3- toxicity mechanism Flashcards
what is the deal with glutathione (GSH)
it is the most abundant low molecular weight thiol
what makes up glutathione
glutamate, cystein and glycine
how does glutathione act is an antioxidant
the thiol groups of the cysteine can donate a hydrogen to reduce other targets and itself become oxidized
what happens once glutathione donates a hydrogen
two of them conjugate to form GSSG (glutathione disulfide)
what can happen to glutathione disulfide
it can be reduced back to GSH by glutathione reductase
what is glutathione reductase
an enzyme that can convert glutathione disulfide back into glutathione
why is glutathione a good antioxidant
it donates hydrogens to other things so they are less likely to interact with other substances
how can some cancer cells protect themselves against anti-cancer drugs
they upregulate glutathione synthesis and glutamate-cysteine ligase (enzyme that is used to make GSH)
what is a way that we can increase effectiveness of some chemo agents
by inhibiting GSH synthesis (Cause they make more GSH to combat the anti cancer drugs)
what are 2 enzymes that are involved in glutathione synthesis
glutamate-cysteine ligase and glutathione synthetase
what does glutamate-cysteine ligase do
uses ATP to join glutamate and cysteine
what does glutathione synthetase do
uses ATP to add glycine (to glutamate-cysteine) to make glutathione
what do glutathione S-transferase enzymes do
conjugates glutathione with xenobiotics
how does glutathione conjugate with a xenobiotic (with help of S-transferase enzyme) (mechanism)
nucleophilic attack of the thiolate anion (GS-) with an electrophilic carbon, oxygen, nitrogen or sulfur
what happens to glutathione conjugates formed in the liver
they are transported into bile and excreted
where is most glutathione found in the body
in the liver
what does sequential cleavage of glutamic acid and glycine lead to
formation of mercapturic acids
what happens to glutathione levels in the cell when 4-HNE is added + why
glutathione levels drop (and so does 4-HNE levels)
because they conjugate with eachother
how does 4-HNE get detoxified (with GSH)
either spontaneous or glutathione-S-transferase mediated conjugation
what are 4 general ways that 4-HNE can be detoxified
- with GSH
- aldo-keto reductase
- reduction by alcohol dehydrogenase
- oxidation by aldehyde dehydrogenase
what happens to the 4-HNE metabolites
they are transported out of the cell
what is the breakdown product when GSH is broken down by aldehyde dehydrogenase
HNA
what is the breakdown product when GSH is broken down by alcohol dehydrogenase
DHN
what is another name for the antioxidant response elements (ARE)
the electrophile response element (EpRE)
what is another name for the electrophile response element (EpRE)
antioxidant response elements (ARE)
what is the antioxidant response elements (ARE) / the electrophile response element (EpRE)
a DNA sequence that controls the proteins that can be up-regulated to combat oxidative stress
what controls the proteins that can be upregulated to combat oxidative stress
ARE / EpRE
what does Nrf2 do
it binds to ARE / EpRE in response to oxidative stress and activates downstream genes
what is Nrf2 (not function)
a nuclear factor, protein
what does Keap 1 do
binds to nrf2 and prevents its movement into the nucleus
and positions Cul3 in place to aid in ubiquitination
what does cul3 do
allows for interaction of ubiquitin ligase with nrf2
what composes the electrophile response pathway protein clump (# amounts too)
2 cul3 proteins
2 Keap 1 proteins
1 nrf2 protein
(3 diff proteins, 5 in total)
what happens to nrf2 under basal conditions and no oxidative stress
nrf2 is targeted for proteasomal degradation (half life of 20 mins)
how is ubiquitin added to nrf2
added to the cysteine sulfhydryl groups by ubiquitin ligase
what does cul3 do
helps to position ubiquitin enzymes
how many cysteine residues does keap1 have
26
where is ubiquitin added to
cysteine sulfhydryl groups in nrf2 by ubiquitin ligase
does nrf2 have cysteine residues
yes
what 2 things can cause the modifications of sulfhydryl groups
- oxidation so that sulfhydryl groups form disulphides
- electrophilic attack of sulfhydryls resulting in covalent modifications (like by 4-HNE)
what happens once the sulfhydryl groups are modified (in that pathway thing)
Cul3 dissociates and ubiquitination no longer occurs, nrf2 is free to do its job and it targets the nucleus
what is sMaf
a transcription factor that pairs with Nrf2 to activate downstream genes (attached to electrophile response element)
what are 6 categories of responses via the electrophile stress response pathway
- xenobiotic detox
- ROS detox (GSH)
- iron sequestration (ferritin)
- GSH synthesis
- export
- protein removal and repair
think GRIPEX
what is the role of membrane transporters (general)
distribute things to and away from targets
what is the role of membrane transporters (toxins)
some accumulate toxins in cells, some pump them out
what are the 2 main families of membrane transporters
active transporters (ABC) facilitated transporters (SLC)
what does ABC stand for
ATP binding cassete
what is the defining feature of ABC transporters
use ATP to transport
what is the defining feature of SLC transporters
use electrochemical gradient to transport
what are the most abundant and important receptors in the SLC family (in humans)
OATP1A2 and OATP2B1
whats the big deal with OATP1A2 and OATP2B1
they are the most abundant and important receptors in the SLC family (in humans)
what is the deal with PEPT1 + which class
highly expressed in gut, uptake peptide and peptdie like drugs (antibiotics, like beta lactams)
SLC
how do transporters vary around the gut
the relative abundance of transporters vary
where is MDR1 most + which class expressed
increasing from duodenum to colon (ABC)
where are most of the uptake transporters in the GIT
highest in duodenum and decrease towards the terminal ileum and colon
what type of transporter is MDR1
ABC transporter
which transporter increases expression from duodenum to colon
MDR1
what is a main mechanism that opposes the distribution to target
export from cells
what is an example of exporter away from cells
multidrug resistant proteins (mdr) are ATP dependent and pump chemicals out of cells
what is the typical structure of ABC family protein
1 single protein, 2 domains, 6 helices in each domain and 2 catalytic regions
how did the structure of the ABC transporter likely arise
via gene duplication events
what type of family is the OAT pumps
SLC
what is an allocrite
something that is transported from one side to another
what happens in step 1 of ABC transporter mechanism
allocrite binds to interior of cell (Resting state, open conformation)
what happens in step 2 of ABC transporter mechanism (3 in this one)
ATP binds to 2 domains, transporter clamps down (closes) and does a 90 degree turn, helices open to outside to release allocrite
what happens in step 3 of ABC transporter mechanism
ATP is hydrolyzed
what happens in step 4 of ABC transporter mechanism
Pi and ADP is released, transporter back to origional state
when is ATP needed in the ABC transporter mechanism
to return it to original state (not even needed to release the allocrite)
which family do DAT SERT and NET belong to
SLC6 family
how do SLC6 family transporters usually work
use an ion gradient to facilitate transporter (usually Na+, sometimes Cl-)
which family do OCT and OAT belong to (more specific)
SLC22
what does OCT stand for
organic cation transporter
what does OAT stand for
organic anion transporter
What is the structure of SLC family protein typically like
2 similar 5-helical domains, substrate binding domain with Na+ and Cl- associations
do all SLC family proteins have the same amount of helices
no, some have more and some have less
what is a weird structural thing about the SLC family proteins
they have a bendy bit, it seems to be an important HINGE
do the helices occur in order in SLC family proteins
no
what are the 3 known SLC mechanism models
- rocker switch (banana)
- gated-pore
- elevator
how does the rocker switch model work
the N and C halves of the transporter oscillate back and forth from an outward facing state to an inward facing state along the symmetry axis perpendicular to the membrane
what is step 1 of rocker switch
open to exterior to get allocrite
what is step 2 of rocker switch
occluded state, not open to inside or outside
what is step 3 of rocker switch
open to the interior to release allocrite
what is the structure of the gated-pore model like
the binding site of the transporter is enclosed by two gates
what are the 2 domains like in the gated-pore model
one domain is static and one domain does the moving
what is step 1 of gated pore model
opening of outward facing fate lets substrate bind
what is step 2 of gated pore model
it binds, closes outside facing gate
what is step 3 of gated pore model
the inward gate opens to face the cytosol, substrate releases
what is the extracellular gate in the gated-pore model
a salt bridge
what is the intracellular gate in the gated-pore model
TM1 (transmembrane 1)
do SLC family transporters require ATP
no
what is the elevator mechanism
the domain containing the binding site (transport domain) moves along axis to transport
what are the domains like in the elevator mechanism
transport domain moves along axis perpendicular to membrane, the oligomerization domain remains static
what is step 1 in the elevator mechanism
transport domain open to exterior, substraet binds
what is step 2 in the elevator mechanism
transport domain is closed to both sides
what is step 3 in the elevator mechanism
loop opens to exterior side to release the substrate
what is common in step 1 of all the SLC mechanisms
it is open to the exterior and the allocrite binds
what does the elevator likely form structurally
trimer
which SLC forms a trimer
the elevator model
what is common in step 2 of all the SLC mechanisms
it is closed off to the the interior and exterior
what is common in step 3 of all the SLC mechanisms
it is open to the exterior and releases the substrate
what is fibrosis
excessive deposition of extracellular matrix
what is the extracellular matrix
polysaccharides linked to proteins to form proteoglycans
what makes up the fibrosis scar tissue
type 1 collagen that replaces healthy tissue
what seems to be responsible for fibrosis + how
TGF B interacting with its cell surface receptor
where is TGF B stored
inactively in the ECM
what kind of things release TGF B
enzymatically by ROS
What are the 3 mechanisms that promote fibrogenesis (big)
- inhibit MMPs and increased TIMP
- increase myofibroblast production
- induction of matrix component gene transcription
what is MMP
matrix metalloproteases
what is TIMP
tissue inhibitor of metalloproteinase
what does TGF B do to matrix metalloproteases
inhibits
what does TGF B do to tissue inhibitor of metalloproteinase
increases its production
what does TGF B do to myofibroblast production
induction
what do myofibroblasts do
key in collagen production
what does TGF B do to matrix component gene transcription
induces it
what does TIMP do to MMP
inhibits
what does TGH B do to the ability to break down ECM
decreases
what do myofibroblasts product
collagen
simply put, what are 3 things that TGF B does
- Less degradation
- Increase cells that make collagen
- Increase in production of proteins we find in ECM
which family is mdr
ABC
which family is mrp
ABC
which family is oat
SLC
which family is pept
SLC
which family is bcrp
ABC
how to remember if its ABC or SLC
if theres a t in the name, its SLC. if not, then its ABC
