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L2 Mol Cell > Mol Cell > Flashcards

Mol Cell Flashcards

1
Q

Altered cell behaviour can be a response to?

List 3 possibilities

A

1) Changing structure of existing protein (such as ion channel)

2) Changing post translational modification (such as phosphorylation)

3) Changing protein levels (via gene expression)

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2
Q

Making & immediately destryoing proteins to achieve a rapid turnover is a wasteful process.

How can we fix this? (List 2 possibilities)

A

1) Signalling by phosphorylation

2) Signalling by GTP-binding

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3
Q

Name 3 essential functions of ion channels, and what they’re useful for

A

1) Transport ions across membrane - secretion/absorption of fluids

2) Regulate membrane potentials - nerve & muscle cells

3) Ca2+ influx into the cytoplasm - secretion & muscle contraction

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4
Q

Define these words:

Alpha helices

Beta sheets

Subunits

A

Alpha helices - a right hand-helix conformation

Beta sheets - Beta strands connected laterally by at least 2 or 3 backbone H bonds, forming a sheet

Subunits - Single proteins that forms with others to form protein complex

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5
Q

Define these:

Transmembrane domain

P-loop / Pore

A

Transmembrane domain - Protein that spans the width of the membrane from extracellular to intracellular sides, usually a helical shape

P-loop/Pore - Pocket where ion will bind

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6
Q

Describe a simple K+ ion channel (KcsA)

(Transmembrane, Cytoplasmic)

A

Transmembrane helicase structures form a p-loop/pore

On the cytoplasmic side, transmembrane domains are more tightly packed, creating a gate

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7
Q

What are the 2 main functions of Voltage gated ion channels?

A

Na+ and K+ create action potentials in excitable cells

Ca2+ transported into cytoplasm, where secondary messenger elicits a cellular response

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8
Q

Describe the STRUCTURE of Cyclic nucleotide-gated channels:

A

Tetramer

6 Transmembrane domains

S5 & S6 alpha helical domains line the central pore with P loop controlling filter

Added regulatory domains to intracellular N & C domains

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9
Q

Describe binding to Cyclic nucleotide-gated channels

A

Cyclic nucleotide binding domain is on the intracellular C terminal, this domain opens a pore permeable to Na+ and Ca+

Ligand must bind to 3 out of 4 of the sites for the channel to open (Gives a sharp conc/response curve)

Ca+ binding to an N terminal associated calmodulin, provides negative feedback

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10
Q

What is the role of Na+/K+ selective channels?

A

To control membrane excitability - to depolarise cells

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11
Q

What is the role of Cl- selective channels?

A

Control membrane excitability - to reduce resistance & hyperpolraise cells - reducing action potential firing

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12
Q

What do extracellular ligand gated ion channels with added permeability regulate?

A

Activity of calcium sensitive proteins

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13
Q

What type of receptor is NAChR?

A

Cys-loop type receptor - Nicotinic acetylcholine receptor

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14
Q

Where can α4β2 be found? What do they have a high affinity for?

A

Abundantly expressed in cortex & hippocampus

High affinity to agonists nicotine & varenicline

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15
Q

What do genetic studies show about targeting nicotinic receptors for nicotine addiction?

A

Specific polymorphisms in nACh⍺4 & 6 are linked to tobacco dependence

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16
Q

Describe 2 ways how a mutation in nAChR can cause Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE seizures)

A

Mutations in the M2 region of the human α4 neuronal nicotinic subunit

Use-dependent potentiation & delay in rising phase caused by a slow unblocking of closed receptors - Enhanced receptor function –> Increased nicotinic-mediated transmitter release

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17
Q

Describe the structure of Glutamate receptors

A

Tetramer

Similar structure to KcsA, except pore is inverted

Ligand binding site is a cleft that closes when occupies

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18
Q

Multiple genes, alternative splicing & RNA editing contribute to the diversity of glutamate receptors. Name 3 and their basic roles:

A

AMPA receptors - mediate fast excitatory synaptic transmission in CNS

NMDA receptor - involved in learning & memory

Kainate - similar to AMPA, lesser role at synapses - linked to Schizophrenia, Depression & Huntington’s

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19
Q

Describe what is meant by ‘Flip & Flop’ in RNA splicing

A

Each subunit exists as 2 splicing isoforms - Flip & Flop

Alternative spicing of 2 exons in the primary transcripts means you have 2 protein isoforms with different domains in the extracellular loop

Flip & Flop have different kinetic properties

Flop has faster desensitisation rate & reduced current responses to Glutamate than Flip

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20
Q

Describe the structure of P2X receptors

A

ATP gated ligand channel

3 subunits with 2 transmembrane domains

Large extracellular domain

3 ATP molecules required to open the channel

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21
Q

Name the Extracellular Ligand(s) and an example disease/physiological condition for each of the following:

P2X/Trimeric

Glutamate/Tetrameric

Cys-loop/Pentameric

A

P2X - ATP - P2X2 hearing loss, P2X4 pain, P2X7 inflammation, neurodegenerative disease

Glutamate - Glutamate - Excess NMDA in Stroke –> Neuron death

Cys-loop - Nicotinic, Acetylcholine, GABA, Glycine, seratonin - Epilepsy

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22
Q

Describe the structural features of GPCRs

A

7 transmembrane alpha helices

TM3 centrally located next to the binding pocket - crucial for ‘transduction’ of ligand binding

Other TMs & extracellular N terminus also contribute to ligand binding

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23
Q

Describe an example of GPCRs in action

A

Protease-activated receptors (PAR) in platelets

Receptor activated by the cleavage of the N terminal, which in turn acts as a tethered ligand (part of the receptor itself acts as the agonist)

Receptors work togehter to elicit a response - 3 independent stimuli activate platelets - Thrombin, ATP, Basal Lamina

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24
Q

What are G-proteins?

A

Guanine nucleotide-binding proteins

Belong to GTPase family

Act as molecular switch to transmit cells from extracellular stimuli

Regulated by ability to bind & hydrolyse GTP (on) to GDP (off)

Exist as heterotrimeric complexes made up of alpha, beta and gamma subunits

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25
Q

Describe the mechanism of actions for G proteins

A

Inactive state - GDP bound to alpha subunit

1) Ligand binding - conformational change in receptor activates G-protein

2) GDP released & alpha subunit seperates from others and binds GTP (now active)

3) Binds to target protein in membrane to elicit a response within a cell

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26
Q

How is G-protein signalling controlled?

A

G-proteins are timers

Duration of signalling by activated G-protein is regulate by the rate of GDP hydrolysis by Gα

RGS proteins stimulate GTPase activity in the α subunit

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27
Q

Activated G proteins regulate the activity of enzymes that control the levels of secondary messengers

Name 3 examples

A

Hydrophobic lipids confined to the membrane in which they are generated

Small soluble molecules that diffuse through the cytoplasm (cAMP, cGMP)

Calcium ions

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28
Q

Give an example of mutations in GPCRs

A

Uveal melanoma - GNAQ & GNA11

Over 90% of uveal membrane have mutations in Gα α subunit

Leads to blocking of GTP hydrolysis - so subunit is always active, causing permanent signal transmission

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29
Q

How can adenylyl cyclase be inhibited or stimulated?

A

Inhibited - Gi alpha subunit

Stimulates - Gs alpha subunit

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30
Q

Describe the 6 steps in cAMP secondary messenger system:

A

1) Ligand binds to receptor activating G protein

2) Alpha subunit moves and binds to adenylate cyclase in the membrane

3) This activated enzyme catalyses the formation of cAMP from ATP

4) The cAMP (2nd messenger) activates protein kinase A (PKA)

5) PKA phosphorylates/activates protein

6) Initiates a response within the cell

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31
Q

How can glycogen metabolism be activated?

A

Beta2 Adrenoreceptor regulation of metabolism in liver & skeletal muscle

Binding of a single Epinephrine molecule to a receptor

Sets of signalling cascade resulting in phosphorylation/activation of enzymes controlling glycogen metabolism

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32
Q

How can cAMP secondary messenger signalling be switched off? (Example Beta2 Adrenoreceptor)

A

1) Agonist dissociating from receptor

2) GTPase activity of Gαs

3) cAMP breakdown by phosphodiesterase

4) Dephosphorylation of enzymes

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33
Q

cGMP secondary messenger system is similar to cAMP, except for 2 things:

A

1) Enzyme is guanylate cyclase - which can be receptor bound or ‘free’ in the cytoplasm

2) Converts guanosine triphosphate (GTP) to 3’,5’-cyclic guanosine monophosphate (cGMP)

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34
Q

Give an example of a Hydrophobic lipid in the membrane, what 2 kinds of secondary messengers can it generate?

A

Phospholipase Cβ

Generates - IP3 - water soluble, diffuses through cytoplasm

Generates - DAG - hydrophobic molecule, remains in membrane

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35
Q

Protein kinase C (PKC) are Ser/Thr kinases

What are they activated by?

A

DAG (C1 domain) & Ca2+ (C2 domain)

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36
Q

What is PMA phorbol ester?

A

An analogue of DAG used in research to activate PKCs

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37
Q

Describe the regulation of Ca2+ as a secondary messenger

A
  • Calcium influx into cytosol is regulated by channels in the ECM & ligand gated ion channels on the ER

-Store operated channels made up of ORAI and gated by STIM - responsible for store refilling & maintaining ER calcium levels

-Has an important role in activation of T-lymphocytes

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38
Q

What does a LOF mutation in ORAI1 do?

A

Causes severe combined immunodeficiency (SCID)

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39
Q

Name 2 consequences of Overstimulation of GPCRs

A

1) Tachyphylaxis - appearance of progressive decrease in repsonse to a given dose after repeated use of substance

2) Disease - uncontrolled growth in cancer

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40
Q

Describe an experimental strategy to test what - Factor X does to cell type Y

A

1) Extract mRNA & convert to cDNA

2) Prep a sequencing library containing all cDNA molecules in each biological sample

3) Sequenceon an Illumina Next Generation Sequencing (NGS) machine

4) Run a series of computational steps and make statistical comparisons

Note- cDNA counts reflects mRNA expression levels

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41
Q

What is a fold-charge?

A

How much gene expressed is increased/decreased by a treatment

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42
Q

What can combining disease risk-associated genetic variant data with gene expression can…

List 4 points

A

1) Identify the gene whose expression leels are liked to the SNP allele

2) Identify the cell type in which the genetic variant have functional consequences

3) Reveal how those variants might regulate gene expression

4) Big data integration reveals & refines insights into biological processes

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43
Q

What is an SNP

A

Single nucleotide polymorphisms

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44
Q

Describe why budding yeast is used to study cell polarity, growth & division?

A

-Yeast undergoes significant morphological changes in response to both internal & external signals

-Yeast is genetically tractable, the entire genome sequence is known & annotated

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45
Q

What is the model organism used to study cell polarity in Eukaryotes?

A

Saccharomyces cerevisiae

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46
Q

Discuss the 4 steps to generate cell polarity in order to grow and divide

A

1) Marking the site - where on the cell surface

2) Decoding the site - signalling

3) Establishing the site - Recruitment of ‘machinery’

4) Maintaining the site - Remebering where the machinery is and keeping it in place (feedback loops)

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47
Q

Discuss the Genes required for yeast axial budding pattern:

A

BUD10, BUD3, BUD4 & the septins

Products from these genes are involved in marking the mother bud neck during one cycle as a site for budding in the next cycle

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48
Q

Discuss the genes required for the yeast bipolar budding pattern

A

BUD8, BUD9, RAX2 & components of actin cytoskeleton

Products from these genes mark the end of diploid cells

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49
Q

Discuss genes required for both yeast axial & bipolar budding

A

BUD1, BUD2 & BUD5

Proteins encoded by these genes decode the axial & bipolar marks and signal the machinery involved in generating the polarity axis

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50
Q

Regarding yeast budding. How is the site decoded?

A

BUD1, BUD2 & BUD5 function together to signal to the polarity establishment machinery the position of the bud site cortical landmarks

They function together in a GTPase cycle

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51
Q

Regarding budding yeast, describe how the site is established

A

1) Cell integrates spatial cues from budding landmarks

2) This info is fed to the polarity establishment material which is responsible for polarisation of cell cytoskeleton

3) Rho-GTPase family are important proteins, in yeast Cdc42 is the most important (highly conserved across evelution)

4) Cell screens found that some mutants were blocked becasue cells couldn’t direct their growth to forma new bud

These set of mutants involved Cdc24, Cdc43 & Cdc42

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52
Q

How does Cdc42 function to establish cell polarity?

A

1) Cdc42 is regulated through cycles of activation & inactivation by it’s binding partners Cdc24 (a GEF) and several GAPs

2) The GEF for Cdc42 (Cdc24) binds to the active form of Bud1 at sites marked for budding. Cdc24 then binds Bud1 and can activate Cdc42 to allow the polarity site to become established

Outcome - A polarised yeast cell with machinery in place for inheritence of genetic material & movement of cytoplasmic organelles and other material from mother to daughter cell

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53
Q

What is the role of Myosins in asymmetric growth & organelle inheritance in yeast?

A

Myosins (Myo2 & Myo4) are required for the asymmetric inheritance of specific factors (proteins and mRNAs)

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54
Q

What are the 2 main routes of diversity regarding polarity & cell fate decisions:

A

1) Polar mother cells could divide to generate daughters that have inherited different compounds

2) Daughters could be equal at birthm but become different by exposure to different environmental signals

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55
Q

Describe Drosophila neuroblast cell division

A

1) In drosophila CNS, progenitor cells called neurblasts are found within the ventral neuroectoderm (epithelial monolayer)

2) They delaminate from this position and undergo repeated rounds of asymmetric division

3) Each division gives rise to a small basal daugther cell and a larger apical daughter cell

4) The GMC only divides once more to give rise to a neuron & glia cell

5) The apical daughter continues to divide asymmetrically

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56
Q

Regarding cell migration depending on the actin-rich cortex beneath the plasma membrane. What are the 3 main activities required for movement?

A

1) Protrusion - pushing out of plasma membrane infront of the cell

2) Attachment - the actin cytoskeleton inside the cell is attached via interacting proteins across the plasma membrane to the substratum

3) Traction - the bulk of the cell body is drawn forward through a process of contraction

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57
Q

Describe one difference between Filopodia & Lamellipodia

A

Filopodia - Dense core of bundled actin filaments

Lamellipodia - Sheet-like broad structures of actin

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58
Q

Name the 3 small Rho GTPases that are involved in establishing cell polarity in cell migration

A

Cdc42, Rho & Rac

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59
Q

Regarding the Epithelial polarity program (EPP), How do epithelial cells adhere to each other?

A

Lateral sides of epithelial cells adhere to each other through homophilic adhesion molecules such as E-cadherin

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60
Q

Regarding the Epithelial polarity program (EPP), what is the role of a polarised actin cytoskeleton on epithelial cells?

A

Allows apical surface to constrict - important for gastrulation and tubulation

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61
Q

What is EMT? How is it triggered?

A

Epithelial to mesenchymal transition

Involved conversion of epithelial apical-basal polarity axis into a migration axis with front-rear polarity

Triggered by signals that lead to a loss of E-cadherin, there’s also asymmetric activation of small Rho GTPases (Cdc42 & Rac1 at the front & RhoA at the back)

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62
Q

What are the 4 points of Rothman’s SNARE hypothesis?

A

1) SNAREs for each transport step within the cell

2) SNAREs should provide specificity for vesicle transport

3) SNAREs should be sufficient to drive bilayer fusion

4) Proposed that NSF & ATP hydrolysis catalyses membrane fusion (THIS IS WRONG)

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63
Q

What is the ratio of R to Q SNAREs?

Give 2 examples of Q SNAREs

Give a basic description of R SNAREs

A

3 Q SNARE domains to 1 R SNARE domain

Q SNAREs - SNAP & Syntaxin

R SNAREs - VAMP2 type molecules, each have Arganine in a certain position

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64
Q

Describe the basic structure of SNARE proteins

A

Generally small - 14-40 kDA

At least 1 coiled-coil or SNARE motif

Generally C-terminally anchored

N terminal extension on syntaxin - Some R SNAREs also have regulatory domains

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65
Q

What does Syntaxin do?

A

Q SNARE

Regulatory domain which regulates its function

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66
Q

How can you observe SNARE proteins in membrane fusion?

A

TIRF microscopy

Lipid bilayer on glass slide - put SNARE molecules on it

Make vesicles or liposomes & put R SNARE on it

Observe

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67
Q

Describe how recombinant SNAREs can drive membrane fusion of purified liposomes

A

1) Zippering process in synapse

2) Influx of Ca2+, partially regulated by proteinsynaptotagmin

3)Go from Trans to Cis SNARE complex

4) NSF comes in with Alpha SNAP - forms 20S complex

5)ATP hydrolysis occurs, SNAREs recycled

6)VAMP and synatxin are now on the same membrane, they have to be recycled to be back on different membranes

7) NSF unwinds the SNAREs - hydrolysis pulls them apart (req alot of energy) then SNAREs are free to be recycled

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68
Q

Describe an experiment to show the inhibition of membrane fusion in animals

A

1) Mutagenise the flies

2) Look for ones paralysed at room temperature

3) Must have a mutation in machinery important for muscular function or synaptic transmission

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69
Q

Regarding KO studies in mice to study SNARE function. What happens if you remove:

VAMP2

SNAP25

A

VAMP2 - Die at birth - loss of synaptic transmission

SNAP25 - Die at birth - loss of synaptic transmission

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70
Q

Regarding KO studies in mice to study SNARE function. What happens if you remove:

Syntaxin 1A

Syntaxin 1B

A

Syntaxin 1A - No gross defects, subtle defects in synaptic transmission

Syntaxin 1B - Die after birth, reduced synaptic transmission

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71
Q

Give at least 2 examples of human diseases caused by mutation in SNARE proteins:

A

VAMP2 - Neurodevelopmental disorder with hypotonia & autism

SNAP25b - Neurodevlopment disorder with seizures & intellectual disability

SNAP29 - Cerebral dysgenesis, neuropathy

Syntaxin11 - Familial hemophagocytic lymphohistiocytosis type 4 (FHL4)

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72
Q

Describe the symptoms of Familial hemophagocytic lymphohistiocytosis type 4 (FHL4)

A

Overproliferation of T cells, NK cells, B cells & Macrophages

Can cause a cytokine storm - life threatening

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73
Q

What is Syntaxin 11 an unusual Q SNARE?

What can loss of Syntaxin 11 cause?

A

It doesn’t have a transmembrane domain

Loss of STX11 causes defective degranulation from cytotoxic T-cells

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74
Q

Regarding Clostridial neurotoxins

What is clostridium tetani & clostridium botulinum?

A

Clostridium tetani - Tetanus - Acts on inhibitory neurons to stop overexcitation of neurons - Spasms so hard you can fracture your bones

Clostridium botulinum - Botulism - Muscles completely relax & go floppy

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75
Q

Briefly describe the role of the 3 domains of clostridial neurotoxins

A

Targeting domain - Binds to neurons

Translocation domain - Gets from inside the endosome, released by a translocation domain (makes a pore in the endosome)

Protease domain - Cleaves the SNARE molecules (Some only cleave certain SNAREs)

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76
Q

Tetanus & Botulinum toxins have similar modes of action but intoxicate different neurons.

Describe at least 1 similarity & 1 difference:

A

They both initially enter the NMJ

Tetanus - able to get into inhibitory neurons, paralyses action at inhibitory neurons

Teatnus - Cleaves VAMP like BoNTs

Botulinum - Paralyses action at NMJ

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77
Q

Briefly describe the major features of the following membrane trafficking pathways:

Secretory/Exocytic pathway

Endocytic pathway

A

Secretory pathway (biosynthetic pathway) -
-ER to Golgi to Endosome/Lysosome

Endocytic pathway (recycling) -
-Cell surface to Endosome to Golgi/ER/Lysosome

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78
Q

How can proteins be modified as they transit the ER & Golgi?

A

Glycosylated by the addition of oligosacchardies & proteolytically cleaved

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79
Q

What is the purpose of glycosylation?

A
  • Assists folding
  • As a ligand
    - Intracellular for trafficking/sorting
    - Outside the cell for interactions with
    the ECM & proteins on other cells
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80
Q

Give at least 2 advantages & 2 disadvantages for sing Yeast as a model organism for understanding cell membrane trafficking.

A

+ Amenable for genetic studies (Can be haploid or diploid)
+ Entire genome sequence fully known & annotated
+ Limited gene diversity
+ Fundamental pathways conserved

  • Limited cell-cell contact - unlikely to be informative about multicellularity
  • Small, so high resolution imaging studies of intracellular compartments is difficult
  • Has a cell wall (can preclude some studies)
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81
Q

Regarding Novick & Schekman’s experiment (1980) using yeast to identify genes involved in the secretory pathway

What was the key aim of the experiment?

What was the rationale for the approach?

A

Aim - To investiage the secretory pathway in yeast

Rationale - If proteins couldn’t be secreted, the cell would increase it’s density as the vesicles varrying the proteins accumulate

They can also look at the changes in proteins that are normally secreted

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82
Q

Regarding Novick & Schekman’s experiment (1980) using yeast to identify genes involved in the secretory pathway

What methods of experimental analysis were used?

A

1) Asssays looking for global defects in secretion (but not the stage of defect) - analysed cells for their ability to secrete enzymes (invertase & acid phosphatase) at permissive and restritctive temperatures.

They defined secretory mutants as those which fail to export active invertase & acid phosphatase, but continued to synthesise protein under restrictive growth conditions

2) Electron microscopy alterations in the normal ultra-structure of cells could be observed

e.g. accumulation of vesicles or aberrant membranous structure

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83
Q

Regarding Novick & Schekman’s experiment (1980) using yeast to identify genes involved in the secretory pathway

What genes were identified? What can this say about secretion?

A
  • 23 genes identified by grouping mutants with similar phenotypes
  • At least 23 distinct gene products are required to ensure the transport of proteins from the ER to plasma membrane
  • Mutant groups placed in sequential order by combining mutants from different classes & using more detailed analysis of protein modifications
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84
Q

Give 3 possible reasons why all the genes/protein involved in the exocytic pathway were identified by Novick & Schekman?

A

1) They only identified temperature sensitive mutants

2) They only considered secretion to the plasma membrane, so defects in transport to endosome/vacuole won’t be identified

3) Andy ‘redundantly’ functioning genes wouldn’t be identified

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85
Q

What is endocytosis?

Why is it important?

A

Endocytosis - The process which the plasma membrane invaginates into the cell, resulting in the production of a vesicle that is able to fuse with endosomes & enter the endo-lysosomal membrane system

Important for:
1) Retrieval of molecules that formed part of the secretory vesicle for recycling

2) Downregulation of signals

3) Remodelling of cell surface lipid & protein composition

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86
Q

What is the function of Vacuolar/Lysosomal protein sorting?

What do these organelles contain that needs to be kept seperate from the rest of the cell?

A

Degradation of extracellular material taken up by endocytosis aswell as certain intracellular components by autophagy

Contain degradative/proteolytic enzymes

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87
Q

Briefly describe the process of Lysosomal/Vacuolar sorting

A

Lysosomes resident enzymes transported to the lysosome through the secretory pathway

At the last Golgi compartment (Trans Golgi network), they are sorted into a pathway destined for lysosomes rather than the plasma membrane

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88
Q

Describe Vacuolar protein sorting (VPS) screens found vps genes (Experiment)

A

Carboxypeptidase Y (CPY) is normally transported to the lysosome having been trafficked through the ER & Golgi

Labs generate mutagenised cells and looked for cells which secreted CPY (Using a colour-based assay)

Cells which secreted CPY were investigated using microscope and biochemical techniques

Over 60 VPS genes have been identified this way, vps mutant strains can be combined to determine the order of action of the genes

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89
Q

What are the 4 possible destinations of molecular determinants being trafficked from the late Golgi?

A

1) Plasma membrane

2) Early endosome

3) Late endosome / MVB (Multivesicular Body)

4) Vacuole

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90
Q

Describe the CPY pathway (Sorting to the late endosome)

A

1) CPY is synthesised in a prepero form

2) Transported through the ER to the Golgi - The transport step requires Clathrin & Gga1 & Gga2 (cytoplasmic factors)

3) Sorting - in the late Golgi, CPY is specifically recognised by a receptor Vps10 (receptor mediated sorting)

4) CPY dissociates from Vps10 at the late endosome/MVB and is transported to vacuoles

5) Here it is cleaved to the mature form

6) Vps10 is retrieved to the late Golgi through a specific aromatic-based signal in its protein sequence (YSSL, FYVF)

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91
Q

Give a basic description of the Nuclear Pore

-Where it’s formed

-What it’s made of

-What it looks like

A

Formed at junction of inner & outer membranes of nuclear envelope

Nuclear pore complex consists of around 30 different nucleoporins

Each complex appears to be made up of 8 subunits with a central plug

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92
Q

What do Nuclear Pore complexes do?

A

Involved in moving substances across the nuclear envelope

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93
Q

Give an example of Nuclear Pore complexes in action

A

In DNA synthesis
-Histone molecules required to package the new DNA, they’re transported from the cytoplasm

Protein production
-Ribosomal subunits formed in the nucleolus have to enter the cytoplasm

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94
Q

Substances transported by pore complexes occur by 2 processes…

A

1) Diffusion (Under 60,000 molecular weight)

2) Active diffusion

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95
Q

Give proof of experimental evidence that Transport is Active at nuclear pore complexes:

1) In Cells

2) In vitro

A

1) mRNA transport out of the nucleus is inhibited on cooling to 4 degrees (ATP hydrolysis required)

2) Importation of proteins into nucleus
In absence of ATP, protein binds to the pore complex but remains outside the nucleus
Add ATP and the proteins start to appear inside the nucleus

96
Q

Describe the signalling process for getting proteins into the mitochondrial matrix

A

1) N-terminal signal sequence is recognised by the TOM complex

2) The protein translocates through TOM & TIM23

3) Translocates through TIM23 into the matrix

4) Signal is cleaved off

97
Q

Name 3 essential components for all transport vesicle formation…

A

1) GTPase

2) Adaptor proteins

3) Coat

98
Q

What is the function of Guanine nucleotide exchange factors (GEFs)

A

It’s a small GTPase (molecular switch)

Turns transport vesicles from GDP inactive –> to GTP active

99
Q

What is the function of GTPase Activating Proteins (GAPs)

A

It’s a small GTPase (molecular switch)

Turns transport vesicles from GTP active –> to GDP inactive

100
Q

Regarding Tranpsort vesicles, Describe how reconstitution experiments allow us to understand what makes a COPII vesicle

A

1) ER membranes containing ribophorin

2) Add Cytosol, ATP & GTP

3) Observe COPII vesicles containing p58

4) Centrifuge them, vesicles are less dense than ER so can observe them floating higher

101
Q

What do the following do?

GDP mutant

GTP mutant

Sar1GDP

A

GDP mutant - Sequester GEFs

GTP mutant - Can’t hydrolyse GTP

Sar1GDP - Inhibits COPII formation

102
Q

Regarding vesicular trafficking, what do Adaptor proteins do?

A

-Recgonise & select cargo - ensuring specificity

-Link the coat to the membrane

-Recgonise motifs in the cytoplasmic domains of membrane proteins

-Adaptor complexes show a precise subcellular localisation

103
Q

Regarding membrane trafficking in disease, what do in vitro studies of F382L SEC23A mutants show?

A

Shows binding of mutant SEC23A to liposomes is unaffected

However COPII coated vesicle formation is affected

104
Q

What does COPII stand for?

A

Coat protein complex 2

105
Q

Discuss the mutations observed in the patients of this case study:

Cranio-lenticulo-sutural dysplasia is caused by a SEC23A mutation leading to abnormal ER to Golgi trafficking

A

-All 6 of the affected individuals were homozygous for the 114 T -> C transition in exon 10 of SEC23A
-Not present in 600 controls
-Protein sequence allignment shows that F382 is invariably conserved in at least 10 species

106
Q

Regarding the case study on: Cranio-lenticulo-sutural dysplasia is caused by a SEC23A mutation leading to abnormal ER to Golgi trafficking

Describe the methodology and observations

A

Wt & Mutant fibroblasts observed with PDI immunofluoresence

Shows-
-Cells visualising pro-collagen COL1A1, shows similar structures with clear colocalisation of PDI & COL1A1

Observe -
-Fine reticular appearance of ER in Wt
-Marked dilation of ER in mutants

-Immunofluoresence with SEC31 antibody produced punctuate staining mostly in perinuclear region of Wt, and diffuse cytoplasmic mislocalisation in mutants

107
Q

Briefly describe the Rab family

A

-Member of Ras superfamily

-Distinct subcellular localisation

-Cycle between membrane and cytosol

-Req for fusion & trafficking functions

108
Q

Give an example of Rab in disease

A

1) Charcot-Marie-Tooth 2B
-Rab7a missense mutations
-Leads to excessive activation, reduce autophagic flux, inhibition of neurite growth

2) Harnessing of RabGEFs by pathogens
-Legionella pneumophila can recruit Rab1 to cell surface
-Creating an ER-like compartment where it can replicate

109
Q

what colour is radar

A

orange

110
Q

What are the roles of Smooth ER & Rough ER

A

Smooth ER - Lipid synthesis, role in calcium storage

Rough ER - Protein synthesis

111
Q

Regarding advances in microscopy:

What is CLEM? What does it do?

A

Correlative light & electron microscopy

Allows localisation of fluorescently labelled proteins to MCS (Inter membrane contact sites)

112
Q

Regarding the molecular machinery of inter membrane contact sites (MCS)

What are Tethers?

Give an example

A

Tethers - Protein lipids that inhibit fusion

Obsp -
-A tether & lipid transfer protein
-Define protein & lipid proteome, raft-like & enriched in sterols

113
Q

Name a calcium sensor in the ER

A

Stim1

114
Q

What does LDL stand for?

A

Low-density Lipoprotein (Cholesterol)

115
Q

Give an example of a disease linked to inter membrane contact sites (MCS)

A

TDP-43 - pathologically linked to ALS - regulates ER-mitochondria contacts

Diseases associated mutations in Hereditary spastic paraplegia - linked REEP1 - lead to disruptions of ER-mitochondria contacts

Presenillin involved in calcium exchange between ER & mitochondria

116
Q

Why do cells need degradation?

A

Homeostasis

Remove damaged components

Signalling

Recycling nutrients

Differentiation

117
Q

Name 4 mechanisms of degradation

HINT - 3 Autophagy’s

A

1) Ubiquitin/proteasome system (UPS)

2) Macroautophagy

3) Microautophagy

4) Chaperone-Mediated Autophagy

118
Q

Describe how Autophagy interacts with nutrient recycling:

A

Autophagy is rapidly upregulated under amino acid starvation

Causing non-selective bulk degradation of the cytosol

Cells lacking autophagy die under starvation

119
Q

Given an example of how Autophagy can cause cellular remodelling

A

-Ertyhropoiesis - red blood cell differentiation

-Removal of sperm-derived mitochondria

120
Q

How is autophagy linked to ageing & neurodegenerative disease

A

-Cells continuosly acquire damage

-Lysosomal capacity decreases as we age

-Reduced autophagy - major reason for age-related degradation

-Long-lived or highly metabolic cells, such as neurons & muscle, are the most susceptible

121
Q

Regarding the question - Can autophagy make you live longer?

What is the dietary restriction hypothesis

A

Starvation/Exercise
–>
Increase in Autophagy
–>
Increase in damage repair

122
Q

Identifying Atg (autophagy-related) genes allowed…

A

1) Disruption of autophagy to investigate its functions

2) A start on dissecting how machinery works

3) Observation of autophagy in live cells

123
Q

What does Neuronal-specific autophagy disruption cause in mice?

A

Accumulation of ubiquinated aggregates

Increased apoptosis

124
Q

What mutation is responsible for Huntington’s disease?

A

Polyglutamine (polyQ) expansion in Huntington protein

If the genetic code has Q<18 - healthy
If the genetic code has Q>35 - disease causing

125
Q

What is the function of small GTPases?

A

-Member of Ras supefamily

-Change conformation upon activation

-Bind & Activate downstream effectors

126
Q

Regarding Rho GTPases in Cell migration

Describe what happens with catalytic glutamate mutants (positioning of attacking water)

A

Active mutants - Q61L catalytic mutant

G12V pushes Q61 out of position & disturbs P-loop

Mutants reduce hydrolysis 10-fold

G12V conflicts with transition state geometry

127
Q

What is the downstream pathway of the GTPase - GTP-RhoA

A

GTP-RhoA
->
Rho Kinase
->
Myosin light chain
->
Actomyosin contraction

128
Q

DNA is packaged into chromatin fibres - a flexible substrate that enables:

A

1) Selective gene expression

2) Faithful replication & transmission of the genome to progeny cells

129
Q

Describe how individual chromosomes can be easily distinguished at metaphase of mitosis:

A

1) Diploid eukaryote cells contain 2 copies of each chromosome

2) Each chromosome pair differs in size & DNA sequence

130
Q

What is meant by ‘The Karyotype of the parent organism’?

A

The organised replication of all the chromomes in a eukaryotic cell at metaphase

131
Q

How does chromatin (of chromosomes) look under a microscope?

A

Interphase chromatin resembles ‘beads on a string’ - the beads are nucleosomes

132
Q

The protein subunits of the nucleosome are core histones. Why does this matter?

A

The N-terminal tails of the 8 core histone subunits project out from the nucleosome core

They’re free to interact with other proteins, facilitating regulation of chromatin structure & function

133
Q

In addition to genes, each linear chromosome has…

What do these do?

A

2 Telomeres - prevent the loss of DNA sequences from chromosomal ends

A centromere - mediates chromosome attachment to the mitotic spindle via the kinetochore

Origins of replication

134
Q

What is the nuclear periphery made up of in interphase cells?

A

Composed of transcriptionally inactive DNA - RNA transcripts excluded from the periphery

135
Q

Describe what Fractal globules look like from 3 perspectives (nucleus level, higher resolution & zoom, even higher zoom)

Why is this organisation important?

A

Nucleus level -see distinct patterns of chromatin

Zoom in - Areas of open & closed regions forming these patterns

Zoom in more - Within the closed region there are distinct patterns of chromatin - these are fractal globules

Organisation is fundamental to stabilise regions of inactive chromatin, it allows flexibility socells can react to certain cues (suchas progenitor cells differentiating into specialised cells)

136
Q

Chromosomes contain specialised DNA sequences that facilitate…

A

Reliable & Complete DNA replication

Segregation of duplicated chromosomes during cell division

137
Q

What are Telomeres?

A

Specialised repetitive DNA sequences at chromosome ends

Single-stranded 3’ overhaning TTAGGG repeat arrays are synthesised by specialised DNA polymerase (Telomerase enzyme)

Telomeres define chromosome ends and maintain chromosome integrity

138
Q

Regarding chromosome structure .What to Kinetochore Inner and Outer plate proteins bind to?

A

Inner - bind to chromatin containing alpha-satellite DNA

Outer - bind to protein components of the mitotic spindle (e.g. microtubules)

139
Q

Increasing biological complexity is accompanied by…

A

Increasing numbers of protein coding genes

Increasing amounts of non-protein coding DNA, for regulating transcription & organising access to protein coding genes

140
Q

Name the 3 types of Transposon

A

DNA Transposons

Retroviral retrotransposons

Non-retroviral polyA retrotransposons

141
Q

How do DNA transposons move?

A

By a cut & paste mechanism - without self-duplication

Requires Transposase (transposon-encoded enzyme)

DNA transposons are powerful mutagents

142
Q

How do Retroviral retrotransposons move & replicate?

A

Replicate via RNA intermediates

Producing new DNA copies that integrate at new genomic locations, using self-encoded Reverse Transcriptase

143
Q

How do Non-retroviral polyA retrotransposons replicate?

A

Via an RNA intermediate using it’s own retrotransposon-encoded Reverse Transcriptase

Copy & Paste mechanism

144
Q

How is DNA synthesis initiated?

A

By creating a replication fork where the DNA strands are seperated

145
Q

DNA polymerase can’t start making a DNA chain without a a pre-existing chain or short stretch of nucleotides.

What is this called? What does it do?

A

Short RNA primer - synthesised using template and NTPs by DNA primase

Once the primer is in place, DNA polymerase ‘extends it’

146
Q

Regarding DNA synthesis:

What do the following enzymes do?

Ribonuclease H

DNA Ligase

A

Ribonuclease H - Extends across the gap

DNA Ligase - Seals the nick

147
Q

What does DNA Helicase do?

A

Uses ATP to seperate parental DNA strands at the replication fork and move the replication fork forward

148
Q

The processivity of DNA Polymerases is greatly enhanced by their association with a sliding clamp.

What is the sliding clamp?

A

Encircles the DNA (like nut on bolt) to help move DNA polymerase forward

ATP dependent

Positioned close to Primer

149
Q

Briefly describe the role of Single-stranded DNA Bindind Proteins (SSBs)

A

Expose single-stranded DNA in the replication fork

Making it avaliable for templating synthesis of the new DNA strand and easing replication fork progression

150
Q

What do DNA Topoisomerases do?

A

Prevent DNA from becoming tangled during DNA replication

Enhance processivity of DNA polymerase

151
Q

What does Helicase unwinding of parental DNA strands at the replication fork introduce?

A

Superhelical tension into the DNA helix

152
Q

What are the key differences between Type I and Type II topoisomerases?

A

Type I - nick & reseal one of the 2 strands - No ATP required

Type II - nick & reseal both DNA strands - ATP req

153
Q

Initiation of DNA replication in eukaryotes is biphasic - What happens during G1 phase? What happens during S phase?

What is the role of Temporal seperation of these 2 events?

A

G1 - Replicator selection occurs - Formation of a pre-replicative complex

S - Origin activation - Unwinding of DNA & recruitment of DNA polymerase

Temportal seperation - ensures that each origin is used and each chromosome is only replicated exactly once per cell cycle

154
Q

Briefly describe how Eukaryotic replicator selection in G1 leads to the formation of a Pre-Replicative Complex (pre-RC)

A

Origin Recognition Complex (ORC) binds to replicator sequence

Helicase-loading proteins Cdc6 & Cdt1 bind to ORC

The Helicase Mcm2-7 binds to complete the formation of pre-RC

155
Q

What is the role of high levels of Cyclin-dependent kinase (Cdk) activity in S-phase

A

Activates existing pre-RC

Prevents formation of new pre-RCs

156
Q

What is the function of Ribonuclease H?

A

Removes RNA primers, further shortening the newly synthesised DNA strands at 5’ ends of chromosomes

Risks loss of valuable coding information

157
Q

Telomeres contain an RNA component that specifies telomere sequence

A

Telomerase is a ribonucleoprotein with an intrinsic RNA component that acts as a template

On which telomere repeat sequences are synthesised in a step-wise process - the Telomerase Shuffle

Telomerase RNA allows the addition of multiple TTAGGG repeats to the 3’-OH at each telomere

158
Q

Name 2 Endogenous & 2 Exogenous sources of attack towards cells

A

Endogenous -
1) Reactions with other molecules within the cell
2) Hydrolysis, oxygen species, by-products of metabolism

Exogenous-
1) Reactions with molecules outside the cell
2) UV, X-rays,Carcinogens, chemotherapeutics

159
Q

Name 3 types of DNA damage

Hint - Effects one strand of DNA helix

Hint - Spontaneously occurs within the cell

A

1) Depurination (Abasic sites)

2) Deamination

3) Methylation

4) Replication errors

160
Q

Name 3 types of Exogenous DNA damage

How many strands of the DNA helix do they effect?

A

1) Pyrimidine dimers - Effect 1 strand

2) Double strand breaks - Effects both strands

3) Interstrand crosslinks - Effects both strands

161
Q

In general, transition mutations are more likely than transiversion mutations

Why is this the case?

A

Because substituting a double ring structure for another double ring is more likely than substituting a double ring for a single ring

162
Q

What are the consequences of Depurination (Abasic site)

A

Results in a frameshift mutation
–>
Generate missense proteins

163
Q

What is an Abasic site (AP)

A

A location in DNA that has neither a purine nor a pyrimidine base, either spontaneously or due to DNA damage.

164
Q

What are the consequences of UV light damage?

A

Can cause interstrand DNA crosslinks & DNA-protein crosslinks - which are highly toxic as they block replication & transcription

Can induce formation of pyrimidine dimers - which distorts the DNA

165
Q

What is the role of Base excision repair (BER)? How does it work?

A

Repairs base damage - such as abasic sites & deamination

Uses a base-flipping stratefy to identify errors

166
Q

What is the role of Nucleotide excision repair (NER)? How does it work?

A

Repars damage when more than one base is involved (e.g. pyrimidine dimers caused by UV)

Involves the excision of short patches of single-stranded DNA to remove the affected bases

167
Q

Translesional DNA polymerases can replicate highly damaged DNA

What do they lack?

What do they cause?

A

Lack-
-Precision in template recognition & substrate base
-Exonucleolytic proof-reading activity

Cause-
-Most base substation and single nucleotide deletion mutations

168
Q

Regarding the 2 mechanisms to repair double stranded DNA breaks

Discuss Non-homologous end joining (NHEJ)

A

Usually results in the loss of nucleotides surroinding their break site

-Error prone
-Restricted to G1 phase
-Important genetic info may be lost

169
Q

Regarding the 2 mechanisms to repair double stranded DNA breaks

Discuss Homologous recombination (HR)

A

Error-free repair

Only occurs in S-phase

Uses intact sister chromatid as template

Double strand break is accurately repaired

170
Q

Name the 3 places where DNA is detected and acted upon to STOP the cell cycle

A

1) G1

2) Entry into S-phase

3) Entry into mitosis

171
Q

How is DNA damage detected?

A

ATM/ATR get activate and associate with the site of DNA damage

171
Q

How is DNA damage detected?

A

ATM/ATR get activate and associate with the site of DNA damage

This activates other kinases to block the cell cycle

p53 is stabilised and activates p21

p21 renders the G1/S-CDK and S-CDK complexes inactive, thus preventing cycle progression

DNA is then repaired (apoptosis if not possible)

172
Q

Give an example of cancer & defects in double-stranded break repair

A

10% of breast cancer is inherited

80-90% of these cases are BRCA1/2 associated

BRCA1/2 carriers have a 80% lifetime risk (10x normal amount)

173
Q

Define the following terms:

Mechanobiology -

Mechanotransduction

Mechanosensing

A

Mechanobiology - The study of how physical forces and changes in cell tissue or tissue mechanics contribute to development, physiology & disease

Mechanotransduction - the conversion of a physical force to a biochemical response

Mechanosensing - when a protein/cellular structure responds to a physical cue to initiate mechanotransduction

174
Q

Describe the steps of mechanotransduction

A

1) Mechanosensing
-Cells test their encironment
-Adhesion receptors, membrane proteins probe the ECM
–>
2) Signal transduction
-Mechanical signal is transduced along a linked network
-Cytoskeleton is often the force conduit
–>
3) Signal integration at nucleus
-Accumulation of signals over time
-Chromatin rearrangement, nuclear pore opening
–>
4) Cellular response
-Shape, motility & growth

175
Q

Give up to 3 examples of Mechanotransduction

A

1) Blood pressure & coronary artery disease - myogenic tone

2) Cells change their cytoskeleton upon fluid flow (e.g. Endothelial cells become strained with fluid flow)

3) Auditory mechanotransduction and hearing

176
Q

Describe how mechanical forces can promote tumour aggression

A

An expanding tumour mass results in increased solid stress

This stress, combined with the mechanical resistance produced by the ECM & Stromal cells - promotes an increase in interstitial pressure

High hydrostatic pressure forces plasma to exit blood and lymphatic capillaries to enter the interstitial space

High solid stress & interstitial pressure can impair lymphatic drainage & drug delivery

177
Q

Define the following terms:

Solid stress

Interstitial pressure

A

Solid stress - force exerted by solid structural components of a tissue experiencing growth

Interstitial pressure - relates to the interstitial fluid occupying the space between cells and containing water

178
Q

How can mechanical stresses be countered?

A

-Tumours develop a desmoplastic response characterised by the recruitment of fibroblasts & immune cells with increased deposition of ECM proteins (including collagen & fibronectin)

-Fibroblasts can stimulate tumour cell growth through paracrine factors, along with tumour cells - to remodel the ECM through cell-generated tesnion and elevated production of ECM molcules and cross-linking enzymes

A linearised and stiffened ECM provides tracks for immune infiltration and may facilitate tumour cell invasion & metastasis

179
Q

How can a stiffened matrix free Beta-catenin to relocated to the nucleus

A

Strengthens cell-ECM interactions in tumour cells

Prompting the disruption of E-cadherin-mediated cell-cell junctions

Thus freeing Beta-catenin to relocate to the nucleus

180
Q

Breast cancer cells cultured on a stiff matrix exhibit SCRIB mislocalisation, leading to…

A

…Nuclear translocation of the Hippo signalling pathway transcriptional coactivator TAZ, to induce stem-like programming of tumour cells

181
Q

Name another consequence of tumour cell interaction within a stiff matrix

A

Integrin receptor clustering & adhesion plaque formation, through the recruitment of vinculin, talin and other focal adhesion components

182
Q

What does focal adhesion maturation stimulate?

A

RHO/ROCK-mediated actomyosin contractility and intracellular signalling through FAK, ERK & PI3K - to enhance cell growth and suvial

183
Q

How can Integrin clustering be further modified?

A

By a bulky glycocalyx, which creates a membrane kinetic trap for integrin complex assembly

184
Q

Mechanical stress on tumour cells also activates…

A

Cellular production and secretion of WNTs

This may drive stem-like phenotypes in tumour cells via Beta-catenin activity

185
Q

The mechanical action of integrin adhesion & cell-generated tension releases latent TGF-Beta from the ECM, allowing it to…

A

Potentially stimulate tumour cell EMT, invasion & metastasis

186
Q

Name 3 mechanosensors

A

Piezo channels

Integrins

Caveolae

187
Q

What causes cystic fibrosis?

A

Mutation of a chloride ion channel that results in thickened mucus that cilia can no loner move –> Results in lung infections

188
Q

How can the body distinguish between the pathogen and ‘self’ cells

A

-Differences between cells & pathogen

Such as Lipopolysaccharides (LPS) - component of gram-negative bacterial cell wall - an AA in bacteria only

These are known as pathogen-associated molecular patterns (PAMPs)

Similar mechanisms are used to identify damaged ‘self’ cells on the basic of damage-associated molecular patterns (DAMPs)

189
Q

What do TLRs do?

A

Toll-like receptors

Detect PAMPs

190
Q

How do TLRs function?

A

They are a molecular signalling cascade

Signal through downstream effectors such as Jun/Fos transcription factors and NFkB and ultimatelu change gene expression

191
Q

What happens to cells when they recognise a PAMP

A

Become activated and secrete molecular ligands which attract additional cells of the innate immune system

192
Q

Activation of the immune system triggers…

A

Inflammation causing dilation of local blood vessels, pain

-Dilated vessels become permeable & endothelial cells become sticky & catach white blood cells, facilitating their access

-Further pro-inflammatory cytokines are released (prostaglandins, histamines & cytokines)

-Fever inhibits pathogen proliferation & speeds chemical reactions used by antimicrobial peptides

-Response appropriate locally can be dangerous systematically - this is shock: loss of plasma volume, crash of blood pressure, cytokine storm, clotting

193
Q

Name and Describe the 3 specialist phagocytic cell types recruited during the innate immune response

A

Neutrophils
-Short lived phagocyte
-Abundant in blood but not tissues
-Migrate to sites of infection

Marcophages
-Long lived professional phagocytes
-Abundant in areas likely to be exposed to pathogens (airway, gut)

Eosinophils
-Specilalists in attacking objects too large to engulf

194
Q

Dendritic cells link innate and adaptive immunity

Describe Dendritic cells, what they do and where they migrate to:

A

Specialist phagocytic cells derived from monocytes

Express a large variety of recognition receptors (TLRs)

Phagocytoses pathogens, cleaves into peptides which are bound to MHC proteins

They migrate to lymphoid tissue, activate & stimulate T cells of the active immune system

195
Q

What are MHC proteins?

A

Major Histocompatability Complex Proteins - group of genes that code for proteins found on the surfaces of cells that help the immune system recognize foreign substances.

196
Q

Describe the function of Adaptive immunity

A

Generate highly specific response to specific pathogens

Can identify, target & destroy a vast range of pathogens/toxins

It’s important to direct Adaptive immunity against foreign targets and not ‘self’ proteins

197
Q

Describe the action of lymphocytes in adaptive immunity

A

1) Lymphocytes develop within the thymus and bone marrow (primary lymphoid organs)

2) They migrate to secondary lymphoid organs (spleen, appendix), where they’re exposed to foreign antigens

3) Lymph drains into bloodstream and cells circulate

198
Q

What are NK cells?

A

Natural Killer cells

-Part of early defence

-Against foreign & autologous cells undergoing various forms of stress (microbial infection & tumour aggression)

-Are lymphoid cells but part of the innate immune response

199
Q

What are the 3 subtypes of T-cell responses?

A

Cytotoxic T-cells - Directly kill infected host cells

Helper T-cells - Activate macrophages, dendritic cells, B-cells & cytotoxic T-cells - by secreting a variety of cytokines & displaying co-stimulatory proteins on their surface

Regulatory T-cells - Use similar strategies to Helper T-cells to inhibit the function of helper T cells, cytotoxic T cells & dendritic cells

200
Q

How does active immunity work?

A

1) Body randomly generates a library of lymphocytes

2) When an antigen is presented by a dendritic/Helper T-cell, they become activated if that have binding affinity

3) Binding of antigen to activated cells leads to their proliferation & clonal expansion

4) Expansion triggers differentiation into effector cells
-An expansion round occurs everytime an antigen is encountered

5) Subsequent encounters can stimulate the memory cells

201
Q

What experimental evidence is there showing that the host can attack self-antigens?

A

Knockout a gene encoding a ‘self’ protein

Allow animal to grow, then reintroduce into host

Host now mounts an immune response as it hasn’t ‘learnt’ that this is ‘self’

Host can mount an attack against self-antigens

202
Q

What experimental evidence is there showing that the immune system can forget what it has previously learnt

A

Remove ‘self’ protein from adult animal

Reintroduce after several weeks/months

Host mounts an immune response to the removed protein

Shows the system can ‘forget’ what is has previously ‘learnt’

203
Q

What is the basic structure of immunoglobulins?

Name the 5 classes

How are they seperated into subclasses?

A

2 antigen binding sites + 2 light chains + 2 heavy chains

IgG, IgM, IgA, IgD, IgE - each has its own heavy chain

Subclasses such as IgG1, IgG2 - each have different hinge & tail structures

204
Q

What do the Constant and Variable domains of immunoglobulins do?

A

Constant - interact with other parts of immune system

Variable - make up antigen binding site

205
Q

Regarding the DNA encoding variable domain

Describe what the following contain:

-a k-light gene variable domain in the germline

-a heavy chain gene variable domain - in B-cells

A

k-light chain - 40 V, 5 J, single C domain

heavy chain B-cell - 40 V, 25 D, 6 J, 5 C domains - they are recombined by VJ recombination

206
Q

Why do developing B-cells join together seperate gene segments in DNA?

A

In order to create the genes that encode the primary repertoire of low-affinity antibodies

207
Q

Regarding VJ recombination - What happens during the development of a B-cell?

A

A coding sequence joining a V to a J segment is assembled by removing the intervening DNA

Transcription starts immediately uptream of the fused V segment, which lies just upstream of a J region

Extra downstream J segments are transcribed but edited out of the mRNA transcript

208
Q

What is the function of VJ recombination

What is the use of VJ recombinase enzyme?

A

-Joins seperate antibody gene segments together to form a functional VL- or VH- region coding sequence

-VJ recombinase (encoded by RAG1 & RAG2 genes) drives DNA splicing

209
Q

What is Junctional diversification?

A

During VJ recombination, nucleotides are lost or inserted from the ends of the recombining gene segments

210
Q

What is Allelic exclusion

A

When developing B/T-cells are diploid but choose just one allele to recombine

211
Q

What is affinity maturation?

A

Progressive increase in affinity of the antibodies over time after initial immunisation

212
Q

What happens after B cells have been stimulated by antigen & helper T-cells?

A

Some activated B cells proliferate rapidly in lymphoid follicles and form germinal centers

213
Q

What is Somatic hypermutation?

A

When B cells mutate at the rate of one mutation per V-region coding sequence per cell generation

Driven by activation-induced deaminase (AID) expressed in germinal centres

214
Q

What should we expect from cells experiencing double stranded breaks (VJ recombination) and high levels of DNA damage (somatic hypermutation)?

A

-To apoptosis via the p53 pathway
-Acts as ‘watch keeper’ to kill cells with potentially oncogenic mutations

215
Q

What is the role of BCL-6 regarding ‘watch keepers’?

A

BCL-6 - transcriptional repressor expressed in germinal centers

BCL-6 binds to sites in the p53 promoter, switching off expression

Leaves the germinal centre without a ‘watch keeper’ oversight

216
Q

Briefly describe how T cells bind to T-cell receptors (TCRs)

A

-T-cells are activated by partly degraded antigens displayed on the surface of antigen presenting cells

T cells bind to MHC proteins via their T-cell receptors

Antigen presenting dendritic cells can either activate or tolerise T-cells

217
Q

Describe the structure of T-cell receptors?

How is TCR diversity generated?

A

Immunoglobulins that contain variable domains & hypervariable loops

TCR diversity is generated by VJ-like recombination & junctional diversification in the thymus

218
Q

Describe the process of cancer ‘immunoediting’

A

1) Elimination phase - tumour cells killed by NK, CD4+ & CD8+ cells

2) Equilibrium between immune & tumour cells

3) When an immune system is unable to destroy tumour cells - ‘escape’ leads to clinically detectable tumours

219
Q

Regarding Cancer immunotherapy

What are:

Checkpoint inhibitors

Cytokines

Immunomodulators

A

Checkpoint inhibitors - take ‘brakes’ off the immune system

Cytokines - stimulate immune cells to attack cancer

Immunomodulators - boosts part of the immune system

220
Q

Regarding cancer immunotherapy

What are:

Cancer vaccines

Monoclonal antibodies

Oncolytic viruses

A

Cancer vaccines - direct immune respoinse to prevent a specific cancer epitope/causing pathogen

Monoclonal antibodies - directed agaisnt cancer specific antigens

Oncolytic viruses - developed in a lab to kill & infect certain tumour cells

221
Q

What is Chimeric antigen receptor (CAR) T-cell therapy?

A

Type of cancer immunotherapy

-Take patients T-cells and infect with a recombinant virus that causes the expression of TCR with an antibody-derived variable domain specific to the tumour antigen

-This generates T-cells able to attach to tumour cells

-These are transfused back to the patient, where they find, attach to and kill the cancer

222
Q

Compare & contrast the structure & function of nicotinic receptors & voltage-gated sodium channels (5 marks)

A

1 mark - both permeable to sodium, cause depolarisation & action potential firing

1 mark - nACHrs made up of 5 protein subunits, each subunit has 4 TMs

1 mark - VGNA one protein has 4 domains, each domain has 6 TMs

1 mark - Nicotinic gated by ligand

1 mark - VGNA gated by change in voltage / depolarisation

223
Q

How does agonist binding to a GPCR lead to increased production of cAMP? (4 marks)

A

1 mark - Ligand binding to receptor causes the affinity of the G protein alpha subunit for GDP to decrease

1 mark - This bring about the exchange of GDP for GTP

1 mark - GTP bound alpha subunit dissociates from beta gamma subunits

1 mark - GTP-bound alpha subunit & beta-gamma subunit are now free to engage downstream effectors, changing the levels of second messenger

224
Q

Briefly describe the function of SNARE proteins (3 marks)

A

1 mark - SNAREs involved in vesicle fusion

1 mark - They form a coiled-coil structure

1 mark - Between vesicles & target membranes forcing membrane merger

225
Q

What is Telomerase & why is it essential for cell viability? (5 marks)

A

1 mark - Specialised DNA polymerase

1 mark - which specifically synthesises multiple copies of a simple sequence hexameric repeat

1 mark - Which are called telomeres, found at the ends of linear eukaryotic chromosomes

1 mark - Telomeres prevent the gradual loss of DNA sequences at ends of chromosomes in proliferating cell populations

1 mark - Which would otherwise occur due to the removal of RNA primer sequences from the Okazaki fragment at each end of a recently replicated chromosome

226
Q

Explain how pyrimidine dimers are removed from DNA and how the damaged DNA is then repaired - Name the 4 main enzymes involved (4 marks)

A

1 mark - Excision Nuclease cleaves the damaged DNA strand on either side of the pyrimidine dimer, several nucleotides away from the dimer

1 mark - DNA Helicase removes the fragment of single-stranded DNA containing the pyrimidine dimer, leaving approx 12 nucleotide gap

1 mark - DNA Polymerase repairs the gap with new DNA synthesis from the primer:template junction

1 mark - DNA Ligase seals the nick

227
Q

Which 2nd messenger does Adenylyl cylase activate?

A

cAMP

228
Q

Describe the structure in terms of protein domains of a GPCR

A

Seven transmembrane domains

Connected by 3 extracellular & 3 intracellular loops

The N-terminal is on the extracellular side and where the ligand binds and the C terminal is in the cytoplasm where an additional helix domain allows the G-protein to bind

229
Q

How many subunits make up P2X receptors?

A

3 subunits make up the trimeric receptor

230
Q

Which 3 stimuli cause platelet activation?

A

Basal Lamina

Thrombin

ADP

231
Q

Name one of the 2nd messengers activated by phospholipase C and state whether it’s membrane bound or cytoplasmic

A

Inositol 1,4,5-triphosphate (IP3) - Cytoplasmic

OR

Diacylglycerol (DAG) - membrane bound

232
Q

How many histones make up the nucleosome

A

8 histones / 2 lots of 4 different histones

233
Q

What 2 features of chromosomes maintain genomic stability?

A

Telomeres & Centromere

234
Q

Describe the proccess of non-homologous end-joining (NHEJ)

A

Double strand break gets resected creating a 3’ overhang

The KU70/Ku80 heterodimer binds to either end of the damage site, along with DNApk, creating a synaptic complex which brings the ends in close proximity to one another

Endonuclease cleaves the 3’ overhang and DNA ligase IV seals the nick

235
Q

How does the kinetochore maintain genetic stability?

A

Connects microtubule to centromemer

Outerplate forms a ring structure that microtubule slots into

Inner plate binds to DNA - recognises centromere specific nucleosomes

Drives equal segregation of chromosomes for daughter cells (unequal segregation leads to instability & promotes tumourgenesis)

236
Q

The deamination of a cytosine base leads to a transversion mutation. True or False? Explain your answer

A

False - C->T is a transition mutation not transversion

Transversion would be if cytosine changed to G/A as pyrimidine is converted into purine

Deamination of cytosine –> Uracil (purine) so it’s a transition

L2 Mol Cell (1 decks)

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