Thomas Lab: Projects
APOPTOSIS IN CANCER CELLS
PACS-2 expression is lost in colorectal cancer. A. IHC shows PACS-2 expressed in the normal epithelium (n) but lost in the adjacent cancer (c). B. and C. Higher magnification of normal and cancer tissue.
In the United States alone, cancer will kill over 500,000 people this year, and most of these deaths will result from cancers of the colon, breast, prostate and lung. An important breakthrough for cancer therapy may be found in the death ligand TRAIL, which selectively kills cancer cells in vivo without harming healthy cells. PACS-2 is an essential TRAIL effector, required for killing colon and breast cancer cells in vitro as well as diseased cells in vivo. Intriguingly, PACS-2 expression is lost in ~50% of late stage colorectal and recurrent breast cancers. In non-apoptotic cells, Akt-phosphorylated PACS-2 mediates cell homeostasis by coordinating the localization of anti-apoptotic ion channels to the endoplasmic reticulum (ER) with ER-mitochondria communication. In response to TRAIL, PACS-2 becomes dephosphorylated, promoting mitochondria membrane permeabilization by integrating apoptotic ER calcium signaling with Bid translocation and activation (see Polycystic Kidney Disease). In cancer cells with elevated Akt signaling, TRAIL-induced apoptosis is blocked at the level of PACS-2. Moreover, TRAIL resistance may be further confounded in late stage cancers by the loss of PACS-2 gene expression. To determine the role of PACS-2 in cancer, we are using mouse models of tumor progression together with cell biology and biochemical studies to determine how TRAIL induces PACS-2 to become apoptotic to integrate ER-mitochondria calcium signaling with Bid activation.
(See Aslan et al., Mol Cell 2009)
POLYCYSTIC KIDNEY DISEASE
TRAIL triggers redistribution of anti-apoptotic TRPP2 from the ER. Bottom cell shows ER localized TRPP2 in a non apoptotic cell. Top cell shows TRAIL triggers redistribution of TRPP2 from the ER.
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common genetically inherited disease, affecting 1 in 500 newborns, debilitating over six million people, and accounting for approximately 10% of all cases of end-stage renal disease. ADPKD is caused by mutations in one of two genes encoding either polycystin-1, which is a multivalent epithelial cell surface membrane receptor, or TRPP2 (formerly called polycystin-2), which is a Ca2+-permeant transient receptor potential (TRP) channel. The sorting itinerary of TRPP2 within the secretory system is complex, highly regulated, and intimately coupled to the biological role of TRPP2 action, including the onset of ADPKD. The trafficking of TRPP2 is regulated by the sorting proteins PACS-1 and PACS-2 and expression of the PACS proteins is dysregulated in the ADPKD kidney. Consistent with this finding, others have shown that misexpression of PACS proteins in zebrafish leads to formation of pronephric cysts. Interestingly, death ligands appear to mediate apoptotic ER-mitochondria calcium signaling (see Apoptosis in Cancer Cells) by controlling the interaction of PACS-2 with components of the trafficking machinery. For example, in healthy cells, binding of PACS-2 to COPI and 14-3-3 is required to localize TRPP2 to the ER. Death ligands trigger dephosphorylation of PACS-2, which releases 14-3-3. Dephosphorylated PACS-2 causes TRPP2 to leave the ER, triggering apoptotic ER-mitochondria calcium signaling. Studies on this project are examining the mechanism by which PACS-1 and PACS-2 regulate TRPP2 trafficking and whether dysregulated expression of the PACS proteins is causal to cystogenesis in mice.
(See Aslan et al., Mol Cell 2009)
HIV-1 IMMUNE EVASION
Despite the enormous effort to control HIV-1, we still do not understand how this deadly pathogen is able to thrive in the face of the integrated host antiviral defense and combined anti-retroviral therapies. Infected hosts have two major responses to viruses: immune surveillance and induced apoptosis of infected cells. Unlike many other pathogenic viruses, HIV-1 relies primarily on a single gene, Nef, to combat these antiviral responses. Our work has identified the mechanism underlying the immune evasive component in HIV-1 permissive cells, which involves the ability of Nef to downregulate the class I major histocompatibility complex (MHC-I), enabling the virus to escape immune surveillance. This pathway is triggered by the binding of Nef to PACS-2, which targets Nef to the Golgi region where it assembles a Src family kinase (SFK)/ZAP-70/PI3K multi-kinase complex that triggers MHC-I internalization. Internalized MHC-I molecules are then sequestered by an AP-1 and PACS-1-mediated process. Current projects include a determination of the role of the multi-kinase complex in Nef-induced pathogenesis and the identification of small molecule inhibitors that prevent assembly of the multi-kinase complex, which may lead to new generation drugs to combat AIDS.
Signaling model of HIV-1 Immune evasion. Step 1: Nef binds PACS-2 in an EEEE65-dependent manner and is targeted to the late Golgi/TGN region. Step 2: at the TGN region, Nef PXXP75 binds and activates a TGN-localized SFK. Step 3: the activated Nef–SFK complex recruits and activates ZAP-70/Syk. Tyrosine-phosphorylated ZAP-70/Syk then binds a class I PI3K. Step 4: Nef-stimulated class I PI3K generates PIP3 on the inner leaflet of the plasma membrane. Step 5: an ARF6 guanine-nucleotide-exchange factor (ARF6-GEF) is recruited to PIP3 at the plasma membrane. Step 6: the recruited ARF6-GEF in turn activates ARF6. Step 7: MHC-I is rapidly endocytosed from the plasma membrane to internal endosomal compartments. Steps 8 and 9: sequestration of newly internalized MHC-I molecules to a paranuclear region requires the Nef Met20 and its interaction with AP-1. The precise step of Nef-mediated MHC-I down-regulation requiring PACS-1 is unclear but the ability of PACS-1 to bind Nef and AP-1 raises the possibility that PACS-1 may contribute to the Met20-dependent internalization of MHC-I molecules.
(See Blagoveshchenskya et al., Cell 2002; Hung et al., Cell Host & Mic 2007; Atkins et al., JBC 2008; and Youker et al., Biochem J. 2009)
The class I PI3K inhibitor PI-103 represses Nef-induced MHC-I downregulation in CD4+ T-cells.
STRUCTURE OF MEMBRANE TRAFFIC REGULATORS
PACS-1 and PACS-2 are multifunctional sorting regulators that integrate secretory pathway traffic with ER-mitochondria communication and apoptotic pathways (Fig. 2). The human PACS-1 gene encodes a 963 residue cytosolic protein, while the human PACS-2 gene encodes an 889 amino acid protein. Based on homology of different PACS-1 isoforms, the PACS proteins can be divided into four regions. PACS-1 contains an N-terminal atrophin-1 related region (ARR) that shares low sequence identity with the atrophin-1 transcriptional repressor. The ARR is followed by a 140-residue furin (cargo) binding region (FBR) identified in the original yeast two-hybrid screen and which binds acidic cluster sorting motifs, a middle region (MR) containing an autoregulatory domain, and a C-terminal region (CTR). PACS-2 shares 54% overall identity with PACS-1, including 75% identity within the FBR. The PACS-1 and PACS-2 FBRs bind cargo proteins, adaptors and signaling molecules with high selectivity but low affinity. Consistent with this finding, CD analysis together with protein secondary and disorder prediction programs (PONDR) suggest the PACS proteins belong to the growing class of proteins known as intrinsically unstructured proteins (IUPs). The IUPs contain regions lacking a definitive protein fold but can attain tertiary structure upon binding to a client protein or ligand. Current studies in the lab are using NMR to investigate the structure of the PACS-1 and PACS-2 FBRs bound to cargo molecules.
Ab initio modelling of the PACS-1 FBR (residues 117–300) was performed using Rosetta++. The tertiary structure is represented as a ribbon diagram surrounded by a semi-transparent surface projection. Acidic residues are coloured red, basic residues are coloured blue, residues required for adaptor binding are coloured orange, residues required for GGA binding are coloured yellow and residues positively selected through evolution are coloured pink. Left-hand panel: top view looking down on the major groove. Right-hand panel: the image is rotated 90° towards the viewer. Images were created using PyMOL.
