Unit 3 Flashcards
Different proteins involved in the canonical and non-canonical NF-kappaB signaling pathways
-Williams
Canonical
NF-kappaB homo/hetero dimers
- RelA
- p105 (degrades to –>) p50
IkappaB proteins: anchor NF-kappaB in the cytoplasm
- IkappaBalpha
IKK proteins
- NEMO
- not a kinase
- component of signalsome
- IKK2
- kinase
- phosphorylates IkappaBalpha to mark for degradation
Non-Canonical
NF-kappaB homo/hetero dimers
- RelB
- p100 (degraded to –>) p52
NO IkappaB proteins
IKK proteins
- IKK1
- kinase
- phosphorylates p100 to mark for partial degradation into p52
Canonical NF-KB signaling pathway
-Williams
- Signal activates signalsome (NEMO, IKK2, IKK1)
- IKK2 phosphorylates IKBa on two serines
- Phosphorylation stimulates IKBa ubiquitylation
- Ubiquitylated IKBa is degraded by protesome and NF-KB (p50, RelA) is free! NLS on RelA is exposed
- RelA is phosphorylated by PKA & CKII
- NF-KB is imported into the nucleus, where it initiates transcription of target genes including IKBa.
To get out of the nucleus:
- IKK1 and MSK1 phosphorylation recruits p300/CBP,PCAF, which acdetylate RelA
- Acetylation reduces transcription activity
- Deacetylation by HDAC3 releases NF-KB from the DNA
- IKBa binds NF-KB and exports it out of the nucleus
Non-canonical NF-KB signaling pathway
-Williams
- Signaling activates NIK, which induces IKK1
- IKK1 phosphorylates p100
- c-terminal of p100 is ubiquitylated & degraded by protesome. Partial degradation leaves p52
- NF-KB (p52 + RelB) imported into the nuclease, where it activtes transcription of target genes
You are investigating the effects of two different drugs (drug A and drug B) on cancer cells that have high activity of both the canonical and non-canonical NF-kB signaling pathways.
You discover drug A inhibits the phosphorylation of IkBa in the cytoplasm of the cancer cells.
a) Do you expect drug A to increase, decrease, or not change the degradation of the IkBa protein compare to untreated cells?
b) Do you epect drug A to increase, decrease, or not change the transcription of the IkBa gene compared to untreated cells?
c) Draw a diagram depicting the changes in protein interaction and PTMs that occur when IkBa is phosphorylated in the NF-kB signaling pathway. Briefly explain why you think drug A might (or might not) affect the degradation of the IkBa protein and the transcription of the IkBa gene.
- Williams
a) decrease
b) decrease
c) see image
Without IkBa phosphorylation, it will not be marked for ubiquitylation and therefore NOT degraded by the protesome. NF-kB (RelA + p50) will not be released and therefore cannot be imported into the nucleus. Ultimately, initiation of target gene transcription will NOT occur INCLUDING transcription of IkBa.
You are investigating the effects of two different drugs (drug A and drug B) on cancer cells that have high activity of both the canonical and non-canonical NF-kB signaling pathways.
You discover the drug B blocks the phosphorylation of p100 in the cytoplasm of the cancer cells.
a) Do you expect drug B to increase, decrease, or not change the amount of p52 in the cells, compared to untreated cells?
b) Do you expect drug B to increae, decrease, or not change the amount of RelB localized in the nucleus of the cells, compared to untreated cells?
c) Draw a diagram depicting the changes in protein interaction and PTMs that occur when p100 is phosphorylated in the NF-kB signaling pathway. Using this diagram, briefly explain why you think drug B might (or might not) affect the amount of p52 in cells and the nuclear localization of RelB in the cells.
- Williams
a) decrease
b) decrease
c) see image
If p100 is not phosphorylated, it is not marked for partial degradation into p52 (p52 is not produced). Additionally, without partial degradation of p100 –> p52, NF-kB (RelB + p52) will not be imported into the nuclease (decreased RelB nuclear localization).
Epithelial-Mesenchymal Transition
-Williams
Epithelial tissue:
- complex, stable junctions
- continuous tissue layer
- apical vs basal cell polarity
- no cell mobility
Mesenchymal tissue:
- loose or no junctions
- no uniform tissue structure
- leading vs trailing edge polarity
- motile
Proteins that increase:
- Vimentin
- Fibronectin
- Snail
- Slug
- Twist
Protiens that decrease:
- E-cadherin
Proteins that accumulate in the nucleus:
- ß-catenin - via Wnt signaling
- Smad 2/3 - via TGF-ß signaling
- Snail - via TGF-ß signaling
- Slug - via TGF-ß signaling
- Twist - via TGF-ß signaling
TGF-ß signaling pathway
-Williams
ß-catenin & Wnt Signaling
-Williams
During your doctoral research, you discover that a certain strain of mice (called the “No Smad4” mouse strain) has a mutation that inhibits the expression of Smad4. This mutant mouse strain lives to adulthood, but has some odd characteristics because it does not express Smad4. You obtain your Ph.D. degree for your pioneering work on the “No Smad4” mouse strain.
You isolate epithelial cells from a normal mouse, and from the “No Smad4” mouse, and compare the responses of the cells to treatment with TGF-ß. You find that treatment with TGF-ß causes Smad2 to localize in different regions of the cells. Compare and contrat the movement of Smad2 through the different regions of normal mouse cell vs the “no Smad4” mouse cell when the cells are treated with TGF-ß.
-Williams
Normal & No Smad4 cells:
- TGF-ß treatment causes type II TGF-ß reeptor to recruit and phosphorylate the type I TGF-ß receptor
- Activated type I TGF-ß receptor recruits Smad2 to membrane
- SARA facilitates recruitment of Smad2 to the activated type I recptor
- Activated type I receptor phosphorylates Smad2 at the SSXS motif
Normal cells:
- Phosphorylated Smad2 leaves the receptor and dimerizes with Smad4
- The Smad2/Smad4 dimer enters the nucleus
“No Smad4” cells
- Phosphorylated Smad2 leaves the receptor but remains in the cytoplasm because it cannot dimerize with Smad4
During your doctoral research, you discover that a certain strain of mice (called the “No Smad4” mouse strain) has a mutation that inhibits the expression of Smad4. This mutant mouse strain lives to adulthood, but has some odd characteristics because it does not express Smad4. You obtain your Ph.D. degree for your pioneering work on the “No Smad4” mouse strain.
You isolate epithelial cells from a normal mouse, and from the “No Smad4” mouse and treat the cells with TGF-ß and examine expression of E-cadherin and vimentin by the cells.
A) Treating the normal cells with TGF-ß will INCREASE, DECREASE or NOT ALTER E-cadherin expression by the cells, compared to untreated normal cells.
B) Treating the “No Smad4” cells with TGF-ß will INCREASE, DECREASE or NOT ALTER E-cadherin expression by the cells, compared to untreated “no Smad4” cells.
C) Treating the normal cells with TGF-ß will INCREASE, DECREASE or NOT ALTER vimentin expression by the cells, compared to untreated normal cells.
D) Treating the “No Smad4” cells with TGF-ß will INCREASE, DECREASE or NOT ALTER vimentin expression by the cells, compared to untreated “no Smad4” cells.
Explain why you expect the changes in E-cadherin and vimentin expression that you predicted.
-Williams
A) decrease
B) not alter
C) increase
D) not alter
In normal Cells:
- Smad2/Smad4 hterotimer binds DNA in the nuclease and binds the Smad-binding element (SBE)
- Initates transcription of HMGA2, which promotes expression of Snail, Slug, and Twist
- Smad2/Smad4 dimer also induces expression of vimentin
In “no Smad4” cells
- Smad2 reamins in the cytoplasm and therefore cannot regulate the HMGA2-Snail/Slug/Twist-E-cadherin pathway or vimentin expression
Draw the scheme for the one-electron reduction of molecular oxygen. Give both the chemical formula and names of the intermediates.
-Hogg
O2-- → H2O2
- SOD
H2O2 → H2O
- CAT
H2O2 → ultimately glucose
- glutathione peroxidase
Describe how hydrogen peroxide can be sensed by a cell to elicit a transcriptional response.
-Hogg
****
OxyR Signaling
-Hogg
Prokaryotic example
SoxRS
-Hogg
Example of iron sulfur cluster oxidation
YAP1/Orp1
-Hogg
Yeast
Keap1/Nrf2
-Hogg
Mammals
ASK1/Trx system
-Hogg
Discuss protective mechanisms that can remove Reactive Oxygen Species.
-Hogg
1) Antioxidants
Vitamin E. Major component is alpha-tocopherol, which can donate an OH group to radicals to form a more stable radicle.
Vitamin C reduces vitamin E radical to form a vitamin C radical, which is outside the membrane and very stable.
2) Enzymes
Superoxide dismutase (SOD) oxidizes superoxide to molecular oxygen. Reduced SOD reduces superoxide to hydrogen peroxide - there are many enzymes that deal with hydrogen peroxide. Ex: Catalase (CAT) reduces 2 hydrogen peroxides into water.
Glutathione reduces hydrogen peroxide into water and initiates signaling pathways (see image)
Peroxyredoxins use thioredoxin system to reduce disulfides by reacting with hydrogen peroxide (see image)
Discuss the concept of ‘vicinal di-thiols’ and ‘resolving cysteine residues’ in the context of redox signaling mechanisms.
-Hogg
Cysteine (SH) oxidized (SOH) by target ROS. Neighboring cysteine ‘resolves’ by forming a disulfide bond (S-S).
Di-sulfide bond directly or indirectly regulates gene expression as in the OxyR, YAP1/Orp1, and ASK1/Trx systems.
Name the three isoforms of nitric oxide synthase and one feature unique to each isoform.
-Hogg
eNOS: has three acetylation sites at its N-terminal for membrane anchoring
nNos: has a N-terminal PDZ domain for membrane anchoring
iNos: does not have an inhibitory loop, reacts with Ca2+ at basal levels
Describe three nitric oxide-dependent modifications of biomolecules that may be associated with its pleotropic signaling mechanisms.
-Hogg
1) NO binds heme of sGC to induce cGMP formation. cGMP interaction with PKG causes dilation of smooth muscle.
2) NO binds heme of complex IV in the ETC, thereby competing with O2 and inhibiting mitochondrial respiration.
3) NO can bind lipid radicals (antioxidant activity) and produce signaling molecules. Ex: LOO• + NO → LOONO
Discuss mechanisms of canonical (cyclic GMP) and non-canonical nitric oxide signaling.
-Hogg
Canonical:
- Endothelial agonist (ex: acetylcholine) increases calcium levels to activate eNOS.
- eNOS produces NO, which diffuses and binds sGC heme group in muscle tissue.
- sGC makes cGMP
- cGMP interacts with PKG to cause dilation of smooth muscle
Non-canonical:
- NO reacts with lipoxygenase to produce liponitride
- S-nitrosation: NO somehow reacts with thiols (mechanism unknown). Post-translational thiol modification changes protein activity.
Describe cellular events that are controlled by the binding of NO to heme groups.
-Hogg
Canonical:
- Endothelial agonist (ex: acetylcholine) increases calcium levels to activate eNOS.
- eNOS produces NO, which diffuses and binds sGC heme group in muscle tissue.
- sGC makes cGMP
- cGMP interacts with PKG to cause dilation of smooth muscle
NO can also bind heme group of complex IV in ETC to inhibit mitochondrial respiration. Occurs when oxygen levels are low (NO better competitor).
Discuss how the formation of NO is controlled at the enzyme level, including a discussion of the different isoforms of NO.
-Hogg
NO is formed by nitric oxide synthase (NOS), of which there are three isoforms: eNOS, nNOS, and iNOS.
All three isoforms convert arginine to nitric oxide and citrulline using oxygen and NADPH.
Two domains:
- Reductase domain: location of NADPH binding. Reaction with flavins causes flow of electrons to:
- Oxygenase domain: electrons bind and activate oxygen
eNOS and nNOS are regulated by an inhibitory loop. Calcium relieves eNOS and nNOS from inhibition. Basal levels of calcium are sufficient for iNOS activation.
Three enzymes mediating ubiquitination
-Joe
E1: uniquitin-activating enzyme
E2: ubiquitin-transferring enzyme
E3: ubiquitin ligase
Mechanism of ubiquitination
-Joe
- E1 activates ubiquitin using ATP
- Activated ubiquitin is transferred to E2 via a theioester bond
- E3 promotes transfer of ubiquitin from E2 to lysine of substrates
There are deubiquitylates (DUB) that remove ubiquitin from substrates.
- As substrate is fed through the proteasome, ubiquitin is released and recycled
Sites of ubiquitin modification
-Joe
There are many different sites on ubiquitin that when modified, mark for different activities. Biggies are kinase modification (lys33), lysosomal degradation (lys29) and protesomal degradation*** (lys48).
Depending on what site is modified on ubiquitin, get different effects.
Role of Ubiquitin linkages in protein quality control
-Joe
Soluble misfolded proteins are tagged with Lys48-linked polyubiquitin, which marks them for proteasomal degradation.
Proteins escape proteasomal degradation, tend to form aggregates. Aggregates are ubiquitinated. Aggregates are collected for lysosomal degradation.
Monoubiquitination & trafficking
-Joe
Monoubiquitination can modulate activity, trafficking, and interaction of substrates in non-degradative processes.
Polyubiquitin chains
-Joe
Lys48 polyubiquitin is most abundant linkage.
Marks for proteasomal degradation
Regulates virtually all biological processes including cell division, transcription, signaling, protein quality control, immunity, etc.
Distinctions between apoptosis and necrosis
-Misra
Necrosis
- mitochondrial swelling
- membrane degradation
- cell structure degradation
- release of contents
- cells seem to EXPLODE
Apoptosis
- fragmentation of cells into membrane encased bodies
- cleared by phagocytosis
Cellular stages of apoptosis
-Misra
Assays and markers for apoptosis
-Misra
DNA Laddering
- Run total DNA on agarose gels
- DNA from apoptotic cells run as distinct ladder pattern, NOT a smear
- Why? Becase activated executioner caspases cleave iCAD, freeing CAD to cut chromosomal DNA between nucleosomes, resulting in the production of DNA fragments that form ladder pattern
Tunel assay:
- cut ends of fragmented DNA labeled with dUDP, which is fluorescently tagged.
- more DNA damge = more ends = increased fluorescence in apoptotic cells
Caspases as effectors of apoptotic cell death
-Misra
Caspases: family of proteases
Inactive procaspase activated into active caspase by cleavage.
Initiator caspases with low level protease activity start the reaction. Leads to explosive activation of caspases.
Cleaves target proteins including nuclear lamins, endonucleases, cytoskeletal proteins, and cell adhesion proteins.
Extrinisic and intrinsic apoptosis pathways
-Misra
Extrinsic
- Fas ligand binds homotrimer fas death receptor and recruits FADD (Fas associated death domain) protein
- FADD recruits initiator caspase to form DISC (death-inducing signaling complex)
- Activated initiator caspases activate executioner caspases
Intrinsic
- Cytochrome C is released from mitochondria intermembrane space
- Cyto C binds and activates Apaf1 via hydrolysis of dATP–>dADP
- Apaf1 and cyto C complex aggregation to form apoptosome
- Procaspase-9 recruited to Apaf1 CARD domain and activated
- Activated caspase-9 activates executioner caspases
- Caspase cascade leading to apoptosis
Role of BCL-2 family in the intrinsic pathway
-Misra
Bcl-2 family proteins regulate apoptosis through mitochondrial function. Can be anti-apoptotic (Bcl2, BclXL) or pro-apoptotic (Bax, Bak).
In absence of apoptotic signals:
- anti-apoptotic Bcl2 proteins bind and inhibit BH123 aggregation
In presence of apoptotic signals:
- Pro-apoptotic Bcl2 proteins activated and aggregate in the outermembrane to promote release of cytoC
There are many Bcl-2 like proteins because various physiological stresses operate through different Bcl-2 proteins.
Release of cytochrome C and assembly of apoptosome
-Misra
When cytochrome C is released from the mitochondria, aggregates with Apaf-1 subunits to form apoptosome. Procaspase 9 recruited and activated.
Trophic factors and apoptosis
-Misra
Trophic factros promote survival
- If there are more cells (ex: nerve cells) produced than can be supported by survival factors released by target cells, apoptosis is used to adjust number of nerve cells.
- In absence of trophic factors:
- BAD binds Bcl-XL and Bcl-2, thereby preventing anti-apoptic proteins from interacting with Bax leading to release of cyto C which binds to Apaf1.
- In presence of trophic factors:
- PI-3 kinase stimulated leading to activation of Akt, which phosphorylates BAD. Phospho-BAD complexes with 14-3-3 and is sequestered in the cytosol, allowing antiapoptotic Bcls to inhibit BAX activity
Distinctions between intracellular, extracellular, and intramembrane cleavage during signaling
-Misra
Intracellular cleavage: NF-kB
Extracellular cleavage: Hedgehog, Notch/Delta, EGF
Intramembrane cleavage: Notch/Delta, APP, SRE-BP
Mechanism of NF-kB signaling-role of proteolysis
-Misra
Mechanism of notch/delta signaling and role of rip
-Misra
Notch synthesized as precursor protein that is cleaved into fragments that stay non-covalently associated and expressed on the surface
Binding of delta leads to cleavage on cell surface by ADAM-10
gamma-secretase complex binds stump and catalyzes intramembrane cleavage to release cytosolic segment
Mechanism of amyloid precursor protein (APP) processing
-Misra
Ways survival factors inhibit apoptosis
-Misra
A) Stimulation of transcription of genes that encode antiapoptotic Bcl2 family proteins such as Bcl2 itself or BclXL
B) Activation of serine/threonine protein kinase Akt, which phosphorylates and inactivates the pro-apototic protein BAD. When not phosphorylated, BAD promotes apoptosis by binding and inhibiting Bcl2. Once phosphorylated, BAD dissociates, freeing Bcl2 to suppress apoptosis. Akt also suppresses apoptosis by phosphorylating and inactivating transcription or regulatory proteins that stimulate txn of genes promoting apoptosis
C) Stimulation of anti-IAP protein Hid phosphorylation. When not phosphorylated, Hid promotes cell death by inhibiting IAPs. Once phosphorylated, Hid no longer inhibits IAPs, which become active and block apoptosis.
True or False
The extrinsic and intrinsic apoptosis pathways can share pathway members
Apaf-1 mediates cytochrome c release
The Bcl-2 protein is an anti-apoptotic factor
Presenelin is part of the gamma secreate complex
The extrinsic and intrinisic pathways both rely on cyt C release from mito
Ubiquitin mediated proteosomal degradation is important for the Notch pathway
Integrin mediated signaling activates a pro-apoptotic signaling cascase
The DISC assembles upon FAS receptor dimerization mediated by the FAS ligand.
True
False
True
True
False
False
False
False
The drug semagaestat, a gamma secretase inhibitor, was developed to treat mild-to moderate Alzheimer’s Disease patients. Trials of the drug were interrupted bc of the observation of detrimental effects on cognition and functionality in patients receiving the drug compared to those receiving the placebo. In phae II trials, AB40/42, responsible for amyloid plaques, concentration in the blood plasma about three hours after applifcation of semagacestat, but an increase of 300% 15 hours after alication.
- Interfereing with notch/delta signaling
- gamma secretase complex cannot bind to stump and so presenelin proease cannot catalyze intramembrane cleavage to release cytosolic segment of Notch.
- Leads to increase of AB40/42
