Unit 3 Exam Flashcards
What are the two large categories of protein-protein interaction methods?
- In the organism
- Immunoprecipitations → Separate proteins using antibodies
- Biotinylation → tag proteins with biotin
- FRET → Fluorescence based approach
- BIFC → Fluorescence based approach - In an unrelated organism system or in a test tube
- GST-Pulldown → test binding in a test tube
- Two-hybrid → bacteria or yeast
What do electrophoresis and western blot allow us to do with proteins?
They allow us to detect proteins in a sample.
Generally:
- Separate proteins by size
- Use a dye that reveals all proteins
- Use antibodies to detect specific proteins
What do Co-immunoprecipitation experiments detect? List methodology. And then connect to Western-blot.
Co-immunoprecipitation experiments detect protein-protein interactions
Methodology:
1. Antibody recognize bait protein
2. Beads take the proteins out of solution (they sink)
3. Remove the unbound stuff
4. The beads in the bottom contain the bait protein with all the interacting proteins
5. Reveal the proteins using western blot
Key - Connection to western blot:
- cdc2 binds cyclinB
- One target at a time, time-consuming
What if there is no antibody available for the protein to fuse to?
If there is no antibody available, fuse the protein to a tag
Antibodies for tags are always available
Ex: His and FLAG
Proximity biotinylation is a new method that involves what?
Proximity biotinylation is a new method that uses a Biotin ligase to mark proteins that are close
Methodology:
1. Fuse the bait protein to a promiscuous biotin ligase (BirA)
2. BirA add biotin (Vit. B7) to exposed lysines on neighboring proteins
- the addition of biotin is called biotinylation
How can we tell where in the cell an interaction is occurring? What are the disadvantages and advantages of FRET & BIFC?
We can use fluorescence methods tell where the interaction happens
- Ex: Split-GFP, FRET (uses 2 proteins)
(-) Disadvantages of BIFC and FRET
- Only two proteins at a time
- Time-consuming
- Need a fluorescence microscope
(+) Advantages of BIFC and FRET
- Single cell resolution or better
- Temporal resolution
A yeast two-hybrid (Y2H) experiment detects the physical interactions of what? Does the yeast change color? Why?
What?
- detects the physical interactions of proteins through the downstream activation of a reporter gene.
Color-change?
- Reporter gene makes yeast blue
- Pick the blue colored yeast
- Sequence the blue colonies to reveal the interacting proteins.
Using knowledge of the yeast two-hybrid (Y2H) experiment, explain how the GAl4 transcription factor protein is used together and apart.
- The GAl4 protein is a transcription factor binds to the UAS sequence
- This protein is cut into 2 parts: AD and BD
- One is fused to the prey protein
- The other is fused to the bait protein
- If together, a functional Gal4 protein will form and activate transcription of the reporter gene
What is a neuron? What’s its task? Why is it discrete?
- Every neuron consists of a cell body (containing the nucleus) with usually one long axon and several shorter, branching dendrites
- Task of a neuron, or nerve cell, is to receive, integrate, conduct, and transmit signals
- Neuronal signals are transmitted from cell to cell at specialized sites of
contact known as synapses - Neurons are discrete cells as they choose to transmit info or not, they also undergo changes in membrane potential
When a neuron is not active, its membrane potential is called resting potential. Explain this concept, compared to action potential.
Resting Potential
- Excitatory signals open cation channels, depolarizing the membrane
- Inhibitory signals open either Cl- channels or K+ channels, suppressing firing and depolarizing the membrane.
Action Potential
- The changes in the Na+ channels, K+ channels, and current flows give rise to a traveling action potential.
Explain how membrane potential propagates through the membrane.
- Voltage-gated Na+ channels transmit signals in a wave through the nervous system.
- High difference = tightly closed
- Low difference = open and ions pass
- Voltage gated Na+ channel starts the depolarization phase by obv. depolarizing the membrane
How are membranes repolarized?
- Repolarization of the membrane requires opening of additional channels, the delayed K+ channels.
- These channels also open in response to membrane depolarization (that is, they are also voltage-gated), but have slower kinetics.
- Voltage gated K+ channel
- Ends the depolarization phase
Explain the 4-step (summarized) process of how neurons communicate at CHEMICAL synapses.
- When an action potential arrives at the presynaptic site, the depolarization of the membrane opens voltage-gated Ca2+ channels that are clustered in the presynaptic membrane.
- Ca2+ influx triggers the release into the cleft of small signal molecules known as neurotransmitters that are stored in membrane-enclosed synaptic vesicles and released by exocytosis
- The neurotransmitter diffuses rapidly across the synaptic cleft and provokes an electrical change in the postsynaptic cell by binding to and opening ligand-gated ion channels
- When the incoming action potential activates a Ca2+ channel in the T-tubule membrane, it triggers the opening of a Ca2+-release channel in the closely associated sarcoplasmic reticulum membrane
intracellular signaling molecules vs extracellular signaling molecules?
Second messengers are intracellular signaling molecules released by the cell in response to exposure to extracellular signaling molecules (aka first messengers)
Explain G-Protein-Coupled Receptors (GPCR)? What is the process of activating a G-protein?
GPCR
- Act by hydrolysis of GTP
- Molecules bind to their ligands, then transmit this signal across the membrane to heterotrimeric G proteins.
Process
- When the GPCR binds its ligand it binds and “activate” the G protein
- The activated G proteins then trigger a cascade of signals inside the cell
- G proteins are only active for a very short period of time
- Ex: some G proteins activate Adenylyl cyclase
Overall: GPCRs act through PKA or IP3
What is cAMP’s role in G-Protein-Coupled Receptors (GPCR)? How does it activate cAMP-dependent protein kinases?
- cAMP is a second messenger of some G proteins
- cAMP activates PKA (a cAMP-dependent protein kinase)
Activation of PKA?
- cAMP binds the regulatory subunits and removes them
- Protein Kinase A (PKA) then phosphorylates many target proteins
Other than cAMP, GPCRs also use ______ as a second messenger. Explain what this messenger activates.
- CPCRs use IP3 as a second messenger
- IP3 activates a Ca2+ channel at the ER
- IP3 receptors = ligand-gated ion channel
Explain enzyme-coupled receptors/protein kinase receptors?
- Activation induces a conformational change exposing the kinases
- Insulin Receptor is an example of tyrosine kinase receptor (has an extracellular binding pocket for insulin, a transmembrane domain and intracellular tyrosine kinases)
The last type of receptor is a nuclear receptor. Explain what steroid hormones are, and what these receptors directly interact with. Use the example of molting hormones in bugs.
- steroid hormone is a steroid that acts as a hormone
- Nuclear receptors often directly interact with DNA
- Nuclear hormone receptors are acting as transcription factors in the cell nucleus. They regulate gene expression of hormonal regulated target genes. The role of hormone in the transcriptional process is to modulate and change the nuclear receptor functionality.
- Ecdysone is a steroid hormone that controls insect molting
Explain a quick overview of the cell cycle.
- In the G1 phase, the cell synthesizes all of the proteins, ribosomes, and other machinery that it will need.
- In S phase DNA is replicated.
G2 is a gap phase After a gap phase called G2 - In M phase the cell divides
In S phase the chromosomes are_______, while in M phase, duplicated chromosomes are ______ into a pair of ________________.
Duplicated, segregated, daughter nuclei
What is flow cytometry? What can it measure? Importance?
- Flow cytometry is a laser-based technique used to detect and analyze the chemical and physical characteristics of cells or particles
- Measures fluorescently labeled cells (forward and side light) and the amount of DNA using a fluorescent dye
Importance?
- The amount of DNA in each cell tells the cycle phase
The control system can arrest the cell cycle at specific checkpoints. List some reasons why cells may be stopped. How do these checkpoints operate generally?
Reasons
1. Incomplete DNA replication
- Entry into mitosis is prevented
- Chromosomes not properly attached the mitotic spindle
- Chromosome separation in mitosis is delayed
General operation:
- Checkpoints generally operate through NEGATIVE INTRACELLULAR signals
What do you know about the cell cycle and cdk (Cyclically Activated Protein Kinases) + cyclin?
- The cell cycle operates based on cdk
- Cdk associates successively with different cyclins to trigger the different events of the cycle.
- Cdk activity is terminated by cyclin degradation.
Name and identify the three (possibly four) main cyclin classes required in eukaryotic cells.
- G1/S-cyclins
- activate Cdks at the end of G1 and commit the cell to DNA replication. - S-cyclins
- activate Cdks during S phase and are required for the initiation of DNA replication. - M-cyclins
- promote the events of mitosis. - G1-cyclin
- which controls start of G1
What does phosphorylation have to do with cdk-cyclin complexes?
- Each cyclin-Cdk complex phosphorylates a different set of substrate proteins.
- Phosphorylation adds an extra layer of control to the Cdk/cyclin complexes
More notes:
- Cdks catalyze the phosphorylation of hundreds of different proteins in the cell.
- Cyclins confer specificity to the Cdk/cyclin complex
What triggers the last stage(s) of the cell cycle? Why is there a drop in M-cyclin?
- the final stages of cell division are triggered by protein destruction (polyubiquitination marks proteins for degradation)
- Anaphase promoting complex (APC) is activated during metaphase and promotes the degradation of M-cyclin (events of mitosis are no longer promoted)
What happens at the end of S phase?
each replicated chromosome consists of a pair of identical sister chromatids glued together.
What is the role of cohesion in the cell cycle?
- Cohesin holds sister chromatids together
- Cohesin is loaded into unreplicated chromosomes, then encircles the new sister chromatid
What is the central idea of M phase?
The central problem for a cell in M phase is to accurately separate and segregate its chromosomes
Describe the process of mitosis?
- Interphase
- Cell increases size, duplicates DNA and centrosome - Prophase
- Replicated chromosomes condense, mitotic spindle assembles outside nucleus - Metaphase
- Chromosomes align at center of spindle - Anaphase
- Sister chromatids separate into 2 chromosomes - Telophase
- chromosomes arrive to the poles, a new nuclear envelope assemble
- Mitosis ends and cytokinesis starts - Cytokinesis
- separates the two cells using a contractile ring (formed by actin & mysoin)
What happens before the cell goes into Metaphase? What is the name of this late step?
- Prometaphase occurs before metaphase
- the nuclear envelope breaks
- w/o the envelope, the microtubules attach to the chromosomes at the kinetochore
Kinetochore microtubules are attached to the __________________ at their ____ ends, and these microtubules also interact with centromeric ________.
sister chromatid pairs, plus, chromatin
What makes up the mitotic spindle? What do microtubules help with? What motor proteins help with assembly of it?
Composition
- Astral and kinetochore microtubules compose the mitotic spindle
Function
- Microtubules pull the daughter chromosomes toward the poles of the spindle (shortening of the microtubules bring the chromosomes the poles)
Motor proteins
- Many Dyneins and kinesins proteins contribute to the assembly and function of the mitotic spindle
- Kinesin-5 slide microtubules oriented in opposite directions
- Kinesin-14 crosslinks antiparallel microtubules at the center and moves one of them
- Kinesin-4/10 are plus directed and push the chromosome to the center
What is Aurora B? What does it tell chromosomes to do? What does it prevent in microtubules?
- Aurora B is a kinase force sensor that tells the cell chromosomes are oriented properly
- Aurora B prevents the stable attachment of microtubules
- When there is tension, Aurora B is inactive
What role does separase play in mitosis? What is it indirectly activated by?
- Separase is a cysteine protease responsible for triggering anaphase by hydrolysing cohesin
- Separase is indirectly activated by the anaphase promoting complex
What would happen to the ‘dividing’ cell if cytokinesis didn’t occur in mitosis? What about if cytokinesis is incomplete?
- Mitosis without cytokinesis makes a cell with many nucleus called syncytium
- Incomplete cytokinesis leaves cells interconnected through intercellular bridges
What does endoreplication mean?
Endoreplication is replication of the nuclear genome in the absence of mitosis leads to polyploidy
Note:
- The nuclear size correlates with the amount of DNA.
What is the proteasome?
- Proteasomes are protein complexes which degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds.
- Enzymes that help such reactions are called proteases.
Note:
- Is a compartmentalized protease with sequestered active sites
Ubiquitin can be covalently attached to target proteins in a variety of ways. Explain what the major form of Ubiquitin does?
The major form of ubiquitin addition produces polyubiquitin chains which
directs the target protein to the interior of a proteasome
What is Ubiquitination?
- tightly regulated, highly specific, and ATP-dependent biological process carried out by a complex cascade of enzymes
- Ubiquitination is an essential player in protein homeostasis, serving to rapidly remove unwanted or damaged proteins.
What enzymes help with ubiquitination? Give an example of what the three do.
- E1,E2, and E3 enzymes handle ubiquitination
Roles:
- E1 proteins activate ubiquitin
- E2 proteins are the carrier protein for ubiquitin
- E3 proteins called ubiquitin ligases that select the target proteins to be modified
What is polyubiquitination?
is a post-translational modification that
targets proteins for degradation
