个人简介

BSc, 1981, Biochemistry, University of Toronto PhD, 1988, Biochemistry, University of Toronto

研究领域

Biochemistry

Nitric oxide (NO) is an important endogenous messenger in a variety of physiological and pathophysiological processes. NO is synthesized by the three isoforms of a homodimeric enzyme, nitric oxide synthase (NOS). Each NOS subunit is composed of a reductase domain that contains the binding sites for NADPH and two flavins. The reductase domain is linked via a 20 amino acid calmodulin (CaM) binding motif to an oxygenase domain that contains the binding sites for heme, L-arginine and tetrahydrobiopterin. The calcium cation (Ca2+) is also an important secondary messenger and the primary protein mediator of calcium is CaM. CaM undergoes Ca2+ concentration [Ca2+] dependent conformational changes that allow it to bind and activate scores of target proteins including NOS isozymes and voltage-gated calcium channels (VGCCs). The long-term objectives of our research program is to understand: 1) the mechanism and control of NO synthesis by NOS; 2) the CaM-dependent activation of its numerous target proteins including NOS isozymes and VGCCs. A) One of our objectives is to understand how calmodulin binds to NOS and affects structure, function and electron transfer within the different domains and the holoprotein. The NOS isozymes are activated by the binding of CaM, the ubiquitous Ca2+-binding regulatory protein. We are investigating the role of calmodulin in the differential regulation of the various NOS isoforms. Recombinant DNA techniques are used to obtain the native and selectively modified forms of the human NOS enzymes. The mechanism of calmodulin binding and activation is being investigated using native and mutant forms of recombinant calmodulin. These enzymes are investigated for enzymatic activity and characterized using a variety of biophysical techniques including various forms of spectroscopy and calorimetry. These investigations will provide a better understanding of the control of NOS enzymes. B) Investigation of electron transfer within the different domains of NOS enzymes. The two mono-oxygenase reactions catalyzed by NOS enzymes require electron transfer within and between molecules that form the active NOS dimer. A fundamental understanding of the electron transfer processes in these enzymes may provide novel methods to control enzyme activity. Biophysical techniques are being used to investigate the rapid transfer of electrons within the molecule. C) Investigation of NOS isoform selective binding. The three mammalian NOS isozyme active sites share nearly full amino acid conservation and structural similarity making the design of isoform specific ligands and inhibitors very difficult. There are numerous examples of the failure of gaining improved potency from modified ligands and inhibitors whose designs were based upon information from the static X-ray structure of the docked complexes. Structure based studies also provide little information on the thermodynamics of binding and desolvation. Furthermore, since most studies have been performed on enzymes that are not of human origin, another reason for the non-availability of NOS isoform-selective compounds for clinical use comes from the inherent differences in the amino acid sequences between human and other mammalian isoforms. We are using a biophysical based approach in the development of molecules capable of isoform-specific binding to the active site of the human NOS isozymes. D) Investigation of Calmodulin Dependent Signal Transduction. Calcium is a ubiquitous intracellular messenger responsible for controlling numerous cellular processes including fertilization, mitosis, neuronal transmission, contraction and relaxation of muscles, gene transcription, and cell death. Many of the cellular effects of calcium are mediated by CaM. Upon binding up to four calcium ions, CaM undergoes conformational changes that enable it to bind and regulate many different protein targets. This process is a key component of a calcium-dependent signal transduction pathway. Our lab is investigating the fundamental properties of CaM that enables it to selectively bind and activate over three hundred proteins. Our investigation of the “Calmodulome” involves the characterization of specifically designed CaM mutants. We are using several biophysical and biochemical methods to map the binding and investigate the activation properties of CaM. The results of our research will provide a better understanding of protein-protein interactions, signal transduction in mammalian cells and may lead to the development of novel therapeutic compounds. E) Regenerative medicine. Regenerative medicine holds the promise of curing disease and organ failure but is challenged by the lack of success in delivering and maintaining cells in implanted grafts. We are working on the development of “biointeractive matrices” that provide a designed and dynamic matrix to support cell proliferation, differentiation and engraftment. Our lab is part of a highly trans-disciplinary group including a protein biochemist, a nanomaterials engineer, a cellular bioengineer, and a physiologist. We are looking for highly motivated students to join this group and work on the development of support matrices that will eventually be used to maintain and direct cell and tissue growth and differentiation in vivo. Individuals joining our lab will be part of a transdisciplinary research team that provide the students with range of concepts not normally encountered in their respective disciplines early in their careers. F) Regulation of VGCCs by intracellular Ca2+ concentration. CaM undergoes Ca2+ concentration [Ca2+] dependent conformational changes that allow it to bind and activate scores of target proteins including NOS isozymes and voltage-gated calcium channels (VGCCs). Cav1.2s are high-voltage-activated or L-type calcium channels that control Ca2+ influx in response to membrane depolarization and are found in numerous cell types including muscle and neurons. These channels are modulated by membrane potential (voltage-dependent inactivation, VDI) as well as Ca2+-dependent feedback mechanisms known as Ca2+-dependent inactivation (CDI). While not fully understood, CDI is suggested to be dependent on the channel-associated CaM that detects intracellular Ca2+ in the vicinity of the channel pore. We are investigating how intracellular Ca2+ concentration regulates VGCCs and more specifically how CaM is involved in this regulation mechanism. G) Investigation of Class II aldolases. Class II aldolases in fungi have a unique active site that is significantly different from the one found in plants and humans making this enzyme a good target for the development of antifungal agents. The recombinant forms of Class II aldolases from diverse plant pathogenic fungi including Phytophthora infestans (responsible for the Irish potato famine) are being used in our investigation with the goal of developing novel antifungal agents. A similar approach is being used for the investigation of the Class II aldolase from Mycobacterium tuberculosis. Tuberculosis is a leading infectious killer worldwide, and new treatments are needed to combat drug-resistant tuberculosis. The investigation of the enzymatic mechanism is being pursued with the goal of gaining insight into the development of novel inhibitors.

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Piazza, M., Futrega, K., Spratt, D.E., Dieckmann, T. and Guillemette. J.G. 2011, Solution NMR structures of CaM bound to NOS peptides: Effect of a phophomimetic CaM mutation. J. Biol Chem. Submitted manuscript number JBC/2011/325035. Labbé, G., de Groot, S., Krismanich, A., Rasmusson, T., Shang, M., Brown, M., Dmitrienko, G.I. and Guillemette, J.G. 2011, Development of Metal-Chelating Inhibitors for the Class II fructose 1,6-bisphosphate (FBP) aldolase. J. Inorg. Biochem. Submitted manuscript number JIB-11-1028 Piazza , M., Duangkham, Y., Spratt, D.E., Dieckmann, T. and Guillemette. J.G. 2011, Expression and purification of an isotopically labeled aggregation prone iNOS CaM-binding protein for use in NMR studies. J. Label. Comp. and Radiopharm 54, 657–663. Spratt, D.E., Duangkham, Y., Taiakina, V. and Guillemette, J.G. 2011, Mapping the binding and CaM-dependent activation of NOS isozymes. The Open Nitric Oxide J. 3, 15-23. Labbé, G., de Groot, S., Rasmusson, T., Milojevic, G., Dmitrienko, G.I. and Guillemette, J.G. 2011, Evaluation of Four Microbial Class II fructose 1,6-bisphosphate aldolase enzymes for use as biocatalysts, Protein Expr. Purif.80, 224-233. Feng, C., Ghosh, D.K., Taiakina, V., Guillemette, J. G., Tollin, G. 2011, Intraprotein electron transfer between the FMN and heme domains in endothelial nitric oxide synthase holoenzyme, Biochimica et Biophysica Acta, in press. Crawford B., Koshy S., Jhamb G., Woodford C., Thompson C. M., Levy A. S., Rush J. W. E., Guillemette J. G., Lillicrap D., Jervis E. 2011, Process Engineering Considerations for Cardiac Decellularization, Can. J. Chem. Eng in press. Sempombe, J., Galinato, M.G.I., Elmore, B.O., Fan, W., Guillemette, J.G., Lehnert, N., Kirk, M.L., Feng, C. 2011, Mutation in the FMN Domain Modulates MCD Spectra of the iNOS Ferric Cyano Complex in a Substrate-Specific Manner, Inorganic Chemistry, 50, 6859- 61. Astashkin, A., Fan, W., Bradley, E., Guillemette, J. G., Feng, C. 2011, Pulsed ENDOR determination of relative orientation of g- and molecular-frames of imidazole-coordinated heme center of NOS, J. Phys. Chem., 115, 10345-10352. Feng, C., Fan, W., Dupont, A., Guillemette, J. G., Ghosh, D.K., Tollin, G. 2010, Electron transfer in a human inducible NOS oxygenase/FMN construct coexpressed with the N- terminal globular domain of CaM. FEBS Letters584, 4335-4338. Astashkin, A.V., Elmore, B.O., Fan, W., Guillemette, J.G., and Feng, C. 2010, Pulsed EPR determination of the distance between heme iron and FMN centers in a human inducible nitric oxide synthase. J Am Chem Soc. 132, 12059-12067. Montogmery, H.J., Dupont, A.L., Leivo, H.E. and Guillemette, J.G 2010, Cloning, Expression and Characterization of a Nitric Oxide Synthase-Like Protein from Bacillus cereus. Biochemistry Research International 2010:489892. Sempombe, J., Elmore, B.O., Dupont, A, Ghosh, D., Guillemette, J.G., Kirk, M., and Feng C. 2009, Mutations in the FMN domain modulate MCD spectra of the heme site in the oxygenase domain of iNOS. J. Am. Chem. Soc.131, 6940-6942. Feng, C., Dupont, A.L., Spratt, D.E., Weinberg, J.B., Guillemette, J.G., Tollin, G., and Ghosh, D.K. 2009, Intraprotein Electron Transfer in Inducible Nitric Oxide Synthase Holoenzyme. Journal of Biological Inorganic Chemistry 14, 133-142 Spratt, D.E., Taiakina, V., Palmer, M., and Guillemette, J.G. 2008, FRET conformational analysis ofcalmodulin binding to nitric oxide synthase peptides and enzymes. Biochemistry, 47, 12006-12017. Spratt, D.E., Israel, O.K., Taiakina, V., and Guillemette, J.G. 2008, Regulation of inducible nitric oxidesynthase by electrostatic interactions in the linker region of calmodulin. Biochimica et Biophysica Acta, 1784, 2065-2070. Spratt, D.E., Taiakina, V., Palmer, M. and Guillemette, J.G. 2007, Differential Binding of Calmodulin Domains to Constitutive and Inducible Nitric Oxide Synthase Enzymes, Biochemistry46, 8288-8300. Spratt, D.E., Taiakina, V. and Guillemette, J.G.2007,Calcium-deficient calmodulin binding and activation of neuronal and inducible nitric oxide synthases. Biochim Biophys Acta. 1774, 1351-1358. Labbé, G., Bezaire, J., de Groot, S. How, C., Rasmusson, T., Yaeck, J., Jervis, J., Dmitrienko , and G.I., Guillemette, J.G. 2007, High level production of the Magnaporthe grisea Fructose 1,6-bisphosphate Aldolase enzyme in E. coli using a small volume Bench Top Fermentor, Protein Expr. Purif. 51, 110-119. Feng, C., Tollin, G., Hazzard, J.T., Nahm, N.J., Guillemette, J.G., Salerno, J.C. and Ghosh, D.K. 2007, Direct measurement by laser flash photolysis of intraprotein electron transfer in a rat neuronal nitric oxide synthase. J Am Chem Soc. 129, 5621-5629.