Cell Biology of Disease Flashcards
(174 cards)
0
Q
Define dynamic instability
A
Rate of GTP hydrolysis
Determines alternation between shrinkage and growth of MTs
Rapidity of the two causes rapid turnover of most MTs
1
Q
Give four diseases (tauopathies) caused by mutations in Tau
A
Alzheimers
Progressive supranuclear palsy
Cortico-basal degeneration
Frontotemperal dementia and Parkinson linked to chromosome 17
2
Q
What are MAPs and what do they play a role in?
A
Stabilisation and destabilisation of MTs
(Needed for efficient cell trafficking)
Determining cell shape and polarity
3
Q
When happens to Tau in Alzheimer’s disease
How can the NFTs be scanned?
A
Tau is hyperphosphorylated
No longer holds MTs together
Tau forms pairs helical fragments which develop into neurofibrillary tangles
Using positron emission tomography, visualisation of a chemical that binds to Tau
4
Q
What is Lissencephaly caused by? What is the physiological consequence because of this?
A
Tubulin mutation Alpha-Tubulin, tuba-3 Results in tubulin misfolding which requires a series of chaperones Absence of normal folds in the cortex Otherwise called smooth brain
5
Q
Give five symptoms of Lissencephaly
What is life expectancy?
A
Abnormally small head Unusual facial appearance Difficulty swallowing Failure to thrive (muscle spasms, seizures, severe psychomotor retardation) Most die before age of 12
6
Q
What was the first discovered mutation for Lissencephaly?
A
Missense
Arg264 to Cys
7
Q
For Lissencephaly dimers of tubulin can form and polymerise normally
What could be the cause of the disease instead
A
Incorrect binding to other MT binding proteins
8
Q
What are MTs made of?
How big are they
A
Alpha and beta tubulin dimers
25nm diameter tubes
9
Q
How many kinesin genes are there in humans?
A
40
10
Q
What is hereditary spastic paraplegia? What mutation is it caused by
A
Kif5a, kinesin-1
Mutations in motor domain
11
Q
Describe symptoms of hereditary spastic paraplegia
What kind of disease is it?
A
Progressive weakness and stiffness in legs
Average age of onset 24 years
Rare disease
Autosomal dominant
Missense mutations, single amino acid change
12
Q
Name different kinds of mutations and what they cause
A
N256S and K253N
Reduced velocity of movement
R280S and K253N
Reduced binding to MTs when attached to cargo
A361V
No change in vitro
13
Q
What drug inhibits mitotic kinesins?
Which kinesins does it inhibit
A
Monastery
Eg5
14
Q
Briefly describe actin
A
Globular monomer that polymerises into filaments 375 amino acid Molecular weight 42 kDa Three main types Alpha in muscle Beta and gamma in non muscle cells
15
Q
Describe structure of F actin
A
Two long pitch helices
One pitch every 5.5nm
72nm long
16
Q
Where and how are actin and titis molecules joined in muscle?
A
Cross-linked in the Z-disc by a Z line protein alpha actinin
17
Q
What is the function of Titin?
A
Provides binding sites for numerous proteins
Acts as a molecular spring for passive elasticity of muscle
18
Q
Give two examples of proteins with mutations that may cause be alone myopathies
What do these cause lack of?
A
Actin and nebulin
Lack of sarcomeres meaning muscle is weak
Death from effects on respiratory muscle
19
Q
Symptoms of nemaline myopathy
A
Facial weakness Scoliosis Floppy babies Can't sit or stand May need ventilation Respiratory problems Multiple joint contractures
20
Q
What do these mutations cause in terms of types of congenital myopathies? G15 D154N V163L I136M D292V
A
Actin myopathy — severe weakness, high mortality
Actin myopathy — some nemaline and IRM, severe, high mortality
Nemaline and IRM — less severe
Nemaline and IRM — less severe
Congenital fibre type disproportion
21
Q
Describe duchenne muscular dystrophy
A
X chromosome gene X- linked recessive Mutations in dystrophin Most affect boys, 100 per year in UK wheelchair bound by 10, death from cardiac problems by 20
22
Q
What is dystrophin?
A
Binds gamma-actin and alpha-beta dystroglycan
Found in muscle costameres (close to plasma membrane in skeletal/cardiac muscle)
23
Q
What is the function of dystrophin?
What do mutations in it cause?
A
Links internal cytoskeleton to extra cellular matrix
Dystrophin loss causes muscle wasting
Affects connectivity between z-discs
Muscle less able to withstand damage upon contraction = membrane ruptures
24
What other mutations in actin based cytoskeleton cause diseases and what diseases do they cause?
Myosin 2a - blood disorders in platelets/WBC
Myosin 7a - deafness and blindness (usher syndrome)
Myosin 5a (griscelli syndrome)
25
Briefly describe intermediate filaments
```
Rope like
10nm diameter
Tough and durable
Form nuclear lamina, also in cytoplasm
Not very dynamic, Central rod domain important
```
26
Name four types of intermediate filaments and their cell location
Keratin - epithelial
Desmin - muscle
Vimentin - fibroblasts
Neuronal intermediate filaments - neurones
27
What are desmosomes?
Localised spot-like adhesions randomly arranged on lateral surface of plasma membrane
Use desmosome in
Help resist shearing forces
28
What are hemidesmosomes?
Also called focal adhesions
Where cells attach to underlying extracellular matrix
Connect basal cell face to basal lamina
Use desmopenetrin
29
Mutations in desmin affect skeletal, cardiac and smooth muscle
Name and briefly describe two main heart problems from this
Hypertrophic cardiomyopathy - thickened portion of myocardium causes sudden cardiac death
Dilated cardiomyopathy - portion of myocardium dilated
Heart becomes weakened and enlarged, less blood pumping efficiency
30
Which genes cause nuclear envelop aphids (lamin loathes)
Lamin A and C
31
What are the roles of nuclear lamin?
Scaffold for protein complexes
| Regulate nuclear structure and function
32
Give an example of a Lamin A mutation
E145K in lamin A central rod
Affects filament formation/polymerisation
Causes down regulation of B lamin
Causes premature senescence
33
How are proteins recognised for translocation into the ER?
N terminal signal sequence
Short and hydrophobic
Cleaved by signal peptidase
Retained for transmembrane proteins
34
What is the Sec61 translocon?
Forms aqueous pore in ER membrane upon displacement of SRP
| translation occurs simultaneously with translocation into the ER
35
What function does the oxidising environment of the ER have for protein folding?
Promotes the binding of cysteine residue and formation of disulphide bonds
Promoted by protein disulphide isomerise
The cytosol is reducing enviromemt
36
What does ER glycosylation entail?
Addition of pre formed oligosaccharide to asparagine residues
2N-acetyl glucosamines, 9 mannoses 3 terminal glucoses
37
Describe protein folding after glycosylation
Trimmed leaving 1 glucose
Chaperone calnexin and ERp57 bind (protein and cysteines) preventing and aggregation
Release of protein removes final glucose
Incorrect folding adds glucose using glycosyl transferase for recognition by Valencia
38
How are unwanted proteins recognised for ERAD
Mannose residues are trimmed
Retro-translocated through sec61 and poly ubiquitinated
Then degraded by cytosolic proteome complex
39
How are escaped membrane protein returned to the ER?
Contain KKXX motif recognised by interaction with coatamer complex - COP1
Transport vesicles return to ER
40
How are escaped luminal proteins returned to the ER?
Contain KDEl motif
| Recognised by coatamer and returned in COP1 vesicles
41
What is Cf caused by? How many affected/carriers?
| What is the most common mutation for CF?
Loss of function of cystic fibrosis transmembrane conductance regulator
Normal function as an ATP binding cassette, couples ATO hydrolysis with Cl- transport
1/27 carry mutation, 1 in 2000 affected
Autosomal recessive
3nt deletion loss of phenylalanine
DeltaF508
At least one mutated in 90% of cases
42
Name and briefly describe 2 other diseases associated with protein folding
Osteogenesis imperfecta
Collagen I mutations prevent folding, cause degradation of pro collagen
Severe defects in bone formation
Alpha1-anti trypsin deficiency
Mutation Impairs folding
Cause protease damage in lungs resulting in emphysema
43
How does the cholera toxin enter the ER starting from outside the cell?
Promotes electrolyte/water movement into intestinal lumen causing diarrhoea
B subunits binds to GM1 on gut surface
Alpha subunit possesses KDEl motif, enters the ER
Dissociates into 2 subunits
Retro translocated avoiding degradation, too few lysines
Binds adenylate cyclase, increases cAMP which activates protein kinase A
PKA Phosphorylated CFTR causing CL- transport
44
How does ricin toxin get to the ER from outside the cell and what symptoms does it cause?
Symptoms = nausea! diarrhoea! seizures and hypotension
Same as ER
B subunit binds via b-galactose residues on glycol iodide
Retrograde transport to ER
A subunit possesses KDEl motif,
A and B dissociate, A unfold and retro translocated across membrane avoiding degradation by too few lysines
Target 28S rRNA and depurinates it by cleavingA4324 and releases adenine
Site of elongation factor binding - inhibits protein translation
45
How does HMCV US2 and US11 avoid detection by the immune system
US2 and 11 are localised to the ERr membrane
They mark MHC1 molecules for ERAD pathway
No MHC1 molecules can then be produced to detect the virus
46
How does adenovirus affect MHCI molecules
Adenovirus E3/19K bind to MHC1
Protein has cytoplasmic tail KKXX motif for retrieval of MHC1 at cis Golgi
Returns to the ER and prevents it from reaching the plasma membrane
47
Give five characteristics of lysosomes
```
All mammalian cells except RBCS
About 1% of cell volume
Roughly spherical
Dense protein rich core
200-400nm diameter
```
48
Lysosomes are involved in...
Degradation of molecules by endocyotosis or auto phage
Apoptosis
Plasma membrane repair
Secretory organelle in immune cells
49
How many hydrolysis do lysosomes contain?
| Normal mode of degradation?
Over 45
Covalent bond hydrolysis
(Nucleases, proteases, glycosidases, lipases)
50
What is the pH of lysosomes and how is it maintained?
| pH of cytosol?
PH between 4.5 and 5.0 maintained by action of H+ vacuolar pump powered by ATP hydrolysis
Cytosol pH 7.2
51
How do lysosomes pump degradation products into the cytosol?
Using secondary active transporters coupled to proton pump coupling
Transport water soluble molecules = sugars! amino acids and nucleosides
Reuse for macromolecules
52
Material for lysosomal degradation is transported in which three ways?
Endocytosis (fluid phase mols, plasma membrane proteins)
Phagocytosis (large extracellular species such as microorganisms)
Autophagy (cytosolic, whole organelles)
53
How do molecules not meant for degradation get back to the plasma membrane
Recognised by the transferrin receptor
| Lysosomal degradation plays an important role the down regulation of receptor signalling
54
What is the best characterised form of autophagy?
| What does it involve?
Macro autophagy
Envelope donated from plasma membrane or organelle surrounds cytoplasmic material forming an auto phagosome which binds with lysosomes
55
Which drug can be used to enhance macro autophagy?
| What disease does it show uses in?
Promoted by starvation of the cell achieved by inhibition of the regulatory protein, the mammalian target of rapamycin/mTOR
Rapamycin promotes clearance of Hungtingtin aggregates in mice and some mutant forms of alpha-synuclein in Parkinson's disease
56
How are lysosomes involved in apoptosis
Apoptosis is indeed by binding to cellular targets of proteases called caspases
Apoptosis cells observe increase permeability of lysosomes membrane
This releases lysosomes proteases called cathepsins that also act on on cellular targets
Can work at cytosolic pH, and trigger mitochondrial intrinsic pathway apoptosis via Bid
57
How are lysosomes involved in plasma membrane repair?
Plasma membrane can be damaged by mechanic damage
Lysosomes help to repair these holes
Damage releases Ca2+ detected by synaptotagmin 7 on lysosomal membrane
58
In which 3 ways are lysosomal molecule accumulation problems associated with diseases?
How many genetic diseases are associated with this?
Insufficient hydrolyse transport
Deficiencies in lysosomes
Inability to deliver breakdown products
Over 50 genetic diseases
59
What are the symptoms of I cell disease
What is the molecular basis I.e. What does the mutation do?
Think in terms of the 3 lysosomes genetic diseases
Also called mucolipidosis type II
Autosomal recessive
Mutation in GNPTAS
N-actylglucosaminidase-1-phosphotransferase
Causes formation of inclusion bodies
Symptoms = facial/skeletal abnormalities, psychomotor retardation, heart failure in first decade
Hydrolyse monosylated Glycans are not modified by the enzyme and so are not transported to endosomes and then lysosomes but instead into the cytosol
60
What are the symptoms of Pompe disease?
| Describe what effect the mutation has on lysosomes and its STORAGE
Autosomal recessive
Hydrolase alpha-d-glucosidase
Symptoms = progressive cardiac/skeletal myopathy, cardio respiratory failure within 1st year
Small %of cell glycogen enters lysosomes for breakdown into glucose
No working enzyme = glycogen accumulation in lysosomes
Enzyme replacement only works in cardiac tissue, taken up by mannose-phosphate receptors
61
What are the clinical symptoms of Fabry disease? What kind of mutation does it cause?
What does it result in the accumulation of?
X-linked disorder, alpha-galactosidase
Symptoms = facial abnormalities, non specific effects for renal/cardiac problems, progressive organ/tissue damage
Renal treatment increases life from 40 to 50
Enzymes usually removes terminal galactose from GB3
Globotriaosylceramide
Accumulates in kidney tubules and glomerular cells, nerves and dorsal root ganglia
Enzyme replacement
62
Describe infantile Sialic acid storage disease and Salla disease
The symptoms and molecular basis
Autosomal recessive mutations in Sialin gene
Symptoms = facial abnormalities enlarged heart/liver/spleen, mental retardation and death within first 2 years
Salla = Finnish population! less severe! life expectancy 50
Normal transport of Sialic acid (9carbon monos) that are breakdown products of glycoproteins/lipids/saminoglycans
Abnormal accumulation in lysosomes
63
Briefly describe the nucleus
| Function, four seperate structures
Contains genetic material
Maintain integrity of genes, control cell activities by regulating gene expression and replication mediation
6 um, 10% cell volume
1) envelope
2) membrane, impermeable to large molecules
3) nucleoskeleton/lamina, meshwork adding mechanical support
4) subnuclear bodies,
64
Briefly describe the nuclear envelope/membrane
Double lipid bilayer
Outer membrane contiguous with rough ER
Inner membrane connected to lamina and intermediate filament network
Peri nuclear space is 20-40nm
65
How thick is the nuclear lamina and what is its function?
Dense fibrillation network, 30-100nm
| Mechanical support, regulate replication and cell division, chromatin organisation, anchors nuclear pore complexes
66
What are laminopathies? What mutations cause?
| Give but don't describe two examples
Mutations in lamin A and C and associated proteins e.g. Emerin
Defects in filament assembly and/or attachment to nuclear envelope, jeopardises nuclear stability
No cure
67
What is Emery-Dreyfus muscular dystrophy? What kind of disease? What are the symptoms?
Affects skeletal/cardiac muscles
Causes joint deformities called contractures restricting joint movements
Slowly progressive muscle weakness/wasting
Abnormalities in heart electric signals, abnormal hearth rhythms
68
What kind of disease of Hutchinson-Gilford progeria syndrome? What mutant protein is its cause?
Physical aspects of ageing accelerated
Point mutation in LMNA gene, LAD50 mutant protein incorporates abnormally into lamina (lacks 50 amino acids)
Mechanical defects, lamina thickening, loss of peripheral heterochromatin, increased DNA damage
69
How is genetic material organised in the nucleus
DNA wraps around histone proteins forming nucleosides called eh chromatin
Multiple histones wraps into a 30nm fibre with nucleosome arrays in compact form called heterochromatin
Higher level DNA packaging occurs at 30 nm fibres into metaphase chromosomes
Insulator elements organise chromatin fibres by establishing separate compartments of higher-order chromatin structure
70
Is euchromatin active or inactive? What does its open form allow?
Active
| Binding of transcription factors
71
Is heterochromatin active or inactive
| What is the difference between constitutive and facultative heterochromatin?
Most inactive
C = never expressed
F = differentially expressed from development or stress
72
Nuclear pore complexes are freely permeable to molecules of what size?
40kDa or less
73
How big is the nuclear pore complex?
| What does it consist of?
125 mega Da
Octagonally organised structure
Approx. 145nm in diameter and 80nm long
Central channel is 69nm (can expand and retract)
74
How many nucleoporins per NPC?
30-50
75
How many rings of proteins in the NPC? How are they connected
8 composite ring of proteins, cytoplasmic and inner membrane surface
Connected by spoke proteins
Filaments from both sides are connected to form a basket like structure
76
How do proteins get into the nucleus through NPCs? (Five step cycle)
1) localisation signal on protein recognised by importin complexed to GTP binding protein Ran
2) importin binds complex to nuclear pore protein in cytoplasmic filaments
3) complex translocated sequentially binding to pore proteins
4) guanine nucleotide exchange factor exchanges GDP to GTP on Ran altering configuration to release the protein
5) importin-Ran/GTP complex is re-exported through the pore and the GTPase activating protein hydrolyses GTP on Ran to GDP
77
What are the 2 types of nuclear localisation signals you can get?
Monopartite (importin alpha)
| Bipartite
78
How do proteins get out of the nucleus?
Nuclear export signals, contain many hydrophobic residues often leucine
Recognised by exportins
Ran/GTP forms stable complexes but dissociates importing and targets
79
Name a disease associated with the nuclear pore complex
Briefly describe its symptoms
What proteins are affected by lack of import
Achalasia-addisonianism-alacrimia syndrome
Symptoms = Autonomic dysfunction, adrenal insufficiency, achalasia, mental retardation
Mutations in NPC component ALADIN
Affects import of aparataxin, DNA ligase I, ferritin heavy chain
All involved in protecting/repairing DNA under oxidative stress
Cells more prone to oxidative damage
80
Why do viruses need to associate with the nuclear pore complex?
Give examples of viruses that are both small and large and the mechanisms they sue
They are too big to diffuse, only if less than 39nm
HBV, 32-36nm, no need for capsid disassembly
HBV capsids possess NLSs accessible to bind importin
HSV, 125nm dock via importin B and release DNA through pore
Adenovirus, 90nm directly associate with cytoplasmic localised nucleoporin CAN/Nup214, then bind Hsc70 and histone H1 initiating capsid disassembly to release DNA
81
How does bulk mRNA exit the nucleus?
1) Large complex approaches NPC and attaches by thin filament
2) reaches pore centre and elongates in 100-150A broad rod
(Transitorily forming dumbbell shaped configuration)
3) material rounds into spherical particle
82
What is hTREX? Where would you find abnormalities of hTREX? (What kinds of cells)
Human transcription/export complex
| Numerous cancers, particularly highly aggressive forms
83
What are the roles of hTREX? What effects do hTREX mutations have?
Normal roles
Stability of mRNA
Efficient nuclear transport
Mutations
Enhance formation of R loops --> DNA-RNA hybrid formed when newly transcribed RNA binds DNA template --> R loops halt transcription and increase DNA damage
84
How much volume of the nucleus does the nucleolus occupy?
25%
85
Name 3 components in the nucleolus and their functions
Fibrillation centres - depot of rRNA genes
Dense fibrillar compartments - maturation of pre-mRNA transcripts
Granular components - assembly of pre ribosomal particles
86
What is the primary function of the nucleolus?
Ribosome biogenesis
87
Describe the five steps in ribosome biogenesis
1) rRNA transcribed by pol I and III to yield long precursor 4S pre rRNA
2) further processing yields small 18S, and large 26,5.8 and 5S
3) RNs modifying enzymes are brought to rRNAs by small nucleolus ribonucleoproteins (snoRNP) and are assembled in GC
4) ribosomal accessory proteins trafficked back in nucleus to form small 40S and large 60S subunits
5) nuclear export and forms functional ribosome
88
Approx. how many proteins are involved in ribosome biogenesis pathways of the nucleolus?
Which two are centred in this mechanism?
4500
| Nucleolin and B23
89
Name 3 ribosomopathies
Diamond-black fan anaemia
Dyskeratosis congenital
Treacher Collins syndrome
90
What mutation causes diamond black fan anaemia and what are the subsequent symptoms?
Mutation in RPS19
Symptoms = Low RBC counts, leads to congenital abnormalities
Craniofacial malformations, thumb/upper limb malformations, cardiac defects
91
What mutation causes dyskeratosis congenital and what are the subsequent symptoms?
Mutation in DKC1 encoding dyskeratosis (nucleolus protein associated with snoRNPs)
Symptoms = premature ageing, skin pigmentation, dystrophy of nails, cancer predisposition
92
What mutation causes teacher Collins syndrome and what are the subsequent mutations?
Mutation in TCOF1 genes (nucleolus protein called Treacle)
Symptoms = Craniofacial deformities e.g absent cheekbones, small lower jaw, malformed/absent ears
93
What are the 3 types of nucleolus protein in terms of time spent in a particular location? Give examples
Mainly in the nucleus - fibrillarin and Nucleolin
Part time in the nucleus - ribosomal protein
Time/condition dependent - p68 and cell cycle factors (blooms in S phase)
94
What 2 morphological changes in the nucleolus and are associated with what 3 types of disease states
Changes in ribosomes synthesis
Apparent sequestration or loss of proteins to the nucleolus
Auto-immunity, viruses and cancer
95
Nucleoli can be bio markers for high grade tumours. What differences would you expect to see between low and high grade tumours in a histology sample?
Normal sample - small and round
| Cancerous sample - enhanced biogenesis, hypo proliferation, larger and less rounded
96
What are 2 alternative links to ribosomes and cancer?
1) increased ribosome biogenesis
Satisfies biosynthesis demand during proliferation
(Evidence from protooncogenes upregulating this)
2) deficiencies in ribosome function
Alterations in protein translation of specific proteins involved in regulating transformation
(Evidence people with inherited ribosomopathies have greater predisposition to cancer development)
97
What proto-oncogene is upregulated related to ribosome biogenesis
C-myc
Variety of leukaemias and solid tumours
Enhances recruitment of pol I and other TFs, histone acetylases to rDNA promoters
98
How many viruses exhibit nucleolar localisation? In what 2 ways do they function?
37 viruses, 67 viral proteins
All 7 classes from Baltimore classification system
Either:
1) use nucleolar protein to enhance virus replication
2) subvert anti-viral pathways
99
What viruses that replicates exclusively in the cytoplasm still requires proteins from the nucleolus? Describe the changes in the nucleolus that come about as a result of this
Coronavirus RNA genome functions as mRNA = exclusive cytoplasmic replication
But one major coronavirus protein localises to the nucleolus
RPL10 - ribosome constituent
RPL14 - RNA binding, ribosomal protein
CELF-1 - alternative splicing, mRNA translation and stability
eIF 5a-1 - protein biosynthesis
PCBP2 - RNA binding and protein biosynthesis
EF-1beta - translation elongation activity
100
Which virus expresses protein that target the nucleolus at different times and cause significant changes to genome replication? Give examples of some of these changes
Herpesvirus
8 proteins
RNA processing/synthesis/modification, DNA synthesis/replication/repair, gene expression, growth and proliferation
101
What 3 sub nuclear structures does the spliced some consist of? What are they involved in?
Nuclear speckles, cajal bodies and Gems
Consist if U1, 2, 4, 5, 6 small nuclear RNPs in conjunction with approx.125 proteins
102
What are cajal bodies?
Spherical, 0.3-1.0 um
Usually 3-5 per cell
Contains proteins for mRNA biogenesis involved in maturation/assembly of spliceosomal sub complexes e.g snRNP
103
What are nuclear gems?
Similar in size and shape to cajal bodies
Do not contain snRNPs
Contain SMN protein functioning in snRNP biogenesis
104
Give an example of a genetic defect in associated with cajal bodies and nuclear gems
Spinal muscular atrophy
SMN1 mutation, essential for snRNP assembly
Symptoms = widespread splicing defects in spinal motor neurones
Death of neuronal cells in anterior horn and atrophy of muscles
Most common genetic cause of infant death
105
What are nuclear speckles?
Structures enriched in pre-mRNA splicing factors acting as storage/modification compartment located in inter chromatin regions of nucleoplasm
Irregular in size and shape, punctuate
106
Why are nuclear speckles always in close proximity to active genes?
Thought to enhance metabolic activity in mRNA maturation/exportation
107
Give an example of a genetic condition caused by mutations in splicing factors
RPR31 mutation cause progressive loss of rods and cones in retinal pigment epithelium, attached to blood vessels that feed retinal cells
Symptoms = vision progresses from night blindness to tunnel visions to complete blindness
108
What are polycomb bodies? Give 2 functions
Hubs for gene expression usually associated with heterochromatin
1) remodelling chromatin inducing epigenetic silencing
2) binding directly to specific DNA sequences facilitating recruitment of complexes to modify local chromatin structure
Overexpression been linked to cancer
109
What are PML bodies? What are their 4 main functions? How can they be categorised into 3 groups?
Spheres of 0.1-1.9um diameter, 10-30 per cell
Promyelocytic leukaemia bodies
DNA damage response, apoptosis, cellular senescence and angiogenesis
1) nuclear storage for protein accumulation
2) 'catalytic surfaces' for protein post-translational modification
3) active sites for transcriptional and chromatin regulation
Regulate p53 dependent apoptosis and cellular senescence induced by cellular stress
Promote acetylation/phosphorylation of p53 to PML-NBs and by inhibiting MDM2 the negative regulator of p53
Recruitment of hits cell proteins to PML bodies can then post-translationally modify and activate p53
110
Briefly describe the formation of PML bodies
1) Dimerise and then multimerise to form nuclear NBs
2) PML sumoylation by SUMO proteins leads to organisation in spherical body
3) SUMO-interacting-motifs-containing partners are recruited by SUMO/SIM of PML into the inner core of the NB
Sumoylation is the post translational modification
111
What is acute promyelocytic leukaemia?
95% cases! PML protein forms reciprocal translocation with RARa gene (retinoic acid receptor alpha)
Causes loss of PML bodies, creates hybrid protein which block transcription/differentiation of granulocytes
112
Name 2 types of cell specific receptors
```
Contact dependent (ligand binding)
Synaptic (neurotransmitter release)
```
113
Name 2 types of cell-TYPE specific receptors
Paracrine (local mediator proteins)
| Endocrine (hormones exit endocrine cell into bloodstream)
114
What are G-protein coupled receptors? Give some examples
Over 800 genes
Often form dimers
Multiple membrane spanning, 7 transmembrane helices bundle to form ligand binding domain
Examples = chemokine, glucagon receptors, glutamate, odorant, GABA
115
How do G-protein coupled receptors caused a response?
Ligand binding cause conformational change
Signal transduction
Dissociation of alpha-beta-gamma complex cause generation of secondary transient messengers
I.e. Alpha activation = activates adenylate cycle = produces cAMP
Switch on genes
116
What mutation in the PTHR cause? What is PTHR's normal function?
Parathyroid hormone receptor
Kidney - regulates calcium/phosphorus concentration
- regulates chondrocyte growth/development
Mutation = constitutive activation by conformational change of histidine to arginine
Jansen's metaphyseal chondrodysplasia
117
Give an example of an autoimmune disease caused by agonist antibodies
Grave's disease
Hyperthyroidism
Autoantibodies cause constant activation of receptor
Excess cAMP generation via Gas subunit
118
Give an example of an autoimmune disease from antagonist antibodies
Hashimoto's disease
Hypothyroidism
Decreased cAMP
Weight gain, fatigue, often post partum
119
How does cholera toxin affect G-protein coupled receptors?
Enters cell via GM1 ganglioside
Catalyse ADP-ribose transfer from NAD+ to arginine residue to Gas subunits
Causes constitutive activation and increases cAMP
Increase in Cl- ions causing diarrhoea
120
How does pertussis toxin affect G-protein coupled receptors?
Prevents activation of Gai subunit which normally functions to reduce cAMP via ADP ribosylation
Causes constant switching off = increase cAMP
Affects ion flux in lung epithelial, life threatening for neonates
121
What is McCune-Albright syndrome?
2 major mutations in Ga subunits causing increase in cAMP
Arg201 in GDP-GTP binding domain of protein
Gln227 required for intrinsic GTPase activity
Increases in melanocyte stimulating hormone cAMP pathway
Causes excess melanin and patchy skin
Fibrous dysplasia - bone dysfunction
Non germline mutations can give sporadic phenotypes
122
What is pseudohypoparathyroidism?
Loss of Gs alpha subunits essential for PTHR signalling
Different phenotype if from mother or father
Monoallelic expression in certain tissues = one chromosome is completely turned off
PHP1b caused by epigenetic mutations, not classic coding mutations
123
How do enzyme-coupled receptor tyrosine kinases cause cell changes?
Signalling by phosphorylation cascades
Include many growth factors:
VEGF, EGF, M-CSF (monocytes/macrophages), Ephrin (neurone migration), insulin
Can be directly/indirectly coupled to enzymes
124
Explain the 2 types of diabetes
Type I - lack of insulin (autoimmune destruction of beta islet cells)
Type II - insulin resistance (down regulation of receptors)
Receptor desensitisation - endocytose and degraded by Clathrin
Uses phosphotyrosine phosphatases switch off down regulation
Downregulation of P13K and IRS (downstream signalling components) by high glucose/free FAs
125
What do adhesion receptors allow for?
```
Spatial patterning
Migration
Differentiation
Guidance
Morphogenesis
```
126
Give 3 categories and examples of adhesion receptors
Cadherins - E-, V-, VE-, flamingo
Integrins - Cd11a/b/c/CD18
Selectins - L, E, P bind sialyl-Lewisx glycosylated proteins
Proteoglycans contain protein core and GAG side chains
Mediate homotypic or heterotypic interactions
127
What are the crucial molecules for a leukocyte adhesion cascade?
Selectins, integrins, ICAMs, JAMs, PECAM/CD31
Mediated through receptors and soluble/matrix bound chemo attractants (selectin L)
128
Describe the role of selectins in capture and rolling of leukocytes
(Stage 1 of leukocyte adhesion cascade)
Bind sialyl-Lewisx sugars on PSGL (p-selectin glycoproteins ligand)
Low affinity, transient in nature
Signal via phosphoinositide-3-kinasey pathway resulting in slow rolling
Mice lacking endothelial expression of P3K show 10x elevated velocities in inflammation
129
Describe the role of integrins in leukocyte activation and arrest
(Stage 2 of leukocyte adhesion cascade)
Mediate arrest via outside-in and inside-out signalling
Activated by chemokines/TNF/chemoattractants inside-out ICAM-a binding on endothelial cells
Increase affinity through clustering/conformational change outside-in
130
Name 3 migration defects in neutrophils
LADI - lacks CD18 (crucial integrin)
LADII - lacks fucosyltransferase that generates selectin ligands
LADIII - integrin activation defect (KINDLIN-3 mutant)
131
What are five embryonic processes that rely on adhesive cell interactions?
1) Segregation of tissues for neural tube formation
2) Cell dispersion from solid tissue
3) Cell migration along adhesive guidance cues
4) Cavity formation requires intercellular sealing by tight junctions/vectorial ion & water transport
5) Cell-cell communication through gap junctions
132
What is metastasis? How does it links to adhesion receptors?
Movement/spreading of cancer cells from one organ/tissue to another
Signalling production of MMP, chemo taxis
Requires epithelial to mesenchymal transition
133
How do metastasis recognise microbial motifs and distinguish between self and non self?
PAMPs recognised by surface pattern recognition receptors
134
What are the major classes of surface pattern recognition receptors?
Toll receptors
| Carbohydrate binding lectins
135
Give an example of a toll mutant causing defects in microbial recognition
Drosophila
Single nt polymorphism in promoter of human Dectin-1
Increased susceptibility to candida infection
136
How does HIV exploit host receptors?
Binds CD4 of T cells
Normal function to interact with MHCII for APC production
Binding allows insertion of fusion protein into membrane
Gp120 and gp41
137
How does listeria exploit phagocytic receptors?
```
Listeriolysin O
Formation of pores
... In host phagosome membrane
Entry of 2 Listeria encoded C-type phospholipases
Cause disruption of membrane
Grow and divide in cytosol
```
138
Describe the mitochondria and its four 'compartments/components'
Approx. 1um
100-1000 per cell
Matrix = enzymes for oxidation/CTA cycle
Inner membrane = proteins for oxidation (ATP synthase)
Outer membrane = permeable to molecules less than 5000 Daltons
(Large pore forming channel porin)
Inter membrane space = enzymes that use ATP passing out of matrix to phosphorylate nucleotides
139
Give a brief overview of what happens in the electron transport chain of the mitochondria
Citric acid cycle produces NADH
Oxidised which releases high energy electrons which pass down ETC
Lose energy as they go allowing H+ pumping into inter membrane space
Builds up electrochemical gradient (proton motive force) of 0.5pH difference = H+ pumped back into matrix coupled to ATP synthase
Electrons oxidised and release water
Known as oxidative phosphorylation
30ATPs created
140
Name the five proteins that electrons pass through in the mitochondrial membrane during oxidative phosphorylation
```
NADH dehydrogenase complex
Ubiquinine
Cytochrome b-c1complex
Cytochrome c
Cytochrome oxidase complex
```
141
Briefly describe the characteristics of DNA that mitochondria encode
Contains 2-10 copies of covalently closed circular dsDNA (mtDNA)
Do not code for ALL protein in mitochondria
Maternally inherited
142
Are mitochondria autonomous in terms of replication?
Requires cooperation with nuclear genome
| Can divide within cells and maintain their number
143
How are proteins targeted to the mitochondria?
Requires N terminal signal sequence
Rich in basic amino acids = serine, threonine
Entry requires unfolded protein bound to chaperone
Energy dependent
After uptake, transit sequence usually cleaved
144
What kind of diseases are usually found in mitochondrial diseases
Usually neuromuscular diseases --- tissues with requirement for high ATP concentrations
High mutation rates in mtDNA
Multiple copies of mtDNA per mitochondria
Homoplasmy or heteroplasmy
145
Name and briefly describe 3 types of mitochondrial diseases
Leber's hereditary optic neuropathy
--- Missense mutation in NADH Co1 reductase
Kearn-Sayers syndrome
--- Large deletions, eye defects and CNS degeneration
Ragged muscle fibre syndrome
--- various mutations (mito Lysine tRNA = decreased mito translation)
146
Are mitochondrial diseases curable?
No
Rare diseases in children
IVF therapy - three person
Unreal edit women donates 'cleanup' egg, shown by craven et al
147
What is apoptosis? How is it caused?
Physiological cell death, orderly and controlled manner
Termed by whole and currie
First identified in C. Elegans
Doesn't provoke immune response as the cell contents are not released
Necrosis in death following injury, trauma or infection
148
How can apoptosis be controlled?
Extracellular activators - death receptors on cell surface
Intracellular activators - withdrawal of survival factors/DNA damage/ metabolic stress/hypoxia
All cause activation of intracellular proteolysis system mediated by caspases
149
What events occurs after activation of apoptosis consenting caspases?
Stimulus releases cytochrome C through activated Bax or Bak causing activation of adapter protein
Adapter protein and cytochrome c Assembles into complex
Recruitment of procaspase9 molecules
Forms apoptosome
Procaspase activation by close proximity and cleavage leaving pro domains
large and small subunit form 1 active caspases
active caspases X produces caspases Y (nuclear lamin)
active caspases Y produce caspases Z (cytosolic protein)
150
How can apoptosis be stopped through the action of the Bcl/2 family?
Bcl-2 is anti apoptosis found in B cell lymphomas
25-26kDa membrane protein
Homologues of Bcl-2 such as Bax forms heterodimers with Bcl-2 and inactivate Bcl-2
151
How do some viruses halt apoptosis?
Necessary part of life cycle
Some viruses contain Bcl-2 homologues
Adenovirus E1B-19kDa protein forms complex with Bax family members and suppresses release of cytochrome c via mitochondrial pores
Similar function also seen in herpesvirus
152
Give 3 functions of pore forming proteins
Establish control of voltage gradient across plasma membrane
Allow free flow of ions down electrochemical gradient
Allows cells to 'react' to external stimuli
153
Briefly describe what happens during an action potential
Na+ channels open, Na+ enter cell
K+ channels open, K+ leaves cell
Na+ channels become refractory so no more enters the cell
K+ continues to leave the cell, causes membrane potential to return to resting level
154
How do ions channels works?
Heteromultimeric integral membrane
Water filled passageway for ions
Physical pore, several subunits, lined with hydrophilic amino acids Narrow region typically charged acts as 'selectively filter'
155
What are the 2 main types of ion channel?
Voltage gated ion - responds to membrane potential
| Ligand-gated ion - chemical stimuli
156
Give 3 examples of new drugs, the frequent targets of which are ion channels
```
Carbamazepine = anti-epileptic drugs (partial seizure control)
Na+ channel blockers
Verapamil hydrochloride = angina
Voltage gated Ca2+ channel inhibitor
Glibenclamide = diabetes
ATP sensitive K+ channel inhibitor
```
157
How can we record ion channel activity?
Patch clamping
Whole cell patch clamp (study of multiple ion channels)
Glass pipette makes tight contact with area
Forms high resistance seal
Suction within pipette disrupts membrane patch
Interior of pipette constant with cytoplasm
Measurements of electrical potentials and currents from entire cell
158
Describe 3 congenital (mutation) disorders caused by ion channel mutations
Cystic fibrosis
Insulin disorders
Cardiac arrhythmia's
159
How does CF relate to ion channel mutations?
Autosomal recessive
Abnormal transport of Cl and Na across epithelium = thick mucous
Most common deltaF508 (in NBD1), loss of triplet loss of phenylalanine
Causes 2/3 of cases
1480 amino acid protein
Member of ATP binding cassette
160
How do insulin disorders relate to ion channel mutations?
ATP sensitive K+ channel
Channel composed of Kir6.x-type subunit and sulphonylurea receptor (SUR)
In pancreatic beta cells, ATP/ADP ratio determine Katp channel activity
Normal conditions - high glucose increases ATP production, channels close, promotes Ca2+ release and insulin is released
In profound neonatal diabetes - channels are over active
Glibenclamide inhibits SUR1 allowing insulin release
161
How are cardiac arrhythmia's linked to ion channel mutations
Long QT syndrome
Delayed repolarisation of heart
Risk of irregular heartbeat originating from ventricles
Increased risk of seizure/sudden cardiac death/ structurally heart and individual are healthy
Mutation at HERG, over 30 identified
HERG encodes a voltage gated K+ channel
162
Explain the acquired channelopathy caused by tetrodotoxin
Blocks action potentials in Na+ channels
Paraesthesis of lips/tongue, sweating, headache, weakness, respiratory failure, coma
Physically binds to channel preventing Na+ flow
163
How is mamba snake venom linked to acquired channelopathies?
Blocks voltage gated K+ channels that control nerve/muscle excitability
Dendrotoxin prolongs actions potential duration
Increases acetylcholine release at neuromuscular junction
Muscle hyperexcitability and convulsive symptoms
164
How is terfenadine linked to acquired channelopathies?
Prolonged ventricular repolarisation
1985 - Seldane drug sold as non-sedating anti histamine for allergic rhinitis
1990 - evidence of ventricular arrhythmia's
1992 - cardiac arrhythmia's for those taking seldane as well as ketoconazole or erythromycin
1997 - FDA removal
165
Give an example of a venom that could potentially be used as as route of therapeutic agents in channelopathies
Ziconotide (synthetic peptide of conotoxin from cone snail)
Blocks voltage gated Ca2+ of spinal cord
Reduces pronociceptive neurotransmitter release in dorsal horn of spinal cord
Inhibition of pain signal transmissions
166
Briefly describe receptor tyrosine kinases
Intracellular catalytic domain and variable extracellular domains (binds to ligands)
Protein kinase = takes phosphate from ATP and adds amino acids in target protein
E.g. EGF, insulin (IGF1), PDGF, VEGF, Eph
167
Briefly explain how RTKs are activate by ligand binding
Causes protein dimerisation (brings C termini into association) and allows transautophosphorylation
Enhances enzyme activity and creates docking sites for high affinity interactions with intracellular signalling proteins
168
Give examples of protein signalling modules and the targets they bind to
SH2 and PTB - tyrosine Phosphorylated sites
SH3 and WW - proline rich sequences
PDZ domains - hydrophobic residues at C-termini of target proteins
PH domains - different phosphoinositides
FYVE domains - specifically bind phosphatidylinositol-3-phosphate
169
Briefly describe the activation of Ras by an activated RTK
Ras = small monomer G protein, similar to alpha subunit of trimeric G proteins. ----- it is an ONCOGENE
Critical for downstream signalling events
Cycles between active and inactive forms
GEF (guanine nucleotide exchange factor) activates
GAP (GTPase activating proteins) inactivate
Activates MAP kinases
170
What are MAP kinases and how are they linked to Ras
Ras-GDP is recruited and converted to Ras-GTP
Activated Ras induces kinase cascade activating MAP kinases
Serine threonine kinases that translocated to the nucleus and phosphorylate many different proteins
E.g. TFs and regulate gene expression
171
How can cancers be caused?
Virus associated
v-Erb2 of AEV (avian erythroblastosis virus) lacks C terminal regulatory domains therefore doesn't require ligand binding for activation
Genetic
Point mutations or truncations can cause ligand independent activation of growth factor receptors
172
Give details on Ras and how it is recognised as an oncogene
2 approaches: introduction DNA from tumours into cell culture and mutated version found in oncogenes of retroviruses
90% of human cancers have mutant Ras
MSG common mutations trap Ras in GTP-bound form for constitutive activation
173
Give 2 examples of how growth factor receptor signalling can be used as a target for anti-cancer therapy
Raf inhibitors BAY 43-9006
Inhibits MAPK signalling in cancer cells
Her2 (RTK) inhibitor Herceptin
Her2 receptors over-expressed in approx. 39% of breast cancer
Herceptin antibody binds to her2 receptors and prevent signalling = reduces rate of cancer cell proliferation
