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Hormone synthesis and degradation Chemistry of hormones • Steroids • Small molecules - NO • Amino acids derivates Receptor inside the cell – Thyroid hormones – Catecholamines • Proteins and peptides • FA derivates - eicosanoids Surface recceptors Steroid hormones • They are produced in cytoplasm from the cholesterol, beginning and end phases are located in the mitochondria • They can not be stored (they are produced de novo after signal) • Transport proteins bind them in the plasma SHBG, CBG, Albumin • Cytosolic receptors • After the receptor binding – Binding of hormone – receptor comlex to DNA – transcription (mRNA production) Steroid hormones Steroid synthesis • Steroid hormones are synthesized from cholesterol. • Cholesterol is mostly derived from plasma, but a small portion is synthesized in situ from acetyl-CoA. • Upon stimulation (e.g. by ACTH), free cholesterol is transported into the mitochondria, where a cytochrome P450 side chain cleavage enzyme (P450scc) converts cholesterol to pregnenolone. Side chain cleavage (removal of the six-carbon fragment) gives the 21-carbon steroid. • An ACTH-dependent steroidogenic acute regulatory (StAR) protein is essential for the transport of cholesterol to P450scc in the inner mitochondrial membrane. Steroid synthesis • All mammalian steroid hormones are formed from cholesterol via pregnenolone through a series of reactions that occur in either the mitochondria or endoplasmic reticulum of the producing cell. • Hydroxylases that require molecular oxygen and NADPH are essential, and dehydrogenases, an isomerase, and a lyase reaction are also necessary for certain steps. Steroid hormone synthesis • C21: – progesterone: directly from pregnenolone – cortisol: from progesterone, hydroxylation at 11, 17 a 21 – aldosterone: from progesterone, 11 and 21 hydroxylation, 18 oxidation to aldehyde • C19: from progesterone or pregnenolone: during 2C shortage at C17 you gain oxogroup, and subsequently OH → testosterone • C18: estrogen: aromatase (cleaves C18) Adrenal steroids A schematic representation of the pathways involved in the synthesis of the three major classes of adrenal steroids. The modifications at each step are shaded. Steroids Regulation of steroid synthesis • 3 regulatory steps: – Cholesterol release from internalized LDL – StAR protein = cholesterol transporter across the inner mitochondrial membrane – SCC = mitochondrial side chain cleavage enzyme • Signal: pituitary hormones (ACTH, LH, FSH) or angiotensine Steroid hormone degradation • Steran core cannot be cleaved in body • In the liver: hydroxylation and conjugation with glucuronides or sulphates • Urinary excretion of their metabolites can be used for the diagnosis. • Small part is excretes inchanged in the urine: UFC (urine free cortisol) Chemistry of hormones • Steroids • Small molecules - NO • Amino acids derivates Receptor inside the cell – Thyroid hormones – Catecholamines • Proteins and peptides • FA derivates - eicosanoids Surface recceptors Nitric oxide NO: produced by NO-synthase NH2 NH NH CH2 CH2 + NADPH + H+ CH2 CH2 HC NH3+ COOArginin NH2 O NH CH2 NADP+ + NO CH2 CH2 CH2 HC NH3+ COOCitrullin Nitric oxide • NO-synthase (NOS) – In neurons: NOS-I: neurotransmission – In macrophages: NOS-II: kills bacteria – Endothelial: NOS-III: vasodilation: difusion of NO toward the smooth muscle, activation of guanylatecyclase → cGMP → relaxation of the smooth muscle cells of the media → vasodilation • Clinical correlation: – Nitrates in the treatment of angina pectoris – Refractory hypotension during septic shock Nitric oxide • Interaction of an agonist (eg acetylcholine) with a receptor leads to intracellular release of Ca2+ via inositol trisphosphate generated by the phosphoinositide pathway, resulting in activation of NO synthase. Nitric oxide • The NO subsequently diffuses into adjacent smooth muscle, where it leads to activation of guanylate cyclase, formation of cGMP, stimulation of cGMP-protein kinases, and subsequent relaxation. • The vasodilator nitroglycerin is shown entering the smooth muscle cell, where its metabolism also leads to formation of NO. Chemistry of hormones • Steroids • Small molecules - NO • Amino acids derivates Receptor inside the cell – Thyroid hormones – Catecholamines • Proteins and peptides • FA derivates - eicosanoids Surface recceptors Thyroid hormones Thyroid hormones • The formation of triiodothyronine (T3) and thyroxine (T4) has many specific steps: • 1) Requires a rare element (iodine) for bioactivation. • 2) Hormones are synthesized as part of a very large precursor molecule (thyroglobulin) • 3) They are stored in an extracellular reservoir (colloid) • 4) There is peripheral conversion of T4 to T3, which is a much more active hormone. Thyroid hormones • In most parts of the world, iodine is a scarce component of soil, and for that reason there is little in food. • A complex mechanism has evolved to acquire and retain this crucial element and to convert it into a form suitable for incorporation into organic compounds. Thyroid hormones synthesis – key steps • 1) Secondary active transport of iodide into colloid • 2) Peroxidase iodidases Tyr residues of thyroglobulin • 3) Endocytosis and breakdown of thyroglobulin (Tgb) provide monoiodotyrosine (MIT) + diiodotyrosine (DIT) to condense to T4 and T3 • 4) Secretion of T3 and T4 is stimulated by TSH • 5) In plasma, T4 and T3 are bound to proteins: – – Albumin TBG (Thyronine binding globulin ) Iodide metabolism in the follicle • Iodide enters the thyroid through a transporter. • Thyroid hormone synthesis occurs in the follicular space through a series of reactions. • Thyroid hormones, stored in the colloid, are released from thyroglobulin by hydrolysis inside the thyroid cell. Hormony štítné žlázy Thyroglobulin (Tgb) • Thyroglobulin is the precursor of T4 and T3. It is a large iodinated, glycosylated protein. • Tgb contains many tyrosine residues, each of which is a potential site of iodination. • About 70% of the iodide in Tgb exists in the inactive precursors, monoiodotyrosine (MIT) and diiodotyrosine (DIT), while 30% is in the iodothyronyl residues - T4 and T3. Iodination of Tgb Thyreoglobulin CO C CH H2 NH HO I HO O I Trijodthyronin (T3) Tyr I HO I Thyreoglobulin CO C CH H2 NH MIT I HO I O I I Thyroxin (T4) I HO I DIT Thyreoglobulin CO C CH H2 NH H C C COOH H2 NH2 H C C COOH H2 NH2 Thyroglobulin (Tgb) • When iodine supplies are sufficient, the T4:T3 ratio is ~ 7:1. • In iodine deficiency, this ratio decreases, as does the DIT:MIT ratio. • Tgb provides the conformation required for tyrosyl coupling and iodide organification necessary in the formation of the diaminoacid thyroid hormones. Thyroglobulin (Tgb) • Thyroglobulin is synthesized in the basal portion of the cell and moves to the lumen, where it is a storage form of T3 and T4 in the colloid (several weeks' supply of these hormones exist in the normal thyroid). • Within minutes after stimulation of the thyroid by TSH, colloid reenters the cell and there is a marked increase of phagolysosome activity. • Various acid proteases and peptidases hydrolyze the thyroglobulin into its constituent amino acids, including T4 and T3, which are discharged from the basal portion of the cell. Iodide metabolism • The thyroid is able to concentrate I– against a strong electrochemical gradient. This is an energydependent process and is linked to the Na+/K+ ATPase-dependent thyroidal I– transporter. • The ratio of iodide in thyroid to iodide in serum (T:S ratio) is a reflection of the activity of this transporter. • This activity is primarily controlled by TSH and ranges from 500:1 in chronical stimulation with TSH to 5:1 or less when there´s no TSH. • The T:S ratio in humans on a normal iodine diet is about 25:1. Iodide metabolism • The thyroid is the only tissue that can oxidize I– to a higher valence state, an obligatory step in I– organification and thyroid hormone biosynthesis. • This step involves a peroxidase and occurs at the luminal surface of the follicular cell. • Thyroperoxidase requires H2O2 (is produced by an NADPH-dependent enzyme). Iodide metabolism • A number of compounds inhibit I– oxidation and therefore its subsequent incorporation into MIT and DIT. • The most important of these are the thiourea drugs. They are used as antithyroid drugs because of their ability to inhibit thyroid hormone biosynthesis at this step. Iodide metabolism • Once iodination occurs, the iodine does not readily leave the thyroid. Free tyrosine can be iodinated, but it can not be incorporated into proteins (no tRNA recognizes iodinated tyrosine). • The coupling of two DIT molecules to form T4 (or of an MIT and DIT to form T3) occurs within the thyroglobulin molecule. • A separate coupling enzyme has not been found, it is assumed that the same thyroperoxidase catalyzes this reaction. • The formed thyroid hormones remain as integral parts of thyroglobulin until the latter is degraded, as described before. Iodide metabolism • A deiodinase removes I– from the inactive MIT and DIT molecules in the thyroid. This mechanism provides a substantial amount of the I– used in T3 and T4 biosynthesis. • A peripheral deiodinase in target tissues such as pituitary, kidney, and liver selectively removes I– from the 5' position of T4 to make T3, which is a much more active molecule. • In this sense, T4 can be thought of as a prohormone, though it does have some intrinsic activity. Chemistry of hormones • Steroids • Small molecules - NO • Amino acids derivates Receptor inside the cell – Thyroid hormones – Catecholamines • Proteins and peptides • FA derivates - eicosanoids Surface recceptors Catecholamine synthesis • Substrate = Phe or Tyr • Synthesis located in: adrenal medulla (A), neural tissue (NA, DA) • Produced in the cytoplasm of the cells • Keeping in preformed vesicles • Free transport in plasma • Surface receptors: – After receptor binding – activation of G-protein • Products: – Epinephrine - adrenaline (hormone) – dopamine, norepinephrine - noradrenalin (neurotransmiters) Catecholamines are made from tyrosine • The major product of the adrenal medulla is epinephrine. This compound constitutes about 80% of the catecholamines in the medulla, and it is not made in extramedullary tissue. • In contrast, most of the norepinephrine present in organs innervated by sympathetic nerves is made in situ (about 80% of the total), and most of the rest is made in other nerve endings and reaches the target sites via the circulation. • Epinephrine and norepinephrine may be produced and stored also in other chromaffin tissues. Catecholamines are made from tyrosine • The conversion of tyrosine to epinephrine requires four sequential steps: • 1) ring hydroxylation • 2) decarboxylation • 3) side chain hydroxylation to form norepinephrine • 4) N-methylation to form epinephrine Catecholamines are made from tyrosine • Tyrosine hydroxylase is rate-limiting step for catecholamine synthesis. • It converts L-tyrosine to Ldihydroxyphenylalanine (L-dopa). • As the rate-limiting enzyme, tyrosine hydroxylase is regulated in a variety of ways. • The most important mechanism involves feedback inhibition by the catecholamines. Catecholamines are made from tyrosine • Dopa decarboxylase is present in all tissues. • This enzyme transforms L-dopa to 3,4dihydroxyphenylethylamine (dopamine). • Compounds that resemble L-dopa, such as αmethyldopa, are competitive inhibitors of this reaction – α-methyldopa is effective in treating some kinds of hypertension. Catecholamines are made from tyrosine • Dopamine-β-hydroxylase catalyzes the conversion of dopamine to norepinephrine. Catecholamines are made from tyrosine • Phenylethanolamine-N-methyltransferase (PNMT) catalyzes the production of epinephrine. • PNMT catalyzes the N-methylation of norepinephrine to form epinephrine in the adrenal medulla. • The synthesis of PNMT is induced by glucocorticoid hormones that reach the medulla via the intra-adrenal portal system. • This special system provides for a 100-fold steroid concentration gradient over systemic arterial blood, and this high intra-adrenal concentration appears to be necessary for the induction of PNMT. Catecholamin synthesis Tyrosine hydroxylase H C C COOH2 NH3+ 1. Phenylalanin H C C COOH2 NH3+ HO 2. HO Tyrosin 3,4 DihydrOxyPhenylAlanin (DOPA) DOPA decarboxylase H H2 C C HO HO Adrenalin H H2 C C 5. HO OH NH CH3 HO N-methyltransferase H C C COOH2 NH3+ HO OH NH3+ 4. 3. HO HO Dopamine βhydroxylase Noradrenalin Dopamin H2 C C + CO2 H2 NH3+ Catecholamines are made from tyrosine • Catecholamines cannot cross the blood-brain barrier – in the brain they must be synthesized locally. • In certain central nervous system diseases (eg, Parkinson's disease), there is a local deficiency of dopamine synthesis. • L-Dopa, the precursor of dopamine, readily crosses the blood-brain barrier and so is an important agent in the treatment of Parkinson's disease. Catecholamine breakdown Two key enzymes: MAO = monoaminooxidase COMT = catechol-o-methyl transferase MAO HO H C 77 COO78 OH CH3O COMT Inhibitors of MAO (IMAO) = antidepresive drugs Chemistry of hormones • Steroids • Small molecules - NO • Amino acids derivates Receptor inside the cell – Thyroid hormones – Catecholamines • Proteins and peptides • FA derivates - eicosanoids Surface recceptors Protein and peptide hormones • Neurotransmiters a neuromodulators: neuropeptides, opioids • Hypothalamic releasing hormones and pituitary peptides • Insulin and glucagone • Growth factors: IGF, CSF, EPO • Intestinal hormones … and many others General steps of peptide synthesis and action • • • • • • • • Expression of “pre-pro” protein Transport to ER Splitting the signaling sequence – we gain „pro-peptid“ Final posttranslational modifications in Golghi (there can be cleavage to definitive peptides) – proinsulin → insulin – POMC - proopiomelanocortine → MSH, ACTH and β-endorfine Their granules are stored in GA Free transport in plasma Surface receptors After receptor binding – Activation of membrane enzyme (insulin) – Activation of G protein Cellular mechanisms for peptide synthesis and release • Proteins synthesised by ribosomes are threaded through the membrane of the rough ER, from where they are conveyed in transport vesicles to the GA. • Here, they are sorted and packaged into secretory vesicles. Cellular mechanisms for peptide synthesis and release • Processing (cleavage, glycosylation, amidation, sulfation, etc.) occurs within the transport and secretory vesicles, and the products are released from the cell by exocytosis. Cellular mechanisms for peptide synthesis and release • Constitutive secretion (plasma proteins and clotting factors by liver) occurs continuously and only little material is stored in secretory vesicles. • Regulated secretion (cytokines or neuropeptides) occurs in response to increased intracellular Ca2+ or other intracellular signals, and material is typically stored in some amounts in secretory vesicles awaiting release. Degradation of peptide hormones • Lyzosomal degradation after endocytosis of complex hormonereceptor • Chemical modification (liver): rearrangement of S-S bridges, cleavage • Renal excretion of small peptides Chemistry of hormones • Steroids • Small molecules - NO • Amino acids derivates Receptor inside the cell – Thyroid hormones – Catecholamines • Proteins and peptides • FA derivates - eicosanoids Surface recceptors Eicosanoids • Derivates of arachidonic acid (20C:∆5,8,11,14). • Paracrine action, low plasma concentration, short halflife, no storage • Many functions: – Immunity/inflamation – Blood clotting – Regulation of the microcirculation… Eicosanoids • General steps of synthesis: – Release of arachidonic acid from membrane PL (enzyme PLA2) • After cleavage of PIP2 by PLCγ • This step is inhibited by glucocortikoids. – Cyclooxygenase (COX) produces prostaglandins and tromboxanes. • Acetylsalicylic acid and non-steroid antiinflammatory drugs (NSAID) inhibit COX. – Lipoxygenase → leukotrienes Prostaglandins 5 8 1 COO20 11 14 Kyselina arachidonová Arachidonate COX Inhibitors: - ASA (Aspirin) - paracetamole COX 1-3 O COO- O OOH Prostaglandin G2 Prostaglandin G2 (PGG2) Thromboxanes Contain oxane ring Synthetized from prostaglandines Role in blood clotting COO- O O OH Thromboxane A2 Leukotriens • Lipoxygenase: adds hydroperoxy (-OOH) group to C2, 12 or 15 of arachidonic acid • HPETE: hydroperoxyeikosatetrenoic acid • Conjugation with Cys or GSH • Antileukotrienes – antagonists of leukotriens receptors (zafirlukast, montelukast) in the treatment of bronchial astma