Fyziologie působení farmak a
toxických látek
Přednáška č. 1
PAS proteiny jako
stresové senzory a
vývojové regulátory
NNááplplňň ppřředmedměětu:tu:
Fyziologie komunikaceFyziologie komunikace
Nízkomolekulární látky přírodního aNízkomolekulární látky přírodního a
antropogenního původu:antropogenního původu:
•• SignalizaceSignalizace
•• ToxicitaToxicita
•• Fyziologické podmínky vs. lidskéFyziologické podmínky vs. lidské
zásahyzásahy
Mechanismy jejich působení na buněčné úrovni
Již na úrovni jednobuněčných organismů je nezbytná schopnost:
1. Přijímat a identifikovat signály z vnějšího prostředí – např. za
účelem výměny genetické informace;
2. Eliminovat toxické látky přijímané z vnějšího
prostředí/vznikající jak vedlejší produkty metabolismu;
Degradace a
exkrece
toxických látek
a vedlejších
metabolických
produktů
Příjem a přenos
specifických
signálů
Modelová forma komunikace – NH4 jako signální
molekula mezi koloniemi kvasinek:
Palková a Váchová, FEMS Microbiol Rev 30 (2006) 806–824
Model of ammonia-triggered differentiation in
Saccharomyces cerevisiae colonies. In first-acidic
phase colonies, ROS and other harmful products
are produced by cells throughout the whole
colonies and induce the regulated yeast cell death
(YCD). To escape damage, yeast cells start to
emit (outgoing violet arrows) and accept (incoming
arrows) ammonia signal, which triggers metabolic
changes that consequently allow cells to lower
their ROS production. Healthy cells located mainly
at the colony border (where the concentration of
ROS is low) can thus escape cell death.
Consequently, at the colony border, there are
mainly slowly growing and dividing healthy cells
(green) in later developmental phases (second
acidic phase), while in the colony centre, dying
cells (red) predominate.Compounds released (red
arrows) from these cells in late stages of YCD
sustain border cell growth and reproduction. The
blue arrow indicates the position of the colony
considered in the model.
U mnohobuněčných organismů (živočichů) se
vyvinuly stovky signálních drah a dalších
mechanismů:
1.Embryonální a postnatální vývoj;
2.Regulace metabolismu a obecně, homeostázy;
3.Pohlavní rozmnožování;
4.Tvorba a degradace signálních molekul i
toxických sloučenin; přenos signálu
Dubrulle, J. et al. Development 2004;131:5783-5793
A model for somitogenesis. (A) Double in situ hybridization of a 2-day-old chicken embryo
with Raldh2 (retinaldehyde dehydrogenase 2) and Fgf8 (fibroblast growth factor 8)
probes. Anterior is towards the top. These genes participate in the establishment of
mutually inhibitory, antagonistic gradients of retinoic acid (RA) and fibroblast growth
factor (FGF) signaling. (B) Molecular mechanisms leading to a segmental pattern. Segment
patterning genes are periodically activated by the segmentation clock, whose main
regulators are the Notch and Wnt signaling pathways. The spatial activation of the
segment patterning genes is defined by the RA and FGF antagonistic gradients: RA
positively regulates their transcription, whereas FGF signaling represses RA activity and
inhibits presomitic mesoderm maturation.
http://www.luc.edu/faculty/wwasser/dev/zebra.mov
Cortisol stimulates the release of amino acids from muscle. These are taken up by the liver
and converted to glucose. The increased circulating concentration of glucose stimulates
insulin release. Cortisol inhibits the insulin-stimulated uptake of glucose in muscle via the
GLUT4 transporter. Cortisol has mild lipolytic effects. These are overpowered by the
lipogenic action of insulin secreted in response to the diabetogenic action of cortisol.
Cortisol also has varied actions on a wide range of other tissues
Endocrinology: An Integrated Approach. Nussey and Whitehead (2001) London: Taylor&Francis
Zásahy z vnějšího prostředí:
1. Produkty sekundárního metabolismu rostlin a
hub;
2. Zásahy člověka – cílené – aplikace chemických
látek jako jsou pesticidy, syntetické feromony;
terapie;
3. Zásahy člověka – nezamýšlené – toxické
sloučeniny; odpad.
Lake trout 4 weeks after hatching. (A) Normal larva with its golden
yellow yolk sac. (B) Dioxin-exposed larva exhibiting a blue yolk sac.
The yolk sac has swelled with water and has numerous sites of
hemorrhage. Such fish often have reduced growth, as well as heart
and facial anomalies. (Photograph courtesy of R. E. Peterson.)
Developmental Biology. 6th ed. Gilbert, Scott F.
Sunderland (MA): Sinauer Associates, Inc.; 2000.
Co všechno se dá najít v odpadních vodách:
(Kolpin et al., ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 36, NO. 6, 2002)
PPerer--AArntrnt--SSimim –– PASPAS superfamily of proteinssuperfamily of proteins
environmental sensors, which mediate
transcriptional responses to various types
stimuli:
 circadian rhythms;
 oxygen sensing;
 sensing of toxicants;
 developmental role/cancer;
These proteins enable adaptation to rapidThese proteins enable adaptation to rapid
changes in the environment.changes in the environment.
There are three main sub-families of bHLH proteins:
(a) those containing only the bHLH domain; and those where the bHLH
domain is contiguous with a second dimerisation domain, either
(b) the leucine zipper (Zip) or
(c) the PER/aryl hydrocarbon receptor nuclear translocator
(ARNT)/single
minded (SIM) (PAS) homology domain.
PAS proteiny jsou souPAS proteiny jsou souččááststíí šširiršíší rodiny bHLH proteinrodiny bHLH proteinůů::
PASPAS proteiny (rodina transkripproteiny (rodina transkripččnníích faktorch faktorůů):):
http://mcardle.oncology.wisc.edu/bradfield/
PAS domainPAS domain
The PAS region consists of two adjacent degenerateThe PAS region consists of two adjacent degenerate
repeats of ~130 amino acids, PAS A and PAS B.repeats of ~130 amino acids, PAS A and PAS B.
The domain is anThe domain is an ancient signalling deviceancient signalling device
conserved through evolutionconserved through evolution, having been, having been
identified in proteins throughout the animal kingdom,identified in proteins throughout the animal kingdom,
in bacteria, fungi and yeast in addition to mammalsin bacteria, fungi and yeast in addition to mammals
and flies, where the most commonly studiedand flies, where the most commonly studied
bHLH/PAS proteins originate.bHLH/PAS proteins originate.
Many bacteria contain PASMany bacteria contain PAS--like proteins that detectlike proteins that detect
light and oxygen (Dos, Aer, FixL, PYP).light and oxygen (Dos, Aer, FixL, PYP).
Similar proteins sense light in plants (phytochromesSimilar proteins sense light in plants (phytochromes
PhyAPhyA--PhyE, NPH1; phytochrome interacting factorPhyE, NPH1; phytochrome interacting factor
PIF3).PIF3).
(Gu et al., Annu Rev Pharmacol Toxicol. 2000;40:519-61.)
bHLH PAS
100 aa
Arnt
protein
N C
A B
TAD
Dimerization
DNA
NLS
AhR
protein
N
C
Variable
lengthA B
TAD
DimerizationDNA
Ligand/HSP90/X
AP2 binding
NLS
Domain structure and function of PAS proteins:Domain structure and function of PAS proteins:
(Comprehensive Toxicology, vol. 14)
The circadian response pathwayThe circadian response pathway
Daily changes in light/dark require physiologicalDaily changes in light/dark require physiological
and behavioral adaptation:and behavioral adaptation:
 CLOCK/MOP3 heterodimer controls expression
of circadian responsive gene products – PER, TIM;
Nature Genetics 26, 23 - 27 (2000)
The hypoxia response pathwayThe hypoxia response pathway
The International Journal of Biochemistry & Cell Biology 36 (2004) 189–204
The ability to maintain O2 homeostasis is essential for survivalThe ability to maintain O2 homeostasis is essential for survival of mammals.of mammals.
The hyperoxic state, or high O2 tension, can result in the generation of reactive
oxygen intermediates and potentially lethal damage to membranes and DNA. The
hypoxic state, or low O2 tension, can result in levels of ATP insufficient to
maintain essential cellular functions. The hypoxic state occurs in a number of
medical conditions, such as cancer and ischemias, inspiring research into
understanding the cellular mechanisms for detecting and responding to low
levels of oxygen. Responses to hypoxia are mediated by three bHLH/PAS
proteins, HIF-1a, HIF-2a (also known as Endothelial PAS domain protein 1, HIFlike
factor and member of PAS family 2), and HIF-3a.
Mutation Research 569 (2005) 87–100
HIF subfamilyHIF subfamily
Biochimica et Biophysica Acta 1755 (2005) 107 – 120
Hypoxia-inducible factor-1 (HIF-1), composed of HIF- and HIF-
(ARNT) subunits, is a heterodimeric transcriptional activator. In
response to hypoxia, stimulation of growth factors, and activation of
oncogenes as well as carcinogens, HIF-1a is overexpressed and/or
activated and targets those genes which are required for angiogenesis,
metabolic adaptation to low oxygen and survival of cells. HIF-1 is critical
for both physiological and pathological processes.
Several dozens of putative direct HIF-1 target genes have been
identified on the basis of one or more cis-acting hypoxia-response
elements that contain an HIF-1 binding site. A variety of regulators
including growth factors, genetic alterations, stress activators, and some
carcinogens have been documented for regulation of HIF-1 in which
several signaling pathways are involved depending on the stimuli and cell
types. Activation of HIF-1 in combination with activated signaling
pathways and regulators is implicated in tumour progression and
prognosis.
HypoxiaHypoxia--inducible factor (HIFinducible factor (HIF--11):):
Masson, N. et al. J Cell Sci 2003;116:3041-3049
World J Gastroenterol 2004;10(8):1082-1087
Masson, N. et al. J Cell Sci 2003;116:3041-3049
HIF-dependent responses to O2 may be
modulated by the cellular environment:
Molecular
Pharmacology Fast
Forward. Published on
August 3, 2006 as
doi:10.1124/mol.106.0
27029
ARNTARNT –– zzáákladnkladníí dimerizadimerizaččnníí partner:partner:
The International Journal of Biochemistry & Cell Biology 36 (2004) 189–204
Jak HIFJak HIF--11, tak ARNT p, tak ARNT přředstavujedstavujíí
proteiny nezbytnproteiny nezbytnéé pro ppro přřeežžititíí –– KOKO
mymyšši odumi odumíírajrajíí jijižž v prv průůbběěhuhu
embryonembryonáálnlníího vývoje.ho vývoje.
Denison et al., Chem. Biol. Interact. 141: 3
The Ah receptor pathwayThe Ah receptor pathway
AhR =AhR =
• ligand-activated transcription factor;
• important mediator of toxicity of POPs;
• regulator of xenobiotic metabolism and activation of
promutagens.
AhR discoveryAhR discovery
• different sensitivity of inbred mouse strains to TCDD and 3-MC –
inducers of CYP1A activity in liver microsomes;
• autosomal dominant Mendelian trait;
• isolation of protein; cloning
Molecular Toxicology,
2nd ed.
Overview of aryl hydrocarbon receptorOverview of aryl hydrocarbon receptor
and dioxinand dioxin--like toxicity:like toxicity:
•• what is AhR;what is AhR;
•• evolution perspective;evolution perspective;
•• activation of AhR; AhRactivation of AhR; AhR--dependent genesdependent genes
•• toxic effects associated with AhR activation;toxic effects associated with AhR activation;
•• AhR interactionsAhR interactions
•• the role of AhR in cell cycle regulationthe role of AhR in cell cycle regulation
AhR domain structure:AhR domain structure:
Denison et al., Chem. Biol. Interact. 141: 3
Evolution of AhR:
AHR1
AHR2
AHRR
ARNT
Hahn, Chem. Biol. Interact. (2002) 141: 131
Toxicity mechanisms;
Liver and kidney
development;
Neuronal
differentiation?
Circadian rhytms?
YesAhR
(AhR1, AhR2)
Vertebrates:
Development;
Regulation of homeobox
genes and dendrite
morphology.
NoSpineless (Ss)Insects:
Drosophila melanogaster
Neuronal development;
Behavioral effects.
NoAHR-1Nematodes:
Caenorhabditis elegans
PhysiologicalPhysiological
function:function:
LigandLigand--binding:binding:Name:Name:Organism:Organism:
Evolution of AhR:Evolution of AhR:
Natural ligands ofNatural ligands of
AhR???????????AhR???????????
 light hypothesis – tryptophane derivatives
Denison & Nagy, Annu. Rev. Pharmacol. Toxicol. 43:309
Natural ligands ofNatural ligands of
AhR???????????AhR???????????
 lipid compounds and flavonoids
Denison & Nagy, Annu. Rev. Pharmacol. Toxicol. 43:309
transcriptional
coregulators
TNGCGTG
Xenobiotic response genes
cytoplasm
nucleus
AhR
hsp90
p23 XAP2
ligand
ARNT
??
((modified frommodified from
Kewley et al.,Kewley et al., 2004)2004)
AhR activation:AhR activation:
Chemico-Biological Interactions 141 (2002) 41–61
+ TCDD
C
ontrol
TC
D
D
5
nM
PC
B
126
100
nM
actin (40 kDa)
CY1A1 (60 kDa)
AhR (93 kDa)
AhR regulated genes:AhR regulated genes:
contain xenobiotic response elements (XRE) or dioxin
responsive elements (DRE) in their promoter region:
• phase I enzymes - CYP 1A1, CYP 1A2, CYP 1B1;
• phase II enzymes - UDP-glucuronosyltransferase,
GST-Ya, NADP(H):oxidoreductase;
• other genes – Bax?, p27Kip1, JunD, TGF- regulation
of cell cycle and apoptosis;
• AhRR.
Denison & Nagy, Annu. Rev. Pharmacol. Toxicol. 43:309
AhR toxicants:AhR toxicants:
Schmidt & Bradfield, Annu. Rev. Cell Dev. Biol. 12:55
Toxic effects of dioxins:Toxic effects of dioxins:
„„NonNon--classicalclassical““ AhR ligandsAhR ligands
Physiological role for AhRPhysiological role for AhR -- AhRAhR--deficientdeficient
mice:mice:
 significant growth retardation;
 devective development of liver and immune system;
 retinoid accummulation in liver;
 abnormal kidney and hepatic vascular structures.
 resistant to BaP-induced carcinogenesis and TCDDinduced
teratogenesis;
 no inducible expression of CYP 1A1 and 2.
Liver defects:Liver defects:
-/-
+/+
PNAS 2000 vol. 97:10447
BaP není karcinogenní v
AhR KO myších:
PNAS (2000) 97: 779–782
AhR je nezbytný pro imunotoxické účinky TCDD:
CTL response
J Biochem Mol Toxicol 16:317–325, 2002;
Interactions of AhR with other proteinsInteractions of AhR with other proteins
AP-1
Biochemical Pharmacology, Vol. 59, pp. 997–1005, 2000.
AhRAhR--ERER crosstalkcrosstalk
Chem. Res. Toxicol., Vol. 16, No. 7, 2003
VyuVyužžitiitiíí AhRAhR--ERa crosstalk v nERa crosstalk v náádorovdorovéé terapii?terapii?
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 356, 239–248, 1998;
CANCER RESEARCH 61, 3902–3907, 2001
Mol Cancer Ther 2004;3(6):715–25
Ohtake et al., 2003, NATURE 423 www.nature.com/nature
Direct AhRDirect AhR--ER interaction?ER interaction?
Regulation of the eukaryotic cell cycleRegulation of the eukaryotic cell cycle
pRB-dephosphorylation
pRB-phosphorylation
cyclin E
+ cdk2
cyclin A
+ cdk2
cyclin A/B
+ cdk1
D-cyclins
+ cdk4 / cdk6
pRB = retinoblastoma protein
cdk = cyclin-dependent kinase
p27p27
Progress in Cell Cycle Research, Vol. 5, 261-267, (2003)
Puga, Elferink Dietrich
ÚÚloha AhR v regulaciloha AhR v regulaci
bunbuněčěčnnéého cyklu jeho cyklu je
pravdpravděěpodobnpodobněě
slosložžititěějjšíší
MCF-7WB-F344
? AhR? AhR--HIFHIF--11 crosstalk ?crosstalk ?
Toxicology Letters 155 (2005) 151–159
JBC 274, 12115–12123, 1999
AhRAhR--retinoid receptors crosstalkretinoid receptors crosstalk
J. Nutr. 133: 277S–281S, 2003.
JBC (2004) 279(24):25284-93.