Gary Thomas, PhD
PhD in Biochemistry, The Biozentrum of the University of Basel (Switzerland)
BSc in Chemistry, Boise State University
Professor, Department of Microbiology and Molecular Genetics
Member, University of Pittsburgh Cancer Institute
Member, Program in Integrative Molecular Biology
Furin inhibitors: Our earlier studies identified the proprotein convertase furin as the first member of a family of secretory pathway-localized endoproteases that catalyze the activation of bioactive proteins and peptide hormones. While furin catalyzes the homeostatic activation of many growth factors, receptors and cell adhesion molecules, microbial pathogens frequently exploit the furin processing pathway. Indeed, furin has an essential role in the processing of viral envelope glycoproteins expressed by pathogenic viruses as well as in the proteolytic activation of many bacterial toxins, suggesting inhibitors of furin may be potential therapeutics. We generated the first potent and selective furin inhibitor, α1-PDX, and showed it could block the furin-dependent processing of envelope glycoproteins from pathogenic viruses as well as the activation of bacterial toxins that require furin for their activation. Our current studies are focused on generating small molecule furin inhibitors that block pathogen activation in vivo and then develop these compounds into potential therapeutics.
HIV-1 accessory proteins: HIV-1 Nef is required for the onset of AIDS and can affect cells in many ways, including alteration of T-cell activation and maturation, promotion of viral infectivity, subversion of the apoptotic machinery and downregulation of cell-surface molecules, including MHC-I. We discovered that HIV-1 Nef directs a temporally regulated program to downregulate MHC-I in virally infected cells. During the first two-days post-infection Nef binds the PACS proteins to assemble a multi-kinase complex that triggers endocytosis and sequestration of cell-surface MHC-I. By day three Nef switches to a stoichiometric mode that downregulates MHC-I by blocking the cell-surface delivery of newly synthesized MHC-I molecules. We identified small molecule inhibitors that block the ability of Nef to assemble the multi-kinase complex and thus downregulate MHC-I. Importantly, recent studies suggest the ability of Nef to assemble the multi-kinase complex is central to its ability to drive disease. Because of the key role of the PACS proteins in Nef action, we have mapped the sites on HIV-1 Nef and the PACS proteins essential for their interaction. Our future studies will determine to what extent assembly of the multi-kinase complex enables Nef to drive disease, identify small molecule inhibitors of Nef action and determine the structure of the Nef-PACS complex.
Mechanism of TRAIL action: The death ligand TRAIL selectively kills cancer cells and virally infected cells, prompting clinical trials examining its potential as a novel cancer therapy. TRAIL induces apoptosis by triggering the permeabilization of mitochondria and lysosomes to activate executioner caspases but little is known about the cellular machinery that coordinates these steps. We determined that TRAIL switches the homeostatic regulator PACS-2 from a secretory pathway trafficking protein to an apoptotic effector that promotes lysosome-mitochondria communication leading to release of apoptotic effectors into the cytosol to induce apoptosis in diseased cells. Molecularly, this switch is manifest by binding of 14-3-3 proteins to a site on PACS-2 phosphorylated by the survival kinase Akt. We identified how cancer cells or an anti-apoptotic herpesvirus protein can block PACS-2 from inducing apoptosis, suggesting key role for PACS-2 in TRAIL-induced apoptosis. Our current studies are investigating to what extent PACS-2 mediates the ability of TRAIL to inhibit tumor metastasis in vivo and how TRAIL signals to PACS-2 to direct membrane trafficking events leading to mitochondria and lysosomal membrane permeabilization and executioner caspase activation.
DNA damage response: Surprisingly, whereas PACS-2 promotes TRAIL-induced apoptosis, this sorting protein has a decidedly different role in DNA damage-induced apoptosis. Specifically, following DNA damage triggered by ionizing radiation or chemotherapeutics, p53-induced transcription of the cell cycle inhibitor p21 is repressed both in PACS-2-/- mice as well as in PACS-2 siRNA knockdown cells. This repressed transcriptional activity correlates with the hypoacetylation of p53 bound to the p21 promoter. Consistent with these findings, PACS-2 interacts with the class III histone deacetylase SIRT1, which blunts p53 action by deacetylating p53 following DNA damage. Our preliminary studies suggest PACS-2 mediates the p53-p21 axis by inhibiting SIRT1. Our future studies will identify the precise mechanism by which PACS-2 regulates SIRT1 enzyme activity, how PACS-2 traffics between the cytosol and nucleus, and how this trafficking is regulated by DNA damage.
Obesity: Consistent with our findings that suggest PACS-2 is a negative regulator of SIRT1 activity, PACS-2-/- mice are resistant to diet-induced obesity but clear glucose more efficiently than WT mice. Indeed, these findings parallel reports of the effect of SIRT1 activators in vivo. Our future studies will rigorously phenotype the PACS-2-/- mice and will determine to what extent PACS-2 regulation of SIRT1 controls endocrine homeostasis.
Jonathan Barroso, Postdoc
Jun Yin, Postdoc
Laurel Thomas, Research Associate
Laura Thomas, Research Assistant
Yujing Zhao, Research Assistant
Areas of Interest
Membrane traffic, HIV-1, cancer, apoptosis, obesity, proteases
Werneburg N. W, Bronk S. F, Guicciardi M. E, Thomas L, Dikeakos J. D, Thomas G. and Gores, G. J. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein-induced lysosomal translocation of proapoptotic effectors is mediated by phosphofurin acidic cluster sorting protein-2 (PACS-2). J Biol Chem. 287: 24427-24437. | View Abstract
Dikeakos J. D, Thomas L, Kwon G, Elferich J, Shinde U and Thomas, G. An interdomain binding site on HIV-1 Nef interacts with PACS-1 and PACS-2 on endosomes to down-regulate MHC-I. Mol Biol Cell. 23: 2184-2197. | View Abstract
Dikeakos J. D, Atkins K. M, Thomas L, Emert-Sedlak L, Byeon I. J, Jung J, Ahn J, Wortman M. D, Kukull B, Saito M, Koizumi H, Williamson D. M, Hiyoshi M, Barklis E, Takiguchi M, Suzu S, Gronenborn A. M, Smithgall T. E. and Thomas G. Small molecule inhibition of HIV-1-induced MHC-I down-regulation identifies a temporally regulated switch in Nef action. Mol Biol Cell. 21: 3279-3292. | View Abstract
Aslan J. E, You H, Williamson D. M, Endig J, Youker R. T, Thomas L, Shu H, Du Y, Milewski R. L, Brush M. H, Possemato A, Sprott K, Fu H, Greis K. D, Runckel D. N, Vogel A and Thomas G. Akt and 14-3-3 control a PACS-2 homeostatic switch that integrates membrane traffic with TRAIL-induced apoptosis. Mol Cell. 34: 497-509. | View Abstract
Aslan J. E. and Thomas G. Death by committee: organellar trafficking and communication in apoptosis. Traffic. 10: 1390-1404. | View Abstract
Atkins K. M, Thomas L, Youker R. T, Harriff M. J, Pissani F, You H, and Thomas G. HIV-1 Nef binds PACS-2 to assemble a multikinase cascade that triggers major histocompatibility complex class I (MHC-I) down-regulation: analysis using short interfering RNA and knock-out mice. J Biol Chem. 283: 11772-11784. | View Abstract
Hung C. H, Thomas L, Ruby C. E, Atkins K. M, Morris N. P, Knight Z. A, Scholz I, Barklis E, Weinberg A. D, Shokat K. M, and Thomas G. HIV-1 Nef assembles a Src family kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-surface MHC-I. Cell Host Microbe. 1: 121-133. | View Abstract