Midterm No. 3, Opus 4 Flashcards
Is the Notch signal pathway good for signal amplification?
No. It’s a high speed train to the nucleus, no room for amplification
What protein connects Notch signaling to Alzheimer’s?
Gamma-secretase
What are the two pathologies to look for when diagnosing Alzheimer’s?
Extracellular amyloid plaques and intracellular (in neurons) neurofibrillary tangles
How is Amyloid Precursor Protein (APP) normally recycled?
It’s a transmembrane protein (single TMD) in the plasma membrane. It’s normally clipped by alpha-secretase and gamma-secretase when its recycled
How are amyloid plaques formed?
APP is cut by beta-secretase and gamma-secretase. This causes a bit of APP’s TMD to be left in the fragment. That hydrophobic section causes aggregation, leading to amyloid plaque formation
(General) how do hydrophilic ligands activate intracellular signaling?
Via a cell surface receptor
(General) how do hydrophobic ligands activate intracellular signaling?
Via simple diffusion straight into the cell, then binding to an intracellular receptor
How do ligands get to nuclear receptors?
Hydrophobic ligands do simple diffusion across the plasma and nuclear membranes
What do nuclear receptors + their bound ligands act as?
Transcriptional regulators
How do signals for nuclear receptors travel through the bloodstream?
They are hydrophobic, so they have to be bound by carrier proteins
What is the role of heat shock proteins in NHRs (nuclear hormone receptors)?
They bind to the NHRs’ hydrophobic binding domains, prevent those hydrophobic residues from being exposed (which could be dangerous)
List the 3 domains present in NHRs
- Ligand binding domain
- DNA binding domain
- Transcription activating domain
What are NHRs?
Ligand-triggered transcription factors
Do NHRs directly affect gene expression?
Yes
Once active (ligand is bound), what can NHRs bind to?
Coactivator proteins
Examples include chromatin remodeling complexes, HDACs, mediator binding sites, etc
What’s the purpose of NHRs’ coactivator proteins?
To directly turn transcription on/off by directly interacting with the DNA
Why were Nobel’s dynamite factories good work environments for people with heart disease?
Because they were exposed to nitroglycerin, a vasodilators (esp for coronary arteries)
What is acetylcholine?
A water-soluble signal molecule, key neurotransmitter
What type of receptor does acetylcholine bind to in heart muscles, and what is its effect?
GPCR, decreases rate and force of contraction
What type of receptor does acetylcholine bind to in salivary glands, and what is its effect?
GPCR, increases cytoplasmic Ca2+ secretion
What type of receptor does acetylcholine bind to in skeletal muscles, and what is its effect?
Ligand-gated ion channels, stimulates contraction
What type of receptor does acetylcholine bind to in (most) smooth muscles, and what is its effect?
GPCR, stimulates muscle contraction
Why does acetylcholine cause vasodilation in smooth arterial muscles?
Acetylcholine binds to a GPCR in the endothelial cells
GPCR → PLC → Ca2+/Calmodulin → NO synthase
NO synthase catalyzes:
Arginine + O2(g) → Citrulline + NO(g)
NO(g) does simple diffusion across the epithelial plasma membrane and smooth arterial muscle plasma membrane, and binds to an intracellular receptor in the smooth muscle cell
The NO receptor is a guanylyl cyclase. It catalyzes GTP → cGMP + Pi. (same family as adenylyl cyclase)
cGMP activates PKG (protein kinase G), which uses its kinase activity to relax the muscle cell (specifically it activates myosin phosphatase)
What are the downstream intracellular effects of acetylcholine binding to a GPCR in endothelial cells?
GPCR → PLC → Ca2+/Calmodulin → NO synthase
What does NO synthase catalyze?
Arginine + O2(g) → Citrulline + NO(g)
When cGMP levels are high, will smooth arterial muscle cells be relaxed or contracted?
Relaxed
cGMP activates PKG, which activates myosin phosphatase to relax the cell
When cGMP levels are low, will smooth arterial muscle cells be relaxed or contracted?
Contracted
No cGMP means PKG is inactive, myosin phosphatase can’t be activated, muscle cell can’t be relaxed
How does the acetylcholine-NO-vasodilation pathway turn off?
PDE1 (a phosphodiesterase) converts cGMP to GMP; this halts the path by removing the signal (cGMP), PKG cannot be activated without cGMP
How long is NO’s half-life?
Very short, only 5-10 seconds
How long is cGMP’s half-life
Very short due to PDE1
What is IC50?
Half of the maximal inhibitory concentration
You want a more potent/effective drug. Should IC50 be low or high?
Low
You want a less potent/effective drug. Should IC50 be low or high?
High
Your drug has a low IC50. How potent is it?
Very potent, very effective
Your drug has a high IC50. How potent is it?
Not very potent, low efffectiveness
Why is it important to run multiple IC50’s to determine your drug’s specificity?
To know if it works on the PDE you want
To know if it works against other PDE’s, may be helpful in determining potential side effects
Why is one of the symptoms of severe sepsis a massive crash in blood pressure?
Immune cells produce NO as part of their defense during sepsis. If the sepsis is too severe, so much NO will be produced that all arteries/veins/vessels will dilate, causing a mass crash in blood pressure
In most cells, where does actin gather?
Around the edges
In most cells, where are the microtubules?
The middle/majority of the cytoplasm
In most cells, where are the intermediate filaments?
They stabilize the nucleus
What is the cytoskeleton? (definition)
A system of filaments that enables cells to interact mechanically with each other and the environment
What are the functions of the cytoskeleton?
Maintain correct cell shape
Physical robustness (not fragile)
Creates internal structure
Involved in motility/movement
Does the cytoskeleton function more like an ant trail or an interstate highway? Explain
Ant trail
Highways exist in the same place once they’re built. Ant trails mostly maintain the same shape, but the individual ants are constantly moving.
Many of these filaments are constantly and dynamically remodeling, even though they seem to be fairly stable.
Size of actin filaments
7-9 nm in diameter
How abundant are actin filaments?
Very
1-10% of total cell proteins
Size of intermediate filaments
10 nm in diameter
Size of microtubules
25 nm in diameter
What NTP is associated with actin?
ATP
What NTP is associated with intermediate filaments?
None!
What NTP is associated with microtubules?
GTP
Which of the 3 filament types (actin, IF, microtubules) are polar?
Actin and microtubules only
IFs are not polar
Implications for IFs not being polarized?
Can’t be associated with motors and motor proteins
How are the polymers of protein subunits that make up actin, IFs, and microtubules held together?
Weak non-covalent bonds
Bacterial homolog to actin
MreB
G-actin
Globular actin, individual monomers
F-actin
Filamentous actin
True or false: actin is an ATPase
True!
G-actin monomers have a cleft in their centers where ATP binds! Hydrolysis happens!
Which process is faster: formation of the G-actin triad or the rest of the polymerization?
The rest of the polymerization
Which process is slower: formation of the G-actin triad or the rest of the polymerization?
Formation of the G-actin triad
Biochem in vitro outcome: low actin concentration
Whole population consists of monomers
Biochem in vitro outcome: increasing actin concentration
There’s a point where filaments form. Monomer levels stay constant, additional actin is polymerized into filaments
Actin treadmilling
Actin is an ATPase. It hydrolyzes its actin-ATP while in filament form, then delplymerizes in it actin-ADP form. Actin can be lost and gained from both ends, but the critical concentrations are different enough that the + end predominantly gains / polymerizes and the - end predominantly loses / depolymerizes
Treadmilling adds to the ant trail concept. A filament could stay the same size, but it’s not always made of the same subunits. The ratioin of monomers to polymers can remain stable even though the filaments are technically new
Based on biochem’s concentration numbers:
Low G-actin concentration
Gain at no end
Loss at both ends (though mostly - end)
Based on biochem’s concentration numbers:
Medium G-actin concentration
Gain at + end
Loss at - end
Based on biochem’s concentration numbers:
High G-actin concentration
Gain at both ends (though mostly +)
Loss at no ends
Low G-actin concentration
Below critical concentration of both ends, both lose
High G-actin concentration
Above critical concentration of both ends, both gain
But! The actual concentration of actin in the cell is WAY over the critical concentration! From the numbers the biochemists generated from in vitro assays, we should expect almost all the cell’s actin to be polymerized, but it’s not! Why?
Profilin, cofilin, and thymosin-beta4 are actin-binding proteins. They maintain a pool of G-actin and keep things dynamic. They’re breaks on the system to make sure its not all polymerized
Profilin
Adenosine exchange factor (AEF)
Adds monomeric actin-ATP to the + end
Cofilin
Breaks off chunks of filament at the - end
Thymosin-beta4
Sequesters monomeric ATP-actin
Stockpiles then hands off to profilin at appropriate times
Formin FH2 domain
Acts as a molecular “rocking ratchet”
Promotes long, linear filaments
Acts in a dimer, binds to a actin dimers
Sits on the + end, stacks actin dimers appropriately
Formin RBD (rho binding domain)
Small GTPase lipid-linked to the plasma membrane
Ensures polymerization happens near the plasma membrane, lococalizes polymerization
GTPase activity regulates the entire formin protein, regulates polymerization
Blocks unregulated function
FH2’s ratcheting+polymerization can occur only when Rho is bound to GTP (aka when Rho is active)
Formin FH1 domain
Recruits and binds to profilin-bound actin-ATP for FH2
Hands off the actin-ATP to be polymerized by FH2
Arp2/3 Complex
When the Arp2/3 complex is activated (by an activator protein, causes a conformational shape change), Arp2/3 is accessible for binding
The Arp2/3 complex becomes the - end for the nucleated actin filament. Actin monomers are added to the + end
The Arp2/3 complex is basically a regulated actin dimer. Its activator protein regulates filament nucleation/organization. Addition of 1 actin monomer (to activate the complex) yields a nucleating timer
Arp2/3 complexes can also bind to the sides of pre-existing actin filaments (70 degree angle between filaments). This allows for networks of filaments fo form, haskmark networks, branching
What are Arp2 and Arp3?
Homologs to actin, structural/molecular cousins
Actin related proteins
What does filament nucleation via an Arp2/3 complex demonstrate?
That nucleation of 3 actin monomers is key to filament polymerization
Why should you never eat death cap mushrooms?
Because they contain phalloidin, a protein that binds to actin filaments and prevents them from depolymerizing. (it’s convenient for cell biologists thought because it’ll label and fix actin filaments when combined with rhodamine)
