TOPIC D: Cells

Question 1

Outline Cdk signalling

Answer 1

Cyclin dependent kinases control the cell cycle

Cdks are activated by binding to a cyclin member

Binding moves the T-loop exposing the ATP for phosphorylation

Cyclin is broken down by Ub-proteasome which then stops Cdk activation

Question 2

Outline Ub-proteasome signalling

Answer 2

Ub-proteasome mediated degradation.

Ub activating enzyme (E1) conjugates to Ub. Ub swaps onto E2. E3 transfers Ub onto another protein to activate it.

Poly-Ub signals for proteasome to degrade protein by using ATP

Question 3

Explain Cdk role in nuclear envelope degradation in mitosos

Answer 3

Cdk phosphorylates the nuclear lamina subunits, changing their conformation, breaking envelope apart.

Allows cystolic condensing protein to contact DNA and condense chromatin

Question 4

Explain Cdk role in microtubules in mitosis

Answer 4

Cdk reorganises the microtubule network from interphase into spindles.

It phosphorylates microtubules associated proteins (MAPs) to inactivate them and allow spindles to form and motor proteins to move centrosomes to the poles

Question 5

Explain Cdk role in anaphase of mitosis

Answer 5

M-Cdk activates anaphase promoting complex (APC) which has Ub ligase activity (E3).

APC targets securin, initially bound to active separase.

Signals Ub-P degradation and allows active separase to cause separation of chromosome arms.

APC then ubiquinates M-Cdk for degradation of the cyclin. Allows phosphatases to dephosphorylate protein in cytokinesis (allows forming of nuclear envelope, interphase, microtubules). This ends mitosis.

Question 6

Explain remodelling of microtubules

Answer 6

heterodimer of alpha and beta subunit form tubulin.

Tubulins form protofilaments which form MTs

Dynamic instability is where tubulin binds to GTP cap causing MT elongation. GTP can undergo hydrolysis which will deplete the GTP cap and cause shrinkage.

Cdk can phosphorylate MAPs which causes them to bind to the side and stop dynamic instability (stabilises MT)

Question 7

Explain MTs at the centrosome

Answer 7

Centrosomes are where MTs radiate out. MT organising centre (MTOC) is where negative end of MT binds to gamma tubulin bound to ring complex and positive end grows to the cell periphery

Question 8

Kinesin and Dynein directions of movement

Answer 8

Kinesins: + end directed (into cell)

Dyneins: - end directed (to the edge of cell)

Question 9

What are the 3 types of MTs

Answer 9

Astral: anchor and position centrosome

Kinetochore: Contact kinetochore of chromosome by dynamic instability until contact

Polar/interpolar: Link to other side polar/interpolar MTs to centralise mitotic apparatus

Question 10

Explain what happens at the spindle assembly cell cycle checkpoint

Answer 10

Mad2 sits at free kinetochores and blocks APC action

Dissociates when MT reaches kinetochore by travelling with dynein to other end.

When all Mad2 gone, APC can work

Question 11

What are the 3 ways protein can be transported around the cell

Answer 11

gated transport by channels
Transmembrane transport through proteins
Vesicular transport

Question 12

Explain nuclear pore complexes

Answer 12

Cystolic fibrils recognise protein

Basket on other side acts as sieve for only less than 50 kDa proteins to diffuse into nucleus

Question 13

Explain the nuclear localisation signal

Answer 13

Basic stretch of amino acids that signals for nuclear import

Importin binds to protein and then to Ran protein inside of nucleus. Then rhe protein is released and Importin-Ran complex dissociates

Question 14

How can the nuclear localisation signal be regulated

Answer 14

Protein binding: TF-BP binds to protein at importin site, not allowing transport

Modification of/around the NLS: cannot be recognised

Question 15

What are TOM and TIM

Answer 15

translocase of the outer membrane

translocase of the inner membrane

transmembrane transporters of the mitochondria

Question 16

Explain protein transport into the mitochondria

Answer 16

Signal sequence is a basic amphipathic alpha helix at N terminus.

Signal sequence binds to import receptors and travels through TOM and TIM

Signal sequence cleaved off and folding occurs once in matrix

Question 17

Explain transport into the ER

Answer 17

Signal sequence is a stretch of hydrophobic aa followed by basic aa.

signal recognition peptide (SRP) binds to signal and brings the tRNA complex to the protein translocator by binding to the SRP receptor

Then translation feeds into the ER

Question 18

How do proteins insert into the ER membrane

Answer 18

ER translocon opens and laterally releases hydrophobic transmembrane anchor

As protein leaves, if it has a stop sequence in it, it will dock to the membrane

Question 19

What does Calnexin do

Answer 19

Holds onto the glycosylation of a protein in the ER membrane to ensure the protein is folded correctly and then released

Question 20

Explain the deficiency resulting in cystic fibrosis

Answer 20

Deficiency in cystic fibrosis transmembrane conductance regulator (CFTR) which is a chloride channel.

F508del mutation causes defect that reduces protein levels due to incorrect folding in ER.

Question 21

Explain treatment of cystic fibrosis

Answer 21

Corrector or potentiator drug treatment

ORKAMBI treament includes

(corrector) lumacaftor: chaperon
(potentiator) Ivacaftor: increases open probability of CFTR channels

Question 22

How does clathrin work

Answer 22

Three legs make up triskelion which form around membrane into spherical shape

pulls membrane and creates vesicle

dynamin pinches the thin neck causing budding

Question 23

Explain soluble ER protein retrieval

Answer 23

All these proteins have same sequence at C terminus

KDEL receptor binds as it leaves the ER and then forms vesicle from the golgi back to the ER

Lower pH out of ER gives receptor high affinity for protein as opposed to when in the ER

Question 24

What are the 3 types of secretion at the trans golgi

Answer 24

Signal mediated diversion to lysosomes

Constitutive secretory pathway (usual secretion out of the cell)

Signal mediated diversion to secretory vesicles (for storage and regulated secretion)

Question 25

Explain protein transport to lysosomes

Answer 25

Lysosomal enzymes are glycosylated in the ER

In the golgi phosphate is added to the mannose to produce M6P. This is by GlcNAc phosphotransferase.

M6P receptors recognise protein and a vesicle transports it to the lysosome

Question 26

What is I cell disease

Answer 26

Lysosomal hydrolases are not targeted to the lysosomes and are secreted.

Cell fills up with inclusion bodies

Results in short stature, and developmental delay

Question 27

What is necrosis

Answer 27

Accidental cell death where contents flows out of cell

Necrotic cells trigger inflammation by cells of innate immune system due to dangers of contents in EC space

This is known as danger associated molecular patterns.

Question 28

What is apoptosis

Answer 28

Purposeful cell death from within

Cell shrinkage

Nuclear blebbing (fragmentation of nucleus)

Blebbing of plasma membrane

Question 29

What are the 2 types of signals that trigger apoptosis

Answer 29

Inside the cell: could be to protect body

Outside the cell: other cells signal for cell to die

Question 30

What are caspases

Answer 30

Proteins that orchestrate and execute apoptosis

Initiator caspases: activated by dimerisation and then activate effector caspases

Effector caspases: activated by cleavage and activate cell death by other proteins

Question 31

What are the 2 ways of caspase activation

Answer 31

Extrinsic death receptor pathway

Intrinsic mitochondrial pathway

Question 32

Explain the caspase extrinsic activation pathway

Answer 32

Death receptors undergoes conformation change when EC ligand binds causing trimerisation of 3 death receptors, recruits adaptor proteins.

FADD binds intracellularly and recruits caspase 8, forming death inducing signalling complex (DISC). Attached caspases dimerise which then activates effector caspases.

Question 33

Explain the caspase intrinsic activation pathway

Answer 33

Apoptosome causes dimerisation and activation of caspase.

Apoptosome becomes activated by Apaf1 binding to cyt c (released from mitochondria) and these subunits coming together to form apoptosome.

Question 34

Explain cyt c release regulation

Answer 34

Bcl-2 acts as guardian and does not allow release of cyt c from mitochondria (thus no apoptosis)

Bax/Bak when active form oligomers to produce membrane pores and cyt c release. Bcl-2 binds to Bak to prevent this.

BH3-only proteins bind to Bcl-2 allowing Bak to work

Question 35

Explain common BH3 mimetics

Answer 35

ABT-737 binds to Bcl-2 like BH3.

Navitoclax ( a derivative of ABT-737) does this but reduces platelet count by binding to Bcl-xl on platelets inhibiting cell survival

Venetoclax binds does not bind to Bcl-xl as much but still binds to Bcl-2. Therefore it targets cancerous cells but not platelets.