Modelové interakce jaderných receptorů a
enzymových systémů
Olefsky J M J. Biol. Chem. 2001;276:36863-36864
JADERNÉ RECEPTORY
JADERNÉ RECEPTORY
http://www.ens-lyon.fr/LBMC/laudet/nurebase/nurebase.html
General design of transcription factors in nuclearreceptor
superfamily. The centrally located DNAbinding
domain exhibits considerable sequence
homology among different receptors and has the
C4 zinc-finger motif. The C-terminal hormonebinding
domain exhibits somewhat less homology.
The N-terminal regions in various receptors vary in
length, have unique sequences, and may contain one
or more activation domains. This general pattern
has been found in the estrogen receptor (553
amino acids [aa]), progesterone receptor (946 aa),
glucocorticoid receptor (777 aa), thyroid hormone
receptor (408 aa), and retinoic acid receptor (432
aa). [See R. M. Evans, 1988, Science 240:889.]
A: Unliganded heterodimerizing
receptors, exemplified here by VDR,
exist as weakly associated
heterodimers with RXR, presumably
bound nonspecifically to DNA
[Haussler et al., 1998]. Binding of the
1,25(OH)2D3 ligand to VDR (1)
promotes high-affinity
heterodimerization with RXR
accompanied by binding of the
heterodimer to its direct repeat
VDRE (2).
B: Unliganded GR, like other
receptors in group (d) (see Fig. 2),
exists as a complex with heat shock
proteins in the cytoplasm. Upon
binding its cortisol ligand (1), GR
dissociates from the cytoplasmic
complex, translocates to the nucleus
and forms a homodimer on its
palindromic GRE (2). Triggered by a
ligand-mediated change in GR
conformation, the AF1 and AF2
domains then synergize to promote a
series of events (3–6) involving the
recruitment of coregulatory
complexes similar to those described
for the VDR-RXR heterodimer, but
with some distinctive features.
Evoluce jaderných receptorů
Many family members have been identified by DNA sequencing only, and
their ligand is not yet known; these proteins are therefore referred to as
orphan nuclear receptors. The importance of such nuclear receptors in
some animals is indicated by the fact that 1–2% of the genes in the
nematode C. elegans code for them, although there are fewer than 50 in
humans.
Syntéza ligandů
Jaderné receptory
Transaktivace
Metabolismus ligandů
Cílové geny
(metabolismus,
kontrola buněčné
proliferace,
diferenciace a
apoptózy)
Toxické látky, farmaka
?????
Nízkomolekulární lipofilní sloučeniny jako ligandy = aktivita jaderných receptorů
je do značné míry závislá na syntéze a degradaci ligandů a naopak
Úloha jaderných receptorů a enzymů katalyzujících
degradaci nebo syntézu jejich ligandů:
• endokrinní regulace - steroidní hormony, thyroidní
hormony;
• regulace signálních drah – eikosanoidy,
metabolismus kyseliny retinové, vitamínu D3;
• metabolismus lipidů;
• metabolismus xenobiotik;
Modelový příklad 1:
Metamorfóza hmyzu
Regulation of insect
metamorphosis. (A)
Structures of
juvenile hormone,
ecdysone, and the
active molting
hormone 20-
hydroxyecdysone.
(B) General pathway
of insect
metamorphosis.
Ecdysone and
juvenile hormone
together cause
molts to keep the
status quo and form
another larval instar.
When there is a
lower concentration
of juvenile hormone,
the ecdysoneinduced
molt
produces a pupa.
When ecdysone
acts in the absence
of juvenile hormone,
the imaginal discs
differentiate, and the
molt gives rise to
the adult
Formation of the ecdysone receptors. Alternative mRNA splicing of the ecdysone
receptor (EcR) transcript creates three types of EcR mRNAs. These generate
proteins having the same DNA-binding site (blue) and hydroxyecdysone-binding site
(red), but with very different amino termini.
Three isoforms of EcR have been identified in insects, each with a different,
stage-specific role in regulation of molting and development. This allows for one
steroid hormone to induce a variety of different tissue responses. In general,
EcR A is predominant when cells are undergoing a maturation response (from
juvenile to adult) and is predominant in imaginal discs, whereas EcR B1
predominates in juvenile cells during proliferation or regression. Little is known
about the function of the EcR B2 isoform.
DNA and hormone binding are similar in the three isoforms of EcR. Little is
known about the crustacean EcR isoforms and how they change during the molt
cycle. However, the EcR that has been cloned from the crab, Uca pugilator
(U31817, GenBank), shares 85–87% homology with that of Drosophila (M74078,
GenBank). The differences are primarily in the region of the molecule involved
with dimerization. Similar sequence similarities are found between the
heterodimeric partner, USP.
There are several ecdysteroids which bind EcR, including 20-hydroxyecdysone,
turkesterone, makisterone A, ponasterone A, and muristerone A. Some
arthropods may use specific ecdysteroids as their principal molting hormone, but
often several ecdysteroids are found within one group. The primary molting
hormone for a range of organisms, including some insects and crustacea, is 20OH
ecdysone (20 HE). Among other examples, makisterone A is an important
hormone for some crustacea and hemipteran insects.
Cholesterol (from diet- a vitamin for insects)
Prothoracic
gland
20-HydroxyecdysoneEcdysone
mono-oxygenase
(fat body, epidermis)
Conjugates
(storage)
Metabolites
(excretion)
Synthesis of molting hormones
Syntéza ekdysteroidů a úloha cytochromů P450:
Modelový příklad 2:
Metabolismus xenobiotik
Metabolismus a detoxikace polutantů
3 fáze metabolismu cizorodých látek:
• 1. fáze biotransformace – konverze – oxidace, redukce,
hydrolýza, hydratace a izomerace;
• 2. fáze biotransformace – konjugační reakce –
glukuronidace, sulfonace, metylace, acetylace, konjugace
s glutathionem, konjugace s aminokyselinami;
• 3. fáze biotransformace – vyloučení konjugovaných
metabolitů z buňky – ABC transportéry a další transportní
proteiny;
Přehled: L. Skálová a kol., Metabolismus léčiv a jiných xenobiotik, Karolinum,
Praha, 2011
1. fáze biotransformace
Oxidace:
• hydroxylace – běžná biotransformační reakce (alifatické,
aromatické uhlovodíky) – snižuje se lipofilita toxikantu,
vzniklé hydroxyderiváty podléhají dalším konverzním
nebo konjugačním reakcím;
• oxidace alkoholů a aldehydů;
• oxidační deaminace;
• dealkylace - sekundární a terciární aminy, alkoxy-,
alkylthiolové skupiny;
• dehalogenace;
• N-oxidace, S-oxidace;
1. fáze biotransformace
Redukce:
• z hlediska množství metabolizovaných látek méně
významné, ale představují hlavní cestu pro detoxikaci
některých specifických skupin látek;
• např. redukce nitrosloučenin a azosloučenin;
• redukce N-oxidů a S-oxidů;
• redukce karbonylových sloučenin a chinonů;
1. fáze biotransformace
Hydrolýza:
• estery, epoxidy, amidy, hydrazidy a karbamáty;
• některé hydrolasy (např. epoxidhydrolasa) jsou schopné
katalyzovat i hydrataci (tj. adici vody);
• hydrolýze mohou podléhat i některé konjugáty xenobiotik;
Enzymy 1. fáze biotransformace
• cytochromy P450;
• flavinové monooxygenasy;
• peroxidasy;
• alkoholdehydrogenasy;
• aldehyd dehydrogenasy;
• aldo-keto reduktasy;
• dehydrogenasy s krátkým řetězcem (např.
karbonylreduktasy);
• hydrolasy – zvl. roli epoxidhydrolasa;
2. fáze biotransformace
Konjugační reakce:
• nejvýznamnější pro – konjugace s UDP-glukuronovou
skupinou, glutathionem a sulfonace (s 3'-fosfoadenosin-
5'-fosfosulfátem);
L. Skálová a kol., Metabolismus léčiv a jiných xenobiotik, Karolinum, Praha, 2011
2. fáze biotransformace
Konjugační reakce:
• nejvýznamnější pro – konjugace s UDP-glukuronovou
skupinou, glutathionem a sulfonace (s 3'-
fosfoadenosin-5'-fosfosulfátem);
L. Skálová a kol., Metabolismus léčiv a jiných xenobiotik, Karolinum, Praha, 2011
2. fáze biotransformace
Konjugační reakce:
• acetylace – cytosolové N-acetyltransferasy;
• bioaktivace heterocyklických aminů – silné genotoxiny;
L. Skálová a kol., Metabolismus léčiv a jiných
xenobiotik, Karolinum, Praha, 2011
2-amino-1-methyl-6-fenylimidazo[4,5-b]pyridin
(PhIP)
Enzymy 2. fáze biotransformace
• UDP-glukuronosyltransferasy;
• gluthathion-S-transferasy – cytosolové, mikrosomální;
• sulfotransferasy;
• N-acetyltransferasy;
• methyltransferasy;
Transport xenobiotik a jejich metabolitů
• někdy označován jako 3. fáze biotransformace;
• hlavní roli hrají specifické transportní proteiny – přenašeče;
• ABC transportéry (z angl. ATP-binding cassette) – BRCP, MDR1/Pgp,
MRP proteiny;
• SLC proteiny - přenašeče hygrofilních látek (angl. solute carrier
family);
Dong et al., 2005, Science 308: 1023-1028.
Aktivace
promutagenů:
Metabolismus léčiv:
Modelový příklad 3:
Steroidní hormony
Pět hlavních skupin steroidních hormonů:
Biosyntéza steroidních hormonů:
Diagrammatic outline of the synthesis of cortisol from cholesterol in the adrenal cortex
Cholesterol is either obtained from the diet or
synthesized from acetate by a CoA reductase
enzyme. Approximately 300 mg cholesterol is
absorbed from the diet each day and about 600
mg synthesized from acetate. Cholesterol is
insoluble in aqueous solutions and its transport
from the main site of synthesis, the liver,
requires apoproteins to form a lipoprotein
complex. In the adrenal cortex, about 80% of
cholesterol required for steroid synthesis is
captured by receptors which bind low-density
lipoproteins (LDL) although recent evidence has
shown that high-density lipoprotein (HDL)
cholesterol may also be taken up by adrenal
cells. The remaining 20% is synthesized from
acetate within the adrenal cells by the normal
biochemical route.
aromatáza
The glucocorticoid receptor and activation by cortisol
1) Unbound, lipophilic cortisol readily crosses
cell membranes and in target tissues will
combine with the glucorticoid receptor (GR).
2) Like the androgen and progesterone
receptors, unliganded GRs are located in the
cytoplasm attached to heat shock proteins
(hsp-90, hsp-70 and hsp-56).
3) When hormones bind to these receptors
hsps are released and the hormone receptor
complexes translocate to the nucleus.
4) These complexes form homo- or
heterodimers and the zinc fingers of their
DNA-binding domains slot into the
glucocorticoid response elements (GREs) in the
DNA helix.
5) Together with other transcription factors,
such as NF-κB or c-jun and c-fos, they
initiate RNA synthesis (activation of RNA
polymerase) downstream of their binding.
Modelový příklad 4:
Metabolismus mastných kyselin
Receptory aktivované
peroxizómovými proliferátory
(PPAR)
The peroxisome proliferator-activated receptors (PPAR , , ) are activated by
polyunsaturated fatty acids, eicosanoids, and various synthetic ligands. Consistent
with their distinct expression patterns, gene-knockout experiments have revealed
that each PPAR subtype performs a specific function in fatty acid homeostasis.
PPAR is a global regulator of fatty acid catabolism. PPAR activation up-regulates
the transcription of liver fatty acid–binding protein, which buffers intracellular
fatty acids and delivers PPAR ligands to the nucleus. In addition, expression of two
members of the adrenoleukodystrophy subfamily of ABC transporters, ABCD2 and
ABCD3, is similarly up-regulated to promote transport of fatty acids into
peroxisomes where catabolic enzymes promote b-oxidation. The hepatocyte CYP4A
enzymes complete the metabolic cascade by catalyzing v-oxidation, the final
catabolic step in the clearance of PPAR ligands.
PPAR was identified initially as a key regulator of adipogenesis, but it also plays an
important role in cellular differentiation, insulin sensitization, atherosclerosis, and
cancer. Ligands for PPAR include fatty acids and other arachidonic acid metabolites,
antidiabetic drugs (e.g., thiazolidinediones), and triterpenoids. In contrast to PPAR,
PPAR promotes fat storage by increasing adipocyte differentiation and
transcription o a number of important lipogenic proteins.
Ligands for PPAR include long-chain fatty acids and carboprostacyclin. PPAR may
affect lipid metabolism in peripheral tissues; it can be antagonized by other small
lipophilic agents, including 22(S)-hydroxycholesterol, certain unsaturated fatty
acids, and geranylgeranyl pyrophosphate.
Syntéza ligandů
Jaderné receptory
Transaktivace
Metabolismus ligandů
Cílové geny
(metabolismus,
kontrola buněčné
proliferace,
diferenciace a
apoptózy)
Toxické látky, farmaka
?????
Nízkomolekulární lipofilní sloučeniny jako ligandy = aktivita jaderných receptorů
je do značné míry závislá na syntéze a degradaci ligandů a naopak