Gary Thomas, PhD
507 Bridgeside Point II
450 Technology Drive
PhD in Biochemistry, The Biozentrum of the University of Basel (Switzerland)
BSc in Chemistry, Boise State University
Our research program focuses on signaling pathways that integrate membrane traffic with the regulation of homeostasis and the onset of disease. These studies were grounded by our identification of the proprotein convertase furin, which is 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. Although furin localizes to the trans-Golgi network (TGN), it can cleave such a diverse array of protein substrates because it moves between multiple processing compartments: the TGN/biosynthetic pathway, the cell surface and early endosomes. Our analysis of the complex intracellular trafficking pathway of furin led us to the discovery of the PACS family of homeostatic regulators, which integrate secretory pathway traffic and interorganellar communication in healthy cells with key steps in death ligand-induced apoptosis in diseased cells. Moreover, the PACS proteins have key roles in the nucleus where they regulate the transcriptional activity of p53 following DNA damage. Together, these findings form the basis for our current studies in cancer biology, viral pathogenesis and metabolism as summarized below:
Furin inhibitors: Using our determination of furin’s cleavage site specificity, we generated the first potent and selective furin inhibitor, α1-PDX. We showed α1-PDX can block the furin-dependent processing of envelope glycoproteins from many 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: We determined that the death ligand TRAIL switches PACS-2 from a secretory pathway trafficking protein to an apoptotic effector that promotes lysosome-mitochondria communication leading to cytochrome c release and death of cancer 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 membrane permeabilization and executioner caspase activation.
DNA damage response: We found that PACS-2 is a key regulator of the DNA damage response in vivo. 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 p21promoter. 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 and do not become insulin resistant. 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.
Research Lab Affiliation
Atkins, K. M; Thomas, L. L; Barroso, J.; Thomas, L.; Auclair, S.; Yin, J.; Kang, H.; Chung, J. H; Dikeakos, J. D; and Thomas G. (2014) The multi-functional sorting protein PACS-2 regulates SIRT1-mediated deacetylation of p53 to modulate p21-dependent cell cycle arrest. Cell Reports (in press). |
Werneburg, N. W; Bronk, S. F; Guicciardi, M. E; Thomas, L; Dikeakos, J. D; Thomas, G; and Gores, G. J. (2012) 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. (2012) 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. (2010) 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. (2009) 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
Atkins, K. M; Thomas, L; Youker, R. T; Harriff, M. J; Pissani, F; You, H; and Thomas, G. (2008) 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. (2007) 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