1

2

2

Vitamin D, calciols

transport proteinsof steroids

27-hydroxycholesterol

conjugation of steroids

© Department of Biochemistry (V.P.),Faculty of Medicine, MU Brno 2011

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Vitamin D, calciols

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cholesterol

( cholest-5-en-3-ol )

HO

3 5

5

7-dehydrocholesterol :

HO

3 5 7

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cholecalciferol, vitamin D3

HO

UV (290 nm) „ 9,10-secosterols “

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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)

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O

HO

HO

O

HO

OH

Brassinosteroid (→ steroid hormones of plants) :

● 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)

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28

ergostane C28 :

C28 - basic steroid hydrocarbon in plants (fytosterol)(the 28th carbon of skelet is in this position marked right as „241“ )

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IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) (1989)The nomenclature of steroids. Recommendations 1989. Eur. J. Biochem. 186, 429-458.

Steroids numbering :

11

HO

28

22

ergocalciferol, vitamin D2

12HO

28

A

DC 22

ergocalciferol, vitamin D2

„unfolded structure“

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Change of vitamin to hormone „calcitriol“: 25 - OH in the liver

1 - OH in the kidneys(influence of parathormone)

HO

OH

OH

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Calcitriol expression of CBP gene (calcium binding protein) synthesis of protein rich in –COOH groups in side chains binding of Ca2+

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1-hydroxylase :

• mitochondrial enzyme in the proximaltubule of kidneys

• hydroxylate 25-hydroxy-vit.D3 (from theliver)

• it is inhibited by higher concentration ofcalcitriol (feedback),by higher concentration of Ca2+ andphosphates in plasma

• stimulated by parathormone and bydecrease of concentration of phosphatein plasma

• at its inhibition is formed the inactive24,25- dihydroxy-vit.D3 (proceeds„alternative“ 24-hydroxylation insubstitution of „common“ hydroxylationat the C-1 )

the liver

• megalin/cubilin mediated reabsorption of „vitamin D binding protein“ is responsiblefor the renal conversion of 25(OH)D3 to 1,25(OH)2D3 in the proximal tubule

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Transport proteinsof steroids

see also: megalin and cubilin !!

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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 higherSHBG),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 thecell (i.e. complex: steroid-protein)

• increase of SHBG in man „estrogenization“(= result of increase of E2 transported into cell – i.e. increase on SHBGbonded E2 ! )

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BLOOD PLASMA:

SHBG (= TEBG) sex hormones binding globulintestosterone/estrogen binding globulin

transfer: testosteroneestrogenes (this hormones do not have the side chain on C-17)

CBG = corticosteroids/cortisol binding globulin(transcortin)

transfer: progesteronecortisol (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 )

Binding proteins of steroids (2):

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Regulating effect of hormones in the males / females :

1/ steroid hormone stimulates / inhibits the synthesis of itstransport (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)

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SHBG

testosterone (TST) estradiol (E2)

the increase of concentrationof binding globulin (supportedby estrogens)

5timeshigheraffinity

estrogens can be bondedin greater extent

the womanhas twiceconcentrationof SHBG

SHBG in woman :

At the sufficient concentration of SHBG is bonded on the proteinboth TST and E2. - SHBG allows the transport of hormons to thecell, because it is a ligand for megalin.

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SHBG

testosterone (TST) estradiol (E2)

the decrease of concentrationof binding globulin(supported by testosterone)

5timeshigheraffinity

estrogens cannot bebonded in greater extent

the man hashalf SHBGconcentration

SHBG in man :

At 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.

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OH

O

HO

OH

testosterone (TST)prohormone !!

estradiol (E2)O

OH

5-dihydroTST

aromatase

( WOMAN )5-reduktase

( MAN )

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.

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Principle of effect of steroid hormones :

steroid - enters the cell, bonded on SHBG (transport: megalin)

receptor protein for steroids in cytoplasm

enter the nucleus

enhancer / steroid responsive element

by bonding of steroid the transcription factor iscreated - different types of protein for differentsteroids, the structure of „zinc “ finger

bonding of steroid receptor.On the SRE are bonded 2 identical structures(homodimer)

activation of promotor

transcription

proteosynthesis

lysosom - steroid hydrolytic is released from the bond on SHBG

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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)

Remark:

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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

Remark:

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paracrine and autocrine effect(effect of E2 near the place of origin)

no endocrine effect !!(man´s body is not under

„flood“ of E2)

Estradiol (E2) in man :

The lack of aromatase (and so estrogens!) can evoke infertility in manand failures of maturation in bones !! Estrogens regulate spermatogenesis in mammals.

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X X ductus Mülleri

AntiMüller hormone (Sertoli cells of testes) :

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In man and in woman are identical control mechanisms :

GnRH(gonadotropin releasing hormone)

hypothalamus

anterior lobe of pituitary(adenohypophysis)

LH(luteinizing hormone)

t1/2 = cca 50 min

FSH(follicle stimulating hormone)

t1/2 = 4 h

corpus luteumovaries

(progesterone)

Leydig cellstestes

(testosterone)

follicular cells.ovaries

(estrogens)

Sertoli cellstestes

effect of both hypophysaryhormones is mediated by cAMP

in target tissues

one hypothalamic hormonecontrols both hormones

of hypophysis

feedback by sexhormones in blood

target tissues(ovaries, testes)

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27-hydroxycholesterol

29

HO

OH

27

27-hydroxycholesterol :

bonds on the same receptors in heart vessels as estrogens inhibits so the production of nitric oxide, which produces

vasodilatation unfavourable circumstances: menopause, hypercholesterolemia

( 2007 )

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Neurosteroids

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Neurosteroids :● specifically progesterone (PROG) and some of its

metabolites (allopregnanolone) are neuroprotective in somebrain 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)● ……

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Conjugation of steroids

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Metabolism of steroids → inactivationand increasing solubility in water :

1/ the reduction of double bonds (includingthe oxo-groups)

2/ conjugation to the glucosiduronates and sulfates3/ the main location: liver, however kidneys too

- the main place of excretion

exception: testosterone 5-dihydrotestosterone(effective metabolite !!)

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Glc-1-P UTP

P PP

O

CH2O

N

N

OH

OO

CH OH

O

2

P

P

O

CH2O

N

N

OH

OO

CH OH

O

2

P

P P

UDP-Glc anhydrid bond

anhydrid bond

Origin of UDP-Glc :

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O

COOH

O

O

CH2OH

O

UDP-Glc UDP-GlcUA

O

COOH

O

Conjugation with glucuronic acid(„GlcUA“) :

steroid-3-glucosiduronate(schematicly)

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O

HO

O

COOH

O

reduction

conjugation

( steroid-3-glucosiduronate )

excretion(urine, bile)

The most common transformation of steroids :

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O

COOH

O

HO

20

( pregnandiole-20-glucosiduronate )

O

OH

inactivation of hormone by reduction of double bonds

conjugation

Steroid skeleton cannot be degraded :

excretion

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O

COOH

O

O

COOH

O

HO

20

Glucosiduronates :

• C-3-glucosiduronates are the most frequent(however the pregnandiol-20-glucosiduronate is the mainmetabolite 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)

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O

N

N

N

N

NH2

CH2 O P

O

OH

O S

O

O

O-

OHO

P

O

OHHO

„PAPS“ :

anhydridbond

= 3´- phosphoadenosin - 5´- phosphosulfate = „active sulfate“

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S

O

O

OHO

Conjugation with sulfuric acid :

Schematicly is depicted alkylsulfate i.e. ester of sulfuric acid and 3-OH steroid.This type of conjugates is less frequent in steroids than glucosiduronate .

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Megalin / cubilin

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Megalin :

● megalin = transmembrane glycoproteinMr ~ 600.000, multiligand transport protein(the member of the LDL-receptors family)

● megalin allowes the way to cell uptake of androgenes a estrogenesbonded on SHBG

● megalin-deficient mice (megalin-knockout) have development disordersof 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 afterblockade of steroid receptors)

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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. B12 is facilitated by the cobalamin-binder (gastric)

„intrinsic factor“.Vit. B12 and intrinsic factor create complex.Intrinsic factor recognises a receptor cubilin, present in epitheliumof intestine and kidney.

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Occurence of megalin :

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Megalin has a plenty of ligands :

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Transport of steroid hormone into cells– yesterday, today and tomorrow ?

● yesterday: transport (plasma) globulin binds a steroidhormone. At the target cell the hormone detachesfrom its transport protein. A free hormone as a nonpolarsubstance passes (also) nonpolar cell membrane.A free (unbound) hormone entering the cell becomesbiologically active (according to this idea)

● today: transport (plasma) globulin is a ligand for the receptormegalin/cubilin. Only the hormone bound to plasmaglobulin enters the cell. A bonded hormone enteringthe cell becomes biologically active.

● tomorrow ? a posssibility of blocking megalin/cubilinreceptor → impact on malignant processes ?

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megalin

cell membrane

cubilin

Megalin and cubilin– multiligand receptors, schematicly :

megalin is a transmembrane glycoprotein

cubilin is a periferal (surface)membrane glycoprotein

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megalin

cell membrane

cubilin

hormones (●) bonding on their transport proteins ()

Megalin and cubilin (2) :

both receptors are able to bond large amount+) of different ligands,which are mainly proteins (transport proteins of unpolar hormones too)

ligands

+) μεγαλος = large, huge → „megalin“ ?

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megalin

cell membrane

cubilin

hormones (●) bonding on their transport proteins ()

internalization (hormone including a protein)

Megalin and cubilin (3) :

the internalization of cubilin bonded ligandsis allowed by colaboration of cubilin withmegalin – only megalin is transmebrane protein !

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megalin

cell membrane

cubilin

hormones (●) bonding on their transport proteins ()

internalization (hormone including a protein)

Megalin and cubilin (4) :

lysosome(releasing hormone from the bond on a protein)

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Impact on tumor cells ?

LHRH = GnRH (see the remark)α- = negation, anti- :α-estrogenes = antiestrogenesα-androgenes = antiandrogenes

REMARK :

GnRH = gonadotrop(h)in releasing hormoneLHRH = LHRF = luteinizing hormone releasing hormone / factor- all the names are of the same significance, however the GnRH shoudbe prefered !!!!!

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Possibility of blockade of megalin receptor ?

„up-regulation“ → carcinoma mammae (estradiol)→ carcinoma prostatae (testosteron)

blue: active receptor megalin white: blockade receptor megalin

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Jakoby-SLOVNÍK (how to read it in „čingliš“ = Czech English)

ABC = ATP binding cassette androgen ….[…'baidiņ kə'sət] ['aendrədžen …]

… BP = ….binding protein progesterone …[… 'baidiņ 'prəuti:n] [prə'džestərəun … ]

CBG = transcortin[ ֽ◌traens'ko:tiņ]

= cortisol / corticosteroid binding globulin[ ko:tisol / ko:tikə'stiəroid '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[tes'tostərəun / i:strəudžən 'baidiņ globjulin]

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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+

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