K1

Question 1

Name six changes/hallmarks which will transform normal cells into cancer cells. List therapeutic targets for three of these hallmarks and justify why you would want to activate or inhibit these targets to reduce the onset of cancer.

Answer 1

A: Activation of invasion and metastasis
R: Resisting cell death –> Pro-apoptotic BH3 mimetics, antagonize Bcl2 famility proteins, induces apoptosis
I: Inducing angiogenesis –> VEGF inhibitors, inhibits growth of new blood vessels
S: Sustaining proliferative growth
E: Evading growth supressors –> CDK inhibitors, to inhibit the cell cycle
E: Enabling replicative immortality

Question 2

What is angiogenesis and why does a tumor need this to thrive?

Answer 2

The forming of new blood vessles, as tumors need that for nutirents to survive and proliferate

Question 3

Which cell types are present in the tumor microenvironment of cancers and indicate for
two how they regulate tumor growth.

Answer 3

Cancer stem cells and non-cancer cells.
Cancer stem cells have the ability to self-renewal so they can give rise to all heterogenous lineages of the primary tumor. Can maintain the tumor.

Fibroblasts can come in contact with other cancer cells to induce growth and form another tumor. (collagen)

Question 4

Name 2 of the 6 original hallmarks and explain how they cause cells to become
cancerous?

Answer 4

Inducing angiogenesis is the growth of blood vessels to provide the tumor with nutrients like glucose. Because of this, the tumor can develop.

Resisting cell death by preventing apoptosis by back-ups

Question 5

What does the term ‘’self-sufficiency’’ in growth signals mean in cells and describe two
manners in which this can take place?

Answer 5

Preventing tumor formation by blocking the gain of function

Question 6

Describe the Warburg effect.

Answer 6

Cancer cells preferentially metabolize glucose by glycolysis, to produce lactate despite the present of oxygen, because they need the amino acids to produce new cells from lactate

Question 7

Does a GPCR function as GEF or GAP? Explain why.

Answer 7

GPCRs function as a GEF as it activates certain proteins.

Question 8

An inactive GPCR is activated by binding to a ligand. The result is a G-protein with two
activated components. Explain how this is realized.

Answer 8

GPCR goes through a conformational change and activates a G-protein. Then GPCR induces the a-subunit of the G-protein to release GDP and that gets replaced by GTP. This causes conformation change activation in the Ga-subunit which releases the G-protein from the receptor and triggers dissociation of the Ga-subunit from the Gby-pair. They will then interact with its targets as they are activated.

Question 9

Explain how GPCR signaling activates protein kinase C and mobilizes calcium.

Answer 9

A ligand binds to the GPCR so it becomes active and activates the G-protein which activates phospholipase C-b. This induces IP3 to let go of PI(4,5)P2 and goes to the Calcium channel. This gets phosphorylated and calcium is released into the cytoplasm. The calcium then binds to protein kinase C to activate it.

Question 10

Explain how GPCR signaling is involved in the production of cAMP and activation of transcription factor CREB.

Answer 10

A ligand binds to the GPCR which activates the G-protein and activates adenyl cyclase protein. cAMP is produced by taking up ATP and activating Pka and is transferred to the nucleus. Pka then induces phosphorylation of CREB to activate it and forms a complex with CBP, this complex then binds to CRE and activates the target gene to induce gene transcription.

Question 11

How is a G-protein activated?

Answer 11

By GTP/ binding of GPCR bound to ligand

Question 12

is calcium release always maximal in GPCR signaling?

Answer 12

It is mediated by IP3 to the calcium induced-calcium release/CTCR -> feedback loop

Question 13

Describe the three modes of adaptation in the desensitization of GPCR?

Answer 13

Sequestration by internalizing the signal-receptor and then emptying the receptor before bringing it back to the membrane

Down-regulation by internalizing and destroying the signal-receptor by lysosomes

Altering the receptor so it can no longer interact with the G-protein

Question 14

How does the arrestin protein contribute to the desensitization process of GPCR?

Answer 14

GPCR is phosphorylated by GRK and binds to the arrestin protein. This protein then prevents the activated receptor from interacting with G-proteins and helps couple the receptor to the clathrin-dependent endocytosis machinery.

Question 15

What is the difference between the homologous and heterologous pathways in relation to the desensitization of GPCR?

Answer 15

Homologous = refers to the loss of response to agonists that act on a particular GPCR

Heterologous = a more general effect where simultaneous loss of the agonist on multiple GPCRs, even in absence of occupancy

Question 16

What is the outcome of the desensitization of GPCR?

Answer 16

Desensitization of GPCR allows the G-protein to not be able to bind to GPCR, so there is no signal. Because the pathway is less sensitive to the hormone, thus reduced signaling

Question 17

How do receptor protein kinases promote RAS activity?

Answer 17

RTKs indirectly bind to Ras-GEF (SOS) which then stimulates the dissociation of GDP from Ras and its uptake of GTP which activates Ras.

Question 18

How does signaling through PI 3-kinase promotes cell survival?

Answer 18

Pi 3-kinase becomes activated through RTK. The signaling of PI 3-kinase causes phosphorylation and activation of Akt by PDK1 and mTor, which then phosphorylates Bad. Bad will become inactivated and will in turn activate the apoptosis-inhibitory protein which stimulates cell survival by inhibiting apoptosis.

Question 19

How is the EGF receptor kinase activated?

Answer 19

The receptor is activated by binding of EGF, this results in a conformational change and you get dimerization. One acts as an activator and the other as a receiver. The activater pushes against the receiver which causes activation of conformational change in the receiver and will phosphorylate multiple tyrosines in the C-terminal tail of the receptors.

Question 20

Name three important modular binding domains involved in RTK signaling pathways. Specify which domains they bind to.

Answer 20

SH2/PTB: can recognize phosphorylated tyrosine residues

SH3: can recognize scaffold proteins and binds to the proline-rich region

PH: Pleckstrin homology domain, this recognizes PIP3 which are important proteins on the plasma membrane

Question 21

How does dimerization cause activation of RTKs? How is this different from GPCRs?

Answer 21

Because RTKs can activate each other as they have the same tyrosine kinase domain. When they form a dimer, a ligand will bind to these RTKs whereafter they will phosphorylate each other (auto-phosphorylation) and thus will activate the kinase domains.

Question 22

How does activation of the EGF receptor vary from the general RTK activation by other external ligands?

Answer 22

For EGF you need one activator and receiver for it to become activated and phosphorylate the tyrosines.

In RTK they can autophosphorylate and don’t need other monomers.

Question 23

Is Ras active or inactive while bound to GTP?

Answer 23

Yes

Question 24

How is the signal of Ras relayed downstream to change gene expression and/or protein activation?

Answer 24

Ras signaling through modular binding domains GEF and GTP. Ras is phosphorylated and relays it to REK for it to become activated, then REK activates MEK and MEK activates ERK.

Question 25

ERK functions to phosphorylate nuclear transcription factors, but also plays a role in the desensitization of the Ras-pathway. How is this realized?

Answer 25

The signal is transduced from REK to ERK, and is phosphorylated through the Scaffold proteins. This means Raf can not bind anymore as the binding site is occupied by phosphate, so you’d get desensitization of the Ras-pathway.

Question 26

What is incorrect with hyperactive mutants of Ras? Explain two mechanisms causing hyperactive Ras in cells.

Answer 26

Mutation of the Ras protein leads to uncontrolled cell growth and it is constantly activated.

Mutated Ras is also resistant to GAP-mediated GTPase stimulation so are locked in the GTP-bound active state.

Question 27

hat are the three steps of activating a CDK?

Answer 27

1. The T-loop will loosen up due to the binding of CDK making it partially active.
2. The T-loop is phosphorylated at tyrosine160 by CAK, this prevents the T-loop from folding back and removes it from the catalytic site. Negative O2 is held by Arg residues from the T-loop.
3. Glutamate from the T-loop comes close to ATP and is hydrolyzed to ADP and fully activates CDK.

Question 28

Following DNA damage, which steps have to take place to inhibit progression from the G1 phase to the S phase?

Answer 28

ATM kinase gets activated and activates CHk1/2. This phosphorylates p53 and binds to P21 for it to become transcribed and translated. P21 then binds to G1/s-CDK and s-CDK to form a complex and blocks the active site of the cDK –> so no progression

Question 29

Explain the proteolysis pathway of the M-cyclin.

Answer 29

Inactive APC/C and activating subunit form a complex so you’d get active APC/C. In turn ubiquitin and its enzymes bind to the M-cyclin/Cdk as a polyubiquitin chain. So that M-cyclin will become degraded in a proteasome.

Question 30

SKP2 (S-phase kinase-associated protein) functions as a receptor protein in phosphorylation-dependent ubiquitination. During which cell cycle stage(s) are SKP2 levels low and why?

Answer 30

SKP2 is low in the G1-stage because it allows p27 to go up and inhibit CDK-S cyclin, so it prevents activation of the S-stage, and stops the cell cycle.

Question 31

How are cell cycle regulating proteins lowered during different phases of the cell cycle, Include in your answer the different proteins that play a role in this process?

Answer 31

The regulation of the cell cycle is determined by cyclin and CDK, cyclins activate CDKs and determine the substrate. So there will be an activity rise and fall during the cell cycle due to the difference in cyclin levels

Question 32

What is the function of the T-loop within cyclin-dependent kinases (CDK) when a CDK gets phosphorylated for full activation? Mention the amino acids which play a role in this process.

Answer 32

The T-loop functions to stabilize the structure of the CDK-cyclin complex, the opening of the T-loop is crucial for substrate recognition and binding during the catalytic reaction.

Cyclin-CDK forms a complex, but phosphorylation of Tyr15 blocks the ATP-binding site. The phosphorylation of Thr160 in the T-loop removes the Tyr15 so that CDK can activate more CDK.

Question 33

What happens between the activation of the RTK and the inactivation of Bad in the PI 3- kinase-Akt pathway?

Answer 33

PDK1 and Akt bind to PI(3,4,5)P3 which phosphorylates Akt. Akt will dissociate and phosphorylate Bad which will come loose from the anti-apoptotic protein and form a complex with 14-3-3 protein and becomes inactive.

Question 34

How do anti-apoptotic factors via receptors promote cell survival? Describe one mechanism in more detail.

Answer 34

Through RTKs Pi 3-kinase is activated and will cause the phosphorylation and activation of Akt by PDK1 and mTOR which will then dissociate. Then active Akt can phosphorylate Bad and inactivate it so the apoptosis-inhibitory protein can disattach from Bad and become active → cell survival

Question 35

When a cell is no longer able to produce the Akt protein, will this cell be able to resist apoptosis? Explain your answer.

Answer 35

No, because Akt is needed for the phosphorylation of Bad. Without Akt, Bad can not be phosphorylated so there will not be an active apoptosis inhibitory protein

Question 36

Describe the intrinsic pathway of apoptosis.

Answer 36

The intrinsic pathway starts with the release of cytochrome C into the cytosol, this is highly regulated. Cytochrome C activates Apaf1 which assembles and recruits caspase-9, which becomes activated and thereby activates the executioner caspases.

Question 37

Which three proteins are involved in the release of cytochrome C and how do they regulate it? Mention their domains.

Answer 37

Bax and Bak promote apoptosis by creating a complex (dimer) through homology domains which opens the functional pore, causing leakage of cytochrome C (BH1,2,3,4)

The anti-apoptotic Bcl2 becomes activated and prevents the aggregation of Bax and Bak (BH1,2,3)

Pro-apoptotic BH3 block the anti-apoptotic Bcl2 and thereby stimulates the aggregation of Bax and Bal (BH3)

Question 38

What happens with STAT following activation by JAK

Answer 38

STAT will become phosphorylated and will migrate away from the receptors, forming a dimer, and then migrate towards the nucleus to control gene expression.

Question 39

How do STAT proteins get phosphorylated in the JAK/STAT pathway?

Answer 39

Cytokines will bind which changes the organization of the JAKs, they will come together and phosphorylate each other. This increases the activity of tyrosine kinase domains. Then the JAKs will phosphorylate the tyrosines in the cytoplasmatic tail of the cytokine receptors which creates a docking site for STAT to be placed, these will then become phosphorylated.

Question 40

Describe the mechanism that has a negative effect on the activity of the JAK-STAT pathway.

Answer 40

Negative feedback: STAT dimers can activate genes that encode for inhibitory proteins that turn off the response.

Dephosphorylation: dephosphorylating tyrosines to inactive JAKs and STATs

Question 41

Where are STAT proteins located and why are they referred to as latent transcription regulators?

Answer 41

STAT proteins are located between the cytosol and the nucleus. They are referred to as latent transcription factors because they regulate the conversion of DNA to RNA and orchestrate gene activity

Question 42

In what life stage of a protein are the HSP70 and HSP60 proteins involved?

Answer 42

Hsp70 is involved in the early stage of a protein and Hsp60 is involved in the stage where a protein is already synthesized.

Question 43

What are all the components of proteasome and what is their function?

Answer 43

Unfoldase ring which unfolds the protein
Protease contains the active sites where the protein will be cut into tiny pieces
The cap which releases the cut protein.

Question 44

Why are there many types of different E3 ligases?

Answer 44

Ubiquitination by E3 ligases regulates diverse areas such as cell trafficking, DNA repair, and signaling and is of profound importance in cell biology.

Question 45

Give two examples of E3 ligase families and describe their mechanism of action. Draw a scheme to explain your answer.

Answer 45

HECT and RING E3 ligase:

RING brings ubiquitin-E2 close to the substrate for them to bind

HECT is bound to ubiquitin and will then bind to the substrate through HECT

Question 46

Describe how chaperones aid incorrect folding of proteins and what happens with incorrectly folded proteins. Chaperons (also known as heat-shock proteins) help in the folding of proteins.

Answer 46

Hsp70 binds to ATP and together with ATP, it can then bind to a protein. During the refolding of the protein, ATP is then hydrolyzed to ADP

You can see this chaperone Hsp60 as a mixer. A wrong protein and ATP go into the chaperone protein. The protein is then reorganized on the inside, this is possible because the hydrophobic parts are recognized and interact. Finally, the correct protein is released.

Question 47

How do BH3-only proteins promote apoptosis in response to an apoptotic stimulus?

Answer 47

BH3-only protein becomes activated and blocks the Bcl2 anti-apoptotic protein which thereby stimulates the aggregation of Bax and Bak complex. So cytochrome C can go through the functional pore and get released into the cytosol thus inducing apoptosis.

Question 48

Mention and briefly explain the 3 mechanisms by which apoptosis can be blocked by extracellular survival factors.

Answer 48

1. Increased production of anti-apoptotic Bcl2 family protein
2. Inactivation of pro-apoptotic BH3-only protein
3. Inactivation of anti-IAPs

Question 49

Which proteins belong to the anti-apoptotic Bcl2 protein family?

Answer 49

Bcl2 and BclXl → BH1,2,3,4

Question 50

What is the role of cytochrome C in apoptosis?

Answer 50

Cytochrome gets released into the cytosol. which binds and activates Apaf1.
Apaf1 assembles an apoptosome which recruits Caspase-9 and thus gets activated and cleaves to the executioner caspases, this leads to apoptosis.

Question 51

What are the 3 main types of p53 inactivations in cancer cells?

Answer 51

1. P53 degradation by MDM2 activation.
2. Preventing p53 from binding to the target genes due to lack of function as the DNA binding domain is mutated/fucked.
3. Preventing the formation of tetramers of p53 by deletion of the carboxy-terminal domain

Question 52

Describe the canonical Wnt signaling pathway with and without Wnt signal. Explain what Wnt proteins are. Use the following terms: Frizzled, Dishevelled, axin, LEF1/TCF

Answer 52

Without: When there is no wnt signaling, Frizzled and Dishevelled do not get activated. So APC and axin will still be bound together which activates GSK3 and CK1. This phosphorylated β-catenin, so it becomes unstable and is then ubiquitinated and degraded by the proteasome, so there is no transcription.

With: When there is wnt signaling, the frizzled receptor is activated and this then activates Dishevelled found in the cytoplasm due to conformational change. This makes the cytoplasm complex fall apart as Groucho and APC let go of the Axin. β-catenin stabilizes and replaces Groucho and binds to LEF1/TCF which activates the transcription of wnt target genes.

Question 53

What is the consequence if APC is mutated in the Wnt signaling pathway?

Answer 53

If APC is mutated in the Wnt-signaling pathway it will form a complex which leads to the destruction of β-catenin meaning it can not be formed anymore.
So you’d get the continuous accumulation of β-catenin and thus the transcription of Wnt target genes. → uncontrolled proliferation

Question 54

How does Wnt signaling play a role in oncogenesis?

Answer 54

The Wnt signaling plays a role in oncogenesis as it makes APC unable to bind to axin, so β-catenin does not get ubiquitinated and accumulates. β-catenin goes to the nucleus and displaces Groucho and associates with the coactivator which starts the transcription of Wnt target genes. Thereby inducing uncontrolled proliferation.

Question 55

Wnt plays an important role in the homeostasis of the intestinal epithelium. How does the degree of Wnt signaling change along the crypt-villus axis for the normal pathway and an APC mutated pathway?

Answer 55

In a normal pathway the wnt signaling decreases along the crypt-villus axis, so passing the crypt the signaling starts to decrease. Whereas in the APC mutated pathway there is no decrease in wnt signaling, so along the crypt-villus axis there is continuous wnt signaling.

Question 56

What makes Lgr5 so important in stem cell colorectal cancer research?

Answer 56

Lgr5 is important because it can be used as a marker that can be manipulated to express GFP. GFP is cloned behind the Lgr5 promoter. These GFP+ cells kept dividing whereas GFP- cells died, the dividing cells can differentiate into other cells of the intestinal epithelium indicating that they were stem cells.