3 steroid metabolism 2


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3 steroid metabolism 2

  1. 1. 2 1
  2. 2. Vitamin D, calciols transport proteins of steroids27-hydroxycholesterolconjugation of steroids© Department of Biochemistry (V.P.), Faculty of Medicine, MU Brno 2011 2
  3. 3. Vitamin D, calciols 3
  4. 4. cholesterol 3 5 HO ( cholest-5-en-3-ol ) 4
  5. 5. 7-dehydrocholesterol : 3 5 7HO 5
  6. 6. cholecalciferol, vitamin D3HO UV (290 nm)  „ 9,10-secosterols “ 6
  7. 7. cholecalciferol, vitamin D3 origin from 7-dehydrocholesterol (skin, UV radiation) open B ring („seco-“ compound) systeme of conjugated double bonds (the first in the position 5, like as in cholesterol) 7
  8. 8. Brassinosteroid (→ steroid hormones of plants) : HO OH HO HO O O● regulate the growth and development in plants (no influence on sexuality in plants !)● skeletone similar to cholesterol (here is next carbon atom in the side chain, the structure of B ring is a lactone, i.e. cyclic ester) 8
  9. 9. ergostane C28 : 28C28 - basic steroid hydrocarbon in plants (fytosterol) (the 28th carbon of skelet is in this position marked right as „241“ ) 9
  10. 10. Steroids numbering :IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) (1989)The nomenclature of steroids. Recommendations 1989. Eur. J. Biochem. 186, 429-458. 10
  11. 11. ergocalciferol, vitamin D2 22 28HO 11
  12. 12. ergocalciferol, vitamin D2 C 22 D 28 „unfolded structure“ AHO 12
  13. 13. Change of vitamin to hormone „calcitriol“: 25 - OH in the liver 1 - OH in the kidneys(influence of parathormone) OH 1 25 HO Calcitriol  expression of CBP gene (calcium binding protein)  synthesis of protein rich in –COOH groups in side chains  OH binding of Ca2+ 13
  14. 14. the liver 1-hydroxylase : • mitochondrial enzyme in the proximal tubule of kidneys • hydroxylate 25-hydroxy-vit.D3 (from the liver) • it is inhibited by higher concentration of calcitriol (feedback), by higher concentration of Ca2+ and phosphates in plasma • stimulated by parathormone and by decrease of concentration of phosphate in plasma • at its inhibition is formed the inactive 24,25- dihydroxy-vit.D3 (proceeds „alternative“ 24-hydroxylation in substitution of „common“ hydroxylation at the C-1 )• megalin/cubilin mediated reabsorption of „vitamin D binding protein“ is responsible 14 for the renal conversion of 25(OH)D3 to 1,25(OH)2D3 in the proximal tubule
  15. 15. Transport proteins of steroidssee also: megalin and cubilin !! 15
  16. 16. Binding proteins of steroids (1):SHBG (= TEBG) sex hormone binding globulin, testosterone/estrogens binding globulin • 1-globulin of blood plasma, transport protein, synthetized in the liver • testosteron has conspicuously higher affinity to SHBG than estradiol (approximately 5times) • estrogens increase the SHBG concentration (woman has app. twice higher SHBG), testosterone reversely decreases the concentration SHBG in plasma • only free hormones can be bonded on proteins, conjugated proteins cannot be bonded (glucosiduronates, sulfates) • steroid, bonded on the protein SHBG, is in this form transported into the cell (i.e. complex: steroid-protein) • increase of SHBG in man  „estrogenization“ (= result of increase of E2 transported into cell – i.e. increase on SHBG bonded E2 ! ) 16
  17. 17. Binding proteins of steroids (2): BLOOD PLASMA: SHBG (= TEBG) sex hormones binding globulin testosterone/estrogen binding globulin transfer: testosterone estrogenes (this hormones do not have the side chain on C-17) CBG = corticosteroids/cortisol binding globulin (transcortin) transfer: progesterone cortisol (they have two carbon side chain on the C-17, total C21 ) Gc-globulin = vitamin D binding globulin transfer: vitamin D and its derivatives (they have voluminous C8 to C9 side chain on C-17, total C27 to C28 ) 17
  18. 18. Regulating effect of hormones in the males / females : 1/ steroid hormone stimulates / inhibits the synthesis of its transport (binding) protein 2/ steroid hormones have different affinity for binding proteins 3/ testosterone (TST) is a prohormone, little effective only. (The effective hormone itself is 5α-dihydrotestosterone) 18
  19. 19. SHBG in woman : testosterone (TST) estradiol (E2) 5times higher affinity SHBG the increase of concentration of binding globulin (supported by estrogens) the woman estrogens can be bonded has twice in greater extent concentration of SHBGAt the sufficient concentration of SHBG is bonded on the proteinboth TST and E2. - SHBG allows the transport of hormons to the 19cell, because it is a ligand for megalin.
  20. 20. SHBG in man : testosterone (TST) estradiol (E2) 5times higher affinity SHBG the decrease of concentration of binding globulin (supported by testosterone) the man has half SHBG estrogens cannot be concentration bonded in greater extentAt the lower concentration of SHBG on the protein is bonded in effect TST only.TST with higher affinity to SHBG takes nearly all binding places.So SHBG (ligand for megalin) allowes the transport of TST nearly exclusively. 20
  21. 21. Transformations of testosterone :In woman goes into cell both TST and E2 together with SHBG.TST (as a prohormone is little effective only !!) is transformated to E2 by aromatase. OH testosterone (TST) O prohormone !! aromatase 5-reduktase ( WOMAN ) ( MAN ) OH OH estradiol (E2) 5-dihydroTSTHO O 21
  22. 22. Principle of effect of steroid hormones : steroid - enters the cell, bonded on SHBG (transport: megalin) lysosom - steroid hydrolytic is released from the bond on SHBG receptor protein for steroids in cytoplasm by bonding of steroid the transcription factor is created - different types of protein for different steroids, the structure of „zinc “ finger enter the nucleus enhancer / steroid responsive element bonding of steroid receptor. On the SRE are bonded 2 identical structures (homodimer) activation of promotor transcription proteosynthesis 22
  23. 23. Remark: Everybody has all existing sex hormones ( his „own“ hormones and the „opposite“ hormones )1/ the man stays man because:  in foetus is influence of antiMüller hormone (from Sertoli cells)  created estradiol act locally only (not endocrinely ! - it may act on nearby cells only)  strong affinity of testosterone to SHBG  occupation nearly all binding places of SHBG by testosterone  testosterone is so (in complex with SHBG) „preferentially“ transported to the cells  under influence of testosterone is synthesis of SHBG considerably limited  „do not remain“ free binding places for estradiol (estradiol „cannot go“ to the cells) 23
  24. 24. Remark: Everybody has all existing sex hormones ( his „own“ hormones and the „opposite“ hormones )2/ the woman stays woman because:  testosteron is nearly present as an intermediate product for synthesis of estrogens ( aromatase)  testosterone alone is little effective (it is prohormone !!)  SHBG with strong affinity to testosterone allowes sufficient transport of testosterone into cells  sufficiency of testosterone as a substrate for transformation to estradiol  under influence of estrogene is the synthesis of SHBG markedly increased  sufficiency of binding capacity for estrogens  by binding of E2 on SHBG is allowed transport estrogens into the cells SHBG = sex hormone binding globulin = TEBG = testosterone / estrogen binding globulin 24
  25. 25. Estradiol (E2) in man : no endocrine effect !!(man´s body is not under paracrine and autocrine effect „flood“ of E2) (effect of E2 near the place of origin)The lack of aromatase (and so estrogens!) can evoke infertility in man 25and failures of maturation in bones !! Estrogens regulate spermatogenesis in mammals.
  26. 26. AntiMüller hormone (Sertoli cells of testes) : X X ductus Mülleri 26
  27. 27. In man and in woman are identical control mechanisms : GnRH feedback by sex hypothalamus (gonadotropin releasing hormone) hormones in blood one hypothalamic hormone controls both hormones LH of hypophysis FSH (luteinizing hormone) (follicle stimulating hormone) anterior lobe of pituitary t1/2 = cca 50 min t1/2 = 4 h (adenohypophysis) effect of both hypophysary hormones is mediated by cAMP in target tissuescorpus luteum Leydig cells follicular cells. Sertoli cells target tissues ovaries testes ovaries testes (ovaries, testes)(progesterone) (testosterone) (estrogens) 27
  28. 28. 27-hydroxycholesterol 28
  29. 29. 27-hydroxycholesterol : ( 2007 ) HO 27 OH bonds on the same receptors in heart vessels as estrogens inhibits so the production of nitric oxide, which produces vasodilatation unfavourable circumstances: menopause, hypercholesterolemia 29
  30. 30. Neurosteroids 30
  31. 31. Neurosteroids : ● specifically progesterone (PROG) and some of its metabolites (allopregnanolone) are neuroprotective in some brain injuries (traumatic, hypoxic, ischemic stroke, …) ● effects are systemic and not limited to CNS injury itself ● multiple beneficial effects cannot be atributed solely to the PROG intranuclear receptor ● actions are result of hormone´s effects on maitenance of mitochondrial functions ● vitamin D deficiency reduces the benefits of PROG treatment neurosteroids e.g. ● protect neurons from ischemic injury ● decrease the size of a lesion ● reduce inflammatory reactions, apoptosis and reactive oxygen species ● stimulate myelin synthesis (remyelination) ● …… 31
  32. 32. Conjugation of steroids 32
  33. 33. Metabolism of steroids → inactivation and increasing solubility in water :1/ the reduction of double bonds (including the oxo-groups)2/ conjugation to the glucosiduronates and sulfates3/ the main location: liver, however kidneys too - the main place of excretionexception: testosterone  5-dihydrotestosterone (effective metabolite !!) 33
  34. 34. Origin of UDP-Glc : OH CH2OH N O O N O P P P P O CH2 O Glc-1-P UTP OH CH2OH N O O N P P O P P O CH2 O anhydrid bond UDP-Glc anhydrid bond 34
  35. 35. Conjugation with glucuronic acid(„GlcUA“) : CH2OH COOH O O O O UDP-Glc UDP-GlcUA COOH O steroid-3-glucosiduronate (schematicly) O 35
  36. 36. The most common transformation of steroids :O reduction HO conjugation COOH O( steroid-3-glucosiduronate ) O excretion (urine, bile) 36
  37. 37. Steroid skeleton cannot be degraded : O inactivation of hormone by reduction of double bonds COOH OH O conjugation 20 O( pregnandiole-20-glucosiduronate ) HO excretion 37
  38. 38. Glucosiduronates : • C-3-glucosiduronates are the most frequent (however the pregnandiol-20-glucosiduronate is the main metabolite of progesterone excreted in urine) • glucosiduronates are present as sodium salts • glucosiduronates are formed in the reaction with glucuronic acid (GlcUA) which is activated by the bond on uridindiphosphate (UDP-GlcUA) COOH O COOH O 20 O O HO 38
  39. 39. „PAPS“ : NH2 N N OH O N N CH2 O P O S O- O O O OH O HO P OH O anhydrid bond= 3´- phosphoadenosin - 5´- phosphosulfate = „active sulfate“ 39
  40. 40. Conjugation with sulfuric acid : O HO S O OSchematicly is depicted alkylsulfate i.e. ester of sulfuric acid and 3-OH steroid. This type of conjugates is less frequent in steroids than glucosiduronate . 40
  41. 41. Megalin / cubilin 41
  42. 42. Megalin :● megalin = transmembrane glycoprotein Mr ~ 600.000, multiligand transport protein (the member of the LDL-receptors family)● megalin allowes the way to cell uptake of androgenes a estrogenes bonded on SHBG● megalin-deficient mice (megalin-knockout) have development disorders of sexual organs from lack of androgenes/estrogenes● probably exist (less important !!) transport of steroids independent on megalin/cubilin, (in experiment elimination of megalin is not fully identical to the picture after blockade of steroid receptors) 42
  43. 43. Cubilin :● Mr = 456.000, glycoprotein, many repeating sequences („CUB“ domains, from their abbreviation the name - acronymus)● periferal membrane protein (= on the surface of cells)● has not the signal sequence for endocytosis● internalization of cubilin (with bonded ligands) allowes megalin (presence of Ca2+ ionts on the bond)● identical to receptor for the complex intrinsic factor with vitamin B12 +) +) uptake of vit. B is facilitated by the cobalamin-binder (gastric) 12 „intrinsic factor“. Vit. B12 and intrinsic factor create complex. Intrinsic factor recognises a receptor cubilin, present in epithelium of intestine and kidney. 43
  44. 44. Occurence of megalin : 44
  45. 45. Megalin has a plenty of ligands : 45
  46. 46. Transport of steroid hormone into cells– yesterday, today and tomorrow ?● yesterday: transport (plasma) globulin binds a steroid hormone. At the target cell the hormone detaches from its transport protein. A free hormone as a nonpolar substance passes (also) nonpolar cell membrane. A free (unbound) hormone entering the cell becomes biologically active (according to this idea)● today: transport (plasma) globulin is a ligand for the receptor megalin/cubilin. Only the hormone bound to plasma globulin enters the cell. A bonded hormone entering the cell becomes biologically active.● tomorrow ? a posssibility of blocking megalin/cubilin receptor → impact on malignant processes ? 46
  47. 47. Megalin and cubilin– multiligand receptors, schematicly : cubilin is a periferal (surface) membrane glycoprotein cubilin megalin cell membrane megalin is a transmembrane glycoprotein 47
  48. 48. Megalin and cubilin (2) : ligands hormones (●) bonding on their transport proteins () cubilin megalin cell membraneboth receptors are able to bond large amount+) of different ligands,which are mainly proteins (transport proteins of unpolar hormones too) +) μεγαλος = large, huge → „megalin“ ? 48
  49. 49. Megalin and cubilin (3) : hormones (●) bonding on their transport proteins () cubilin megalin cell membrane the internalization of cubilin bonded ligands is allowed by colaboration of cubilin with megalin – only megalin is transmebrane protein ! internalization (hormone including a protein) 49
  50. 50. Megalin and cubilin (4) : hormones (●) bonding on their transport proteins () cubilin megalin cell membrane internalization (hormone including a protein) lysosome 50 (releasing hormone from the bond on a protein)
  51. 51. Impact on tumor cells ? LHRH = GnRH (see the remark) α- = negation, anti- :REMARK : α-estrogenes = antiestrogenesGnRH = gonadotrop(h)in releasing hormoneLHRH = LHRF = luteinizing hormone releasing hormone / factor α-androgenes = antiandrogenes- all the names are of the same significance, however the GnRH shoudbe prefered !!!!! 51
  52. 52. Possibility of blockade of megalin receptor ? blue: active receptor megalin white: blockade receptor megalin „up-regulation“ → carcinoma mammae (estradiol) → carcinoma prostatae (testosteron) 52
  53. 53. Jakoby-SLOVNÍK (how to read it in „čingliš“ = Czech English)ABC = ATP binding cassette androgen …. […baidiņ kəsət] [aendrədžen …]… BP = ….binding protein progesterone … [… baidiņ prəuti:n] [prədžestərəun … ]CBG = transcortin [ ֽ◌traensko:tiņ] = cortisol / corticosteroid binding globulin [ ko:tisol / ko:tikəstiəroid baidiņ globjulin]CBG = corticosteroid binding globulin [ ֽ◌ko:tikə ֽ◌stiəroid baidiņ globjulin]Gc-globulin = vitamin D binding globulin [vitəmin/vaitəmin … baidiņ globjulin]GS.. = gonadal steroid ….. [gonədl stiəroid …]SHBG = sex hormone binding globulin [seks ho:moun baidiņ globjulin]SP2 = steroid-binding -globulin [steroid/stiəroid baidiņ bi:tə globjulin] = TEBG = testosterone / estrogen binding globulin [testostərəun / i:strəudžən baidiņ globjulin] 53
  54. 54. GnRH = gonadotrop(h)in releasing hormone [ ,gonədo´trəupin / ,gonədo´trəufin ri´li:sin ´ho:məun ]LHRH = LHRF = luteinizing hormone releasing hormone / factor [ ,lu:ti:i´naizin ´ho:məun ri´li:sin ´ho:məun / ´faektə ] tyto pojmy jsou významově shodné, ale GnRH by mělo být preferováno !!!!! doplněno 12 03 09, vp+ 54
  55. 55. 55