Kinetic models of Ras-GTPases in molecular signaling networks.
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1 Kinetic models of Ras-GTPases in molecular signaling networks. Hugo A. Armelin Instituto Butantan FAPESP week-montevideo
2 Outline 1. CeTICS: institutional status and mission. 2. Signaling networks: static maps and dynamic models. 3. Ras-GTPases molecular switches.
3 Center of Toxins, Immuneresponse and Cell Signaling Grant 2013/ , São Paulo Research Foundation (FAPESP) Governance and management staff Hugo A. Armelin, chief executive Vanessa Rioli, executive manager Leila H. Kussunoki, bookkeeper officer Faculty of Principal Investigators Ana Marisa Chudzinski-Tavassi Maria Carolina Q. B. Elias-Sabbaga Denise V. Tambourgi Fan Hui Wen*** Hugo A. Armelin* Inácio Junqueira de Azevedo Junior Barrera Mônica Lopes Ferreira** Solange M. T. Serrano Wilmar Dias da Silva Yara Cury *Coordinator **Coordinator of Technology Transfer ***Coordinator of Knowledge Dissemination
4 Mission of CeTICS Premise Novel toxins might lead to discovery of molecular signaling networks of potential therapeutic value. Objectives 1 - To discover, isolate and chemically characterize novel toxins. 2 - To analyze systemic responses of cells and/or organisms to novel toxins aiming to uncover molecular signaling networks underlying complex biological phenomena. 5
5 Interdisciplinary Group of Molecular Cell Biology, Bioinformatics and Computer Biology Principal investigators: Hugo A. Armelin (Group leader) and Junior Barrera Associate investigators: Marcelo S. Reis and Milton Y. Nishiyama-Jr Postdoc fellows: Matheus H. Dias and Vincent Noël PhD students: Cecília S. Fonseca and Eduardo Lopes
6 Present critical challenge Transformation of molecular static maps into functionally predictive dynamic models of metabolic reactions and molecular signaling networks based on low- and high-throughput quantitative omics data plus biological knowledge of heuristic value.
7 Map of the EGF/EGFR signaling axis (Oda & Kitano, 2005)
8 Ras-GTPase Subfamily Ras-GTPases paralogs. A G1 G2 G3 HRAS MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMR NRAS MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMR KRAS MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMR G4 HRAS DQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVKDSDDVPMVLVGNKCDLAA NRAS DQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPMVLVGNKCDLPT KRAS DQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPS G5 HRAS RTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREIRQH^ESGPGCMSCKCVLS NRAS RTVDTKQAHELAKSYGIPFIETSAKTRQGVEDAFYTLVREIRQY^DGTQGCMGLPCVVM KRAS RTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKH^KKKKKKSKTKCVIM B KRAS Splicing variants: KRAS2B (C-terminal):KKKKKKSKTKC**VIM* KRAS2A (C-terminal):KTPGC*VKIKKC**IIM* ** Farnesylation site * Palmitoylation site * Three C-terminal residues eliminated by proteolysis.
9 Ras superfamily of small monomeric GTPases 5 Families: Ras Rho Ran Rab Arf 7 Ras subfamilies: Ras Ral Rit Rap Rheb Rad Rhot-Miro Ras subfamily: HRas KRas NRas All Ras superfamily members possess a G domain core, which provides latent GTPase and G nucleotide binding activities.
10 [GTP] GEF [GDP] Active Inactive Ras-GTP Ras-GDP Efector P i 2- GAP ON / OFF molecular switch.
11 wt-kras/amplification-driven mouse Y1 adrenocortical tumor cell line Y1 tumor cells display a surprising association of phenotypes*: a) high basal levels of [K-Ras-GTP] and b) irreversible cell cycle arrest by FGF2 Simulations with K-Ras kinetic models suggested a molecular basis for this unexpected phenotypic association, which was experimentally validated. *Costa et al, Cancer Res (2008)
12 K-Ras-GTPase cycle module 1 Ras-GTP+ SOS k 1 k 1 SOS allo Ras-GTP k cat 4, K 4m k cat 5, K 5m SOS allo Ras-GDP k 2 k 2 SOS +Ras-GDP GTP positive feedback Ras-GTP Ras-GDP positive feedback GDP GAP k cat 3, K 3m P i d[sos] d[sos allo Ras-GTP ] d[sos allo Ras-GDP ] d[ras-gdp] = k 1 [SOS][Ras-GTP] + k 1 [SOSallo Ras-GTP ] = + k 1 [SOS][Ras-GTP] k 1 [SOSallo Ras-GTP ] = + k 2 [SOS][Ras-GDP] k 2 [SOSallo Ras-GDP ] = kcat 4 [SOSallo ][Ras-GDP] Ras-GTP k cat 5 [SOSallo ][Ras-GDP] Ras-GDP + K + [ Ras-GDP] 4m K + [ Ras-GDP] 5m k 2 [SOS][Ras-GDP] + k 2 [SOSallo Ras-GDP ] + k 3 cat[gap][ras-gtp] k K + [ Ras-GTP] 2 [SOS][Ras-GDP] + k 2 [SOSallo ] Ras-GDP 3m d[ras-gtp] = + k 4 cat[sosallo ][Ras-GDP] Ras-GTP K + [ + k cat 5 [SOSallo ][Ras-GDP] Ras-GDP Ras-GDP] 4m K + [ + Ras-GDP] 5m kcat 3 [GAP][Ras-GTP] K 3m + [ Ras-GTP] k 1 [SOS][Ras-GTP] + k 1 [SOSallo Ras-GTP ] (M.S.Reis, M.H.Dias, et al., unpublished)
13 Computer simulation 1 Low [SOS]: (M.S.Reis, M.H.Dias, et al., unpublished.)
14 K-Ras-GTPase cycle module 2 Ras-GTP+ SOS k 1 k 1 SOS allo Ras-GTP k cat 4, K 4m GEF k6 cat, K 6m k5 cat, K 5m SOS allo Ras-GDP k 2 k 2 SOS +Ras-GDP GTP positive feedback Ras-GTP Ras-GDP positive feedback GDP GAP k cat 3, K 3m P i d[sos] d[sos allo Ras-GTP ] d[sos allo Ras-GDP ] d[ras-gdp] = k 1 [SOS][Ras-GTP] + k 1 [SOSallo Ras-GTP ] = + k 1 [SOS][Ras-GTP] k 1 [SOSallo Ras-GTP ] = + k 2 [SOS][Ras-GDP] k 2 [SOSallo Ras-GDP ] = kcat 4 [SOSallo ][Ras-GDP] Ras-GTP k cat 5 [SOSallo ][Ras-GDP] Ras-GDP + K + [ Ras-GDP] 4m K + [ Ras-GDP] 5m k 2 [SOS][Ras-GDP] + k 2 [SOSallo Ras-GDP ] + k 3 cat[gap][ras-gtp] K + [ k Ras-GTP] 2 [SOS][Ras-GDP] + k 2 [SOSallo ] Ras-GDP 3m kcat 6 [GEF][Ras-GDP] K 6m + [Ras-GDP] d[ras-gtp] = + k 4 cat[sosallo ][Ras-GDP] Ras-GTP + k cat 5 [SOSallo ][Ras-GDP] Ras-GDP + K + [ Ras-GDP] 4m K + [ Ras-GDP] 5m k cat 3 [GAP][Ras-GTP] k K + [ Ras-GTP] 1 [SOS][Ras-GTP] + k 1 [SOSallo ] + Ras-GTP 3m kcat 6 [GEF][Ras-GDP] K 6m + [Ras-GDP] (M.S.Reis, M.H.Dias, et al., unpublished)
15 Computer simulation 2 (M.S.Reis, M.H.Dias, et al., unpublished.)
16 Expression of RasGEF families in Y1 cells Parental Y1 cells RasGEF families: 1. SOS 2. GRF 3. GRP GRP4 qrt-pcr (M.S.Reis, M.H.Dias et al., unpublished)
17 FGF2-Resistant Y1 sublines (Y1FRs) (C.S.Fonseca, M.H.Dias et al., manuscript in preparation, 2016)
18 Thank you
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