Professor Emeritus, Department of Biochemistry
Irving Listowsky-Recent Research Interests
[Anti] Oxidants, S-Thiolation of Proteins and Glutathione Transferases –functions in chemopreventive mechanisms and oxidative stress.
Cancer Prevention: Our laboratory has been studying the structure and biological functions of glutathione-S-transferases (GSTs) and mechanisms of regulation of expression of their genes. Mammalian GSTs are products of gene superfamilies and function catalytically to conjugate glutathione (GSH) with a wide variety of electrophilic substrates. GSTs also may serve as intracellular stoichiometric binding proteins for structurally diverse lipophilic ligands. The proteins are considered to serve in a detoxification capacity to protect cells from many types of noxious substances. Dietary components or substances from the environment that induce elevations in levels of GSTs and other phase II drug metabolizing enzymes, are therefore thought to represent chemoprotective or anticarcinogenic agents for human beings. By virtue of GST increases, these inducer compounds may indeed ameliorate the activity of cytotoxic agents but could also adversely affect responses to administered drugs and cancer chemotherapeutic agents. Effects of hormones and various dietary components that selectively elevate levels of specific GSTs in different tissues are analyzed. We believe that some of these interactions are governed by the redox status of cells and involve S-glutathiolation of specific cysteine residues of proteins. Accordingly, our central hypothesis suggests that the GST can recognize, and bind to, the glutathionyl moiety and thereby influence the activity of these proteins by direct protein-protein interactions. GSTs can also influence thiolester formation and exchange reactions with GSH. GSTs could therefore be involved in cellular processes such as protein turnover by the ubiquitin pathway of proteasomal degradation, SUMOylation, and other biological thiolester mediated systems.
Fundamental concepts derived from studies on GSTs: The GST system is exploited to serve as a prototypic model for ligand-protein interactions, cotranslational protein folding and subunit assembly, and as a paradigm to probe fundamental questions about cell type-specific, or oxidative and chemical stress induced regulation of expression of mammalian genes. The human Mu-class GSTs have been crystallized for structural analyses by X-ray diffraction, and many different mutants generated to understand catalytic specificities and binding mechanisms. A coexpression system has also been devised to determine subunit assembly and compatibility of interactions as well as cotranslational folding of the subunits. NMR, microcalorimetry and various spectroscopic methods are employed to study protein-ligand interactions. Topographies of the binding pockets of different GST isoforms are analyzed to predict catalytic and binding mechanisms, and to design ligands that interact with GSTs.
GST functions in reproductive Biology: A distinctive mammalian GST subclass encoded by highly homologous single copy genes in humans, rodents and other mammalian species and exemplified by the mouse mGstm5 gene, has been characterized in our laboratory. Mice with a targeted disruption of the mGstm5 gene were produced to determine functions of this GST subclass. The null mice are viable, fertile and exhibit no overt distinguishing phenotype. However, the mGstm5 -/- mice become prematurely infertile after 4-6 months of age. Moreover, mice bearing the targeted disruption of the mGstm5 gene revealed a greater susceptibility to oxidative stress. For instance, all null mice expired almost twice as soon as WT cohorts after administration of paraquat (PQ). PQ is primarily a pulmonary toxin, but inflammatory lesions in many organs were more severe in the KO animals. In particular, notable cortical necrosis of the adrenal was observed only in the KO animals. Gene expression profiles in response to non-lethal doses of PQ administered to mice of different ages over short periods were studied by cDNA microarray analyses. The most striking effects on gene expression profiles were in testis. Thus, PQ treatment resulted in significant changes in more than 800 genes in testis of WT animals including transcription factors (particularly PHD domain and Zn finger proteins), histone deacetylases, and redox sensitive signaling and growth factors. By contrast, testicular gene expression patterns were largely unchanged in null animals. Evidently this subclass has specialized functions that differ from those of other GSTs and the mGstm5 gene product can govern global responses to oxidative challenges in testis.
Transgenic mice expressing EGFP under control of a 2 kbp promoter sequence in the 5’-flanking region of the mGstm5 gene were produced to study transcription mechanisms. The transgene expression was limited to germ cells in testis, indicating that the mGstm5 proximal promoter is sufficient for its targeting to testis. Real-time quantitative PCR (qPCR) data were consistent with alternate transcription start sites in which the promoter region of the natural mGstm5 gene in somatic cells is part of exon 1 of the germ cell transcript. Thus, the primary transcription start site for mGstm5 is upstream of a TATA box in testis and downstream of this motif in somatic cells. Both the null and transgenic animals are employed to study functions of this GST subclass.
References NCBI PubMed search of "I. Listowsky” –selected recent publications
1- Y. Patskovsky, L. Patskovska and I. Listowsky “Functions of His-107 in the Catalytic Mechanism of Human Glutathione S-Transferase hGSTM1a-1a Biochemistry 38, 1193-1202 (1999).
2- Y. Patskovsky, M.Q. Huang, T. Takayama, I. Listowsky and W.R. Pearson “Distinctive Structure of the Human GSTM3 Gene-Inverted Orientation Relative to the Mu-Class Cluster”. Arch. Biochem. Biophys. 361, 85-93 (1999).
3-Y. Patskovsky, L. Patskovska and I. Listowsky, “An Asparagine-Phenylalanine Substitution Accounts for Catalytic Differences Between hGSTM3-3 and other Human Class Mu Glutathione S-Transferases”. Biochemistry 38, 16187-16194 (1999).
4- Y. Patskovsky, L. Patskovska and I. Listowsky, “The Enhanced Affinity for Thiolate Anion and Activation of Enzyme-Bound Glutathione is Governed by an Arginine Residue of Human Mu Class Glutathione S-Transferases”. J. Biol. Chem. 275, 3296-3304 (2000).
5-Tchaikovskaya, T., Patskovsky, Y., Almo, S. Girvin, M. and Listowsky, I. A basis for the broad substrate specificities of the glutatione S-transferases. Chem. Biol. Interactions 133, 170-172 (2001).
6- Cheng, H., Tchaikovskaya, T., Tu, Y-S., Chapman, J., Qian, B., Ching, W-M., Tien, M., Rowe, J.D., Patskovsky, Y.V., Listowsky, I. and Tu, C.P. Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox reactive cysteine 115 residue that forms mixed disulfides with glutathione. Biochem J. 356, 403-414 (2001).
7-Andorfer, J.H., Tchaikovskaya, T. and Listowsky, I. Selective expression of glutathione S-transferase genes in the murine gastrointestinal tract in response to dietary organosulfur compounds. Carcinogenesis 25, 359-367 (2004). .
8- Tchaikovskaya, T. Fraifeld, V, Wolfson, M., Asraf, H.,Urphanishvili,T., Andorfer, J.H., Davies, P. and Listowsky I. Glutathione-S-transferase hGSTM3 and aging-associated neurodegeneration in Alzheimer’s disease. Mechanisms of Aging and Development 126, 309-315 (2005).
9- Listowsky,I., Proposed intracellular regulatory functions of glutathione transferases by recognition and binding to S-glutathiolated proteins. J. Peptide Res. 65, 42-46 (2005).
10-Chico, D.E. and Listowsky, I. Diverse Expression Profiles of Glutathione-S-Transferase Subunits in Mammalian Urinary Bladders. Arch. Biochem. Biophys. (2004) 435, 56-64 (2005).
11- Listowsky,I. A subclass of Glutathione transferase selectively expressed in brain and testis. Methods in Enzymology-Conjugation Enzymes 401, 278-87 (2005)
12- Mannervik, B., Board, P. G. ,Hayes, J.D. Listowsky,I, and Pearson, W.R., Nomenclature for Rodent Glutathione S-Transferases. Methods in Enzymology 401:1-8(2005).
13- Arias, I. M. and Listowsky, I. Glutathione transferases and ligandin; historical milestones. In “Toxicology of Glutathione Transferases”, YC Awasthi ed. CRC (2006) – p1-9.
15- Patskovsky,Y., Patskovska, L., Almo, S.C. and Listowsky, I. Transition State Model and Mechanism of Nucleophilic Aromatic Substitution Reactions Catalyzed by Human Glutathione S-Transferase M1a-1a. Biochemistry 45, 3852-3862 (2006).
The Proximal Promoter Governs Germ Cell-Specific Expression of the Mouse Glutathione Transferase mGstm5 Gene Mol. Reprod. Dev.. 76,379-88 (2009).
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