Signal transduction Flashcards
Explain the steps of the signal transduction pathway.
- Synthesis
- Release of signaling molecule
- Transport
- Binding of ligand to receptor (agonist vs antagonist)
- Activation of intracellular signal transduction cascade(s)
- Specific changes in cellular function
- Termination of response, e.g. by removal of ligand
What are the types of extracellular signaling?
- Endocrine signaling
- Paracrine signaling
- Autocrine signaling
- Contact-dependent signaling
Insulin is secreted in pancreas but travel through the blood. What type of signaling is this?
Endocrine signaling, which associated with long-distance signaling.
Neurons release neurotransmitters that can act on an adjacent neuron.
Paracrine signaling because they are associated with close proximity signaling.
How does autocrine signaling allow cells to behave as a group?
In a group of identical signaling cells, each cell receives a strong autocrine signal. An example of this are cancer cells.
What is contact-dependent signaling?
This happens when signaling molecules are membrane bound.
What are the two types of cellular responses?
- Rapid short-term (physiological) => Changes in activity in preexisting enzymes and proteins
- Slower long-term (developmental) => Changes in gene expression (e.g. pregnancy)
Name a laboratory technique to isolate ligands.
- Ion-exchange chromatography
Separation based on electrostatic interaction between charged ligands and charged stationary phase in a column; the molecules interacting within the column is then eluted
Ligands are separated based on their net charge - Gel filtration chromatography
Size-exclusion chromatography separates ligands based on their size, as larger molecules travel more quickly and smaller molecules travel more slow
SDS-Page can be performed after to separate bands based on molecular size
How can I identify a ligand using mass spectrometry? Is there a limitation?
After running gel, I can identify the ligand by the band that is the fattest, since the protein of interest accumulates over time, as you perform isolation chromatography for it.
We digest it with trypsin, a protease that cuts after lysine and arginine.
With mass spectrometry, we can get the size of the cut peptides.
Because each amino acid has a different weight, we can determine the composition of the peptide.
However, we cannot determine the ordering of the peptide. To do this, we need to check against existing data, previously sequenced genome.
How can I isolate a receptor in the lab?
- (For receptor) Affinity chromatography
Ligand is isolated, purified and bound to column beads
Receptor will bind to the ligand within the column beads, allowing it to be isolated and further identified - (For receptor) Antibody-affinity chromatography
How can I quantify the interaction between ligand and receptor?
Kd, dissociation constant.
Kd = Koff/Kon = [LR] / [L] * [R]
When is Kd = [L]?
When 50% of the ligand is bound.
What does a low Kd suggest?
Strong interaction. High affinity between ligand and receptor. Slow off rate, meaning that LR complexes are more stable. Slow termination
What does a high Kd suggest?
Weak interaction. Low affinity. Fast off rate. Quick termination. Higher [L] required.
Between the total binding curve, the specific binding curve and the non-specific binding curve, which one is of interest if we are looking into one particular ligand?
Specific binding curve. Obtained by subtracting non-specific from total.
In the relay step of a signal transduction pathway, what are two possible actors?
Second messengers and signaling proteins
What is one characteristic of a second messenger? Name an example.
Hydrophillic. cAMP that activates PKA.
What are two types of signaling proteins that can act as molecular switches?
- Phosphorylation covalent (kinases + phosphatases)
- GTP-binding non-covalent (GDP -> GTP)
I am interested in determining the role of kinase substrates, what experiment can I run?
Do mutagenesis.
Non-phosphorylatable mutations
* Mutate serine/threonine to alanine, and tyrosine to phenylalanine. Alanine lacks the OH group, making it incapable of being phosphorylated
* If phosphorylation of these specific residues sites is important, then mutating them may affect the protein
Phosphomimetic mutations
* Mutate serine/threonine to aspartate, and tyrosine to glutamate; aspartate and glutamate have negative charges, mimicking the negative charge introduced by phosphorylation
* The mutated protein will mimic the phosphorylated state at the specific residues; therefore, if phosphorylation is functionally relevant, then it might activate or alter the protein
Explain the concept of signal amplification.
As one signaling protein gets activated, it will activate multiple other signaling proteins, leading to the signal to be relayed by many molecules.
What is the result of the signal transduction pathway? What do we expect to see?
Leads to altered metabolism, cell shape or gene expression.
How does signal termination in he pathway work?
Binding of a signal molecule to receptor initates cascade of events within an endosome, leading to downregulation by lysosome or inactivation of pathway by inhibitor.
Can I use biochemistry to identify components of the pathway following ligand-receptor binding?
No because binding happens in low affinity, creating a lot of noise, and there are too many downstream targets.
Explain the steps of forward genetics and its goal.
Goal: Identify components of the pathway.
1. Mutagenesis to generate mutants
2. Screen more mutated phenotype of interest
3. Rough mapping to chromosome
4. Now, need to figure out if the selected mutants have mutations are on the same genes or not. Do this by complementation (cross over mutants). If they result in WT, then not same gene. If they result in mutation, same gene.
5. Classify mutant by crossing deletion and mutant, and comparing it with homozygous mutant
When I cross a deletion with my mutant, I see that it has the same phenotype as a homozygous mutant. What can I conclude?
Mutant is null.
When I cross mutation and deletion, and I have a hypomorph, what would be the expected phenotype?
Crossed has more severe phenotype than homozygous mutant.
What is saturation? What can it tell you?
Saturation is whether you have mutated all genes of interest. You can evaluate it by looking at the number of hits of mutants to a gene. The higher the better.
What is double mutant analysis? What can it tell you?
- Make mutant with both mutations
- Analyze outcome
- If double mutant = single mutant then they are in the same pathway. (Knocking one upstream will turn off the entire pathway)
- If double mutant is worse than the single mutant, then they are in different pathway.
It tells you whether your mutations of interest are in the same pathway or not.
I am doing a double mutant analysis with a hypomorph. I see that double mutant = single mutant, what can I conclude?
Nothing because need to have null mutants for this to work.
