Glycoproteins - protein + a few sugars - CHO is specific region for ID by other proteins - e.g.

, MHC class I and blood group antigens - also binding sites for Ag (viruses/ parasites) Proteoglycans (sugars with a little protein) - e.g., mucopolysaccharide, glycosaminoglycans - sugars + a few proteins - cell surfaces, ECM, CHO = anionic network - anticoagulation, compression, adhesion, structure Blood Group antigens - O: minimal CHO, ending in fucose - A: glycosyl transferase extends chain by adding Gal-N-Ac - B: glycosyl transferase extends chain by adding Gal Targetting - Mannose-6-P lysosome - Sialic acid secretory (or membrane bound if containing v. hydrophobic region) I cell disease - mucolipidosis - acid hydrolases necessary for proper lysosomal function are missing - N-acetyl glucosamine phosphotransferase is deficient o Enzymes lacks M-6-P (no targeting!) Synthesis of N-linked glycoproteins + protein targeting - targeting begins in the ER (cytoplasm proteins dont have CHO) - dolichol phosphate = carrier of core oligosaccharides to be added - sugar is attached to every amide N of asparagines; must be Asn XThr or Asn X Ser - partially processed in ER where 3 glucose and 1 mannose are removed - then, Golgi, sugars are added or removed Sunthesis of O-linked glycoproteins - Unlike N-linked, sugars are added sequentially instead of all at once! - Added to Ser or Thr Amino Acid Metabolism - 20% daily energy goes to resynthesizing proteins - N is lost from humans as HCO3- and NH4+ - -KG ALWAYS participates in transamination

transamination o removes AAs amino group as an intermolecular redox reaction o carbon that receives amino group is at aldehyde or ketone oxidative levels o carbon that donates amino group is at alcohol level Phenylalanine NH3 NH3-Glutamate + KG Phenylpyruvate +

o transamination to -KG to form glutamate occurs with 17 AAs AA + KG _______ + glutamate Exceptions are glu, lys, thr

Role of pyridoxal phosphate (Vitamin B6) - ping-pong reaction o this is why transamination doesnt deplete CAC of integral intermediates!! st - 1 step = Ping o Phe + enzyme-B6 ketoacid (phenylpyruvate) + enzyme-B6NH2 o AA donates is amino group to enzyme-bound B6 nd - 2 step = Pong o enzyme-B6-NH2 + KG enzyme-B6 + Glu o enzyme catalyzes the reverse reaction using a different ketoacid Basically, you use one amino acid and Vit B6 to make one Kacid; then KG can be used to make a new AA never running out of ketoacids make one, use one! Important Reaction: Oxaloacetate + Glutamate aspartate + KG b/c aspartate is the amino donor in urea synthesis and adenine synthesis

Glucose-Alanine Cycle - another way to transaminate - in post-absorptive state (eating), net release of AA into blood o Ala and Glu are > 50% total - Excess Ala is formed

o Branched chain AAs + KG ketoacids + Glu o Glu + pyruvate + ALT alanine + KG When Ala gets to liver, ALT reaction runs in reverse o Get pyruvate for glucose synthesis o Get amino group for urea synthesis o Different from Cori Cycle, where lactate instead of Ala is exported from muscle Glutamate needs an oxidative deamination catalyzed by glutamic dehydrogenase o Glutamate + NAD KG + NADH + NH4 o v. unfavorable o Equilibrium overcome in 2 ways: Consumption of NH4 by urea synthesis Can also form KG by an alternate route = purine nucleotide cycle GTP hydrolysis makes this rxn favorable!

Essential Amino Acids - nine are essential o Methionine, Threonine, Phenylalanine, Histidine, Isoleucine, Leucine, Lysine, Valine, Tryptophan MT. PHILLy VT (thanks, Matt!) Nonessential Amino Acids - 10 of the 11 can be synthesized by glucose - tyrosine cannot (is synthesized from phenylalanine) - phenylketourics have no phenylalanine hydroxylase o have to avoid excess consumption Phe o tyrosine has to be supplemented Synthesis of Nonessential AAs 1. minor chemical transformations of Krebs cycle intermediates a. e.g., glutamate 2. chemical modification of intermediates of glucose metabolism a. e.g., serine, glycine 3. chemical transformation of dietary essential AAs a. e.g., tyrosine ** cysteine synthesis requires #2 and # 3; modification of serine, but sulfur atom is derived from methionine Cysteine biosynthesis requires SAM - SAM is also used to methylate carboxylate side chains of Asp Glu and CG doublets in genome

Homocysteine donates its CH3 group but then needs to be remethylated Requires THF 1C pool A 1C moiety is transferred from THF to homocysteine Methylation of homocystrine also requires methylcobalamin o 5-Me-THF + homocysteine methionine o methylo cobalamin

Degradation of AAs - glucogenic can give rise to a net production of glucose - ketogenic The 1C THF Pool - His, Ser, Gly, and formate donate 1C fragments to the pool o Since serine can be derived from glucose, there is a virtually unlimited supply - Poll functions o Methylate metabolites (e.g., homocysteine) o Thymine side chain o Purine biosynthesis Metabolsim of NH4 released from AAs - urea is nontoxic; NH4 is though! - Urea synthesis is compartmentalized o Only occurs in liver - glutamate provides both NH4 molecules o one directly, the other through aspartate - the higher the NH4 concentration, the higher the synthesis of urea - urea synthesis is also regulated by N-acetylglutamate = allosteric activator of carbomyl phosphate synthase o more arginine more enzyme activity - urea synthesis consumes 4 ATP + in transporting citrulline into mitochondria o but, malate shuttle (+2ATP) net ATP loss of 2.25 ATP ** when in urea synthesis failure: if treated w/ large doses of benzoic acid, the THF, HCO3-, NH4+ can be excreted by means other than urea: hippuric acid Synthesis of creatine and creatinine - creatinine is synthesized at a constant rate in muscle o not reabsorbed in the glomerular filtration!! :. The amount of creatinine in the urine = filtration rate

creatinine synthesized in muscle by a first order nonenzymatic cyclization of creatine phosphate o more creatine PO4 more creatinine, more muscle mass, more creatine, and, again, more creatine PO4

Hormones - HRE = hormone reponse element - lipophilic hormone and its receptor bind to the HRE (transcriptional activating region ) of a gene - Protein Kinase catalytic domains o ATP-binding lobe | substrate binding lobe Catalytic center

Activation Loop Polypeptide hormones are synthesized as inactive pre-pro-hormones - pro-hormone = after signal sequence is claved off in RER - serine proteases cleave pro-hormone at dibasic AAs o e.g., Lys-Arg, Lys-Lys, or Arg-Arg o cleave on carboxyl side of site - processing begins in trans-Golgi o continues in secretory vesicles o final step can occur in extracellular fluid after secretion One gene = multiple copies of a single hormone - e.g., several Met-ENK, one Leu-ENK One gene = several different hormones - e.g., pre-pro-opiomelanocortin (POMC) 7 or 8 diff. Hormones - pre-pro-glucagon - major hormones with opposite effect o glucagons v. GLP1 o expression depends on specific proteases and + stimuli One gene = inactive precursor of a single hormone - e.g., insulin o mature = 2 chains connected by sulfide bonds o immature = B chain C peptide A chain Receptor Tyrosine Kinases - integral membrane proteins - hormone binding to EC domain dimerization - dimerization results in auophosphorylation

Three classes of tyrosine kinases - I: monomers in the absence of ligands - II: ligand binding causes conformational change in receptor dimers, autophosphorylation of cytoplasmic domain and IRS (docking site) - III: hormones are dimers themselves; crosslink monomers Intracellular signaling by tyrosine kinase receptors 1. IRS as a docking site for signaling components a. In class II receptors 2. Conserved domains are the building blocks of these complexes a. SH2, PTB: phosphotyrosine b. SH3: proline rich sequence c. PH: phospholipids, membranes a, b, and c for signaling proteins 3. A given receptor binds to multiple signaling molecules Intracellular Relays - G protein activation of protein kinase cascade - E.g., Ras = monomeric G protein + adapter protein o Activates GEF (guanine exchange factor) o Activates Raf = protein kinase MAPK kinase cascade Raf has a negative region domain at its N-terminus Ras binding causes Raf to assume an open conformation Exposes catalytic domain = ACTIVE Raf phosphorylates 2 serines in the activation loop of MAPK Or, - 2nd messengers generate effector enzymes o activate cytoplasmic protein kinases - PI3 kinase has SH2 domain o Binds to activated receptor o Phosphorylates IP3 at 3rd position o Causes recruitment of PKB from cytosol to membrane *** can use either/or, but usually both are needed to simulataneously activate different relays Insulin Receptor Signal Pathway - in liver, insulin stimulates MAPK pathway and targets PHAS-1 - phosphorylation of pHAS-1 activates eIF4e (protein synthesis) o change b/w glycolysis v. gluconeogenesis - insulin also changes the phosphorylation/dephosphorylation of enzymes

o changes enyzymatic activation sites (and, therefore, function) 1. insulin binds to the insulin receptor 2. activated receptor phosphorylates IRS 3. IRS-P serve as docking sites for: PI3K, tyrosine phosphatase, adapter molecule (Grb2 + Ras) a. *** PI3K translocation of GLUT4 receptor b. each of PI3K pathway and Grb2 and Ras pathway are needed for activation of glycogen synthase Type II diabetes 1. impaired insulin receptor function a. decreased tyrosine kinase activity in target cells b. inhibitors in cells c. interference w/ IRS phosphorylation and docking 2. impaired glucose uptake in fat and muscle cells a. remember: IRS-dependent activation of PI3K translocation of GLUT4 to plasma membrane b. so, decreased IRS receptor function dec. GLUT4 and :. dec. glucose uptake *** TNF negatively regulates IRS phosphorylation ** IRS2 -/- mice develop Type II diabetes b/c of failure of increased cell mass during dvelpmt G protein Receptors - receptors are serpentine o 7 transmembrane spanning domains 3 EC loops, 3 IC loops - ligand-binding pocket on EC surface composed of helices IV, VI, and VII - C-terminal tail contains sites that can be phosphorylated o Desensitization to stimulatory ligand heterotrimeric G proteins = , , and subunits o subunit = GTPase when receptor binds ligand, GTP exchanged for GDP when active, G dissociates from G subunits

G protein families - Gs stimulate adenylate cyclase - Gi inhibits adenylate cyclase - Gq stimulates phospholipase C - G12 stimulates ion channels

Free subunits (G or G) bind to effector enzyme + trigger production of 2 nd messenger Gs stimulates adenylate cyclase 1. GTP-Gs binds to adenylate cyclase (effector enzyme) 2. Adenylate cyclase stimulation causes an increase in camp 3. camp reveses negative regulation on PKA a. PKA was under pseudosubstrate inhibition 4. PKA phosphorylates specific cellular proteins Gq stimulates phospholipase C 1. GTP-Gq binds to phospholipase C (effector enzyme) 2. Phospholipase C catalyzes the hydrolysis of PIP2 into IP3 and DAG 3. IP3 release of Ca++ from the ER 4. DAG stimulates PKC 5. PKC phosphorylates substrate proteins GI inhibits adenylate cyclase (Gi stimulates PLC, same as Gq) 1. GTP-GI binds to adenylate cyclase and inhibits it 2. decreased adenylate cyclase maintained negative reg. of PKA 3. PKA cannot phosphorylate its cellular substrates Glucagon - acts almost exclusively on the liver 1. glucagons binds to Gs receptor 2. G s stimulates adenylate cyclase 3. adenylate cyclase increased camp PKA activity 4. catalytic subunit of PKA controls transcription factor CREB When your G-protein-linked signaling pathway goes WRONG! 1. Failure of pathway function by hormone resistance a. Reduced expression of receptor, G protein, or effector enzyme b. Impaired activation of receptor, G protein, or effector enzyme 2. Constitutive Pathway Function by hormone independent response a. Overexpression of receptor, G protein, or effector enzyme b. Inappropriate activation of receptor, G protein, or effector enzyme c. Increase in the activated time of receptor, G protein, or effector enzyme Cholera toxin (inappropriate activation) - ribosylates Gs w/ ADP - GTPase is inhibited - Gets trapped in active state!!

Elevated levels of camp cause intestinal cells to secrete large volumes of fluid

Pertussis toxin (impaired activation) - ribosylates GI w/ ADP - inhibits the exchange of GDP for GTP - prevents GI from inhibiting adenylate cyclase - increased camp Familial Precocious Puberty (inappropriate activation) - Asp578 residue important for H bonding w. adjacent helix o Leads receptor to off conformation - mutation subs glu for asp o no H bonding - LH receptor is now parially active even w/o ligand/hormone Pseudohypothyroidism (reduced expression) - decreased production of Gs o target cells are therefore unresponsive Steroid Biosynthesis - Five major classes of steroid hormones 1. progestins i. 21C from corpus luteum ii. stimulated by LH 2. glucocorticoids i. 21C from adrenal cortex ii. stimulated by ACTH 3. mineralocoticoids i. 21C from adrenal cortex ii. stimulated by AII/AIII 4. androgens i. 19C from testis ii. stimulated by LH 5. estrogens i. 18 C from ovary iii. stimulated by FSH ***Progesterone = intermediate for the formation of all of these hormones *** Mito. or ER enzymes convert cholesterol to these steroid hormones e.g., for cortisol - cholesterol mitochondria - intermediate ER - intermediate mito

secretion

hydroxylation reactions convert cholesterol to steroid hormones - require NADPH and O2 - catalyzed by monooxygenases o members of the cytochrome P450 family - also requires specialized O2-transport chains Adrenal Hormones Zona glomerulosa = mineralocorticoids Zona fasciculate = glucocorticoids Zona reticularis = androgens/ DHEA

Male gonadal hormones Leydig cells: testosterone Female gonadal hormones Theca cells: androgens (which will then be converted to estrogens by granulosa) Granulosa cells: estrogens Corpus Luteum: progesterone Cortisol secretion (molecular action) - ACTH binds cell membrane receptor - This binding activates Gs - This activation increases cAMP (from Gs) and Ca++ (from Gi) - cAMP activates PKA Renin/Angiotensin (aldosterone secretion) - angiotensin II and III bind to their cell surface receptor - activates Gq - increased IP3, DAG, and Ca++ - DAG and Ca++ activate PKC - PKC phosphorylates key proteins for aldosterone secretion Steroid Hormone Receptors - HBD = hormone binding domain at C terminus o Transcriptional activation domain is adjacent o May bind heat shock protein (HSP) when hormone is not bound - DBD = DNA binding domain o Centrally located o Cys-X2-Cys = zinc fingers x2 ZF1 = DNA binding ZF2 = dimerization - Variable domain = N terminus - Each receptor recognizes a specific HRE in the transcriptional control region - concensus HRE = 2 short repeats = half sites o separated by ,5 base pairs o can be inverted (homodimers) or direct (heterodimers) Function of Cortisol - stress response - increased blood glucose - immune suppression - mechanism o transcriptional regulation of gene expression

o suppresses immune system by inhibiting NFkB increases production of negative regulation products also cortisol binds to NFkB to prevent its action Function of Aldosterone - acts on distal convoluted tubule o Na+ retention o K+ and H+ excretion - mechanism o changes flux of ions across target cell membranes o stimulates phospholipase and fatty acid synthesis and acyltransferase

Disturbances in Adrenal Corticosteroid secretions - CAH = congential adrenal hyperplasia o Masculization of females (male 2nd ary sex characteristics) - Increase in ACTH production causes the hyperplasia - Diminished cortisol and aldosterone (because no 21 means no pathway to those two!) - Increased androgens (only way to go) - 21 -hydroxylase and 11 -hydroxylase = 2 most common defects o porter-silber color reaction will distinguish looks for 11-hydroxycortisol Cushings Syndrome = ACTH overproduced o Oversecretion of cortisol and androgens