Julian Grusovin

Insulin stimulates movement of sorting nexin 9 between cellular compartments: a putative role mediating cell surface receptor expression and insulin action

SNX9 (sorting nexin 9) is one member of a family of proteins implicated in protein trafficking. This family is characterized by a unique PX (Phox homology) domain that includes a proline-rich sequence and an upstream phospholipid binding domain. Many sorting nexins, including SNX9, also have a C-terminal coiled region. SNX9 additionally has an N-terminal SH3 (Src homology 3) domain. Here we have investigated the cellular localization of SNX9 and the potential role it plays in insulin action. SNX9 had a cytosolic and punctate distribution, consistent with endosomal and cytosolic localization, in 3T3L1 adipocytes. It was excluded from the nucleus. The SH3 domain was responsible, at least in part, for the membrane localization of SNX9, since expression of an SH3-domain-deleted GFP (green fluorescent protein)-SNX9 fusion protein in HEK293T cells rendered the protein cytosolic. Membrane localization may also be attributed in part to the PX domain, since in vitro phospholipid binding studies demonstrated SNX9 binding to polyphosphoinositides. Insulin induced movement of SNX9 to membrane fractions from the cytosol. A GST (glutathione S-transferase)-SNX9 fusion protein was associated with IGF1 (insulin-like growth factor 1) and insulin receptors in vitro. A GFP-SNX9 fusion protein, overexpressed in 3T3L1 adipocytes, co-immunoprecipitated with insulin receptors. Furthermore, overexpression of this GFP-SNX9 fusion protein in CHOT cells decreased insulin binding, consistent with a role for SNX9 in the trafficking of insulin receptors. Microinjection of 3T3L1 cells with an antibody against SNX9 inhibited stimulation by insulin of GLUT4 translocation. These results support the involvement of SNX9 in insulin action, via an influence on the processing/trafficking of insulin receptors. A secondary role in regulation of the cellular processing, transport and/or subcellular localization of GLUT4 is also suggested.

Cellular munc18c levels can modulate glucose transport rate and GLUT4 translocation in 3T3L1 cells

Munc18c has been shown to bind syntaxin 4 and to play a role in GLUT4 translocation and glucose transport, although this role is as yet poorly defined. In the present study, the effects of modulating the available level of munc18c on glucose transport and GLUT4 translocation were examined. Over‐expression of munc18c in 3T3L1 adipocytes inhibited insulin‐stimulated glucose transport by approximately 50%. Basal glucose transport rates were also decreased by approximately 25%. In contrast, microinjection of a munc18c polyclonal antibody stimulated GLUT4 translocation by approximately 60% over basal levels without affecting insulin‐stimulated GLUT4 levels. Microinjection of a control antibody had no effect. These data are consistent with the likelihood that antibody microinjection sequesters munc18c enabling translocation/fusion of GLUT4 vesicles. Mutagenesis of a potential proline‐directed kinase phosphorylation site in munc18c, T569, that in previous studies of its neuronal counterpart munc18a caused its dissociation from its complex with syntaxin 1a, had no effect on munc18cs association with syntaxin 4 or its inhibition of glucose transport, indicative that phosphorylation of this residue is not important for insulin regulation of glucose transport. The over‐expression and microinjection sequestration data support an inhibitory role for munc18c on translocation/fusion of GLUT4 vesicles. They further show that altering the level of available munc18c in 3T3L1 cells can modulate glucose transport rates, indicating its potential as a target for therapeutics in diabetes.

Chapter Four – Structure and Function of Ecdysone Receptors—Interactions with Ecdysteroids and Synthetic Agonists

Abstract The binding of ecdysteroids and the bisacylhydrazine insecticide, tebufenozide, to recombinant ecdysone receptor ligand-binding domains from pest insects points to conserved and variable features of the receptors ligand-binding pocket. Fluorophores conjugated to the terminus of the ecdysteroid alkyl chain have surprisingly little effect on receptor binding, permitting the development of a fluorescence polarization chemical library screen that has led to the discovery of a new class of ecdysone receptor ligands, the methylene lactams. X-ray structures of ecdysone receptor ligand-binding domains have allowed identification of the conserved and variable features within the binding pocket. The structures offer explanations for the lepidopteran selectivity of the bisacylhydrazines, the effect of amino acid replacements on the binding of ecdysteroids and other chemistries, and the preference of a phytophagous pentatomomorphan for makisterone A; indeed, they speak to the control spectra of future ecdysone receptor-targeting insecticides. Possible ligands for nematode ecdysone receptor orthologs are also considered.

Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection.

Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.

Definition of a minimal munc18c domain that interacts with syntaxin 4

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein-protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c(1-592), munc18c(1-139) and munc18c(1-225), but not munc18c(226-592), munc18c(1-100), munc18c(43-139) or munc18c(66-139), interacted with the cytoplasmic portion of syntaxin 4, Stx4(2-273), as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by beta-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx4(29-157), failed to interact with full-length munc18c(1-592), indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein-protein interaction studies between Stx4(2-273) and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c(1-592), munc18c(1-139) and munc18c(1-225) interacted with Stx4(2-273) whereas munc18c(1-100) did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

Unprecedented conformational flexibility revealed in the ligand-binding domains of the Bovicola ovis ecdysone receptor (EcR) and ultraspiracle (USP) subunits.

The heterodimeric ligand-binding region of the Bovicola ovis ecdysone receptor has been crystallized either in the presence of an ecdysteroid or a synthetic methylene lactam insecticide. Two X-ray crystallographic structures, determined at 2.7 Å resolution, show that the ligand-binding domains of both subunits of this receptor, like those of other nuclear receptors, can display significant conformational flexibility. Thermal melt experiments show that while ponasterone A stabilizes the higher order structure of the heterodimer in solution, the methylene lactam destabilizes it. The conformations of the EcR and USP subunits observed in the structure crystallized in the presence of the methylene lactam have not been seen previously in any ecdysone receptor structure and represent a new level of conformational flexibility for these important receptors. Interestingly, the new USP conformation presents an open, unoccupied ligand-binding pocket.

Determination of molybdenum in plant tissue by graphite furnace atomic absorption spectrophotometry (GFAAS)

Abstract The analysis of molybdenum in plant tissue using graphite furnace atomic absorption spectrophotometry (GFAAS) is described. The method involved wet digestion using a mixture of sulphuric, nitric and perchloric acids followed by a solvent extraction procedure. Molybdenum was extracted into di‐iso butyl ketone (DIBK) as the iron‐thiocyanate complex. The extract was then analysed for molybdenum by GFAAS. The results of analyses of the reference plant materials (orchard leaves and citrus leaves) of the National Bureau of Standards (NBS) compared very well with the certified values. Other types of plant tissue were also analysed and the results correlated well with those obtained by an alternative method.

Abstract 5371: PRMT5 inhibitors as novel treatment for cancers

Increased expression or dysregulation of protein arginine methyltransferase 5 (PRMT5) activity is associated with poor prognosis in many cancers. Through increased methylation of epigenetic and non-epigenetic targets, the aberrant activity of PRMT5 has been associated with many pro-tumourigenic cellular changes such as, increased levels of protein synthesis, dysregulation of cell cycle, cellular adaptation to hypoxic conditions, and suppression of normal cell death pathways. Genetic studies suggest that suppression of PRMT5 activity can reverse many of these pro-tumourigenic effects making PRMT5 an attractive drug discovery target. We screened a library of 350,000 lead-like compounds with a biochemical assay measuring the methylation of a histone H4 peptide by the recombinant human PRMT5/MEP50 complex. Biochemical and biophysical profiling of the inhibitory compounds indicated that several distinct binding modes were exhibited by the different chemical scaffolds. Inhibitors displayed competitive, noncompetitive or uncompetitive interactions with respect to S-adenosyl methionine and the peptide substrate. Medicinal chemistry developed several classes of potent, highly selective inhibitors of PRMT5 methyltransferase activity from the hit set. The optimised tool compound, CTx-034, is a potent inhibitor of PRMT5 methyl transferase activity (KD = 2 nM), which is highly selective (>100-fold) versus a panel of 18 methyltransferases (including 6 PRMT family members), 11 lysine demethylases, and 15 safety related targets (GPCRs, ion channels, enzymes). Treatment of cancer cell lines with CTx-034 reduces cellular levels of symmetrically dimethylated H4 Arginine 3 (H4R3me2s), in a dose dependent manner (IC50 = 4 nM) to levels undetectable by Western blot. Furthermore, within this chemical series the ability of compounds to reduce cellular levels of H4R3me2s closely correlates with PRMT5 inhibitory activity supporting PRMT5 as the cellular target of these compounds, and suggesting that PRMT5 is the major writer of this histone mark in many cancer cell lines. CTx-034 also inhibits the symmetric dimethylation of arginine on other histone and non-histone cellular substrates of PRMT5, including H3R2me2s and SmD1. Conversely, CTx-034 treatment does not reduce levels of H4R3 asymmetric dimethylation, a histone mark catalysed by PRMT1. Finally, CTx-034 has good oral bioavailability and pharmacokinetic properties in rodents and twice-daily dosing (10 - 100 mg/kg) over 10-14 days produces a dose dependent reduction of the H4R3me2s mark in bone marrow cells and peripheral white blood cells. This treatment is well tolerated by the mice, with no significant reduction in body weight or changes in haematological parameters observed. CTx-034 provides an excellent tool compound for cellular and in vivo proof of concept studies. Citation Format: Hendrik Falk, Richard C. Foitzik, Elizabeth Allan, Melanie deSilva, Hong Yang, Ylva E. Bozikis, Marica Nikac, Scott R. Walker, Michelle A. Camerino, Ben J. Morrow, Alexandra E. Stupple, Rachel Lagiakos, Jo-Anne Pinson, Romina Lessene, Wilhelmus JA Kersten, Danny G. Ganame, Ian P. Holmes, Gill E. Lunniss, Matthew Chung, Stefan J. Hermans, Michael W. Parker, Alison Thistlethwaite, Karen White, Susan A. Charman, Brendon J. Monahan, Patricia Pilling, Julian Grusovin, Thomas S. Peat, Stefan Sonderegger, Emma Toulmin, Stephen M. Jane, David J. Curtis, Paul A. Stupple, Ian P. Street. PRMT5 inhibitors as novel treatment for cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5371. doi:10.1158/1538-7445.AM2015-5371

Abstract 4029: BL-011256 is a novel VEGFR3 selective inhibitor, which suppresses tumor lymphatics and lymph node metastasis in an animal model of melanoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The role of VEGFR3 in lymphangiogenesis has been well established. Targeting VEGFR3 has been shown to curtail tumor progression mediated via lymphatic dissemination. More recently VEGFR3 was shown to play an important role in the mediation of tumor-induced immune cell tolerance. We have identified BL-011256, a novel inhibitor of VEGFR3 that exhibits 17-fold selectivity over VEGFR2 and a narrow tyrosine kinase inhibition spectrum. A 7-day b.i.d repeat oral dose study showed that BL-011256 is well tolerated in mice. Mouse plasma exposure experiments demonstrated that BL-011256 attains free drug plasma concentration levels that exceed the concentration required for IC50 activity on VEGFR3 in vitro but are considerably lower than the in vitro IC50 required for activity on VEGFR2. In the B16F10 mouse melanoma model, animals bearing melanoma tumors displayed considerably attenuated signs of tumor progression when treated with BL-011256. BL-011256 caused a 70% reduction in primary lesion growth and a 50% reduction in metastasis to the draining lymph node. Furthermore, BL-011256 was active in reducing the number of satellite in-transit metastases. Immunohistochemical whole mount analyses on ears with primary tumor lesions derived from BL-011256 treated and vehicle-treated mice using Lyve-1 for the identification of lymphatic vessels and CD31 for the identification of blood vessels was conducted. Tumors in vehicle-treated mice displayed a peri-tumoral area densely populated by lymphatic vessels. In contrast, tumors derived from BL-011256 treated mice were devoid of peri-tumoral lymphatic vessels. Notably, both vehicle-treated and BL-011256-treated animals displayed similar staining for peritumoral blood vessels, suggesting no effect on blood vessels (this is consistent with no activity on VEGFR2). Furthermore, PK sampling during the last day of dosing in a 14-day dosing schedule demonstrated that there is no compound accumulation during the repeat dosing schedule utilised in the B16F10 tumour efficacy experiment. In conclusion BL-011256 has been identified as a selective inhibitor of VEGFR3 that supresses both primary tumor growth and lymph node metastasis. Citation Format: Annabell Leske, Richard Foitzik, Donna Beaumont, John Bentley, Ylva Bergman, Chloe Brown, Michelle Camerino, Susan Charman, Neil Choi, Melanie De Silva, Matthew Chung, Hendrik Falk, Danny Ganame, Alison Gregg, Julian Grusovin, Andrew Harvey, Catherine Hemley, Ian Holmes, Belinda Huff, Daniel Inglis, Wilhelmus Kersten, Tina Lavranos, Romina Lessene, Gillian Lunniss, Brendon Monahan, Benjamin Morrow, Marica Nikac, George Nikolakopoulos, Dharam Paul, Tom Peat, Justin Ripper, Michaela Scherer, Paul Stupple, Karen White, Ian Street, Gabriel Kremmidiotis. BL-011256 is a novel VEGFR3 selective inhibitor, which suppresses tumor lymphatics and lymph node metastasis in an animal model of melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4029. doi:10.1158/1538-7445.AM2014-4029