and a kinetic approach. In the third chapter, novel acyl-protein thioesterase inhibitors are applied to define their roles in organizing cell junctions and suppressing metastatic transformation. One of the fundamental goals in this thesis is to address the limitations of current chemical tools of protein palmitoylation and provide a framework for the development of selective pharmacological agents to accelerate the study of this modification. From a physiological standpoint, this work offers novel insights into the in vivo functions of palmitoyl transferases and de-palmitoylases, highlighting the intricacies of the regulatory system governing the palmitoylation state of a given protein.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116725/1/davdad_1.pd