ETOX 202

Macromolecular Target

 

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Clofibrate

Macromolecular Target

 

Clofibrate = PPAR

Macromolecular Target

 

Lead

Macromolecular Target

 

Lead = ALAD

Macromolecular Target

 

Pertussis Toxin

Macromolecular Target

 

Pertussis Toxin = G-protein Gα-i

Macromolecular Target

 

Cholera Toxin

Macromolecular Target

 

Cholera Toxin = G-protein Gα-s

Macromolecular Target

 

Dioxin/TCDD

 

Order Sensitivity

Macromolecular Target

 

 

Dioxin = Ah Receptor

 

must bind with AHR/HSP90/ARA9

 

HIF is competitor

 

weightloss is correlation not caused by p450 induction

 

Amino acid Ile 332/ser makes more sensitive,
ILE/ala, Val/ala

Macromolecular Target

 

 

Botulinum A Toxin

Macromolecular Target

 

 

Botulinum A = VAMPS

Macromolecular Target

 

Parathion

Macromolecular Target

 

 

Paration = AchE

Macromolecular Target

 

 

Muscarine

Macromolecular Target

 

 

Muscarine = Mucarinic Receptor

Macromolecular Target

 

 

Nicotine

Macromolecular Target

 

 

Nicotine = Nicotinic Receptor

Macromolecular Target

 

 

Tubocurare

Macromolecular Target

 

Tubocurare = Nicotinic neuromuscular acetylcholine receptors

G-Protein linked Receptor Sequence

Ligand binds to GPCR causing activation and conformational change

 

GDP bound to Gαs is replaced by GTP and G-beta and G-gamma dissociates from G-alpha

 

G-alpha binds to adenylyl cyclase to activate it causing it to catalyze formation of cAMP, raising cAMP levels

 

cAMP dependant protein kinase causing activation of pka

 

pka goes into the nucleous, Atp ->ADP activates CREB by phosphorylation

 

CBP binds to CREB and then binds to cAMP response element causing transcription

 

G-beta activates phospholipase C which cleaves IP3 from biphosphate and activates Ca2+ channels in ER

 

Ca2+ activates protein kinase C

Diptheria Toxin Sequence

Two Domains:

A = Catalytic Domain

B = Transmembrane/Receptor Domain

 

Low iron, uses siderophores to sequester and produces toxin. High iron no toxin made

 

A & B Toxins are cleaved by protease but stay together due to a disulfide bond. The cleaveing activates dip. tox. 

 

 binds to HB-EGF and enters cell through endocytosis

 

The proton pump by V-type ATPase causes acidification 7 to 6 in vacuole resulting insertion into the membrane

 

A domain is fully cleaved from B domain, A domain crosses the membrane and affects EF2 function by inhibiting the translocation of tRNA from A-site to P-site

Critical Amino Acids in epoxide Hydrolase and why

Y381-OH and Y465-OH hydrogen bond with the O on epoxide faciltates nucleophilic attack by

 

ASP333-COO- attcks epoxide ring leading to formation of hydroxylacyl intermediate

 

His523 and ASP495  hydrolyze the intermediate allowing ASP333-COOH to disassociate

Cholinergic neuromuscular junction sequence

Ca2+ enters and binds to SNARE complex (VAMP & SNAP on vesicles) ,opening Ca2+ ion channels

 

Once bound, the vesicle is docked on presynaptic membrane and releases neurotransmitters. Ach binds to AchR on post synaptic vessicle and triggers transmission of signal flow.

 

Na+ enters cells and depolarizes, if threshold is reached and action potential triggers.

 

Sarcolema repolarizes as K+ flows, NA/K pump restores ions.

 

AcHE cataylzes degradation of Ach in synapse.

CYP1A1 regulation

Dioxin binds to AhR,

 

AhR and Arnt attach to Dioxin repsonse element

 

CYP1A1 expressed

CYP3 Expression

 

CYP3A4

Steriod (Rifampicin) binds to PXR, cis-9-retinoic acid binds to RXR

 

PXR and RXR attach to XREM

CYP4 Expression

colfibrate binds to PPAR, Cis-9-retinoic acid binds to RXR

 

PPAR and RXR attach to PPRE

 

CYP4 expressed

CYP7 Expression

fatty acids/cholesterol binds to LXR, CXR

 

LXR and CXR attach to LXRE

CYP1A1 expressed

CYP2B Expression

Phenobarbital binds to CAR, cis-9-retinoic acid binds to RXR,

 

CAR and RXR attach to Phenobarbital repsonse element

 

Important Amino Acids in Glutathione

Normally GSH is pH7 then when bound to GST pH 5-6, GSH is not very active but is in presence of GST. Alpha is most predominate

 

Gsite and binds to GSH, H site is lipophilic and binds to substrate

 

Y-6 on G site forms the electrophile pocket, its acidic and allowing gsh to be ionized and perform nucleophilic attack on epoxide,

 

S-209 forms hydrophobic pocket, facilliating Y115

 

Y-115 acidic h site, forms the substrate pocket, is electron acceptor

BAP Carcinogens

(+)7,8 diol 9,10 epoxide-2 (anti)

(-)7,8 diol 9,10 epoxide-1 (syn)

(+)7,8 diol 9,10 epoxide-1 (syn)

(-)7,8 diol 9,10 epoxide-2 (anti)

 

 

Reporter bashing
Take regulatory part of gene and attach a reporter gene. Remove parts of regulatory gene and monitor reporter to know which part of gene is important
mEH and sEH
mEH – Cis, microsomal epoxide hydrolase, ER metabolism

sEH- Trans, soluble epoxide hydrolase, cytoplasm metabolism

Arachidonic acid
Cox pathway -> pain
LOX pathway -> Leukotriene = allergies
CYP2 Epoxygenase -> EETs = hypertension adds epoxide to any double bond
CYP4 hydrolase -> Hetes Adds adds an OH to CH3
Leukotrine Steps
Leukotrine A4 -> LTB4 by hydrolase no acyl group

or A4-> C4 Glutathioe transferase loses glutamic acid

c4-> d4 CGP takes off glycine and becomes acetyl group

d4->e4 acetyl group is transfered becomes amine

e4 -> f4 formation mercapturic acid

UDGP Pathway
Toxin enters membrane, gets metabolized by P450, goes to UDPGA, UGT->UDP, and then leave cell as toxin-glucuronide,

UDP -> UDPse -> UMP -> Translocase trades cytoplasm UDPGA into ER from UMP to cytoplasm

Target for pyrethroids
Na Channel
Target for diazapines
sodium/potasium pump
P-type pumps, f-type pumps, v-type pumps, abc transporter
p = ion pump ATP to pump out
f-type = H+ pump builds ATP and makes acidic
V-type = same as f type but in vacuole
ABC uses 2 ATP to pump molecule out
Dimerization of AHR/ARNT
bHLH is DNAbinding with PAS-A, secondary dimerizization is PAS, B is ligand binding,
Clostridium c2 toxin target
actin
Picolo Toxin target
GABA a
Tetanus Toxin and botulism toxin mechanism
binds Bot A. binds to SV2, bot. B binds to synaptotagmin, inside vesicle before it buds

buds and retrograde transport into the cell,

acidification inside vesicle due to atpase proton pump

conformation change allowing inserting into membrane

A domain is release in cytoplasm

Bot A. cleaves SNAP-25
Bot B. cleaves synaptobrevin
Tetanus cleaves VAMP

leads to no vesicle docking and no neurotransmitter release

Parasympatheic
Sympathetic
Parasympathetic: Long pre, short Post, Ach
Sympathetic: short pre long post,Ach, Ne
Muscarinic receptor
Nicotinic receptor
Muscarinic = gprotein receptor
Nicotinic = ligand gated
Atropine target
Muscarinic receptor
Types of receptors

GABA A & C
GABA-B

Transmitter gated channel = GABA A&C

GPCR – GPCR

Important Amino Acids of ACHE
Peripheral site – allow the substrate to enter, determine affintiy, W279,Y121,F330
Catalytic site – charged residue of Ache, this is required for binding, S200, Hist440, E327
AchE ageing
organophoste binds to ACHE, ethylene is removed, permanant addition
GSH reacts with dibromide
GS is – and H is +

GS attacks Br//Br -> SG//Br- -> GS+ triangle

SULT
HIST 108 extract proton from OH and Sulfate, another OH attacks sulfur in SULT producing and intermediate

-OH on substrate becomes -OSO3

Nicotine pathway
Nicotine -UGT-> add Glu onto empty N

Nicotine -CYP2-> add =O on CH2 to the right of N-CH3 -UGT-> to add glu onto emtpy N

Nicotine -CYP2-> add =O on CH2 to the right of N-CH3 -CYP2-> -OH to right of =O -UGT-> -OH to -OGlu

BAP Pathway
BAP is 2 on top of 3 rings

P450 add epoxide on bottom left or top left and its either down or up. Epoxide hydrolase makes them OH’s the one closest to the bay region matches up/down of epoxide other -OH is opposite. then P450 act on which ever it didn’t act on fisrt making another epoxide. Epoxide hydrolase can break and then It can then react with N7 on DNA and then be removed by aicd hydrolysis.

If GST/GSH breaks 2nd you get -oh -SG