ADVANCED METABOLISM BCH 6206
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References for Gluconeogenesis Lecture Glucose 6 Phosphatase Burchell A 1990 Molecular pathology of glucose 6phosphatase FASEB J A29782988 Waddel I D Scott H Grant A and Burchell A 1991 Identi cation and characterization of a hepatic microsomal glucose transport protein T3 of the glucose 6phosphatase system Biochem J 363667 Lei KJ Shelly L L Pan CJ Sidbury J B and Chou J Y 1993 Mutations in the glucose 6 phosphatase gene that cause glycogen storage disease type la Science 580583 Argaud D Kirby T L Newgard C B and Lange A J 1997 Stimulation of glucose6phosphatase gene expression by glucose and fructose26bisphosphate J Biol Chem m 1285412861 Hemmerle H Burger HJ Below P Schubert G Rippel R Schindler P W Paulus E and Herling A W 1997 Chlorogenic acid and synthetic chlorogenic acid derivatives Novel inhibitors ofhepatic glucose6phosphatase translocase J Med Chem 137145 Arion W J Can eld W K Callaway ES Burger HJ Hemmerle H Schubert G Herling A S and Oekonomopulos R 1998 Direct evidence for the involvement of two glucose 6phosphate binding sites in the glucose 6phosphatase activity of intact liver microsomes Characterization of T1 the microsomal glucose 6phosphate transport protein by a direct binding assay J Biol Chem m62236227 Li Y Mechin MC van de Werve G 1999 Diabetes Affects Similarly the Catalytic Subunit and Putative Glucose6Phosphatase Translocase of Glucose6Phosphatase J Biol Chem m 3386633868 Massillon D 2001 Regulation of the Glucose 6phosphatase Gene by Glucose Occurs by T 391 39 39 n t 39 39 39 39 39 7 Biol Chemamp 40554062 1 Phosphoenolpyruvate Carboxykinase PEPCK Imai E Miner J N Mitchell J A Yamamoto K R and Granner D K 1993 Glucocorticoid receptorCAMP response elementbinding protein interaction and the response of the phosphoenolpyruvate carboxykinase gene to glucocorticoids J Biol Chem M53535356 Patel Y M Yun J S Liu J McGrane M M and Hanson R W 1994 An analysis ofregulatory elements in the phosphoenolpyruvate carboxykinase GTP gene which are responsible for its tissuespeci c expression and metabolic control in transgenic mice J Biol Chem 5619 5628 Granner D Andreone T Sasaki K and Beale E 1983 Inhibition of transcription of the phosphoenolpyruvate carboxykinase gene by insulin Nature 549651 Hod Y and Hanson R W 1988 Cyclic AMP stabilizes the mRNA for phosphoenolpyruvate carboxykinase GTP against degradation J Biol Chem m77477752 FranckhauserVogel S AntrasFerry J Robin D Robin P Forest C 1997 Transcriptional and l 391 39 39 39 39 of 39 39 39 39 u diat d repression of phosphoenolpyruvate 39 39 39 gene 1 39 quotI J Cell Biochem 386393 Role of PFKz Wu C Okar D A Newgard C B and Lange A J 2001 OvereXpression of6phosphofructo2 kinase26bisphosphatase in mouse liver lowers blood glucose by suppressing hepatic glucose production J Clinical Investigation m 9198 Role of Substrates Digirolamo M Newby F D and Lovejoy J 1992 Lactate production in adipose tissue A regulated function with eXtraadipose implication FASEB J 24052412 METABOLITE CONTROL OF TRANSCRIPTION N EUKARYOTIC CELLS Michael S Kilberg Department of Biochemistry amp Molecular Biology R3116 ARB 22711 mkilbergufledu THE BACTERIAL LACTOSE OPERON Transcnpllon mocked t 9 Lame Negative control of the lac opelon nacnve repvessor The detection and signal transduction mechanisms are different in mammals CHARACTERIZING METABOLITE CONTROL REQUIRES IN VITRO MODEL SYSTEMS De cient diet in Vivo Nutrient changes Hormonal changes Assay transcription CHARACTERIZING METABOLITE CONTROL REQUIRES IN VITRO MODEL SYSTEMS De cient diet in Vivo Nutrient de cient culture medium in Vitr0 Nutrient changes Hormonal changes Assa transcri tion Assay transcrlptlon y p CARBOHYDRATE CONTROL PROCESSES IN EUKARYOTIC CELLS 1 Genes Induced by Carbohydrate Starvation anolded Erotein Response U PR or ER Stress 2 Excess CarbohydrateInducible Genes A positive feedforward pathway ACTIVATION OF TRANSCRIPTION BY CARBOHYDRATE STARVATION THE UNFOLDED PROTEIN RESPONSE UPR IS A SIGNAL TRANSDUCTION PATHWAY RESPONSIBLE FOR THE INDUCTION OF GENES AFTER GLUCOSE DEPRIVATION AND OTHER CONDITIONS THAT CAUSE ENDOPLASMIC RETICULUM STRESS IN LEASL Few untoldd plotelns In ER Many unfolded proteins in ER cytoplasm 39 Acume c re1p en a 2 Transpcn and I I HAG1quotmRNA IIansIaIicn an 63 RNA lignss m HAC1mRNA Q m ranscanion cl genes encoding ERIasIdBnl chapemnes MECHANISM BY WHICH UNFOLDED PROTEINS ARE DETECTED IN THE ER 731 opia m Em twlasmic Reticulum BiP GRP78 is a resident chaperone protein in the ER that is normally bound to stress transducer proteins and therefore blocks their activation THE YEAST UNFOLDED PROTEIN RESPONSE UPR IS MEDIATED BY THE Hac1 TRANSCRIPTION FACTOR ed proteins In ER GLUCOSE LIMITATION Transcnplion cl genes encoding ERresidem chapewnes n Illuuei nu 39 r v 1 n 39 39 39 a mu h 39 39 activated by accumuialion oi unIoIded proIeins in me ER Actlvalad causes attenu oi translat and Ihe HACiv Ivan 791 p ciaavas lha intvon irom HAC1quot mRNA The spiicing reaction is accumulates int me that 39 39 39 a danirlnll r I 39 quot 39 r 39 39 39 39 nuclear details transcription oi ganss encoding ERresidenl chaperones In ihe THREE SUBGROUPS OF GENES ARE INDUCED BY Hac1 1 ER chaperones to refold proteins 2 A protein degradation system 3 Proteins that induce apoptosis UPRE GENOMIC SEQUENCES RECOGNIZED BY THE YEAST Hac1P TRANSCRIPTION FACTOR 1 2 Yeast UPRE Consensus LH57 ACT KAR FKB PDI SC U6 EUG mRNA Induction I m 9 T quot XLO XEJ KZJ x33 x23 x32 X13 1000 iGafactostdase Acmny Units o gtlt KARZ 459 GGAACTGGACAGCGTGTCGAAA i s gtlt FKBZ an CATTACTGCCAGCGCATCTTCA 4m gtlt x PDIIa 4m CCETTTGCCACCGTGTAGCAT 77 gt34 PDIb A247 CCTG I CGGGCGGCGCCTCTTTT 7226 04 SCJ7 gt155 Cgl gGTAATCAGCGTAGTACTT 217 41a CTTTTATAACAGCGTGTTCGAT 405 0 a EUGI 7123 TTC AAGGCACGCGTGTCCTTT dc CAnCnTG TM tunicamycin which blocks glycoprotein biosynthesis and thus activates the UFR pathway THE UPR IN MAMMALIAN CELLS IS MORE COMPLICATED amp IS ALSO REFERRED TO AS THE ER STRESS RESPONSE Identification of the endoplasmic reticulum stress response element ERSE GRP78 ERSEl human 61 CCAATCGGCGGCCTCCACG ERSEl marine CCAATCGGAGGCCTCCACG ERSEI rat 98 CCAATCGGAGGCCTCCACG GRP94 ERSEl human 72 CCAATCGCGCCGCACCACG ERSEl Chicken 74 CCAATGGGAGCGCACCACG ERSES human 191 CCAATCGGAAGGAGCCACG ERSEB Chicken 204 CCAATCGACGCCGGCCACG CRT ERSE3 human Z4 CCAATGATGGTCGACCACG ERSES marine 207 CCAATGAGGGTCGACCACG Yoshida H Haze K Yanagi H Yura T and Mori K J Biol Chem 1998 273 3374133749 Characterization of the endoplasmic reticulum stress response element ERSE ERSE1 I Relative Activity Fold Induction 39 65 464300 05 10 15 i wouxlmulbwwia Yoshida H Haze K Yanagi H Yura T and Mori K J Biol Chem 1998 273 33741 33749 Binding proteins for the endoplasmic reticulum stress response element ERSE GRP78 ERSEl human 61 CCAATCGGCGGCCTCCALG ERSEl marine CCAATCGGAGGCCTCCACG ERSEl rat 98 CCAATCGGAGGCCTCCACG GRP94 ERSEl human 72 CCAATCGCGCCGCACCACG ERSEl chicken 74 CCAATGGGAGCGCACCACG ERSES human 191 CCAATCGGAAGGAGCCACG ERSE3 chicken 204 CCAATCGACGCCGGCCACG CRT ERSE3 human 284 CCAATGATGGTCGACCACG ERSES marine 29 CCAATGAGGGTCGACCACG AFFINITY PURIFICATION USING THE ERSE CONSENSUS SEQUENCE or 5 CCAAT N9 CCACG 3 THE UNFOLDED PROTEIN RESPONSE IN MAMMLIAN CELLS FREE But mammalian cells have an re1p protein How is it involved And what about XBP1 the other ERSE binding protein THE UNFOLDED PROTEIN RESPONSE IN MAMMLIAN CELLS RE1 ATFG unlolded unfolded pro ems proleins I gt U gt lumen G 6MB W WW pATF6P l unspliced XBP1 mRNA spliced XBP1mRNA pATF6N 0 T pXBP1U ease4m pXBP1S ERSE ER chaperone UPRE XBP1 mrget mNm gtNm xwuNmmqumltm ltgtltltgtgtz Omzmm gt DO 20 OOZgtZ m mmmm 002mmzmcm mmOcmZOm VAN uk m I 40900400 w THE UNFOLDED PROTEIN RESPONSE IN MAMMLIAN CELLS HE1 unlolded unfolded ATFG protems proteins gt U O gt W lumen O pATF6P unspliced XBP1 mRNA spliced XBP1 mRNA pATF6N O V T pXBP1U ew pXBP1 S ERSE ER chaperone UPRE XBP1 target THREE GROUPS OF GENES ARE INDUCED BY THE UPR PATHWAY IN A SPECIFIC TIMEDEPENDENT MANNER 1 ER chaperones to refold proteins 2 A protein degradation system 3 Proteins that induce apoptosis THE UNFOLDED PROTEIN RESPONSE IN MAMMLIAN CELLS F6 IRE1 unlolded unlolded AT protems proteins 0 gt U O gt W lumen i O pATF6P unspliced XBP1 mRNA spliced XBP1 mRNA pATF6N 0 T pXBP1U easem pXBP1S ease upne ER Stress Response Element ERSE Mammalian UPRE 5 CCAAT N9 CCACG 3 5 TGACGTGGIA 3 For comparison Yeast UPRE Consensus 539 CAnCnTG 339 ACTIVATION OF TRANSCRIPTION BY THE EXCESS OF CARBOHYDRATE GENE TRANSCRIPTION INDUCED BY THE PRESENCE OF CARBOHYDRATE 1 LType Pyruvate Kinase Gycoysis 2 Acetyl COA Carboxylase Fatty acid synthesis 3 Fatty Acid Synthase Fatty acid synthesis 4 ATP Citrate Lyase Fatty acid synthesis Carbohydrateinduced enzymes associated with fatty acid curate COA blosyntheSIS l Citrate Lyase Acetyl CoA Oxaloacetate HCO339l l Acetyl CoA Carboxylase l Malonyl COA l lFattyAcid Synthase l Fatty acids TRANSCRIPTIONAL CONTROL OF LType PYRUVATE KINASE BY CARBOHYDRATE The ChRE within the LPK promoter 1ao 7160 7140 12u gt100 I l I I I I I 1 I GATCCAGCAGCATGGGC I CGGGGCACTCCCGT TTCCI39GGACTCTGGCCCCCAGTGTACMGGCWCCGI39I39GGCAAGAGAGATG Frc 1 Sequence of the glucose response element of the LPK gene 188 to 97 Determination of ChRE in several genes led to the identification of a consensus sequence Similar to two Ebox CACGTG GCACT CACGTG CAnnTG sequences separated by 5 bp The Ebox sequence is recognized by the basichelixloophelixleucine zipper family of transcription factors GLUCOSE SENSING GENOMIC ELEMENTS IN MAMMALIAN CELLS GLUCOSE EXCESS ChRE 539 CAnnTG N5 CAnnTG 339 GLUCOSE DEFICIENCY ERSE 539 CCAAT N9 CCACG339 UPRE 539 TGACGTGGIA 339 IDENTIFICATION OF A CARBOHYDRATE RESPONSE ELEMENT BINDING PROTEIN ChREBP BY ELECTROPHORESIS MOBILITY SHIFT ANALYSIS EMSA ChRE radiolabeled DIA prabe used ta test for pratein binding CAnnTG N5 CAnnTG Fat Cho ChFIEBF k USF GFIBP Hg 4 A W mm my m mum nmeanmamm remg39uzetm wr LPKEWE meerenarvemar h aneIlandgummuemwre eda mum mum mm WWIer cammmng usFarme gmch rewame mm mmmg DMem swam a ammm by warm Yamaslma at al znn1 PNAS 933115 THE DNA BINDING ACTIVITY OF ChREBP IS REGULATED BY PHOSPHORYLATION l A a NLS Prorich bHLH ZIP ZIPlike B l 2 3 4 5 6 PChREBP 3 IE 1 v E lt E 5 s47 lt 5 an 577 2 5 a 3 939 E 30 i 0 PKA ATP 4 PPZA PKI on Fig 4 A ChREBP contains several recognizable protein motifs including a bipartite nuclear localization signal NLS a basic helix loop helix leucine zipper bHLHZIP o i and three consensus PKA p osphorylation sites P including one sequence nun 39 39 quot39quot Hre 39 quot39 39 quot rmquot quot39 quot39 quot quot39 ofChREBP can be regulated by phosphorylation in vitro Incubation of active ChREBP lane 1with both A P and PKA abolished DNAbinding activity lane 2 Omission of ATP from the 39 39 r 39 39 39 r 39 L39 L39 39 39 quot 39 39 39 rnl an 4 After PKI addition DNA binding auivily u L A 4 L A 39 L L A 2A PPZA lane 5 Addition of the PPZA inhibitor okadaic acid OKA 10 nM prevents reactivation of the ChREBP DNAbinding a Right Incubation of the protein with ATP in the presence ofPKA leads to radIolabeling of Ch E Yamashita et al 2001 PNAS 989116 REGULATION OF ChREBP FUNCTION BY THE PRESENCE OF CARBOHYDRATE ywonl Nucleuli Hiroiiii vaiiiashita Makoto Takehoshita Masaharu sakurai Rlchard K Eruick William J Hehzel n m Am r 2001 transcription factor that regulates carbohydrate metabolism in the liver Proc Natl Acad Sci 59 51168121 SUMMARY 1 Specific genes are transcriptionally regulated by metabolites so that the cell can respond to changes in nutritional variability 2 Each class of metabolites amino acids sugars lipids etc has its own signal transduction mechanisms and modulate a different set of cellular genes 3 Even for a given metabolite glucose for example the signal transduction pathway that detects an increased level of the metabolite can be different than the one that detects a limiting amount 4 The genomic cisacting elements that mediate the changes in transcription of the target genes are different for each signal transduction pathway Even when the metabolite is the same eg glucose BCH 6206 Lecture Notes on Amino Acid Metabolism Charles M Allen Office R32263 Phone 3923366 email callenbiochemmedufledu AMINO ACID METABOLISM A B Sources and Uses of Amino Acids Nutritional Dietary Requirements for Amino Acids Digestion Adsorption and Excretion Nitrogen Balance Endogenous Amino acid Degradation and Urea Cycle Hepatic Glutamine Metabolism Biosynthesis and Utilization of Various Amino Acids in Specialized Biosynthetic Pathways Sources and Uses of Amino Acids Sources Proteins in the diet provide both essential and non essential amino acids in contrast to microorganisms that for the most part synthesize their own Turnover of endogenous proteins De novo biosynthesis nonessential amino acids Uses Protein synthesis Nitrogen and carbon source of general and special product biosynthesis Energy source a glucogenic those that can be used for the synthesis of glucose b ketogenic those whose metabolism leads to ketone bodies Overview of Nitrogen Metabolism Dietary Protein Intracellular Protein and Amino Acid Pool Blood Amino Acids ll Intracellular Amino Acid Pool amp Carbon Skeletons Energy l Glucose Nutritionally Nonessential v Amino Acids Nonprotein Derivatives Urea Ketone bodies B Nutritional Dietary Requirements for Amino Acids daily adult intake of 08 glkg 25 oz of meatday This will be higher for a child or pregnant woman essential amino acids Leu Trp Lys Met lle Phe His Thr Val Arg Biosynthesis rate inadequate to support normal growth of children Animal proteins are often low in certain important amino acids eg Met and Lys whereas some vegetables are low in specific amino acids legumes are low in Met grains are low in Lys corn low in Try and Lys so diet must be varied to complement the missing amino acids from one food with those from another C Digestion Adsorption and Excretion Digestion Dietary proteins can not be absorbed directly from the intestine They must be hydrolyzed by a group of proteases and peptidases to amino acids dipeptides and tripeptides The rst major site of digestion is in the stomach by the action of pepsin then by the action of pancreatic enzymes eg trypsin and chymotrypsin and intestinal peptidases functioning in the intestine then complete the hydrolytic process Absorption The amino acids and small peptides are transported into the intestinal cells of the brush border by a family of amino acid specific transports many of which require Na The Na dependent transporters are just one class of transporters In this group the amino acid and Na ion are transported together in the same direction symport Transport in this case is driven by the Na gradient high in the intestinal lumen and low in the intestinal cell The cell in turn has active transporters which utilized the energy of ATP hydrolysis to transport Na out of the cell and K into the cells thus maintaining the low cellular Na concentration In the case of the intestinal cell facilitated transporters on the serosal side transport the amino acids into the blood plasma intestinal lumen Amino acid Na p Brush ll border I quot Amino Nafr i acid Active I transporter 5 Na K4 Serosal side Facilitated Amino transporter Cid Portal vein System ASC Examples of Amino Acid Transport Systems lon Dependence Na Na Na independent Na Na independent Na Na Amino Acid Transported Neutral Neutral Branched chain and aromatic Nitrogen side chain GlnAsnHisLysArg Cationic amino acids Aspartic and Glutamic acids Proline Excretion Nitrogen is also constantly being lost from the body in a variety of different forms The principal excretory nitrogen product in mammals is urea about 1220 g urea nitrogenday Other excretory products include ammonium ion uric acid and creatinine The amounts of each of these is dependent of the metabolic state of the individual Ureotelic excrete urea mammals Uricotelic excrete uric acid birds insects Ammonotelic excrete ammonium ion fish Some animals switch from one form to another during development more about that later D Protein Turnover and Nitrogen Balance Most intracellular proteins are undergoing continual breakdown and synthesis The rate of turnover of these proteins is variable and usually will vary depending on the nature of the protein and the metabolic state of the individual Two major pathways are involved in protein turnover one is carried out by proteases in lysosomes and a second major pathway involves a ubiquitin dependent pathway working in conjunction with a macromolelcular protease complex called a proteosome The amino acids released in this process can then enter into the same pathways as the amino acids derived from the diet Nitrogen Balance Nitrogen intake Nitrogen excretion positive nitrogen balance negative nitrogen balance Positive Nitrogen balance growth of children pregnancy wound healing convalescing adult intake gt excretion excretion gt intake Negative Nitrogen balance starvation malnutrition disease burns trauma surgery Mammals cannot select the specific amino acids they have in their diet therefore they take in some amino acids in excess of their needs The mechanisms of utilization of those consumed in excess for the purpose of synthesizing the deficient ones is part of the dynamic metabolism of nitrogen metabolism Of course the essential amino acids cannot be made de novo in the animal cell Amino acids which are not utilized for protein synthesis or other pathways of nitrogen utilization are not excreted in any large amounts but are deaminated in one of several ways The carbon skeletons are oxidatively degraded for the production of energy or stored as carbohyrdrate Ammonia produced is reutilized for new amino acid synthesis or converted to urea for excretion It is important to remember however that just because an individual is in nitrogen balance it does not mean that the dynamic metabolism of the amino acids is nonexistent On the contrary the proteins of the body are constantly in the process of degradation and resynthesis At the same time dietary amino acids are being utilized in a manner that supplies the cells with the needed energy and building blocks for normal function Endogenous Amino Acid Degradation and Urea Cycle Transamination amino transferases transaminases Deamination a Oxidative NAD an FAD dependent b Nonoxidative Ammonia Assimilation a Glutamate dehydrogenate b Glutamine synthetase c Carbamoyl phosphate synthetase l Urea Cycle 1 Transamination amino transferases transaminases Nearly all amino acids except lysine threonine proline and hydroxyproline can be metabolized with the loss of the damino group to give the corresponding dketo acid The immediate acceptor for this amino group is the cofactor pyridoxal phosphate Ultimately the amino group is transferred to dketoglutarate or pyruvate to give the amino acids glutamate and alanine as products Since alanine can also give up its amino group to dketoglutarate all amino acids undergoing transamination can have their amino group transferred to form glutamate O H HO CHZOP Pyridoxal I phosphate H30 Ti H
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