Jaderné receptory (ER, AR, PR, GR, TR, RAR/RXR, PPAR) a jejich Ugandy J. Vondráček Jaderné receptory • jaderné receptory představují největší skupinu transkripčních regulátorů u mnohobuněčných živočichů; • společná architektura - málo konzervovaná N-koncová doména, vysoce konzervovaná DNA vazebná doména (DBD), spojovací oblast, doména vázající Ugandy (LBD) a variabilní C-koncová doména; \ • N-koncová doména bývá také označována jako doména zodpovědná za tzv. aktivační funkci 1 (AF1) - pomocí této oblasti je jaderný receptor regulován dalšími signálními dráhami indukujícími specifické posttranslační modifikace, např. fosforylace; LBD je označována jako AF2 - receptor je aktivován specifickým ligandem; • spektrum ligandů jaderných receptoru je velmi široké, presto je u většiny jaderných receptoru neznáme a označujeme je jako sirotčí (orphan) receptory; JADERNE RECEPTORY Sutinu Ituf/HiHH- RcirfíOfi luiUň A|> IVfíMtiilh-rtnJ S -Sri tvutalVMI ™ VIM* . W ťft 1% *|i •■jt os * A l, eft hí ^ Ch ľ.\i.- a NH2- DNA Ligand HT1 COOH Ligands: Endocrine Receptors Adopted Orphan Receptors Hígh-affinity, hormonal lipids Low-äff í nity. dietary lipids ER o,ß PR AR GR MR RXR tufa PPAR u,ßtY LXR FXR PXR/SXR RAR a.fJ.y TR a,ß VOR EcR CAR Orphan Receptors Unknown SF-1 LRH-1 DAX-1 SHP TLX PMR NGFI-B afij ROR a,ßty ERR u,ß,Y RVRa,ßtY GCNF TR 2,4 HNF-4 COUP-TFu^y J. Cell. Biochem. Suppls. 32/33:110-122, (1999) NLS (ligand dependent) Dimerization (strong) Helix H12 ^ Function AF-1 (Cell and promoter specific) DNA binding Dimerization (weak;, DNA binding induced) Ligand binding _i AF-2 Ligand dependent, cell and promoter specific) Function TRENDS in Endocrinology & Metabolism 1 la+tyirexf-progd.!.-" ;j ;jnc: _I_ [ Dehydrogenase; Isofflfrf-ase PR PR l£' DBNydrrjep-androsterone " Progesterone [ ] ^ toJrosttinol 21 I yJruA/ljJ:^ ^«-nya-DSyposesterone Andr03^dions \ er jLll^'JtLs U- 5 11 -Deöxy-Ksrtisol c TSSteseions Fig. 1. (a) Schematic of the structural and functional organization of NRs. The evolutionary conserved regions C (DBD) and E (LED) are indicated as boxes and a black line represents the divergent regions AifE.. D and F Two transcription AFs have been described in several NRs, a constitutively active (if taken out of the context ofthe receptor) AF-1 in region A/B and a ligand-inducible AF-2 in region E. Within these AFs, ads have been defined, (b) Estrogen receptor DBD complex on a cognate DNA response element, (c) Agonist-induced changes ofthe LBD, allowing binding of coactivators (the bound coactivator-binding peptide is shown). Figures 1 b,c are three-dimensional views derived from the corresponding crystal structures. Abbreviations: See Glossary. 19-Hydrnj*ylase; 'L Ol L^MvdrcjLitjr:!^-: MR MR 1 ^H±ON OI3 Cortisol GR GR t7p-Es.traflia ER | ER Dihydrotestosterone AR AR ►-3nH- TRENDS in Endocrinology & Metabolism 12:460 (2001) Vazebné motivy DNA na které se váží jaderné receptory Fig. 2. Thetypes of DNA-response elements used by nuclear receptors, (a) Symmetric repeats using the consensus ha If-site 5-AGAACA-3' are used by the glucocorticoid receptor (GR]r progesterone receptor (PR), androgen receptor (AR) and mineralocorticoid receptor (MR), each of which is a homodimer. The estrogen receptor (ER) bindssimilar symmetric sites but with consensus 5'-AG GT CA-3' half-sites, (b)A'1-5rule' specifies the use of direct-re peats wi th va r i a b le spaci ngs by RXR and its many partners (depicted in red). Some receptors, such as the vitamin D receptor (VDR) or RevErb, can form homodimersas an alternative to heterodimers. The size of the i nter-h a If-s ite s paci n g (n) can vary from one to five base-pa irs. (c) Sites containingjustone copy of 5'-AGGTCA-3' flanked with specif ic5' sequences (xxx) are used by the nerve g rowth fa cto r i n d uced B (NGFI-B) receptor, RevErb andsomeotherorphan receptors. (a) Symmetric sites AGAACA n TGTTCT n = 3 GR-GR PR-PR AR-AR MR-M R (b) Direct repeats AGGTCA n AGGTCA n = 1 RXR- RXR RAR P PAR COUP RXR- PPAR RevErb-RevErb RXR- VDR VDR-VDR RXR- T R LXR CAR RXR- RAR NGFI-B (c) Monomeric sites xxx-AGGTCA XXX = aaa NGFI-B XXX = act RevErb i.i---- Jaderné receptory bývají také někdy rozdělovány na základě jejich dimerizace: •steroidní receptory - vytvářejí homodimery - receptory pro estradiol (ER), progesteron (PR), androgeny (ARs), glukokortikoidy (GR) a mineralokortikoidy (MR); •receptory vytvářející heterodimery s retinoidnímy X receptory (RXR; receptor pro kyselinu 9-c/s retinovou). Patří sem receptory pro kyselinu all-trans retinovou (RAR),vitamín D3 (VDR), tyroidní hormony (TR), jaterní X receptor (LXR), receptory aktivované peroizómovými proliferátory (PPAR) a další; •skupina receptoru schopných vázat DNA jako monomery - např. NGFI-B, RevErb, RORaSF-1. TRENDS in Biochemical Sciences 26, 384, 2001 Fig. 4.(a)TheDNA- bindingdomains (DESDs) in the nuclear receptor fami ly contain a conserved recognition a helix (shown in blue) and a variable C-terminalextension (CTE) that continues past the core66-residue DBD into the hinge region. Each of the se two e I e ments provides a distinct DNA-binding surface35. (b) The recognition helix recognizes the major groove half-sites, with HzO bridging some of the protein-DNA interactions (water molecules are shown as dark circles). (c) By co ntrast, the CTEs of RevErb, NGFI-BandTR bind alongthe minor groove and backbone of DNA(Refs 32,35.36). I: I DNA Tmns-cription- binding Sceioid regulation domain binding - NH. C D E7F COOH hGRot hGR[i hAR hPRA hPRB hERu hERK hMR 420 4SB 517 777 420 4SB £27 742 ££7 629 669 917 39D46£ £15 762 555 630 927 I I SO 302 345 545 595 I 90 202 465 602 670 734 934 TRENDS in Biochemical Sciences 26, 384, 2001 GR a jeho aktivace kortizolem - modelová aktivace jaderného receptoru Bound hormone Free hormone atmi membrane hip hip U U U U hsp Dimcrization P=3 1. Volný lipofilní kortizol snadno prochází buněčnou membránou a váže se na GR; 2. Podobně jako další steroidní receptory je GR v neaktivním stavu vázán v cytoplazmě na tzv. heat shock proteiny (hsp-90, hsp-70 a hsp-56); 3. Po navázání Ugandu na receptor dochází k uvolnění Hsp a translokaci GR do jádra; Nuclear membrane oooc^^raxxxxxxr- GRE GRE RNA polymerase rna 4. Vznikající homodimer se váže na specifické sekvence DNA -glukokortikoidní responzívní elementy (GRE); 5. Ve spolupráci s dalšími koaktivátory a faktory remodelujícími chromatin iniciuje transkripci cílových genů; Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Regulace transkripce jadernými receptory - komplexní proces Chromatin remodeling complexes HAT coactivators +- Acetyl CoA Mediator-like coactivators TRAP/D R [P RNA pol II complex ŕ- 4 /^TPfŕlfrf, TFIID .íl ľr (mTi im S Srn J N SRC rcA^Ci^ p220/205 PCAF NR LNRJ . f j í|| Nuclear receptors Basal transcription machinery TRENDS in Endocrinology & Metabolism Fig. 6. Multiple physical and functional interactions among nuclear receptors, coactivators, chromatin remodelers and chromatin iQad to an ordered sequence orqvents culminating in the transcription of hormone-regulated genes including: (1) ligand-dependent interaction of coactivators withchromatinbound NRs, (2) ATP-dependant chromatin remodeling by chromatin remodeling complexes, (3) histone acetylation by HAT coactivators and (4) contacts between MRs and the basal transcriptional machinery by the Mediator-like coactivators (e.g. TRAP/DRIP). Whether functional interactions occur between the 5RC-p3O0/CBP-PCAF coactivators and the TRAP/DRIP complex is currently unclear, as is indicated by the question mark. Abbreviations: acetyl CoA, acetyl coenzyme A (the acetyl donor for acetylation reactions); CBRCREB-biinding protein; CREB, cAMP-response element-binding protein; HAT histone acetyltransferase; MR, nuclear receptor; PCAR p3G0/CBP-associated factor; p22u7205. TRAP220/DRIP205; RNA pol II, RNA poly merase II; SRC, steroid receptor coactivator; TFIID. transcription factor IID [which contains tata-binding protein (tbp) and TBP-associated factors]; TRAP/DRIP thyroid hormone receptor-associated proteins/vitamin D receptor-interacting proteins. TRENDS in Endocrinology & Metabolism, 12, 191, 2001 Toxické látky, farmaka Vnitrobuněčná signalizace syntéza ligandu Transaktivacc jad.receptory \ Cílové geny (metabolismus, buň. proliferace, diferenciace a buň. smrt) metabolizace ligandů Jaderné receptory a enzymy: Drug Metob. Pharmacokinet. 21:437-457 (2006) Metabolické dráhy zajištující syntézu a eliminaci ligandů jaderných receptoru Acetyl CoA Retinoic Acids čHasíS Isoprenolds t Lano sterol Diet * 7-Dehydro-Cholesterol ^Fatty Acjd^ t fCYP4A '■|W1«J 1,25-Dihydroxy-Vitamin D3 f CYP24 Cholesteroh 'VSSSľ |CYP7A1 o steroids v f ABCA1,G1,G5,G8 BiieAcids iCYP7A1 *CYP3B1 O C BGB 5 Science 294, 1866, 2001 Jaderné receptory vytvářejí složitou síť regulující funkce _organismu Fig. 5. Schematic diagram of overall xenobiotic responsive systems. iNOS: inducible nitric oxide synthase, GST: glutathione S-transferase NQO: NAD(P)H:quinone oxidoreductase Drug Metob. Pharmacokinet. 21: 437-457 (2006) Hepatocyty- mechanismy regulující transport a metabolismus endogenních látek a xenobiotik islc22a1j/ \ CYP3A Biliary elimination mdri (abce1) , 'bsep -—\^abcb1 l)y MRP?\_ ^ABCCl)/----- OH | BA sohf BCG ,4 ' m0h3 (abcb4) BILE Drug MetaĎ. Pharmacokinet. 21:437-457 (2006) Jaderné receptory hrají zásadní roli v endokrinní signalizaci Local circulation I n c i\ic ri n a Autocrine Ganaia circulation ■ ■ Endocrine Endocrine gland Distant cargec cell e N eu roendocrine Neurosecretory cel q j Distant target cel k Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Steroidní hormony a jejich receptory Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Hlavní skupiny steroidních hormonů: 4-eri4-3-4ne Cortisol Aldosteron« Ppoge-iterone OH OH Tttt«terone Estradiol Shaded boxes show structural requirements for glucocorticoid and mineralocorticoid activity. Hatched boxes show additional structural requirements for specific glucocorticoid or mineralocorticoid activity. Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Syntéza kortizolu z cholesterolu v kůře nadledvinek Choltrterol LDL(HDLT) Cľtulettetxil Lipid droplet Mitochondria Sterol transfer protein StAR Sm«th endoplasmic reticulum Pregnenolone Progetítroňt Cholesterol je syntetizován v těle nebo získáván z potravy v poměru cca 600 mg/300 mg denné. Vzhledem k tomu, že je nerozpustný ve vodě, je z jater (hlavní místo jeho tvorby) transportován ve formě lipoproteins V kůře nadledvinek dochází k zachycení cca 80% cholesterol nezbytného pro syntézu steroidů na receptorech pro nízkodenzitní lipoproteiny (LDL). Zbylých 20% je syntetizováno z acetátu přímo v buňkách nadledvinek. Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Biosynteza steroidnfch hormonu: Chaleicerol Choltsieral fide-chain cleavage C=0 HO I Tijt-OH-Prcgncnolonc Pregnenolone Progesterone I 7(jt-OH-Pro£cstcrene Androgens /\ Testosterone —Estrogens I_I [+ Progesterone) Gonadal steroids Corticostcnonc Cortisol I_ Aldosterone _I Adrenal steroids Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. -o And rosten edion c Teste* Cc rone Di hjfdroticsCoiCcron c OH Qy Cholesterol ICYPII AI I i CH, Side chain drawee CH, HO H CYFI7 \*- H CYPI7 ^ o Pregnenolone I ' DHEA HO' HO' 17*- 0 Prageicerorie ,_i_ l7ci-OH Protei is rane Andre£EÉľi£dÍDŕií CYP2IA2 j CHjOH ® CH.OH =0 ■I © Deoxycorticosterone I I -Dcoxycorosd |CYP IBI J ® + CH^OH HO ■I Corticostcronc ICYPlľäTI i ® t cHjOH c-o HO_ í Hl Cortisol ■o IIC ■.........................1 + Z (úľ 4)-hyd rovyes ma ne I S»-hydroxyes n-ůne HO OH [Cawchölaströjena) i + Prog.cs teron c 1 Estriol Pregnandiol -ŕ 20«-hjHrc^ľprcsoswsnonc Aldostanons Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Biosyntéza pohlavních steroidních hormonů: -SO, □ HEA-S O OH I7[5-H£D s^asK, DHEA Or T " O"**^^^ HO* A nd rů&ten edi on e Testosterone Estradiol j\' reductase OH H 5a- Dihydroteitosteront Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Transport a metabolizace pohlavních steroidních hormonů: jen asi 2% testosteronu v oběhu je ve volné formě, která je schopna vstoupit do buněk. Zbytek je vázán v plazmě na albumin (cca 40%) nebo na sex-hormone-binding globulín (SHBG). Volná forma a vázaná forma je v ronováze. SHBG je syntetizován v játrech a jeho hladina je kontrolována řadou faktorů (estrogen, tyroidní hormony, androgeny, glukokortikoidy, růstovým hormonem a dalšími faktory - stres, obezita apod.). Testosteron je v játrech metabolizován na androsteron, etiocholanolon a ty jsou po konjugaci exkretovány ve formě 17-ketosteroidů. estradiol, je transportován ve formě vázané na albumin (cca 60%) and SHBG (30%). V játrech je rychle metabolizován na estron a poté většinou dále na estriol nebo na 2- or 4-hydroxyestron pomocí katecho-O-methyltransferáz. Metabolity jsou dále konjugovány a vyloučeny s močí. Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Tyroidní hormony štítné žlázy Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Syntéza tyroidních hormonů: Cold Bound + free Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Syntéza tyroidních hormonů: BASA I 1) Active uptake of iodide (I-) in exchange for Na+. 2) Iodide may be discharged from the follicular cell by administration of competing ions such as perchlorate, bromide or chlorate. 3) Iodide uptake, the main control point for hormone synthesis, is stimulated by TSH. 4) Oxidation of iodide by hydrogen peroxide (H202) to form active iodine. The reaction is catalyzed by thyroid peroxidase (TPO). 5) Active transport of iodine across the apical surface of the follicular cell. 6) Incorporation of active iodine into the tyrosine residues of thyreoglobulin molecules to form mono-and di-iodotyrosines (MIT and DIT). 7) Uptake of the thyreoglobulin into the lumen of the follicle and lining of iodinated tyrosine residues. Endocrinology: An Integrated Approach, Nussey S and Whitehead S.; 2001. HO-£^— CHjCHCOOH H O C H 2CH C OOH I NH DIT Uvolňování tyroidních hormonů: 1) Under the influence of TSH, colloid droplets consisting of thyroid hormones within the thyreoglobulin molecules are taken back up into the follicular cells by pinocytosis. 2) Fusion of colloid droplets with lysosomes causes hydrolysis of thyroglobulin and release of T3 and T4. 3) About 10% of T4 undergoes mono-deiodination to T3 before it is secreted. The released iodide is reutilized. Several-fold more iodide is reused than is taken from the blood each day but in states of iodide excess there is loss from the thyroid. 4) On average approximately 100 ug T4 and about 10 ug T3 are secreted per day -¥—' ^ Thyiojlobulin Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Transport a metabolizace tyroidních hormonů: HO Podobně jako steroidní hormony jsou i tyroidní hormony prakticky nerozpustné ve vodě - po uvolnění se rychle vážou na proteiny v plazmě, především transthyretin, thyroxine-binding globulin (TBG) a albumin. Ty mají různou afinitu vůči T3 a T4; cca 70% cirkulujících tyroidních hormonů se váže na TBG. Jen velmi malý podíl (<0.5%) tyroidních hormonů existuje ve volné formě a je v rovnováze s vázanými formami tyroidních hormonů. • tyroidní hormony jsou otherinetaboIizovány pomocí specifických deiodináz (viz čísla v C H, C H CO OH I NH, l,5,3'5'-Tetraiodotliy ronine (thyroxi rte, or Tt) A i,í,, 3' Triiodothyronine 3,5', 3' reverse triiodothyronine ITRIAC 3.3'T, Diiodothyronincs 3' 5'T.j 3'T| Monoiodothyroninc! Thyronine závorkách); • část T4 může být přímo konjugována a vyloučena z organismu; • podobně část T3 může být sulfatována (T3S) nebo přeměněna na derivát kyseliny octové - (TRIAC)', který je ještě ucmejsi nez T3; Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Transport a metabolizace tyroidních hormonů: cca 80% uvolněných tyroidních hormonů tvoří T4, ale ten je relativně málo aktivní a tak je považován za prohormon. Většina T4 je působením deiodináz v cílových tkáních přeměněna na biologicky aktivní T3 a inaktivní rT3; odstraněním I z uhlíku 5' prostřednictvím deiodináz typu 1 a 2 vzniká T3, zatímco odstraněním I z uhlíku 5 vzniká rT3. Dalším odštěpením atomů I vznikají di- a monojódtyroniny a jód organismus znovu využívá, část konjgovaných slučenin je vylučována močí a žlučí. Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Struktura a aktivace tyroidních receptoru Hamodinier* Heieii&dimer RXFí. ratinoic acid X neoeplor k: v uL7Qŕ7QOÍYAi hre-^iŕ^y^^ Promoters m RNA Endocrinology: An Integrated Approach, Nussey S and Whitehead S., 2001. Receptory pro retinoidy (RAR, RXR) 3,4-didehydro-RA Transport a metabolismus retinoidů 9,13-di-c/s-RA FIG. 2. Structures of naturally occurring retinoids. Transport a metabolismus retinoidů Retinoidy a jejich receptory: apoptosis * spermatogenesis nervous system A differentiation Í RETINOIDS 1 - reproduction - immune system skin vision embryogenesis conception placenta ['IG. 1. Kunrtioiis of naturally occurring retinoids. TABLE 1 - LIGANDS AND ISOFORMS OF RAR AND RXR RECEPTORS Receptor . , Chromosomal Isoforms i™«„„ location Ligand RARa RARfi RAR7 a1, oí2 P1 - [32, p3, ß4 Yl,72 17q21.1 3p24 12q13 j a\\-trans RA & I 9-c/s RA RXRct RXRß RXRy a1, c/2 ß1,ß2 71,72 9q34 6q21 1q22-q22 J 9-c/s RA Proteiny podílející se na transportu retinoidů: TABLE 1 Retinoid Binding Proteins Cla ss/Pľol ťin MW (kDa) Primary liga n cis 1 (X i Prospei t.lve fain Lion Kxt.racellular lipld-blndlng proteins (llpocallns) Rill3 ZI Rul inol Serum Rt't.lnol transporter ß-\acl oglobulln 18.3 Retinol? Milk Ret.lnol t.iansportei"! I-. RAUP 18.5 RA = řkRA ] 'epididymis RA/9cRA transporter Intracellular llpld-bindlng prot elns CRBP 14.B Retinol > retinal Many (e.g., liver, I)olo: substrate for LRAT and RoDl 1 kidney, It's lis) apo: stimulates REH; Inhibits LRAT CRBP(II) 14.6 Retinol = retinal Intestlne liolo: substrates for I,RAT and retinal reductase (. RAUP 15 RA > 9cRA > Many (e.g., hole: substrate1 for RA metabolism; 13cRA > 9,13cRA testis, lung, sequesters RA and possibly RA kidney) met abollt.es C RAUP (II) 15.7 RA > 9cRA > Adult skin, Same as for CRABP but with 9cRA > 9,13cRA embryo different affinities for RAs? Others C R AL BP 33 11-cfs-retinal, lief* RPie Protects retinoids from isomerization ret Inol IRBP 1 15 Rcilinol, many others Retina Lipid transporter Struktura a aktivace retinoidních receptoru: A A : B c i D E ! F Transcriptional activation DNA Hinge Ligand-binding domains (AF-1) binding region H eierod imeri zati on domain Transcription activation (AF-2) (RARE) j j N-Cor?SMRT binding domains j B ^ Early response genes Secondary response genes Gene product (e.g. transcription factors STATs, RARs, c/EBP, etc.) Inhibition of cell growth, Induction of differentiation, apoptosis Fig. 1 - Structure and functions of retinoid receptors. A) Schematic representation of retinoid receptor protein depicting various functional domains. B) A molecular model for retinoid action. The liganded RAR forms heterodimer with RXR, binds to specific regulatory sequences (RARE) in the promoter region of target genes. Transactivation of such early response genes is a primary event of retinoid action. In addition to this, the products of early response genes can activate the transcription of secondary genes. Transactivation of these genes therefore represents secondary action of retinoids since their transcription requires protein synthesis. This cascade of gene events leads to secondary and tertiary events that eventually produce a phenotype that is characteristic of retinoid action. Struktura a aktivace retinoidních receptoru: J, Basfíen, C. Rockette-Egly I Gene 328 (2004) 1-16 CorapratMfi eomptaxM ......... RAR/RXR B Htatanu/AcatytoHon/ mathyltrtlon /photphorylaUon Raprasisd Transcription f CoKttvatora complexw Ac P Ae Racruttmant of tha mdlutar and trwwcriptton machinery i Transcription Fig. 5. Three-step mechanism of retinoid receptor action. (A) In the absence of ligand, retinoid receptors hound to response elements located in the promoter of target genes are associated with hi stone deacetylase-containing fHDAC) complexes tethered through compressors and repress transcription. fB) Upon ligand binding, the compressors dissociate, allowing the recruitment of coaotivators associated with complexes displaying hist one ace ryl transferase (HAT), methyl transferase, kinase or ATP-dependent remodeling (SW1/SNF) activities that decompact repressive chromatin. (C) In the third step, the coaotivators dissociate and the SMCC mediator complex assembles. Then the mediator expedites entry of the RNA Pol Jl and the general transcription factors to the promoter, resulting in transcription initiation. Fig. 2. Scheme illustrating cell-cycle regulation by certain nuclear receptors. The cell cycle phases GO. Gl, S, G2and M are depicted in (a]r together with a schematic illustration of the corresponding levels of the various Cdk-cyclin complexes Same steroid receptors (ER, AR and PR) stimulate expression of the gene that encodes cyclin Dl, which interacts with and activates Cdk4. The activated cyclin-Cdk complexphosphorylates pRB, which dissociates from the dp-E2F complex., thus allowing transcription of cell cycle regulatory genes. In an opposite regulatory mode, vitamin Daandretinoic acids can induce expression of the CKI p21r which blocks Cdk activity, resulting in Gl arrest of treated cells, such as U937 Abbreviations: See Glossary. TRENDS in Endocrinology & Metabolism 12, 460, 2001 4010 Classic NRs Orphan receptors Response element principles Reverse endocrinology, orphan ligpnds, and new drug pipeline Cofactor concepts DBD-LBD crystal structures Chromatin remodeling, transcriptome, proteome Virtual physiology and digital medicine 1ÖB5 36 87 88 89 90 91 92 93 94 95 98 97 9G 99 2000 02 04 06 08 10 12 14 —}- First MR doned (GR) I RXR isolated T=P1 j l l Cotransfectian assay established, domaine structure elucidated VDF1. RAR '." q red Concept of NR suoerTamily Direct repeats as response element revealed (3-4-5 rule.) I- rsr P PAH isolated Estrogen receptor cloned ERR1/2 first orphan NRs First orphan receptor ligand {9-Cis RA;RXR) X-ray structure Of fiXR-TR on direct repeat DMA I XR, FXB i&oiated CBPrp.300 are NR coactivators Xenobiotic receptora PXR, CAR isolated Thyroid hormone -eceptor donad Concept Of RXft heterodimer Cloning of N P colaclors SRC family and N-CoR/SMRT H Lim ani zed First NR HHWblotic cistrome mouse 1 Atlas profiling of NR expression g the Ring of Physiology X-ray structure of intact receptor on DNA N-Cc-R/SMRT bind H D AGs First orphan ligand clinically approved (RXFt agonist) Cell 157,255-266(2014) Receptory aktivované peroxizómovými proliferátory (PPAR) Struktura a aktivace PPAR: Lt gand - índepend ent activation domain (AF-1) DBD DNA Binding Domain (2 zinc fingers) Li gand Li gand -depe ndent activation domain (AF-2) LBD-Dim. Li gand Binding and Dimerization Domains AGGTCAMAGGTCA TCCAGTNTCCAGT 9-ci j-iietinoic add r CH, I I 1 NH CT sl Ciglitazone CH, r jfj^ x „.;co * CT Rosiglitazonc FIG. 1. General structure and mechanism oí action of PPARs. PI1 ATI isoforms share a co ui m on do in a hi structure anil molecular mechanism of action. Membrane phospholipids Pfiospholipase A2 CO^H linoleic acid Arachidonic acid Linoleic acid OxLDL Cycloxygenase Lipoxygenases Lipoxygenases 15-deoxy PG-J2 LTB4, S-HETE, 1S-HETE 9-HODE, 13-HODE Gütazones PPAR /-COM H 0H 15-deoxy-AlJ14-PGJ2 HO 9-HODE 8(S)-HETE 'COüH OH 13-HODE Úloha PPAR v organismu: PPARs jsou aktivovány vícenenasycenými mastnými kyselinami, eikosanoidy a radou syntetických látek. Vzhledem k různé expresi isoforem PPAR hraje každý typ PPAR unikátní roli v organismu: PPARa je hlavní regulátor katabolismu mastných kyselin - kontrola exprese řady proteinů podílejících se na transportu a metabolismu mastných kyselin, především v játrech - liver f atty acid-binding protein, ABC transportéry, ABCĎ2 and ABCĎ3 - transport mastných kyselin do peroxizómů - (3-oxidace mastných kyselin; jaterní CYP4A enzymy -katalýza in-oxidace ligandů PPARa. PPARy je klíčový regulátor adipogeneze a hraje významnou roli buněčné diferenciaci, citlivosti na inzulín, rozvoji aterosklerózy a nádorových onemocnění. Mezi jeho ligandy patří mastné kyseliny, metabolity kyseliny arachidonové, triterpenoidy a některá léčiva (např. thiazolidinediony). Na rozdíl od PPARa, PPARy napomáhá ukládání tuků prostřednictvím posílení diferenciace adipocytů a indukce syntézy lipogenních proteinů. Funkce PPARÔ jsou méně známé. Jeho ligandy zahrnují mastné kyseliny s dlouhým řetězcem, karboprostacyklin a předpokládá se, že ovlivňuje metabolismus lipidů ve periferních tkáních. Ugandy PPAR: P. Escher, W. Wahli f Mutation Research 448 (2000) 121-138 127 H N CO,H 01 Wy 14,843 ETYA CT* Clofibrate O COjH A Bezafibrate ry C02H Indomethacin GW2331 Ci GW2433 BRL49653 (Rosig litazone) -i N H B ,C0£H Linolcnic acid HC 9-HODE COnH Arachidonic acid COjH Leukotriene B4 Eicosapcntaenoic acid ,CO;H Linoleic acid 8(S)-HETE CO,H 15-deoxy-A12,14 -Prostaglandin J2 Fig. 4. Natural and synthetic PPAR ligands, (A) Synthetic PPAR agonists comprise peroxisome proliferators (Wy 14,643), fatty acid analogs (ETYA), fibrates (Clofibrate, Bezafibrate. GW2331, GW2433), non-steroidal anti-inflammatory drugs {Indomethacin) and thiazolidine-diones (Rosiglitazone), (B) Natural PPAR agonists comprise polyunsaturated fatty acids and their metabolites. Pregnanový X receptor (PXR) aktivuje expresi genů obsahujících tzv. PXR responzívní elementy (PXRRE); nejznámější cílový gen je CYP3A4, indukuje ale i další enzymy I. a II. fáze biotransformace - ALDH, CYP2B, SULT, UGT; n* I CYP3A podrodina - nejrozšířenější CYPy v játrech a střevní tkáni s velmi širokou substrátovou specifitou, které hrají zásadní roli při odbourávání celé řady léčiv, ale i toxických látek; Cytochromy P450 RH + 02 + NADPH + H+ -> ROH + H20 + NADP+ Cytochromy P450 Table 2 | Functions of human CYP enzymes Family Number of subfamilies Number of aenes Substrates/function CYPl 2 3 Metabolism of eicosanoids* and xenobiotics; in addition, CYP1A2 metabolizes melatonin, oestrogen, uroporphyrin and -24 drugs CYP2 13 16 Metabolism of eicosanoids*, xenobiotics and many drugs CYP3 1 4 Metabolism of eicosanoids*, xenobiotics and many drugs CYP4 6 12 Metabolism of eicosanoids* xenobiotics and few drugs gypT 1 1 Throm box a neA2 synthase ^CYP7 2 2 Cholesterol, bile acid synthesis CYP8 CYPll CYP17 cyp19 CYP20 cyp21 cyp24 CYP26 CYP27 2 3 1 1 1 1 1 3 3 Prostacyclin synthase, bile acid synthesis Steroidogenesis Steroid 17-hydroxylase, 17/20-lyase Oestrogen aromatization Expressed in gastrula, neural patterning and somitogenesis, organogenesis, fetus and nasopharynx Steroid 21-hydroxylase Vitamin 24-hydroxylase Retinoicacid hydroxylation Bile acid biosynthesis, vitamin D? hydroxylations Box 2 I The CYP gene families Cytochrome P45C1 (CYP) proteins are arranged into families and subfamilies, which are derived from percent a mi no-acid sequence identity. Proteins that have roughly >40% sequence similarity are members of the same gene family, whereas those with >70% similarity are members of the same subfamily. The development and application of this delightfully logical system of nomenclature3 has eliminated the confusion that often plagues the naming of gene families and superfamilies. The fact that there are 20 CYP genes in Mycobacterium tuberculosis and seven in BociHus subtil is emphasizes the likelihood that the CYPsuperfamily existed long before the prokaryote-eukaryote split some 2.1 billion years ago65, and the earliest ancestor probably arose around the time that the partial pressure of oxygen increased dramatically on the planet. The CYP51 gene exists in bacteria, plants,fungi and animals, indicating that thiswas probably the first eukaryotic CYP gene. The human, mouse and rat genomes contain 57,102 and 87 CYP genes, respectively2, Orthologous Cyp genes between the mouse and rat generally show 90-92% sequence similarity, human CYP orthologues of rodent Cyp genes are usually 76-62% similar. cyp39 1 1 24-hydroxycholesterol7-hydroxylase cyp46 1 1 Cholesterol 24-hydroxylase in the central nervous system \yP51 1 1 Lanosterol 14-demethylase ^/ Nature Reviews Cancer 6, 947, 2006 Cytochromy P450 a bioaktivace prokarcinogenu Possible pathways of activation of suspected human carcinogens Heterocyclic amines e.g. IQ, PhIP Aromatic amines e.g. 4-ABP PAHs eg- BMP, DMBA 4-ABP 7,12 DMBA 8) ► H2-RC7-OH f88) Dihydroxy ^ Tetrol sult (56) metabolite DNA-reactive products of metabolic activation NH+ Dihydrodiol epoxide Electrophile B[«]P Carcinogenesis 22,209,2001 PXR ligandy PO