Week 9 Flashcards
Two ways cell death occurs
- Apoptosis
- Necrosis
Characteristics of apoptosis
- Actively regulated
- Cells shrink and condense
- Cytoskeleton collapses
- Nuclear envelope disassembles
Characteristics of necrosis
- Typically in response to insult or acute trauma
- Cells swell and burst, spilling their contents out, and elicit an inflammatory response.
T-cells and apoptosis
- The immune system relies on apoptosis
- T cell detects cell it believes is infected, so it forces the infected cell to commit apoptosis
How was C. elegans used to study apoptosis?
Adult has 959 somatic cells but that’s after some cells (131 to be exact) have died via apoptosis.
Can screen for mutants in which apoptosis fails to occur.
Ced-3 and ced-3
Ced-3 is a central mediator of apoptosis. ced-3 is a caspase (its mammalian homolog is called caspase 3)
How do the sites of caspases work?
Caspases have a cysteine at their active site. And their targets have aspartic acid at their cleavage sites.
Targets of caspase 3
Greater than 1000 targets, including nuclear lamins, cytoskeleton,
cell-cell adhesion,
DNA endonuclease inhibitors
Caspases
Enzymes with a cysteine residue in the catalytic core. The target of the enzyme has an aspartic acid. Goal of the caspase is to cleave the target protein.
CAD and iCAD
CAD is an endonuclease (cuts DNA).
iCAD is an inhibitor of CAD
Process of DNA fragmentation during apoptosis
- A DNA endonuclease will cleave DNA
- iCAD inhibits CAD
- iCAD gets cleaved by caspase-3
- The activated CAD can then cleave DNA
(Simply stopping this process would not stop apoptosis)
During DNA fragmentation where does cleavage of DNA occur?
Between nucleosomes
Purpose of TUNEL labeling
To visualize apoptotic cells during DNA fragmentation by staining them.
TUNEL
TdT-mediated dUTP nick end labeling
TdT
Terminal deoxynucleotidyl transferase
Role of TdT
Adds chains of labeled deoxynucleotide (dUTP) to the 3’-OH ends of DNA fragments (3’ end has hydroxyl group)
When using TUNEL labeling, why do apoptotic cells fluoresce more than normal cells?
The large number of DNA fragments in apoptotic cells results in their bright fluorescent labeling.
Cytochrome C
Cytochrome C is a soluble cytochrome that is part of the electron transport chain. Its normal job is to transfer electrons from QH2 (in complex III) to O2 (complex IV).
Release of cytochrome C during apoptosis
Cytochrome C is released out into the cytosol in UV-treated cells that are undergoing apoptosis
In the intrinsic pathway of apoptosis, what does cytochrome C bind to?
Apaf1. This then triggers dATP to come and bind to Apaf1.
In the intrinsic pathway of apoptosis, what happens after cytochrome C binds to Apaf1?
When cytochrome C binds to Apaf1, it undergoes a conformational change, exposing an oligomerization domain (CARD). This allows multiple domains of Apaf1 to come together using CARD domains to interact and form an oligomer.
In the intrinsic pathway of apoptosis, what is the role of the oligomer?
The oligomer serves as a docking platform for the initiator caspase.
What process does apoptotic stimulus induce?
Apoptotic stimulus induces pore formation in mitochondria in a process called mitochondrial outer membrane permeabilization (MOMP), which lets out cytochrome C
How is cytochrome C released from the mitochondria?
Large pores in the outer membrane of the mitochondria form, allowing cytochrome C to leak out. When the cell receives an apoptotic stimulus, proteins will come together and form these pores. This process is called MOMP.
What is the critical event that triggers apoptosis?
MOMP–this is a highly regulated process.
Three classes of proteins that control apoptosis
- Anti-apoptotic Bcl2 family proteins
- Pro-apoptotic Bcl2 family effectors
- Pro-apoptotic BH3-only proteins
Role and targets of anti-apoptotic Bcl2 family proteins
Anti-apoptotic proteins antagonize pro-apoptotic Bcl2 family proteins. Prevent MOMP.
Targets:
- BH4
- BH3
- BH2
- BH1
Role and targets of pro-apoptotic BH3-only proteins
Pro-apoptotic BH3-only proteins add an extra layer of regulation by antagonizing the anti-apoptotic proteins
Targets of pro-apoptotic Bcl2 family effectors
- BH3
- BH2
- BH1
Why is MOMP regulated with three layers?
Once MOMP is formed, apoptosis will proceed and cannot be reversed.
How does BH3 mimetic venetoclax work in cancer therapy?
Drugs that promote apoptosis can be used in cancer therapy. BH3 mimetic venetoclax binds to anti-apoptotic protein Bcl2 to tip the balance towards apoptosis.
Extrinsic form of apoptosis and example
- Another cell tells the cell to undergo apoptosis
- Lymphocyte detects infected cell and forces it to commit apoptosis
Intrinsic vs extrinsic forms of apoptosis
The extrinsic form uses the same executioner caspase (caspase 3) but has its own initiator caspase (caspase 8)
In the extrinsic form of apoptosis, what does the Fas ligand do?
The Fas ligand forms a homotrimer complex. This complex will bind to the Fas receptor, bringing three molecules into close proximity that can then recruit others.
What leads to the phagocytosis of dead cells?
Dying cells emit an “eat me” signal, which in combination with the loss of the “don’t eat me” signals leads to phagocytosis of dead cells
Role of executioner caspase
Executioner caspase inactivates PS and PE flippase while simultaneously activating scramblase.
Result of executioner caspase activity
- Results in high levels of PE and PS on the outer leaflet, which serves as an “eat me” signal.
- Executioner caspase also inactivates proteins on the membrane that signal “don’t eat me”
Four types of intercellular signaling
- Contact-dependent
- Paracrine
- Synaptic
- Endocrine
How are nuclear hormone receptors an example of intracellular receptors?
Besides containing a ligand-binding domain, nuclear hormone receptors also have a DNA binding domain that allows them to regulate transcription.
Three main classes of cell-surface receptors
- Ion channel coupled receptors (rapid signaling)
- G protein coupled receptors
- Enzyme coupled receptors (mostly kinases)
Characteristics of GPCRs
- Largest family of receptors (~800 in humans)
- 1/3 of all prescription drugs target GPCRs
- All contain 7 transmembranes
- All use G proteins
- Odorant receptors (smell)
and rhodopsins (vision) are GPCRS
How does the trimeric GTP-binding protein (G protein) relay signals from the GPCR?
- Activated alpha subunit usually transmits the signal (but beta/gamma complex can also signal)
How is the alpha subunit of a G protein activated?
Alpha subunit can only be activated if it is part of the G protein complex.
How is the beta/gamma complex of a G protein activated?
By disassociating from the alpha subunit.
Why is an activated GPCR a GEF?
GEFs help turn ON GTPases while GAPs help turn them OFF. The alpha subunit of the G protein is a GTPase.
Why are GPCRs so widely used for signaling?
The switch-like ability to control alpha subunit activity is why GPCRs are so widely used for signaling
Gs second messenger and cellular response
cAMP, stimulatory
Gi second messenger and cellular response
cAMP, inhibitory
Gq second messenger and cellular response
IP3 and diacylglycerol, not sure if q stands for anything. Phospholipid based second messengers. Very important because signaling ultimately leads to increased calcium levels, which have a wide variety of effects.
Golf second messenger and cellular response
cAMP, olfactory
Gt second messenger and cellular response
cGMP, transducin
What does an activated alpha subunit of Gs activate?
An activated alpha subunit of Gs activates adenylyl cyclase to promote production of cAMP from ATP.
What does Gi inhibit?
Adenylyl cyclase
What does Golf activate?
Golf activates adenylyl cyclase in olfactory receptors neurons
Olfactory pathway
- Odorant molecule (ligand)
- Odorant receptor (GPCR)
- Golf
- cAMP
- cAMP-gated cation channels
- Na2+ influx
- Neuronal firing
Basis of olfaction
- Each olfactory receptor neuron expresses only one type of odorant receptor.
- Each smell activates a unique combination of olfactory receptor neurons.
What does Gt activate?
cGMP phosphodiesterase, though this is an inhibitory pathway.
What triggers rod cells?
Rod cells fire in the dark and are inhibited by light
Rod cell activation pathway
- Photon (ligand)
- Rhodopsin (GPCR)
- Gt
- cGMP phosphodiesterase
- cGMP
- cGMP-gated cation channels open
- Cation influx
- Rod cell depolarizes
- Cell fires
What happens in the rod activation pathway when cGMP levels decrease?
When cGMP levels decrease, cation channels close, cation influx stops, cells become hyperpolarized, and cell firing is inhibited.
What does the Gq mediated activation of phospholipase C-beta produce?
IP3 and diacylglycerol (both are lipid-derived). Alpha subunit and the beta/gamma complex are both involved in PLC-beta activation.
Retinotopic map
Systematic arrangement of visual information in the brain that corresponds to the spatial layout of the retina
RTKs
- Receptor tyrosine kinases (RTKs) are a major class of
enzyme-coupled receptors and includes the insulin receptor - Many RTKs promote growth, proliferation, and differentiation and hence are implicated in cancer (in contrast, GPCRs mostly regulate physiological responses)
Why do RTKs dimerize?
Binding of ligand to GPCRs results in conformational change. But single transmembrane domain in RTKs unlikely to transmit conformational change across the membrane. They therefore dimerize.
Main function of extracellular side of RTKs
Promotion of protein-protein interactions.
Main function of cytosolic side of RTKs
Cytosolic domain has kinase domain that phosphorylates tyrosines on its target
How many transmembrane domains do RTKs have?
A single domain.
Trans-autophosphorylation of RTKs vs cis-phosphorylation
Trans-auto-phosphorylation is used to phosphorylate each other’s domains and activate the kinase domains vs cis which means they would phosphorylate themselves
After phosphorylation, what is the role of the activated tyrosines?
- The phosphorylated tyrosines serve as docking sites to recruit scaffolding molecules
- Typically need SH2 domain to bind and recognize phosphorylated domain
- Also have SH3 domain to recruit further downstream effectors
Which proteins do RTKs mostly signal by?
RTKs mostly signal via Ras and Rho GTPases
What do Rho GTPases regulate?
Cell shape, polarity, motility, and adhesion
How do RTKs activate their respective GTPases?
By recruiting GEFs. SH3 domains are used to recruit a GEF.
How are GEFs activated in the RTK pathway?
GEF has a Ras-GTPase swap out the GDP for a GTP to become activated
How do RTK-activated GTPases like Ras propagate their signal?
Through a MAP kinase cascade. Uses three steps in a highly conserved cascade with three kinases. Any one of the steps can be hyperactivated even in the absence of an upstream ligand, leading to cancer due to the constant signaling
EphA
A receptor tyrosine kinase that signals through a Rho GTPase
Role of ligand Ephrin A
Ligand Ephrin A repels neurons expressing EphA
Notch/delta signaling in flies
Notch/delta signaling is used widely to determine cell fate. First well characterized in flies. Gives rise to mechanosensory bristles. The cell that ends up expressing Delta becomes the neuron.
Delta/notch competition via lateral inhibition
- Notch receptor activation inhibits neural fate.
- Cells with notch receptor activation also inhibit delta function in cis.
- Notch signaling is required for cell fate choice in the gut of mammals (and many other instances where competition dictates cell fate)
