KEY SIGNALING PATHWAYS THAT CONTROL METABOLISM and their modulaon by protein post translaonal modificaons. And an introduc,on to systems biology. CELL HOMEOSTASIS • Cells are constantly monitoring their environment and deciding whether they can divide, repair, go into senescence or should die. • In ,ssues cell death occurs when possible by a process that does not alert the immune system to respond…..there are 2 forms of this programmed cell death called apoptosis and necroptosis CELL STRESS MONITORING • CELL VIGILANCE AND RESPONSE IS PROVIDED BY A SET OF CELL SIGNALING PATHWAYS WHICH MODULATE METABOLISM DEPENDING ON CELL CONDITIONS. • THIS IS A VERY DYNAMIC PROCESS, OFTEN OCCURING RAPIDLY AND CHANGING REGULARLY. SUCH REGULATION INVOLVES REVERSIBLE POST- TRANSLATIONAL MODIFICATIONS OF KEY PROTEINS IN THE DIFFERENT METABOLIC PATHWAYS. THE KEY PROTEIN MODIFICATIONS THAT REGULATE CELL METABOLISM ARE: • Phosphorylaon • Acetylaon • Ubiqui,nylaon Longer term regulaon of metabolism also occurs through gene regulaon to alter the levels of enzymes in various pathways. Phosphorylaon/ dephoshorylaon by kinases and phosphatases respec@vely regulate enzyme pathway func@ons. KEY SITES OF PHOSPHORYLATION ON PROTEINS. • 1). Tyrosine • 2). Serine • 3). Threonine 30% OF ALL PROTEINS ARE MODIFIED. THERE ARE 518 KINASES IN HUMAN CELLS. OF THESE, MUTATIONS IN 100 CAUSE CANCER. Protein lysine acetylaon Acetylaon and deacetylaon is par,cularly important in regulang metabolic pathways as acetyl coA is a key metabolic intermediate. Acetyla@on of key proteins of metabolism inHepG2 cells aQer inhibi@on of SIRT3 (a deacetylase). All 5 oxphos complexes are acetylated to control electron flow and ATP synthesis. Deacetylaon of metabolic enzymes is through a set of proteins caled sirtuins 1-7 with 3,4 and 5 located in mitochondria. UBIQUITINYLATION Ubiqui,nylaon of proteins controls their levels by modulang their degradaon rate Ubiqui,n ligase Transcrip,onal regul. FBW7 PUMA c-Myc Ubiq Displaces NOXA MCl1 Mcl-1 NOXA De-ubiqui,nases Promotes de-ubiq. USP9X BIM Release from Mule MOM Upregulates p53 NOXA Apoptosis: regula@on of the levels of the an@- apopto@c protein Mcl-1. CELLS MONITOR THE ENVIRONMENT AND INTRACELLULAR FUNCTIONING CONTINUOUSLY AND RESPOND AS NEEDED TO ANY STRESS BEING IDENTIFIED. External stressors are:hormones substrate availability cell-cell contacts. • Internal stressors are: Reduced ATP/ADP rao NADH/NAD rao Acetyl coA levels ROS. NOS Illuminating Drug Toxicity • Try repair e.g UPR or mitophagy or autophagy • Not successful • Try senescence and wait for beer condions • Not successful • Iniate cell death. Illuminating Drug Toxicity • UPR (from the ER) • UPR from mitochondria • Mitophagy • Autophagy • Senescence • Apoptosis • Necroptosis All include morphological changes in the cell as well as pathway changes induced by the different signaling pathways. GLUCOSE Glycogen storage ribose glycogen nucleodes G6Pi 5-Pi Pentose Phosphate Glycolysis Pathways Gluconeogenesis lactate PYRUVATE amino acids Urea Cycle Pyruvate Dehydrogenase Complex B Oxidaon hormones sterols ACETYL CoA fay acids Oxidave Citric Acid Fay Acid Synthesis Phosphorylaon Cycle ketone bodies triacylglyercol CO2 ATP SHORT TERM REGULATION OR METABOLISM PHOSPHORYLATION ACETYLATION UBIQUITINYLATION Short term Cellular Control of Energy Metabolism AMP-ac,vated Kinase Glycogen glycogen creane Synthesis synthase G6Pi kinase Creane Pi pyruvate Sterol Synthesis HMG-coA reductase Acetyl-coA Fay Acid acetyl CoA carboxylase Synthesis GPAT Triacylglyerol ATP Fay Acid Synthesis Oxidaon LONG TERM REGULATION Transcription factors Transcription factor activators Transcription factor activator activators (sirtuins!) AMP kinase PGC 1 α(β) AMP kinase PGC 1 α(β) PPARs Fay acid oxidaon NRF 1 & 2 ERR α FOXO GR HNF4 α Tfam gluconeogenesis mt DNA nuclear mt replicaon genes Oxidave stress SOD, glutathione AKT SIGNALING ATP citrate P lyase PI3K PI3 P PDK1 P53 P AKT P AKT P Ikk P ac@ve MDM2 P Bad P inac@ve P FOXO3 FOXO3 inac@ve P mTOR Casp 9 P inac@ve GSK3 P P GSK3 Bax S6 S6 inac@ve ac@ve inac@ve Cell Survival Cell survival/proliferaon RAS/MEK PATHWAY P PI3K PI3 PDK1 RAS P AKT AKT P ac@ve P RAF P FOXO3 inac@ve FOXO3 P MEK 1/2 P GSK3 GSK3 ERK1/2 inac@ve P ACTIVE Cell survival/proliferaon mTOR SIGNALING AKT P PKCalpha SGK1 AMPK TSC2 RICTOR mTOR TSC1 mTORC 2 GbetaL RAPTOR mTOR P mTORC GbetaL S6ribosomal SK61 1 P Cell Growth ATG1 4E-BP1 Senescence Autophagy SELECTED MAPK PATHWAYS THAT CONVERT STRESS TO SURVIVAL OR DEATH Ox Stress TRAFs RAS PI3-K TRX RAF P AKT JNK U ASK1 P inac@ve ERK1/ERK2 (P42/44MAPK) Apoptosis P P ac@ve C-Jun BLOCKED P38MAPK ac@ve p53 ATF2 CHOP autophagy apoptosis CHOP DEATH TRADD ASK1 TRAFs CASP 3 Pro- RIP 1 Casp FADD 8 CASP 8 NIK BID ac@ve PI3K AKT OXIDATIVE IKK gamma RADICALS IKK beta P P IKK IKK PROTEOLYTIC IKK alpha alpha DEGRADATION alpha NFkappaB NFkappaB INACTIVE NFkappaB ACTIVE survival Apoptosis Amino acids ATP/ADP BCl2 mTOR. AMPK LC3 p150 ATG13 ATG4 BECLIN ULK1 LC3-A ATG7 ATG14 Membrane nucleaon. Sequestraon LC3-B LAMP2 Autophagosome lysosome AUTOPHAGY PATHWAY Insulin signaling • BUT WHAT HAPPENS IF YOU ARE INSULIN INSENSITIVE i.e. YOU HAVE TYPE 2 DIABETES AMP kinase meormin U thiazolidinediones Type II PGC 1 α(β) Diabetes PPARs Fay acid oxidaon NRF 1 & 2 ERR α FOXO GR HNF4 α Tfam gluconeogenesis mt DNA nuclear mt replicaon genes Oxidave stress SOD, glutathione Illuminating Drug Toxicity The MetProf Methodology HEAT MAP OF PROTEIN CHANGES INDUCED BY THIAZOLIDINEDIONES Arrows show protein changes unique enough to TRO among the glitazones in HepG2 (and not seen in cardios) to be related to toxicity. FA Protein a.a.met Mito/ FAO Glu met Krebs synth Ox stress apoptosis ER stress autophagy signaling synthesis oxphos Confiden,al .