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CEDB30002 Cell Biology > Core Cellular Functions - Proteins, Signalling and Polarity > Flashcards

Core Cellular Functions - Proteins, Signalling and Polarity Flashcards

1
Q

What causes protein misfolding disease?

A

Caused by misfolding of proteins into beta-sheet aggregated structures (become sticky)

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2
Q

What leads to the formation of amyloid plagues?

A

• B sheet aggregation lead to intermolecular aggregates → oligomers → protofibrils → fibrils → amyloid

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3
Q

Why might protein misfolding occur?

A
  • Gene mutation
  • Somatic mutation
  • Transcription/translation errors
  • Failed folding and chaperone machinery
  • Post-translational modification/trafficking mistakes
  • Clearance failure (too much, e.g. hard to ub)
  • Environment (e.g. toxin)
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4
Q

What are the features of prion disease?

A
  • Neurodegenerative
  • Fatal
  • Long IP
  • Host encoded prion protein (PrP) accumulates
  • Transmissible
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5
Q

What are the three forms of human prion disease? What is the occurrence like for each?

A

• Sporadic (85%) , inherited (15%) , acquired (rare)

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6
Q

What caused Scrapie in the 1930s?

A

• Sheep
• Brains of affected sheep used to make Louping ill virus vaccine (antigen present because brain virus)
• 2.5 years, vaccinated flocks infected
o Infective agent in brain, spinal cord, spleen
o Withstand high conc of Formalin (kill conventional infectious agents)
o 2 year IP

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7
Q

What is a prion?

A

Proteinaceous Infectious Particle
• Resistant to inactivation by procedures that modify nucleic acids
• mRNA in infected and non-infected

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8
Q

What is the basis of the infectious protein/prion hypothesis?

A
  • Host DNA makes normal protein

* Mutation, sporadic event causes infectious disease associated protein to arise

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9
Q

How do PrPc and PrPSc compare?

A 
PrPc
Post translational modification of normal cellular protein
Protease sensitive
Soluble
Alpha helix
Many tissues
Required for infection

PrPSc
27-30kDa protease resistant protein that co-purifies with infectivity
Concentration correlates to infectivity 
Required for PrP transmission, pathogenesis 
Protease resistant
Insoluble
Beta sheet
Disease specific
Infectious
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10
Q

What are the methods for studying prion disease?

A
  • Epidemiology
  • In vivo animal models
  • In vitro cell culture
  • Biochemical systems
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11
Q

Can a PrP knockout get prion disease?

A

No

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12
Q

What is the process for cell free propagation of a prion?

A
    1. Put PrPc(uninfected) and PrPsc(infected) in material (brain)
    1. Shake up (300 rpm, overnight, 37degrees)
    1. PK treat, run on gel
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13
Q

What does efficient prion propagation require? What features of cell free prions demonstrate this?

A
  • Propagation: partially purified PrPsc can induce conformational change in mammalian and recombinant PrPc
  • Infectivity: spontaneously generated from mammalian and recombinant PrPc and from recombinant protein in presence of cofactors (RNA and Lipid induced misfolding of recombinant PrP)
  • Efficient prion propagation requires PrP and host derived factors
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14
Q

How do HY and DY strains of hamster scrapie differ?

A

o HY: short IP, brain stem and cerebellum, hyperactivity
o DY: long IP, pyramidal layer near hippocampus, lethargy
o Different electrophoretic mobility, PK digestion, FTIR spectroscopy profile
o Different conformation of PrPSc = strain variation

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15
Q

How can you work out the features of prion strains since they have no genome?

A 
Biochemical and biophysical properties 
•	PK digestion then electrophoretic mobility to show conformation, differences in beta sheet content
•	Glycosylation 
•	Polymorphism in prion protein gene
•	Region of brain affected
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16
Q

What are the acquired prion diseases?

A

Kuru (humans)
BSE (cows)
Variant CJD (humans)
Latrogenic

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17
Q

What are the features of Kuru?

A
  • Cannibalism
  • Cerebellar ataxia and cognitive decline
  • No vertical or domestic transmission
  • Long IP
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18
Q

What are the features of vCJD?

A
  • Exposure to BSE

* Clinical and neuropathology different to CJD but similar to BSE

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19
Q

How is latrogenic prion disease acquired?

A
  • Medically acquired
  • Surgical transmission (contamination)
  • Biological matter contamination (organs, hormones)
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20
Q

How can prion disease infection be controlled?

A
  • Sterilisation kills living things
  • Disinfection renders transmissible agents inert
  • WHO recommends chemical disinfectant , followed by steam sterilisation
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21
Q

What directs proteins to the right place in cells?

A

Signal sequences

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22
Q

What structures are important in gated transport?

A

• Nuclear envelope (double membrane, nuclear pores, continuous with ER)
NPCs

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23
Q

What is the size limit for free transport through NPC? How do bigger proteins get through?

A

60 kD.

Active Transport

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24
Q

How do cargo proteins get through NPC?

A
  1. Cargo proteins bind specific nuclear IMPORT receptors (NIR) via nuclear LOCALISATION signals (NLS)
    o Important for nuclear localisation
  2. NLR interact with NPC proteins, transfer cargo in or out
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25
What controls the directionality of nuclear transport?
Ran GTPase o Nucleus = on o Cytosol = off • Ran-GEF in nucleus
26
What is the process of nuclear import?
1. Cargo with NLS binds NIR 2. NIR into nucleus via NPC 3. Ran GTP binds BIR, cargo released 4. NIR leaves nucleus 5. Ran-GAP causes Ran-GTP to Ran-GDP hydrolysis (cytosol) 6. Ran-GDP enters nucleus using own NIR and converted back to Ran-GTP via Ran-GEF
27
What is the process of nuclear export?
1. Ran-GTP causes cargo with nuclear EXPORT signal to bind to nuclear EXPORT receptor (NER) 2. Ran-GDP release cargo from NER Import and export: NIR and NER cycling independent of Ran-GTP and cargo
28
What is required for transmembrane transport?
Energy, transmembrane proteins
29
What is the process that directs ribosomes to the ER?
1. Translation starts, free ribosome 2. Signal sequence binds SRP, translation pauses 3. SRP binds SRP receptor on ER membrane, directs ribosomes there 4. Ribosome pore docks with translocator, SRP releases 5. Translation continues into ER lumen
30
What makes up the translocator?
• 4 Sec61 complexes
31
What is the translocation process?
* Ribosome and nascent peptide binds, pore opens, polypeptide fed through * ER signal peptide = start transfer signal * ER signal is cleaved peptidase, leaves translocator, degraded * Mature protein released to lumen
32
How are some proteins integrated into the ER membrane?
* Second stop transfer sequence (HYDROPHOBIC) * Stops transfer, not synthesis * C tail in cytoplasm not lumen
33
How are multi-pass membrane proteins possible?
* Stop transfer and start transfer sequences * Internal signal peptide binds translocator, N terminal left in cytoplasm, middle region feeds through * Second stop transfer signal stops translocation (not synthesis) * C tail in cytoplasm, not ER lumen (double pass)
34
What is glycosylation? When and where does it occur?
* Occurs in rough ER once protein enters lumen | * Transfer precursor oligosaccharide by transferase enzyme associated with translocator
35
What are the three types of vesicular transport?
bio synthetic secretory pathway, endocytosis, retrieval pathways
36
What are the types and roles of coat proteins?
Clathrin: endocytosis COPI: retrograde COPII: anterograde
37
What makes up a clathrin coat?
triskelion (3 heavy chains, 3 light chains)
38
How are clathrin coats assembled and disassembled?
* Adaptins bind clathrin triskelions and cargo receptors | * Budding due to Dynamin
39
What is dynamin? What is ti’s role?
o GTPase o Destabilises neck, lipid bilayers flow together Budding
40
What is involved in membrane fusion?
* Rab proteins guide vesicles * SNARES mediate fusion * Selectivity due to complimentary V and T SNARES
41
What are v snares and t snares?
* V-SNARE: vesicular SNARE | * T-SNARE- Target SNARE
42
How does the recycling/retrograde pathway work?
* Retain resident proteins in ER * Soluble ER proteins have KDEL sequence * Membrane bound receptor in cis Golgi binds ER retention signal, shunts proteins into special transport vesicles and returns them to ER * COPI
43
How do cis and trans Golgi networks vary?
* Cis golgi: receive | * Trans golgi: secrete
44
What are the roles of the Golgi complex?
• Carbohydrate synthesis, sorting, dispatching products of ER
45
What are the types of exocytosis?
* Constitutive | * Regulated
46
What is the process of endocytosis?
• PM → early endosome → late endosome → endolysosome →lysosome
47
What happens during the receptor mediated endocytosis of LDL?
o LDL binds, endocytosis via clathrin, endosome fusion, LDL receptor and LDL separated, LDL degraded, receptor recycled o Illness where LDL receptors can be mutant: heart attack (receptors don’t bind clathrin)
48
What is the role of the extracellular matrix in connective tissue?
Main stress bearing component
49
What are the four functional classes of cell junctions found in mammals?
Anchoring Junctions Occluding Junctions Channel-Forming Junctions Signal-relaying junctions
50
What are the types of anchoring junctions?
• Actin filament attachment sites o Cell-cell (adherens) o Cell-matrix (actin linked cell-matrix adhesions) • Intermediate filament attachment sites o Cell-cell (desmosomes) o Cell-matrix junctions (hemidesmosomes)
51
What are the types of occluding junctions?
* Tight | * Septate (invertebrates)
52
What are the types of channel-forming junctions?
* Gap | * Plasmodesmata (plants)
53
What are tight junctions?
• Transcellular transport • Barriers to diffusion (solutes, membrane proteins: proteins directed to specific regions) Meshwork of sealing strands of transmembrane proteins • Focal connections are the bits that come together • Strands of claudin and occluding proteins (sealing proteins) which associate with other cytoplasmic proteins
54
Which direction do the junctions go, from apical to basal?
Occluding (tight) Cell-Cell anchoring (adherens then desmosome) Channel Forming (gap) Cell-matrix anchoring (hemidesmosome)
55
What proteins make up the tight junction?
Strands of claudin and occludin proteins (sealing proteins) which associate with other cytoplasmic proteins
56
What are anchoring junctions? What types are there?
Allow cytoskeleton to adhere to ECM or other cells Adherens junction Desmosomes Hemidesmosomes
57
What makes up an anchoring junction?
* Intracellular plague attaches to cytoskeleton, transmembrane proteins bind to adjacent proteins on other cells/ECM * Adhesion molecules linked to cytoskeleton so adhesion is robust and strong between cells/ECM
58
How do cell-cell and cell-matrix junctions compare?
Both anchoring. Cell-Cell: adherens, desmosome. Adhere to cadherin Cell-matrix: hemidesmosome, adheres to intergrin a6b4
59
What are adherens junctions?
connects to actin • Classical cadherin • Form continuous belt below tight junction, bring actin filaments to alignment • Cadherins form transmembrane linkages
60
How can myosin interact with actin? What can this form?
actin bundle contraction in adhesion belts, cells narrow at apex, form tubes by pinching off
61
What are the features of cadherin binding?
o Homophillic binding, dependent upon calcium conc | o Ca2+ binds to binding regions and allows rigidity of cadherin
62
What is compaction?
o E-cadherin = strong adherence at 8 cell stage (compaction)
63
How can cadherin link to development?
o Homophillic adhesion, differential expression of classical cadherins = cell sorting, tissue formation (different binding strength etc.)
64
How are cadherins linked to actin?
• Catenins
65
What are desmosomes?
connect to intermediate filaments • Cadherin (desmoglein, desmocollin) • Spot weld cells together, distribute tension • Connect intermediate filaments between cells to form strong continuous network • Bind SIDE of filaments, interact with cytoplasmic plague attached to adjacent cell via cadherin
66
How are hemidesmosomes different to desmosomes?
connect to intermediate filaments • Integrin (α6β4)- link filaments via plectin, dystonin anchor proteins to extracellular laminin • Attach to ECM via integrin and to ENDS of intermediate filaments • Chemically, functionally distinct from desmosomes
67
What are gap junctions?
Communication between cells (not structural role) Gated Channels = Connexons • Six subunits of connexin
68
What are the types of connexons?
* Homotypic – same type of connexins | * Heterotypic – different types of connexins
69
What can pass through connexons?
• Molecules up to 1000 daltons (not proteins. Ions, sugars, AA’s nucleotides, vitamins)
70
What can cause connexons to be shut or open?
* Shut: ↑Ca2+ ↓pH | * Open: ↓Ca2+↑pH
71
What is polarity and how can it link with junctions?
* One part if the cell behaves differently to another * E.g. cell movement towards mating partner * Involves signals, interactions with cytoskeleton and sometimes cell junctions
72
What are the 3 major forms of cell polarity?
* Apico-basal * Asymmetric cell division * Planar cell
73
What parts of the cell are affected by apical basal polarity?
* All cellular components involved | * Proteins, membrane lipids, cytoskeleton, organelles, vesicles, cell-cell/cell-matrix junctions
74
What makes up the ECM? Where can a lot of it be found?
* Everything between cells * Not much in epithelial tissue (Basal lamina) * Lots in connective tissue * Meshwork of proteins and polysaccharides * Structural proteins (collagen, elastin) * Cell adhesive ligands (laminin, fibronectin) * Cell anti-adhesive ligands
75
What does cellular adhesion to ECM depend on?
* Cell adhesive ligands (laminin, fibronectin) | * Cell anti-adhesive ligands
76
What are GAGs?
Glycosaminoglycans (GAGs) Chains • Take up lots of space, lots of negative charge, attract water • Swell with water, occupy space Structure
77
What makes up GAGs?
* Unbranched, polysaccharide chains, repeating disaccharide units * Amino sugar then uronic acid sugar * Very negative acidic side chains (hydrophilic)
78
What is hyaluronan?
* Simple GAG | * Cell migration, joint fluid
79
What are proteoglycan?
* GAG chains attach to proteins via a link tetrasaccharide * Bind to secreted growth factors, enhance/inhibit them * Can be massive
80
What are collagens?
* Major ECM proteins (structure) * Triple stranded helices, three α chains * Tensile strength
81
What is laminin?
* Major part of ECM and basal lamina * Adhesive protein * Cross shaped trimer * Many domains, interact with ECM proteins and receptors (integrin)
82
What is the basal lamina?
* Special ECM underneath epithelia * Determinant of apico-basal polarity * Organised mesh-work * Integrins allow interaction with epithelial cells
83
What are the differences in junctions between drosophila and mammals?
Most apical in drosophila is adherens (not tight) | Have septate instead which are lower down.
84
How is the primitive adherens junction formed? What does it lead to the establishment of?
* Earliest event involves formation of primitive adherens junction * Mediated by homophillic interactions with E-cadherin
85
When is aPKC inactive? Why?
• Par complex baso lateral. o aPKC inactive (binding Par3) o wants to be apical
86
What is the role of PI3 Kinase?
• PI3 Kinase leads to PIP3 (basal)
87
What is the role of PTEN?
• PTEN Phosphotase leads to PIP2 (apical)
88
How does the positioning of Pip relate to polarity?
PIP 3 basal, PIP 2 apical
89
What is the role of Pip2?
1. PIP2 binds annexin | 2. Recruits small GTPase CDC42
90
What are the roles of CDC42?
3. Binds GTP when apical and is activated 4. Recruits Par complex 5. CDC42 activates aPKC which phosphorylates par3 6. Par3 goes to tight junction 7. aPKC and par6 bind crumbs complex in cell apex
91
What is the significance of apico-basal polarity in cancer?
A-B polarity usually regulates: • Localisation and activity of signalling factors (growth factors, intracellular signalling) • Orientation of mitotic spindle o Polarity helps determine direction for cells to divide by orientating spindle o Mis-orientation means cells can divide into lumen • Metastasis o Require cells to lose polarity and cell-cell adhesion molecules to occur o Leads to epithelial-mesenchymal transition (EMT)
92
How can a stem cell divide to produce daughters with different fates?
* Environmental asymmetry | * Divisional asymmetry
93
How does the apical Par6/aPKC complex influence asymmetric division in stem cells?
o Drosophila neuroblast o Apical complex for cell division o Apical: stem cell factors NB o Basal: Differentiation factors (ganglion mother cell for differentiated neurons) 1. Apical Par6/aPKC complex phosphorylates Mir and Numb • Mir brings Brat and Prospero proteins • This with Numb promotes daughter cell to differentiate to GMC • GMC gives rise to neuron and glial cell 2. Both move to basal side and only incorporated into one daughter cell (this cell differentiates)
94
What allows environmental asymmetry to occur?
o One cell not in ideal environment for stem cell so divides instead o Stem cell bound to stromal cell via Kit ligand on stromal cell o Stem cell divides, stromal cell kit ligand only available for one stem cell to bind o The other transit amplifying cell is stuck to differentiate or die
95
How is A-B polarity maintained via feedback?
* aPKC phosphorylates par 1 to ensure it remains basolateral * Par 1 phosphorylates substrates like aPKC to make them go apical * Scribble complex kept basal due to aPKC phosphorylating Lgl

CEDB30002 Cell Biology (10 decks)

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