1
Q
State the 6 general steps in a signaling pathway
A
- triggering of response
- transfer of information across a membrane
- amplification of signal
- location of signal
- divergence of one signal to a range of response proteins
- termination of response
2
Q
State the different modes of signaling between cells
A
- diffusible signal, long range
- diffusible signal, short range (paracrine and autocrine)
- signaling by cell contact
- signaling by cytoplasmic connection
3
Q
Define: paracrine
A
acts on something nearby
4
Q
Define: autocrine
A
signals act on the same cell producing them (harder to terminate the signal compared to paracrine)
5
Q
Define: chemotaxis
A
the phenomenon by which cell movement is directed by a molecular concentration gradient (e.g., B and T cells)
6
Q
Define: secondary messengers
A
molecules released into the cytosol in response to the primary signal outside of the cell
7
Q
Explain the process of adding and removing a phosphate PTM
A
These are reversible PTMs in which kinases transfer the terminal phosphate group from ATP onto an amino group of a protein, releasing an ADP. This will be specific and defined by the amino acids surrounding the residue to be phosphorylated. Phosphatases will then cleave the phosphate group off when the signal needs to be changed.
8
Q
Describe the advantages of phosphorylation
A
- rapid
- direct use of cellular energy source
- addition specifically catalysed by an enzyme - kinase
- reversible but requires an enzyme to remove the phosphate - phosphatase
- both enzymes show specificity
9
Q
Give the 3 key sites of phosphorylation
A
- amino acid side chains with OH (e.g. serine and threonine - sometimes tyrosine)
- lower eukaryotes also have histidine phosphorylation
- OH groups on lipids e.g., inositol ring on the headgroups of phospholipids
10
Q
What can SH2 domains interact with? Give an example pathway.
A
Phospho-tyrosine residues can interact with these domains on proteins. e.g., PIP2 —> IP3 + DAG pathway via autophosphorylation of the tyrosine kinase receptor
11
Q
Describe PH domains
A
These can interact with inositol lipids e.g., PIP2 —> PIP3 which releases PH-PKB(hydroxyl)
12
Q
Describe the addition and removal of G proteins
A
The protein will contain a binding site for the guanine nucleotide (either GTP or GDP). If inactive, GDP is bound. Guanine nucleotide exchange factor (GEF) allows the GDP to diffuse out and replaces it with GTP, causing a conformational change in the protein and its activation. To terminate the signal, the proteins have intrinsic GTPase activity to remove the GTP, but GTPase activating proteins (GAPs) can assist.
13
Q
What are the two classes of G proteins? Describe their structures.
A
- small monomeric GTPase proteins
- single subunit
- conserved GTPase domain - heterotrimeric G proteins
- 3 subunits: alpha, beta, and gamma
- alpha subunit is GTPase
- activated by GPCRs
2. heterotrimeric G proteins
14
Q
Describe one component systems and give examples
A
Input and output are on the same molecule, driving conformational change that changes the ability to bind to either DNA or RNA polymerase.
E.g., the lac repressor (allolactose)/Trp repressor (tryptophan)/CAP (cAMP)/FNR (oxygen)
15
Q
Describe two component systems and give examples
A
Involves two molecules such that there is an input molecule and an output molecule.
E.g., the Ntr system and the chemotaxis system in control of the flagellum motor: sensor is histidine protein kinase and output is the response regulator protein
16
Q
Give the location and describe the structure of most histidine protein kinases.
A
- most are membrane proteins, but all are dimers that undergo transautophosphorylation
- the bond formed between the phosphate and the histidine is easily hydrolyzed and thus unstable
- contains three key domains:
1. N-terminal sensor domain
2. DHp domain that contains the phosphorylated histidine and is responsible for dimerization
3. C-terminal kinase domain
17
Q
Describe response regulator proteins.
A
A response regulator is a protein that mediates a cell’s response to changes in its environment as part of a two-component regulatory system. Response regulators are coupled to specific histidine kinases which serve as sensors of environmental changes. Response regulators and histidine kinases are two of the most common gene families in bacteria, where two-component signaling systems are very common.
- aspartate becomes phosphorylated, by removing the phosphoryl group from the unstable histidine kinase
- phosphoanhydride bond (not phosphoester) which is also unstable
- majority have two domains:
1. Receiver (REC) that contains the aspartate
2. Either DNA binding domain (69%) or enzymatic domain (8%)
18
Q
Give an overview of G protein coupled receptors. Include structure and how they transmit a signal through the membrane.
A
- 7 transmembrane domains
- Compact protein with relatively small movements in helices 5 and 6. These can rotate around one another, leading to conformational changes that cross the membrane to transmit the signal
- Loop between 5 and 6 interacts with the G protein, thus the GPCR acts as a GEF
19
Q
What is the structure of tyrosine kinase receptors? How do they transmit a signal? What proteins bind them?
Give an example pathway.
A
- single TM domain and intrinsic TK activity at the C-terminal
- form dimers upon ligand binding, allowing for transphosphorylation of tyrosines in the C-terminus
- SH2 or PTB domain proteins can bind phosphorylated tyrosines (also dependent on the surrounding amino acids)
Example pathway: ligand binds TK receptor –> autophosphorylation –> Grb2 (adaptor protein) binds via SH2 domain —> SH3 domain of Grb2 binds to SOS protein. SOS is a GEF which activates Ras by exchanging GDP for GTP. Once GTP-bound, Ras can trigger transmission of the signal.
20
Q
What are scaffold proteins?
A
Bind several proteins within a cascade, localising the signal and providing specificity but limits amplification
21
Q
What is the function of PI3 kinase? What domains does it contain? How does it interact with PKB? Give an example pathway that it’s used in.
A
- lipid kinase that phosphorylates the 3’ position of inositol rings (e.g., PIP2 into PIP3)
- contains SH2 domains and PP domains (which interact with SH3)
- PI3K activates PKB via phosphorylation of serine and threonine residues (activation also requires phosphorylation by PDK1 and mTOR2)
Example pathway: cell growth
- IGF activates TK receptor
- IRS1 binds SH2; PI3K binds SH3 of IRS1
- PI3K cleaves PIP2 into PIP3
- PIP3 binds Akt and PDK1
- Akt allows for mTORC1 to phosphorylate further proteins and increase protein synthesis for more growth
22
Q
What is the function of mTORC? What inhibits/regulates mTORC1 and how?
A
A protein kinase complex that’s implicated in cell growth and metabolism downstream of PKB and also of nutrient status e.g., amino acids to integrate complex growth responses.
mTORC1 is inhibited by a complex of rapamycin and FKBP12.
It’s regulated by amino acids on lysosomes.
23
Q
What property allows most signals to travel in the bloodstream? How is this overcome for hydrophobic signals?
A
Most signals are hydrophilic to be able to travel in the bloodstream.
Some are hydrophobic, and so they tend to be made close to their target site to prevent movement over long distances.
24
Q
Give an example where the same signaling molecule can be used to trigger 3 different pathways.
A
Acetylcholine can trigger:
1. decreased firing rate in heart pacemaker cells
2. secretion in salivary gland cells
3. contraction in skeletal muscle cells
25
How can kinase activity be monitored?
1. Immunoprecipitate the kinase of interest
2. Mix with protein of interest for different time periods.
3. Western blot for each time period
4. Use a phosphospecific antibody for the substrate and compare to another WB that only shows the amount of protein present from each time period.
26
How can chimeric receptor experiments show specificity of GPCRs and heterotrimeric G proteins?
Chimeric receptors are made by swapping intracellular portions at the level of the gene. When these chimeric receptors are expressed in cells, they activate the 'wrong' pathway in response to a ligand.
This showed that specificity is defined by the 5-6 intracellular loop.
27
What are the two functions of B-arrestin?
In response to the activation of a GPCR, beta-arrestin is recruited to the receptor and binds its phosphorylated intracellular domain. This binding event causes the receptor to be internalized and desensitized, leading to the termination of the initial signaling response.
It can also recruit other signaling proteins to the receptor, such as MAPKs and Src kinases.
28
How can toxins be used to identify which G proteins are being activated in a pathway?
If we know which G-proteins are targeted by certain toxins, treatment of systems with these toxins and seeing their outcomes can tell us which G-proteins are involved.
E.g., cholera toxin promotes the activation of Gas, so if addition of this toxin increases the response, the pathway likely involves Gas.
29
What enzymes are involved in the synthesis and removal of cAMP?
Synthesis: adenyl cyclase
Removal: cAMP phosphodiesterase
30
Describe the pathway triggered by epinephrine that causes glycogen breakdown, including the role of cAMP.
1. Epinephrine activates GPCR
2. GPCR activates a-subunit of Gs protein
3. a-subunit activates adenyl cyclase
4. AC uses ATP to synthesize cAMP
5. cAMP activates PKA
6. PKA phosphorylates phosphorylase kinase
7. PK phosphorylates glycogen phosphorylase
8. GP leads to glycogen breakdown
31
How can PKA lead to changes in transcription of glucose transporter genes?
Activates PKA phosphorylates transcription regulatory CREB (cAMP response element binding protein). CREB activates the transcription of the glucose transporter genes.
32
How do antagonist, agonist, and biased agonist drugs work?
Antagonist: block binding of ligand
Agonist: mimic effects of endogenous ligands
Biased agonist: selectively activate some pathways whilst blocking others
33
Morphine is a strong analgesic, but with addictive side effects. How are new drugs being designed to stop this side effect?
By creating a bias to G protein responses the lead of pain relief, whilst not interfering with the tolerance/dependency pathways that are caused by beta-arrestin interference.
34
What experiment showed that mitogenic responses rely on tyrosine kinase activity?
Cells were transfected with a 'novel' RTK that don't normally have this receptor. The cells responded to the RTK mitogen.
The kinase was then mutated to destroy kinase activity, and this resulted in no response. This showed that the response had to be due to kinase activity of the RTK.
35
What is the function of Ras?
Ras is a small GTPase protein, activated by RTKs and GPCRs. When activated it binds to and activates downstream effector proteins, including Raf and PI3K.
36
Give 3 receptors/pathways that lead to direct regulation of gene expression. What roles do the genes have that these pathways activate?
1. Cytokine receptors + JAK/STAT pathway --> immune response
2. Steroid hormone receptors (cortisol) --> anti-inflammatory genes
3. TGFB receptors --> apoptosis, G1 arrest
37
What is the role of the Hedgehog pathway?
Regulation of embryonic development and tissue homeostasis in animals.
38
What is the role of the canonical Wnt signaling pathway? Name some potential drug targets in this pathway.
Embryonic development and tissue homeostasis.
Targets:
- porcupine inhibitors
- WNT5 mimetics
39
What is the role of the non-canonical Wnt signaling pathway? How does it differ from canonical pathways?
Embryonic development and tissue homeostasis.
Unlike the canonical Wnt signaling pathway, which activates β-catenin-dependent transcription, the non-canonical Wnt signaling pathway activates a range of β-catenin-independent signaling pathways.
40
How do we know PI3 kinase mediates PKB activation, and that phosphorylation is essential for PKB activation?
PI3K inhibitors (wortmannin) and point mutations that block PI3K activation also blok PKB activation. This was assayed using immunoprecipitation experiments.
Incubation of PKB with Ser/Thr phosphatases results in activity loss.
41
What are phospho-specific antibodies? How are these used in Western blots and immunoprecipitation experiments?
A type of antibody designed to recognize and bind to phosphorylated proteins.
In WB's, these can be used to detect changes in protein phosphorylation levels in response to different treatments.
In immunoprecipitation, phospho-specific antibodies can be used to selectively enrich for phosphorylated proteins in a complex mixture.
42
How is mTORC1 regulated by glutamine?
One proposed mechanism for how glutamine regulates mTORC1 involves the formation of the Rag GTPase complex. The Rag GTPases are small G proteins that are tethered to the lysosomal surface and play a critical role in regulating the localization and activation of mTORC1. When the Rag GTPases are in their active GTP-bound form, they recruit mTORC1 to the lysosomal surface and promote its activation. Glutamine has been shown to activate the Rag GTPases by increasing the ratio of GTP-bound to GDP-bound Rag proteins, leading to the recruitment of mTORC1 to the lysosomal surface and subsequent activation.
43
Describe the signaling cascade that occurs in photoreception, when 1 photon of light hits 1 receptor.
1 receptor activates 500 G proteins, called transducins. Each transducin actives a phosphodiesterase that breaks down cGMP.
cGMP normally keeps the ion channels in photoreceptor cells open, so its degradation results in channel closing. This hyperpolarizes the photoreceptor cell membrane and causes a decrease in glutamate release.
Reduction of glutamate causes other cells in the retina to begin processing the visual information.
44
Why are calcium levels low in the cell? What are the pumps and stores that maintain these levels?
High calcium levels in the cytosol can cause precipitation of negatively charged polyphosphates, such as NTPs, DNA and RNA.
Maintained by PMCA and SERCA, using ATP. Calcium is stored outside the cell, in the ER (SR in muscle cells) and small amounts in lysosomes.
45
What is YC-Nano, and how is it used to study calcium signaling?
YC-Nano is a genetically-encoded protein complex of two fluorescent proteins, connected by a calcium-binding protein (calmodulin).
The binding of calcium to calmodulin causes FRET to occur between the two fluorescent proteins and this can be detected to monitor changes in calcium levels in living cells.
46
How are IP3 receptors involved in generating calcium waves?
IP3 receptors are calcium channels located on the ER, which acts as a store of calcium ions within the cell. When IP3 binds as a secondary messenger, the sensitivity of the channel to calcium increases, allowing it to open at background levels of calcium.
The channels near this receptor will now 'see' a higher level of calcium and open, triggering a wave of calcium.
47
Give two examples where calcium release activates a response.
1. secretion of neurotransmitters
2. muscle contraction
48
Stim1 and Orai are proteins that play an important role in the regulation of calcium signaling in cells. Explain how.
Stim1 is a TM protein on the ER that senses calcium levels. When these levels deplete in the ER, Stim1 changes conformation and interacts with Orai channels.
Orai channels are calcium channels on the plasma membrane that allows for influx of extracellular calcium ions into the cell.
This is important in immune cells for store-operated calcium entry (SOCE). SOCE is important for allowing these cells to respond to extracellular calcium levels.
49
What is the function and key domains of NF-kB?
NF-kB is a transcription factor. Upon release, it upregulates genes involved in the inflammatory response.
- N-terminal Rel Homology Domain (RHD) that binds DNA/dimerizes
- nuclear localization sequence
50
What two methods are used to sequester NF-kB in the cytoplasm?
p100 and p105 are inhibited through binding of the C-terminal repressor domain to the nuclear localization sequence.
Other NFkB homo/heterodimers are inhibited by IkBs via their Ankyrin repeats.
51
How does NFkB function in the following pathways/cells:
- Canonical
- Non-canonical
- Neuronal cells
- Glial cells
1. Functions in innate and adaptive immune responses to activate the IKK complex for NF-kB release, triggering genes for inflammation and cell survival.
2. Functions in B-cells via activating of NFkB initiating kinase.
3. NFkB signaling is constant - doesn't require activation. It promotes synapse growth and enhances excitatory function.
4. NFkB signaling is inducible in response to inflammatory signals.
52
What are 4 signals that can activate NFkB pathways?
PAMPs
DAMPs
Cytokines
Interleukins
53
The TNF superfamily are receptors, and their function depends on their domain structure. What function do the following domain structure confer for TNF receptors:
1. Death domain
2. No death domain
3. No cytoplasmic domain
1. Can activate intrinsic apoptosis
2. Interacts with TRAFs to activate NFkB
3. Functions as a decoy receptor
54
What is the role of TRAFs (TNF receptor-associated factors)?
These are adaptor proteins that can bind TNF receptors, despite having no death domains. Many are E3 ligases to mediate non-degradative ubiquitin chain formation.
55
What is the function and domain structure of Toll-like receptors? What adaptor protein does it bind to?
Senses DAMPs and PAMPs
LRR motif and intracellular TIR domain.
Myd88
56
What is the role of LUBAC in inflammatory signaling?
LUBAC is an E3 ubiquitin ligase that ubiquitinates NEMO, promoting assembly and activation of the IKK complex to activate the NFkB pathway.
57
What is the primary role of the IKK complex?
To phosphorylate the IkB complex, leading to its degradation and release of NFkB.
58
How do DUBs control the ubiquitin signal in inflammation?
DUBs can positively or negatively regulate inflammatory signaling pathways by cleaving ubiquitin chains from key signaling molecules.
For example, the DUB CYLD negatively regulates the NF-kB pathway by cleaving K63-linked chains from TRAF2 and TRAF6, which are required for the activation of the IKK complex.
59
How can TNFR1 signaling result in either cell survival OR cell death?
Cell survival:
TNFR1 activation leads to activation of the NFkB pathway, promoting cell survival by inducing expression of anti-apoptotic genes, such as Bcl-2.
Cell death:
TNFR1 can recruit FADD through its death domain and caspase-8, initiating the caspase cascade. This occurs when NFkB activation is inhibited.
60
List ways that specificity can be achieved in signaling pathways.
1. Relative levels of secondary messengers
2. Duration of signals
3. Activation of particular receptors
4. Location of signal through anchoring domains
5. Specificity of protein-protein interactions
6. Scaffold proteins
61
What is cross talk of signaling pathways?
When the signal of both pathways together is different to the sum of the signals from the two pathways individually.
62
Describe the signaling pathway that activates platelets.
Agonists activate GPCRs on the surface of platelets, leading to the activation of PLC.
PLC cleaves PIP2 into IP3 and DAG.
IP3 binds its receptor to release calcium ions, activating Ras and protein kinase C.
PKC leads to the activation of FAK, which results in integrin activation by talin.
Activation of integrins on the surface of platelets leads to the formation of platelet aggregates and the initiation of blood clotty.
63
What is the function of thromboxane A2? What blocks its production?
Produced by platelets in response to injury or inflammation, acting as a potent vasoconstrictor and platelet aggregator.
Aspirin
64
How can biochemistry be used to mimic the 'off' and 'on' states of kinases?
Off: use chemical inhibitors to block the ATP-binding site or catalytic activity of the kinase.
On: phosphomimetics, such as aspartate or glutamate which mimic the negative charge of a phosphate group. These mutants can activate the kinase, without being phosphorylated.
65
What 4 methods are used to terminate a signal through dephosphorylation?
1. Third-party phosphatases
2. Intrinsic phosphatase activity of the kinase
3. Instability of the phosphate bond
4. Phosphate sinks
66
What is a phosphate sink?
A method for speeding up dephosphorylation as part of terminating a signal response.
If a kinase can phosphorylate two proteins, there's an equilibrium such that the phosphate can be passed backwards when the signal is removed, and only one of the proteins elicits a response, then when the signal is removed, there will be some transfer back onto the other protein, depleting the output i.e., increasing the flux through the system to speed up the auto-dephosphorylation.
67
What happens in a phosphorelay?
In a phosphorelay system, a sensor kinase protein detects an external signal and transfers a phosphate group from ATP to a specific aspartate residue on its own protein or to another protein called a response regulator. The phosphorylated response regulator then undergoes a conformational change, enabling it to interact with downstream effector proteins and initiate a cellular response.
NO SIGNAL AMPLIFICATION
68
How are pp-G-pp and cyclic-di-GMP used as secondary messengers in bacteria?
Nucleotides are used as secondary messengers within bacterial cells.
Guanine pento-phosphate is produced by the ribosome/enzyme associated with the ribosome, generating the starvation response if tRNAs are becoming uncharged. This turns down protein production and turns on genes to combat the stress and scavenge amino acids, etc.
Cyclic-di-GMP (c-di-GMP) is a second messenger molecule that plays a critical role in regulating biofilm formation in many bacterial species.High levels of c-di-GMP are associated with an increase in biofilm formation, while low levels promote planktonic (free-floating) growth.
- Also a potential signal integrator.
69
How is membrane potential important for cell signaling in bacteria? What are the key ions that establish the potential?
Some bacterial species can use changes in the membrane potential to generate electrical signals that can be detected and used for intercellular communication or biofilm formation
K+ ions
Molecular Processes
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