Summary Molecular and Cellular
toxicoloog
and transporters
Lecture 2 :
signaaltransductie .
ubiquiti nation
Defence induced stress
against drug -
metabolisme
Drug
-
Antioxidant response
-
Apoptosis
Stress responses
-
Induction protective enzymes
protein rekking
-
protein degradatie / resynthes.is
-
ftp.optosis
'
BIOCKACK Cev
Cycle
'
'
DNA -
repair
ROS Causes and responses
chemo -
therapy SOD
NF KB
Radiation
Is
-
#
(ataiase
PPAR AD -1
Growthf
\
1- Glutathion
Yq 9 /
.
_
ROS STA-13 -
ROS Nrfz
# -1# peroxiredoxin
-
uv
Cytokines / f. ]
Thiooedo✗in reductor
kypoxiape.ro .
HIF '×
-
gp , p53
✗ idase
Major een stress
signaiing response's
oxidatie stress ER stress In Flammarion DNA damage Heat shock Heavy metal Hypoxia
.SI!
/keameffectOrS
IKBX
÷
53BPI MTF -
1 VHL
Nrf 2 ATFY / XBPI Rel A P53 HSFI / 2 MTF -
1 HIER
Srxnl (HOP / Bip ICAMI pzl USD70190 MTS HUMMR
Sensors transducers and effectors
.
Sensor :
receptor of signa/
-
Transducer of
Signals relay
-
:
Effector ion Factor :
transcript
signa/ molecules
-
-
Survival factors :
promoties Cev Survival
by Suppressing apoptosis
blocks in cel Cycle
primary by overreding
stimulate cel division
Mitogens
-
:
.
-
Growth factors : stimulatie
ceugrowth b promoting Synthesis and inhibiting
the degradaties of protein's and other macromolecules
↳
there always be Cross talk
possiblyactivatesmore
-
one molecule
win .
signa
than one
transductionpathway
ij
,Example : TNF x - -
enhanced diclofenac toxi
City
-
Hep62 Cells ( Ii ver cells . but no metabolism)
for basic toxi
City of compounds
↳ look
TNFX is a
cytokine used to Mimic an inham Motion reaction in the een
-
-
Use of a non -
toxic dose Of TNFX
diclofenac
Adding TNFX increases the
toxicity of
-
-
Common tool to Mimic certain situations in the Cev
kinase -
linked receptor
signa/
of molecule 50
GG
~
phosphorylation
§ Jr
- '
Auto -
in trans or (is
trans :
phosphoryiate eachother
-
itself
< is :
phosphoryat.es
-
ËËËË .
Dominant
negative
mutant by -
inhibitors
RTK :
no kinase activator in trans
IS this kinase important :
Inactie RTKS Cross
phosphoryiation tyrosine
-
-
activated kinase domains transfect and out
b a mutant kinase
compote the wild type -
stress -
activated protein -
kinase pathways
Stimuli Growth factors etc . oxidatie stress ,
cytokines
RTK
b. ↳ !
'
MAPYK Ras
;
t 6 b.
MAP 3K Raf ASKI MEKKI , ,
MLK}
↳ b b.
MAPZK MEK MKK3/6 MKK417
to b t t
MAPK ERK p38 JNK
6 1 t t
ceil responseS proliferatien proliferatiein
Differentialion -
differentiaties and apoptosis
ceil
Migration Inflammatoire responses
from
Dominant
negative mutant : mutant kinase over express in ce 11 preventS WT
being active
-
, ,
↳ serine to alanine Can . on ever be
phosphorylated .
blocks
pathway
Dominant active mutant :
always phosphorylated
-
-
charge surface op the protein →
introduces a
negative charge
→
mutate serine to
( as partie / acid)
a
negativey charged amino acid giutamic
Example phospho specific: -
antibodies
JNK effector : (
Jun
-
IPJNK is activated.it is
phosphorylated
-
Take cells -
seen if there is
phosphorylated JNKI
-
phospho specific antibodies recognize phosphor lated :
JNKI
-
-
phosphorylated
↳
but the
it is not the aMount of JNKI that Changes . aMount of JNKI
ij ij
, Regulation of MAPKS b GSTS
•
Certain GSTS can bind to certain MAPK
and
Regular inactivate
b binding to JNK
<
Stress leads to the disruption of these interaction5 and the activator of the
signaiing pathway
-
Kinase -
specific inhibitors
-
IS the kinase that is
phosphorylated also really important for the effect we want to
study
•
Solution : kinase -
specific inhibitor
Example Imatinib / : Gieevec
-
The BCR ABL ONCOprotein
phosphoryiates tyrosine
-
-
-
you get leukemie
-
Inhibitor birds in the pocket and preventS this
Example :
JNKI and APAP toxicity
APAP activate 5 JNK →
phosphoryiated c-
Jun
-
•
with an inhibitor -
no
phosphorylation
Serum ALT of protein from liver
Ieakage
•
:
that leads to inhibited JNK inhibitor
Downstream
signaling of JNK Cev
damage is
by a
'
Ubiquiti Nation :
proteasemal protein degradaties
El ( UB activa
ting enzyme ) activates UB
-
onjugating enzyme) cor ugates ligase)
-
Ez ( UB ( with E3 ( UB
Degraded b proteasome
-
UB Coupled via its
is
always lysine
'
-
C terminus
-
ub to
lysine residues
-
Dependant on E3
youget different Shapes
FunctionS Of ubiquitination
•
protea50 Mes : K-48 / K -
11
Endocytoseis / DNA repair 10calizatiON (Multi) mono ub
•
. : - -
signalling Ms / K -63
.
:
•
Functions unclear : K -6/-271 29 / - -
33
Example :
proteasome inhibitor
In
hibiting proteasome leads to increased proteotoxic stress
•
- can Kit1 tumour cells
ou
.
HOW can we see this →
accumulatieN Of Ub
protein 5 -
have protectOxic stress because of imbalance the
Leukemia cells
genome by
giving
-
in .
compound this cells cannot Cape with the stress and die
Study of transporters
↳
specific transporter inhibitors
ij ij
ijij
, Example : Saabte
yeast xenobiotic receptor
Drug activate 5 transcriptin factor
'
In Crease in level of ABC transporter pdrsip and others
many
-
-
Lower level of the due to export
drug in cel1
'
Importance of transporters
•
-
knock-out and look at growth
-
over expression of transporter
BSEP
-
transports bileacids
.
drugs wilI be tested For BSEP inhibitor
Al 1
↳
ifinhibitedyougetchoestas.is
Transporter vesicles
ex
pressing
-
over express transporters
•
IP make a veside
youdisrupt the membranen
you
•
.
↳
vesice can be in 2
Ways
.
Insideout vesíce : what was in síde .
is now outside
vesides that in an effect
only 90 give
-
reduced
-
efficiency is
but the rest does not
giveanynoise
-
In vivo
imaging
Build luciferase in Mouse (Nrfz)
in
gene
•
can detect nrfz stress response5 and selectie
Organ Eoxicity
-
-
( isplatin in
Kidney high amount OF OCTZ
-
-
paracetamol in li ver needs bioactiva tion
high level of
Cyp450 in river
•
-
.
Lecture 3 :(en modeIs for toxic
ity testing
Cancer (en limes
-
characterstics
abnormaal
karyotype
-
-
mutation in p53 in more than 50% of an Cancer cells
-
Immoreel . expression of telomerase or Stabilization of te10Mers
-
Loss of contact inhibítion
-
Immortalization
spontaneons
-
( papillomavirus)
virus in
during Cancer
-
-
L imitation S
over expression of MDR
-
Lock of metaboli
zing enzymes
-
Mare
oncogen es tess
suppressorgenes
-
.
-
Abnormat behavior . no controlled Cev Numbers
toxicoloog
and transporters
Lecture 2 :
signaaltransductie .
ubiquiti nation
Defence induced stress
against drug -
metabolisme
Drug
-
Antioxidant response
-
Apoptosis
Stress responses
-
Induction protective enzymes
protein rekking
-
protein degradatie / resynthes.is
-
ftp.optosis
'
BIOCKACK Cev
Cycle
'
'
DNA -
repair
ROS Causes and responses
chemo -
therapy SOD
NF KB
Radiation
Is
-
#
(ataiase
PPAR AD -1
Growthf
\
1- Glutathion
Yq 9 /
.
_
ROS STA-13 -
ROS Nrfz
# -1# peroxiredoxin
-
uv
Cytokines / f. ]
Thiooedo✗in reductor
kypoxiape.ro .
HIF '×
-
gp , p53
✗ idase
Major een stress
signaiing response's
oxidatie stress ER stress In Flammarion DNA damage Heat shock Heavy metal Hypoxia
.SI!
/keameffectOrS
IKBX
÷
53BPI MTF -
1 VHL
Nrf 2 ATFY / XBPI Rel A P53 HSFI / 2 MTF -
1 HIER
Srxnl (HOP / Bip ICAMI pzl USD70190 MTS HUMMR
Sensors transducers and effectors
.
Sensor :
receptor of signa/
-
Transducer of
Signals relay
-
:
Effector ion Factor :
transcript
signa/ molecules
-
-
Survival factors :
promoties Cev Survival
by Suppressing apoptosis
blocks in cel Cycle
primary by overreding
stimulate cel division
Mitogens
-
:
.
-
Growth factors : stimulatie
ceugrowth b promoting Synthesis and inhibiting
the degradaties of protein's and other macromolecules
↳
there always be Cross talk
possiblyactivatesmore
-
one molecule
win .
signa
than one
transductionpathway
ij
,Example : TNF x - -
enhanced diclofenac toxi
City
-
Hep62 Cells ( Ii ver cells . but no metabolism)
for basic toxi
City of compounds
↳ look
TNFX is a
cytokine used to Mimic an inham Motion reaction in the een
-
-
Use of a non -
toxic dose Of TNFX
diclofenac
Adding TNFX increases the
toxicity of
-
-
Common tool to Mimic certain situations in the Cev
kinase -
linked receptor
signa/
of molecule 50
GG
~
phosphorylation
§ Jr
- '
Auto -
in trans or (is
trans :
phosphoryiate eachother
-
itself
< is :
phosphoryat.es
-
ËËËË .
Dominant
negative
mutant by -
inhibitors
RTK :
no kinase activator in trans
IS this kinase important :
Inactie RTKS Cross
phosphoryiation tyrosine
-
-
activated kinase domains transfect and out
b a mutant kinase
compote the wild type -
stress -
activated protein -
kinase pathways
Stimuli Growth factors etc . oxidatie stress ,
cytokines
RTK
b. ↳ !
'
MAPYK Ras
;
t 6 b.
MAP 3K Raf ASKI MEKKI , ,
MLK}
↳ b b.
MAPZK MEK MKK3/6 MKK417
to b t t
MAPK ERK p38 JNK
6 1 t t
ceil responseS proliferatien proliferatiein
Differentialion -
differentiaties and apoptosis
ceil
Migration Inflammatoire responses
from
Dominant
negative mutant : mutant kinase over express in ce 11 preventS WT
being active
-
, ,
↳ serine to alanine Can . on ever be
phosphorylated .
blocks
pathway
Dominant active mutant :
always phosphorylated
-
-
charge surface op the protein →
introduces a
negative charge
→
mutate serine to
( as partie / acid)
a
negativey charged amino acid giutamic
Example phospho specific: -
antibodies
JNK effector : (
Jun
-
IPJNK is activated.it is
phosphorylated
-
Take cells -
seen if there is
phosphorylated JNKI
-
phospho specific antibodies recognize phosphor lated :
JNKI
-
-
phosphorylated
↳
but the
it is not the aMount of JNKI that Changes . aMount of JNKI
ij ij
, Regulation of MAPKS b GSTS
•
Certain GSTS can bind to certain MAPK
and
Regular inactivate
b binding to JNK
<
Stress leads to the disruption of these interaction5 and the activator of the
signaiing pathway
-
Kinase -
specific inhibitors
-
IS the kinase that is
phosphorylated also really important for the effect we want to
study
•
Solution : kinase -
specific inhibitor
Example Imatinib / : Gieevec
-
The BCR ABL ONCOprotein
phosphoryiates tyrosine
-
-
-
you get leukemie
-
Inhibitor birds in the pocket and preventS this
Example :
JNKI and APAP toxicity
APAP activate 5 JNK →
phosphoryiated c-
Jun
-
•
with an inhibitor -
no
phosphorylation
Serum ALT of protein from liver
Ieakage
•
:
that leads to inhibited JNK inhibitor
Downstream
signaling of JNK Cev
damage is
by a
'
Ubiquiti Nation :
proteasemal protein degradaties
El ( UB activa
ting enzyme ) activates UB
-
onjugating enzyme) cor ugates ligase)
-
Ez ( UB ( with E3 ( UB
Degraded b proteasome
-
UB Coupled via its
is
always lysine
'
-
C terminus
-
ub to
lysine residues
-
Dependant on E3
youget different Shapes
FunctionS Of ubiquitination
•
protea50 Mes : K-48 / K -
11
Endocytoseis / DNA repair 10calizatiON (Multi) mono ub
•
. : - -
signalling Ms / K -63
.
:
•
Functions unclear : K -6/-271 29 / - -
33
Example :
proteasome inhibitor
In
hibiting proteasome leads to increased proteotoxic stress
•
- can Kit1 tumour cells
ou
.
HOW can we see this →
accumulatieN Of Ub
protein 5 -
have protectOxic stress because of imbalance the
Leukemia cells
genome by
giving
-
in .
compound this cells cannot Cape with the stress and die
Study of transporters
↳
specific transporter inhibitors
ij ij
ijij
, Example : Saabte
yeast xenobiotic receptor
Drug activate 5 transcriptin factor
'
In Crease in level of ABC transporter pdrsip and others
many
-
-
Lower level of the due to export
drug in cel1
'
Importance of transporters
•
-
knock-out and look at growth
-
over expression of transporter
BSEP
-
transports bileacids
.
drugs wilI be tested For BSEP inhibitor
Al 1
↳
ifinhibitedyougetchoestas.is
Transporter vesicles
ex
pressing
-
over express transporters
•
IP make a veside
youdisrupt the membranen
you
•
.
↳
vesice can be in 2
Ways
.
Insideout vesíce : what was in síde .
is now outside
vesides that in an effect
only 90 give
-
reduced
-
efficiency is
but the rest does not
giveanynoise
-
In vivo
imaging
Build luciferase in Mouse (Nrfz)
in
gene
•
can detect nrfz stress response5 and selectie
Organ Eoxicity
-
-
( isplatin in
Kidney high amount OF OCTZ
-
-
paracetamol in li ver needs bioactiva tion
high level of
Cyp450 in river
•
-
.
Lecture 3 :(en modeIs for toxic
ity testing
Cancer (en limes
-
characterstics
abnormaal
karyotype
-
-
mutation in p53 in more than 50% of an Cancer cells
-
Immoreel . expression of telomerase or Stabilization of te10Mers
-
Loss of contact inhibítion
-
Immortalization
spontaneons
-
( papillomavirus)
virus in
during Cancer
-
-
L imitation S
over expression of MDR
-
Lock of metaboli
zing enzymes
-
Mare
oncogen es tess
suppressorgenes
-
.
-
Abnormat behavior . no controlled Cev Numbers
