Undone III Flashcards
(98 cards)
1
Q
Nuclear receptors
A
Direct binding to DNA to regulate Gene expression
2
Q
Nuclear receptors main structure
A
- DNA binding domain (2 Zn fingers binding HREs)
- Hinge domain (NLS)
- Ligand binding domain (AF-2 site act. transcription, ligand dep.)
- N-terminal domain (AF-1 site)
- C-terminal domain
3
Q
Zinc Finger
A
- Fold of 30a.a
- Zn2+ interacts with 4a.a cysteines
- Forms short a-helix & 2 B-pleated sheats
- P-box: Recognition of the RE of DNA
- D-box: Dimerization upon ligand binding
4
Q
Nuclear-R conformation after Ligand binding
A
Last a-helix (12) altered position causing a hydrophobic patch on the surface of the receptor which acts as a dimerization core where Co-activators will bind
5
Q
Type I Nuclear receptors
A
In the Cytoplasm & Bind palindromic repeats
- Steroid receptors
(homodimers)
6
Q
Type II Nuclear receptors
A
Retained in Nucleus & Bind direct repeats
- Thyroid receptor
- Retinoid Receptor
(homo / heterodimers)
7
Q
Mech. of Type I Nuclear receptors
A
Hsp90 & p23 maintain receptor
1) Ligand binds, Hsp90 dissociates
2) Complex transported by Immunofilin to nucleus (through pore)
3) SR binds DNA in nucleus
4) Co-activators collected, acetylate histones, DNA unwinds, Transcription
8
Q
Mech. of Type II Nuclear receptors
A
Nuclear receptor (e.g. Thyroid) always bound to the DNA
- No ligand: Co-repressors bound, Deacetylation
- Ligand: Switch to Co-activators, Acetylation, DNA unwind, transcrip.
9
Q
Orphan Receptors
A
- Nuclear receptors without known ligands
- Form heterodimers with other receptors to improve DNA targeting
- Control basal transcription levels by directly recruiting coactivators or corepressors
- Regulated by expression levels, stability, and modifications
10
Q
Xenobiotics
A
Biologically active chemical substances that are not naturally produced or present in the body (medicines, poisons, pollutants)
11
Q
Xenobiotic Elimination
A
- Phase-I: Cytochrome-p450 add polar functional groups by oxidizing (-OH, -COOH)
- Phase-II: Conjugated with charged particles by UDP-glucuronosyl transferase (glutathione, sulfate, glycine)
- Excretion in Bile / Urine as water-soluble
12
Q
Xenobiotic Issues
A
- Polyaromatic hydrocarbons (PAH)
- Halogenated PAH
= cyt-P450 related cause cancers, birth defects, some endocrine disrup.
13
Q
AhR receptor structure
A
Aryl Hydrocarbon Receptor (cytoplasm)
- Ligand binding domain
- Hs binding site (maintenance)
- DNA binding domain
- Activation of transcription domain
- NLS
Helix-loop-helix structure (TF family)
14
Q
AhR receptor subunits
A
- Type I: AhR (xenobiotics)
- Type II: ARNT (+ HIF-1B)
15
Q
Mech. of AhR receptor
A
1) Anchored in cytoplasm waiting for ligand (Hsp90 & p23)
2) Ligand binds (Hsp90 & p dissociates)
3) Complex transported by XAP-2 to Nucleus
4) Receptor dimerizes with Type II subunit ARNT/HIF-1B
XRE / DRE on DNA (xeno/drug)
5) Coactivator to acetylate histones
16
Q
What helps Maintain Nuclear receptor strcuture
A
- Hsp90
- p23
17
Q
Benzo(a)pyrene dangers
A
Xenobiotic that forms reactive intermediates Diol & Epoxides throughout oxidation by p450
- Can intercalate bw DNA bases
- Cis can be fixed by NER
- Trans cant be fixed = mutation
18
Q
Types of Signalling
A
- Endocrine (blood)
- Paracrine (tissue)
- Autocrine (itself)
- Juxtacrine (close)
19
Q
Framework of signal transduction
A
1) Ligand
2) Receptor Protein
3) Signal transducers
4) Second messenger
5) Regulatory protein
6) Target proteins
= Biological response
20
Q
Classification of PM receptors
A
- Ligand gated ion channels (nAChR)
- GPCRs (mAChR)
- Enzyme linked (GC, TK, Ser/Thr K)
- R connected to enzymes (cytokine-R)
21
Q
Types of GTP-binding proteins
A
- Heterotrimeric GTP binding protein (G protein): Large
- Small GTPases (Ras): Monomeric
22
Q
Heterotrimeric GTP binding protein
A
- Anchored to PM, activated by 7TM GPCR
- a, B, y subunits (3x polypeptide chains)
- a binds GDP/GTP, intrinsically activated
(same mech from physio, types Gs/i)
23
Q
Small GTPases
A
- Anchored to PM
- Single polypeptide chain
- GDP to GTP mediated by GEF
- Inactivation by GAP (GTPase AP)
24
Q
Serine/threonine-specific protein kinases phosphorylate what residue
A
OH group on A.A
(use ATP)
25
cAMP function path
1) PKA has 4 subunits (2 regulatory & 2 catalytic)
2) Regulatory.s with pseudosubstrate sequence binds cAMP
3) 4x cAMP needed for full activation
4) Catalytic subunit of PKA released
= phosphorylates ser/thr side chains
26
Abnormal Kinase
Urokinase (uPA)
Doesn't transfer a phosphate group, but is a serine protease used in blood coagulation
27
What can PKA phosphorylate
- Pyruvate Kinase L (INACT)
- Glycogen Synthase (INACT)
- Phosphorylase Kinase (ACT)
(phosphorylase kinase can phosph. glycogen phosphorylase, activating it)
28
CREB proteins mechanism
1) PKA enters nucleus phosphorylating CREBP (cAMP RE BP) binding CRE
2) CREB-P recruits CBP
3) CBP has acetyltransferase activity
4) Transcription factors & RNA polymerase 2 recruited = transcription
29
Classification of Protein Kinases
- Based on side chain
- Allosteric activation
- Activation by Phosph.
- Inactivation by Phosph.
- EC Ligand (TGF-B)
30
Protein domains in Signal
- Writer (AC: ATP to cAMP)
- Reader (SH2 domain)
- Eraser (PDE: cAMP to AMP)
= Adaptor, Docking, Scaffold proteins
31
Phospholipases (groups)
Break ester bonds
- PLA1: 1st ester
- PLA2: 2nd ester (usually arachidonic.a)
- PLC: 3rd ester bw glycerol & Pi
- PLD: Ester bw Pi & side chain
32
PLC role
Cleaves PIP into IP3 & DAG
33
PLC isoforms
- PLC-B (Gq pathway)
- PLC-δ (signal amplifier)
- PLC-y (GF signal on RTK, SH2/SH3 domain)
34
PLC common features
- Catalytic domain cleaves PIP2
- PH (targets PLC to PM)
- EF hand domain (senses Ca2+)
35
Phorbol Esters & issues
Produced by plants and can activate PKC similar to DAG
- PKC phosphorylates everything including TFs which cause increased proto-oncogenes so more cell division
- Long exposure causes PKC wearout, PKC dissapears = promotes cancer
36
PKC groups
- Classical (cPKC): a,B,y isoforms & activated by DAG & Ca2+
- Novel (nPKC): δ, ε, η, θ iosforms & activated by DAG and phospholipids (no Ca)
- Atypical (aPKC): λ, ι, ζ isoforms & activated by phospholipids only (no DAG or Ca)
37
PKC structural similarities
All have catalytic part with ATP & Substrate binding sites
Different regulatory parts:
- cPKC: C1 binds DAG, C2 binds phospholipids in Ca2+ presence
- nPKC: C1 & shorter C2 domain (non-funct)
- aPKC: short C2 & PB1 domain (protein factors)
38
PI3K structure & similarities
3 classes, class 1 for signal transduction (1A/B)
- Catalytic subunit p110 & Ras domain
- 1A: p85 kDa regulatory sub, with SH2 domain activating RTK
- 1B: p101 kDa regulatory sub, binds heteromeric G-protein to By-subunit
39
PI3K function
1) PIP2 to PIP3 recruits PDK1 (PH)
2) PDK1 phosphorylates Akt/PKB
3) PKB phosphorylates pro-apoptotic members, GSK3, translation factors
4) GSK3 releases inhibition of SREBP & eIF2B = synth of macromolecules
5) BAD phosphorylated by PKB releasing anti-apoptotic Bcl-2
40
PI3Ka gene & regulation
Proto-oncogene that if mutated causes unregulated growth
- PTEN is the only protein that can switch PI3K off (PIP3 to PIP2) to induce apoptosis
41
NFκB info
Nuclear Factor kappa-light-chain enhancer of activated B-cells
- Goes to nucleus where it regulates gene expression of 200 genes involved in immune response, survival, proliferation, inflammation, angiogenesis, apoptosis
- Activated by Cytokines, GFs, Bacteria/Virus, Ox. stress
42
NFκB active structure
Heterodimer when active
- p50 (kDa) consists of NF-κB1 or NF-κB2
- p65 (kDA) consists of RelA, RelB, & c-Rel = Transcriptional Activator Domain
43
NFκB when no stimulus
NFκB is inactive
- Retained in cytosol by IκB
- IκB masks NLS of NFκB proteins
44
C-Rel NFκB
Transcription activation domain which interacts with co-activators
- Without signal is retained in cytosol by IκB (inhibitor) masking the NLS of the NFκB proteins
45
Other components of NFκB Signaling Pathway
- IKK: ser/thr-K (IKKa, IKKB, IKKy/NEMO) phosphorylate IκB
- TAK1: ser/thr-K phosph. & activates IKKB using canonical route
- NIK: ser/thr-K phosph. & activates IKKa using non-canonical route
- Ubiquitin
- Ubiquitin ligases
46
Cannonical NFκB Activation
1) Inf. mediators like TNFa bind receptors gathering adaptors, scaffold-P, and Ubiq-ligase from Cytosol
2) Ubiq-ligase polyubiquitinates scaffold making signaling platform
3) Polyubiq. protein recruits IKK & TAK1
4) TAK1 posph. IKKB = active
5) IKKB posph. IκB releasing NFκB & IκB gets polyubiq.
6) IκB degraded & NFκB activates genes in nucleus
47
Non-Cannonical NFκB Activation
1) No stimulus, adaptors & ubiq-ligases polyubiq. NIK, so continuously degraded
2) Other inf. mediators bind receptors & polyubiq. itself so complex is degraded & NIK levels rise
3) Active NIK activates IKKa
4) p100 phosphorylated and then polyubiq. and undergoes limited proteolysis
5) Leftover p53 goes with Rel = NFκB2 goes to Nucleus
48
NFκB Negative feedback
Activation of NFκB transcription also activates IκB transcription to switch signaling off
49
NFκB Trans-repression
Steroids/GC
- When administered they activate steroid-R which steals same co-activators since regions are close on genes
- This causes less inflammation when taking steroids which messes with immunity
50
TGFβ
Transforming GF
- First messenger polypeptide (25kDa), water soluble
- Forms Dimer
- Inhibits cell prolif, Immunosuppression, induction of apoptosis
51
TGFβ cytokines major groups
- Bone Morphogenic Proteins
- TGFβ/Activin proteins (opposites)
(activin for FSH)
52
TGFβ Receptors
Serine/Threonine protein kinases & always act together
- TGFβ I (55kDa) GS domain where 5 residues can be phosph.
- TGFβ II (70kDa)
53
Target protein of TGFβ-R (& types)
SMAD proteins (TFs) have a DNA binding domain (MH1) & Dimerization domain (MH2)
- Receptor modulated SMAD (R-SMAD): contains phosphorylation site of ser/thr K (1,5,8 BMP/2,3 TGFB activin)
- Common SMAD: Smad4 dimerized with any R-SMAD
- Antag/Inhib SMAD: no DNA binding domain (6,7)
54
TGFβ mechanism SMAD
1) TGFβ ligand arrives causes dimerization of Type I/II
2) Autophosphorylation (II to I) on GS domain
3) SARA will help SMAD find receptor and phosph. R-SMAD
4) Phosphorylated R-SMAD meets co-SMAD4 in cytosol forming Dimer to nucleus
5) Activates gene expression of inhibitors of cell cycle, or induce apoptosis
55
TGFβ & Cancer mutations
- TGFβ-R1: head, neck, lymph, breast
- TGFβ-R2: GI
- Smad4: first in pancrease
(loss of any speeds up prolif: cancer)
56
RTK structure
Transmembrane Receptor
- EC ligand-binding domain
- Hydrophobic TM domain
- IC TK-domain
- C-terminal with tyrosine
57
What domains do proteins bind on RTK
- SH2 domain
- PTB domains
58
RTK Proliferation Mechanism
1) GF binds GFR
2) Homodimerization & autophosphorylation of PTB domains on IRS
3) SH2 domain phosphorylated, binds SHC protein
4) Grb2 bound & binds SOS by SH3 domain
5) SOS (GEF) activates Ras
6) Ras triggers Raf, Mek, Erk1/2 (MAPK pathway)
7) ELK & S6K activated = FOS / JUN / MYC activation and mTOR
59
What are RAF/MEK/ERK
- RAF: Ser/Thr Kinase
- MEK: Thr/Tyr Kinase
- ERK: Ser/Thr Kinase
60
RTK cell growth mech
1) PI3K binds tyr-residues of IRS-1 via SH2
2) phosph. PIP2 to PIP3
3) PDK-1 & PKB (PH domains)
4) PKB phosph. TSC1/2 leaving mTOR so mTOR is active
5) mTOR activates IFs like eIF4E & S6K activating eIF4B/2
6) PKB inhibits GSK3 (inh. glycogen synthase, SREBP1, eIF2B) so more macromolecule synth.
7) Inh. BAD/BIM/BAX/Caspases so no Apoptosis
61
RTK cell survival
PKB phosphorylates BAD protein holding death-inhibitory protein so cell lives
(BAD, BIM, BAX, Caspases)
62
Sh2 domain
Src homology 2 domain
= phosphorylated tyrosine residues
63
Sh3 domain
Src homology 3 domain
= proline rich domain
64
PH domain
Pleckstrin homology domain
= PIP2 / PIP3
65
Insulin receptor (& difference vs GF-R)
Tyrosine Kinase
- Dimer in resting state unlike GF where R is in monomer state while resting
66
Insulin pathway
Same exact as GF but uses IRS on PTB domain (insulin receptor substrate)
67
Effects of Insulin pathway (glucose)
GLUT4 trafficking
1) PKB phosph. GAP (AS160)
2) Rab-GTP forms
3) Induces formation of GLUT4 vesicles in ER
4) Traffic towards PM
68
AS160
GAP in GLUT4 trafficking
- Inhibited by PKB, so GLUT4 synth.
- AMPK from AMP levels inhibits AS160 GAP so more GLUT4 through Rab-GTP
- Also Ca-calmodulin inhibits it also leading to more GLUT4
69
GLUT 4 fusion to PM
1) Docking protein Cbl binds RTK
2) Adaptor protein Crk binds Cbl by Sh2 & C3G-GEF by Sh3
3) C3G-GEF activates small GTPase TC10 inducing fusion of GLUT4
70
Inactivation of Glycogen Synthase
Presence of Glucagon/Adrenaline
1) cAMP, PKA act
2) PKA phosph Glycogen synth so partially inactive
3) GSK3 fully inactivates it by phosph.
= less glycogen made
71
Activation of Glycogen Synthase
Presence of Insulin
1) PKB activation through RTK
2) PKB inactivates GSK3
3) Glycogen synthase relieved from inhibition
4) PP1G (phosphatase) also activated by insulin so further activation
72
How Gluconeogenesis is stopped
PKB phosphorylates FoxO1 TF removing it from gene in nucleus so less Gluconeogenesis enzymes
73
How Lipid storage is stimulated
PKB translocates SREBP1c TF to nucleus to transcribe lipid synthetic enzymes
74
Roles of PKB in metabolism
- AS160 (GLUT4)
- GSK3 (glycogen synth)
- FoxO1 (no gluconeogen)
- SREBP1c (lipogenesis)
- mTOR (protein synth)
75
Main Cytokine pathway name
JAK - STAT
76
Cytokine-R Mechanism
1) Ligand binds & activates R
2) JAK autophosphorylation on tyr residues
3) STAT recognize phosphotyrosines & bind (SH2)
4) JAK phosph. STAT
5) STAT transported for gene expression (importins / exportins)
76
JAK domains
- Kinase phosphorylates STAT
- Pseudokinase
- SH2 recognizes phosphotyrosine
- FERM binds non-covalently to IC of cytokine-R to anchor it
77
STAT domain functions/parts
- ND (for stat dimerization)
- CCD (coiled-coil)
- DBD (gene exp)
- LD (links DBD & SH2)
- SH2 (recognizes phosphotyrosine)
- TAD (activates transcription & recruits co-acti)
78
IFN induced JAK-STAT
Protection in viral infection
1) JAK-STAT activation
2) IRF9 binds dimer
3) Complex binds ISRE, makes:
- PKR: phosph. eIF2a blocks protein synth
- OASE: in presence of viral DNA makes oligoadenylate pol. activates RNaseL to degrade viral RNA
79
JAK-STAT Natural regulators
- Phosphotyrosine Phosphatases (PTP) inactivates main components
- Protein inh. of STAT (PIAS) like SUMO
- Supression of cytokine signaling: blocks promotor or sends to proteosome by ubiq
80
JAK-STAT Artificial regulators
- Recombinant Cytokines
- Cytokine antibodies & R
- JAK inhibitor
- Decoy oligonucleotide
81
JAK inhibitors
- Tofacitinib (1,3)
- Baricitinib (1,2)
(both have similar structure to adenine)
82
mTOR name
Mammalianm Target of Rapamycin
(rapamycin has immunosup. & anti-inf. properties)
83
mTOR description
- Ser/Thr Kinase
- PI3K family
- Serves in mTORC1 & mTORC2
- 289kDa + other subunits
84
mTOR complex 1 & subunits
Cell nutrient, Protein regulator of homeostasis, redox sensor, promoting cell growth & survival
- Raptor (ripamycin-sensitive) for complex assembly & localization
- Tit1/Tel2
- GBI (positive)
- PRAS40 (negative)
- DEPTOR (negative)
85
mTOR complex 2 & subunits
Cytoskeletal Regulator via Rho-family GTPases
- Rictor (non-ripamysin sensitive) for complex assembly & localization
- Tit1/Tel2
- GBI (positive)
- Protor (binds Rictor)
- DEPTOR (negative)
- mSIN1
86
Activation of mTORC1
- GTP-binding Rheb: In response to GF, activated by TSC1/2 phosph.
- GTP-binding Rag: In response to Amino Acids, Binds Raptor, Translocated to Lysosome to Rheb-GTP = activation
87
mTORC1 targets
- S6K1
- 4E-BP1
88
Main idea of AMPK
In high AMP:ATP ratio AMPK is activated promoting catabolism and inhibition of ATP consumption
89
AMPK Structure & Activation
- Ser/Thr Kinase
- Nucleus & Cytoplasm
- Heterotrimeric protein
- 1 Catalytic subunit
- 2 Regulatory subunits
90
To activate AMPK
Threonine172 needs to be phosphorylated on a-subunit & y-subunit needs to be allosterically activated by AMP
(inhibited if ATP is bound to y)
91
AMPK Activators/Inhibitors
- Activators: LKB1, CaMKKB, TAK1
- Inh: PP2A+PP2C, PKA, PKC
(Phosphorylation & Depohosphorylation of thr172)
92
AMPK/mTORC1 in Autophagy
When AMPK is active in absence of nutrients, mTORC1 is phosphorylated & inhibited, ULK1 activated and leads to Autophagy
93
Cellular Oxygen Sensor
a-KG Dependent Dioxygenases
- Very low affinity for O2
- Catalyze incorporation of O2 into substrates using Fe2+, VitC, a-KG
- Most common on proline residues making Hydroxyproline by Prolyl Hydroxylase
- 1 O2 added = Oxidation of a-KG to Succinate & CO2 formation
- When prolyl hydozylase is active it inhibits HIFa
94
HIF1a
Hypoxia Inducible Factor 1
- TF in response to Hypoxia
- Heterodimer of O2-sensitive a-subunit & constitutive B-subunit
95
HIF1a Normal O2/Normoxia
1) Prolyl Hydroxylase hydroxylates a-subunit on HIF1
2) Recognized by pVHL
3) pVHL ubiquitinates HIF1a causing its rapid degredation
96
HIF1a Low O2/Hypoxia
1) Less HIF a-subunit hydroxylated by prolyl hydroxylase
2) a-sub accumulates & forms heterodimers with B-subunits
3) a/B translocated to Nucleus binding DNA for expression
97
HIF1 Gene expression results
- Glycolytic Enzymes & GLUT1 for energy
- iNOS for NO vasodilation and increased blood flow
- Genes for hematopoiesis
