Cell Biology and Disease Flashcards
(194 cards)
1
Q
What is Post Translational Modification?
A
- Covalent cleavage of proteins
- Occurs during or after protein biosynthesis
- Important component of cell signalling
- Occurs on AA chain, usually at a terminal
- Extends the 21AAs available, adding/removing chemical groups increases diversity, while extending function and stability.
2
Q
What are pluripotent stem cells?
A
- True stem cells or adult stem cells
- Ability to renew and differentiate, potentially into any cell in the human body.
- Unclear how it is maintained and modulated, evidence suggests complex cell signalling networks
- Differentiate into 1 of 3 types of germ layers: endoderm, mesoderm, ectoderm.
- Induced PSC can be created from differentiated cells.
- As development progresses, they lose pluripotency.
3
Q
Names 5 types of PTM.
A
- Phosphorylation
- Glycosylation
- Acetylation
- Methylation
- Disulphide bonds
4
Q
What is phosphorylation?
A
- Reversibly add/remove phosphate, causes a conformational change in protein.
- Activate/deactivate an enzyme.
- 1/3 of cellular proteins thought to be phosphorylated at any given time.
5
Q
Results of phosphorylation?
A
- As P has 2 -ve charges, there can be significant conformational change.
- Activate enzyme = form a site recognised by other proteins
- Deactivate enzyme = can mask a binding site preventing protein-protein interaction.
6
Q
What is glycosylation?
A
A carbohydrate is covalently bound to an N or O (functional group) on a protein, via a glycosidic bond
7
Q
Functions of glycosylation?
A
- Help correct folding
- Increase protein stability
- Cell-cell / cell-environment adhesion
- Immune response
- Hormone activity
- Embryonic development
8
Q
How many reported disorders of glycosylation are there?
A
- 40 disorders
- 13 different monosaccharides involved
- 8 different amino acids
- > 41 different bonds
- 16 enzymes
9
Q
Name 5 types of glycosylation.
A
- N-linked
- O-linked
- Glypiation
- C-linked
- Phosphoglycosylation
10
Q
Bond, location and example of N-linked glycosylation.
A
Bond - Glycan binds to the amino group of asparagine.
Location - ER
Example - Insulin receptor, ECM, regulation
11
Q
Bond, location and example of O-linked glycosylation.
A
Bond - monosaccharide binds to the hydroxyl group serine or threonine.
Location - ER, Golgi, cytosol, and nucleus.
Example - collagen, several pathogenic bacteria secretions to form the ECM
12
Q
Bond and example of glypiation.
A
Bond - glycan core links a phospholipid and a protein
Example - anchors cell surface proteins
13
Q
Bond and example of C-linked glycosylation.
A
Bond - mannose binds to the indole ring of tryptophan.
Example - only mammalian cells, ECM
14
Q
Bond involved in phosphoglycosylation.
A
Glycan binds to serine via phosphodiester bond.
15
Q
What are protein kinases?
A
- Catalyse the transfer of a phosphate group from a high energy donor molecule to a specific substrate - phosphorylation.
- Allow enzymes to be phosphorylated.
- 513 characterised, 478 have a homologous catalytic domain, 35 remaining are atypical.
- Reaction is essentially unidirectional, taking Pi from ATP and adding to protein, due to large amount of free energy released from P-P bond in ATP.
16
Q
What are phosphatases?
A
- Enzymes that catalyse the removal of a phosphate group from a substrate by hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group (dephosphorylation).
- Opposite reaction of a kinase
- Can be specific or broad range
- Broad range controlled by regulatory proteins
- Often in a chain- cell signalling
17
Q
What is acetylation?
A
- Addition/removal of an acetyl group, donated by Acetyl CoA.
- Can be enzymatic or non-enzymatic, enzymes involve acetylase or deacetylase.
- Some proteins are chromosome related and this indicates its importance in gene expression.
18
Q
Name two types of acetylation
A
N-terminal and lysine acetylation
19
Q
What is N-terminal acetylation?
A
- Most common co-translational modification in eukaryotes
- Synthesis localisation stability
- 80-90% of human proteins
- Catalysed by a set of enzyme complexes NATs.
NATs transfer an acetyl group from Acetyl CoA to a-amino group of the first AA residue of the protein.
20
Q
What is lysine acetylation?
A
- Often acetylation and deacetylation cycle is linked to transcription factors
- Activation of gene expression
21
Q
What is antagonistic acetylation?
A
- Acetylation of histones encourages binding of effector proteins, relaxation of chromatin conformation, and an increase in transcription.
- Also in the synthesis, stability, and localisation of other proteins.
- But high-level acetylation associated with transcriptional hyperactivity.
22
Q
Therapeutic application of acetylation:
A
- Targeting HDACs (KDAC) in malignant cells and treatment of neurodegenerative disease.
- Development of small-molecule inhibitors (HDI).
23
Q
What is methylation?
A
- Adds a methyl group, usually at lysine or arginine residues.
- Methyl donated by S-adenosylmethionine.
24
Q
Name 4 types of methylation.
A
- Carbonyl methylation - generally reversible and used to modulate a reaction.
- Nitrogen methylation - generally irreversible creating new amino acids.
- Arginine methylation - regulation of RNA processing, gene transcription, DNA damage repair, protein translocation, signal transduction.
- Lysine methylation - histone function regulation, epigenetic regulation of transcription, lysine methyltransferase.
25
Characteristics of p53 PTM interaction.
- p53 known for its tumour suppressor activity.
- Activated by various genotoxic stresses by regulating: apoptosis, DNA repair, senescence, autophagy.
- Regulates by targeting expression of downstream target genes.
- Significance indicated by p53 being muted in 50% of cancers and pathway disruption in the remaining.
- p53 is rich in Lys and Arg.
- p53 activity is regulated by complex array of PMT, acetylation, phosphorylation and methylation.
- Concentrated in N- and C- terminals.
- Under stress there is an up regulation of kinase activity reducing p53 activity and increasing cofactor activity
- Acetylation stabilises p53 levels increasing DNA interaction.
26
What is passive transport?
- Movement along a concentration gradient (high to low)
- Down a gradient, no energy expenditure (ATP hydrolysis) required.
27
Name 3 types of passive transport.
1. Simple diffusion - movement of small or lipophilic molecules
2. Osmosis
3. Facilitated diffusion - movement of large or charged molecules via membrane proteins (e.g., ions, sucrose, etc.)
28
What is active transport?
- Movement of materials against concentration gradient (low to high)
- To move against the gradient, energy expenditure (ATP hydrolysis) is needed.
29
Name 2 types of active transport.
1. Primary (direct) active transport – involves the direct use of metabolic energy (e.g., ATP hydrolysis) to mediate transport.
2. Secondary (indirect) active transport – involved coupling the molecule with another moving along an electrochemical gradient.
30
What are ion channels?
- Transport ions across the plasma membrane
- Regulates membrane potential – the difference in electrical potential between interior and the exterior of a cell.
31
What is the difference between flux and diffusion?
- Flux – movement, occurring due to the difference in concentration across the membrane.
- Diffusion – principle of moving from regions of high to low concentration.
32
Describe the ion distribution of a typical cell at rest.
- Neuron – sodium, chloride, calcium are concentrated outside the cell.
- Potassium, other anions are concentrated inside.
- Ion distribution leads to a negative resting membrane potential.
- Ion movement requires channel to be in open state, movement governed by potential across membrane.
33
What is membrane potential (Vm)?
- Voltage difference between the inside and the outside of a cell
- Difference in charge only exists at the plasma membrane.
- Arises due to ion movement across the plasma membrane.
- The phase between action potentials is often called the resting membrane potential (V rest)
- ‘Resting’ - cell is at rest (silent, quiescent)
- Usually around -50mV in most mammalian cells
34
What does the sodium-potassium pump do?
-K+ in = -90mV, Na+ out = +60mV
- Na+/K+ pump drives increased negativity inside the membrane.
- Non-gated (leak) potassium channels are open at rest causing potassium to have the highest permeability at rest.
- Other ion channels (chloride and sodium) are also open, but fewer are open than potassium.
- The resting membrane potential of a typical neuron is relatively close to the equilibrium potential for potassium.
- The sodium-potassium pump is responsible for maintaining the electrochemical gradients needed for neuron functioning.
35
What are neurons?
- Responsible for storage and retrieval of data.
- Neurons terminally differentiated and non-proliferative, relatively stable throughout life.
- Communicate through synapses.
36
Name 2 types of synapses.
- Chemical - Intermittent transmission
- Electrical - Continuous transmission, signal between both sides of the synapse is couples.
37
Name some major extracellular cations and anions.
Cations - Potassium, magnesium
Anions - phosphate, some amino acids
38
Name some major intracellular cations and anions.
Cations - calcium, sodium
anions - chloride
39
What is an action potential and when do they occur?
- Sudden/rapid reverse of membrane polarity (charge)
- Transmission of a signal – aka firing, excitability, impulse
- Only excitable cells/tissues respond to action potentials: muscle contraction, neurotransmitter release, secretion
40
What are the stages of an action potential?
1. Resting phase
2. Stimulus initiates depolarisation (-ve MP to less -ve MP): slow rising phase, rapid rising phase
3. Repolarisation
4. Hyperpolarisation (refractory period)
5. Back to resting state
- Action potential is driven by ion flux.
41
Describe depolarisation.
- Change from a (relatively) negative charge to a positive charge.
- Na+ channel open, K+ channel closed, Na+/K+ pump closed.
- As Na+ ions are more concentrated outside of the neuron, the opening of sodium channels causes a passive influx of sodium.
- The influx of sodium causes the membrane potential to become more positive (depolarisation)
42
Describe what the voltage gated Na+ channel does.
- When the membrane potential reaches about -55mV, the voltage-gated Na+ channel opens very rapidly.
- Na+ rushes into the cell through the ‘activation’ gate of the channel.
- Closed to open.
* Fast, voltage gated.
- Open to inactive
* Fast, automatic
- Inactive to closed
* Slow, automatic
43
Describe repolarisation.
- Restoration of membrane potential (after depolarisation).
- Na+ channel closed, K+ channel open, Na+/K+ pump closed.
- Following an influx of sodium, potassium channels open within the membrane of the axon.
- As K+ ions are more concentrated inside the neuron, opening potassium channels causes a passive efflux of potassium.
- The efflux of potassium causes the membrane potential to return to a more negative internal differential (repolarisation).
44
Describe what the voltage gated K+ channel does.
- Opens when the membrane is depolarised, but more slowly than the Na+ channel.
- Closes slowly in response to membrane repolarisation.
45
What is the refractory period?
- Period after an impulse before a cell can fire again.
- Na+ channel closed, K+ channel closed, Na+/K+ pump open.
- In a normal resting state, sodium ions are predominantly outside the neuron and potassium ions mainly inside (resting potential)
- Following depolarisation (sodium influx) and repolarisation (potassium efflux), this ionic distribution is largely reversed.
- Before a neuron can fire again, the resting potential must be restored via the antiport action of the sodium-potassium pump.
46
What is the absolute refractory period?
- Membrane cannot generate another action potential.
- Sodium channels are inactivated.
47
What is the relative refractory period?
- Membrane could generate another action potential (if given a larger than normal stimulus)
- VG-Sodium channels are recovered.
- VG-potassium channels are still open.
48
How does an action potential move down an axon?
- Action potential travels down an axon via current loops- nearby area becomes depolarised by the current AP to initiate the next AP.
- The refractory period prevents the AP from going backwards.
49
What is tetrodotoxin?
Sodium channel inhibitor, blocks pore.
Therefore would effect depolarisation.
50
What is nociception?
- Nociception is the sensory nervous system’s process of encoding potentially harmful stimuli. Signals are sent from nociceptors to the central nervous system.
- Sensory neurons detect stimuli through nociceptors.
51
What are 4 discrete phases of nociception?
1. Transduction – hand on hot plate. Signal transduced through nociceptors.
2. Transmission – sends signal up primary afferent nociceptor.
3. Modulation – into spinal cord where it is modulated, first through inter neuron and then to a neuron which sends the message into the thalamus and into the brain.
4. Perception – reaches brain which perceives the signal.
52
What are the 3 main fibre types and their characteristics?
- Alpha beta fibre: Myelinated – 30-70m/s, large diameter, proprioception – light touch responses, usually mechanical not thermal
- Alpha delta fibre- Lightly myelinated – 2-10m/s, medium diameter, nociception – mechanical, thermal, chemical
- C fibre - Unmyelinated – 1m/s, small diameter, innocuous temperature, itch, nociception – mechanical, thermal, chemical
- Myelinated fibres propagate signals faster. Different nociceptors detect different types of pain.
53
How do the fibres transmit signals?
Transduce signals and relay them into the spinal cord through dorsal root ganglion. Then consolidated in spinal cord and relayed to CNS through projection neurons in dorsal root. This then goes into higher centres e.g., regions of brain that elicit some sort of response to signal.
54
How do ion channels mediate nociception?
- Na+, Na+/Ca2+: propagate an action potential.
- Na+, K+: drive action potential through axon
55
How is pain classified?
- Physiological or pathological.
- Acute pain (<3 months), or chronic pain (>3 months)
56
What is nociceptive pain and what are the 2 main types?
- Pain that arises from actual or potential damage to the body's tissues, detected by nociception.
- Somatic (body) pain and visceral pain
57
What makes something somatic pain?
- Pain arises from PNS (skin, muscles, joints, bones, and connective tissues)
- Localised to specific region.
- Stimulation nociceptive nerve fibres (Aδ and C fibres)
- Muscle cramps, sprains, etc.
- Usually responsive to pain relief
58
What makes something visceral pain?
- Pain arises from the viscera (uterus, intestine, kidneys, stomach)
- Diffuse pain, often difficult to pinpoint to a specific region.
- Nociceptive nerve fibres (Aδ and C fibres)
- Autonomic system symptoms nausea, sweating, changes in heart rate/blood pressure.
- Appendicitis, dysmenorrhea, bowel obstruction
- Poorly responsive to pain relief
59
What are inflammatory mediators of pain?
- Inflammatory molecules that arise from immune cells (APCs etc.,) produce pro-inflammatory mediators. They can bind to nociceptors and trigger a similar stimulation in those receptors.
- Inflammation in tissues driven by immune cells, propagates signal, received by brain as a pain response.
- Highly treatable with anti-inflammatories or opioids
60
What is neuropathic pain?
- Arises because of damage to the neuron (spinal cord injury, thalamic stroke carpal tunnel syndrome etc.,)
- Damage to neuron, lose transducing part of nociception, neuron can fire without a stimulus. Can lead to constant chronic pain.
- Changes in ion channel expression, changes in post translational modifications etc.
- Neurons are highly specialised so not easily repaired, limited in terms of treatment options.
61
Name some potential therapeutic targets.
- Nociceptors themselves
- Ion channels that propagate action potential
- Block how message is sent from PNS to CNS
- Target how brain processes information
62
What is lidocaine?
- Voltage-gated Na+ channel blocker – acts on depolarisation stage of action potential.
- Lidocaine reversibly binds to the inner pore of voltage-gated Na+ channels when they are in their open and inactivated states, most active in firing neurons.
- Lidocaine has greater affinity for inactivated channels compared to resting channels.
- Blocks the inner pore.
- Non-selective
63
Name 4 type of gated ion channels.
- Voltage-gated
- Ligand-gated (extracellular ligand)
- Ligand-gated (intracellular ligand)
- Mechanically gated
64
Name some animals and their respective ion channels.
- Pufferfish – tetrodotoxin, sodium channel blocker (pore)
- Deathstalker scorpion – charybdotoxin, potassium channel blocker (pore)
- Funnel-web spider – w-agatoxin, calcium channel blocker (voltage sensor)
65
How are ion channels specialised and selective?
- Substrate selectivity (such as toxins)
* Separate pathways for the flux of different ions
- Gating
* Control ion channel opening/closing
* Dynamic control
* Voltage/signal/ligand/stretch gated.
66
Name 5 ways of investigating ion channel structure.
1. Amino acid sequence
2. Experimentally - X-ray crystallography and cryo-electron microscopy
3. Computational (predictions)
4. AI
67
How is amino acid sequence used to investigate structure?
- Looking at biochemical properties of specific amino acid sidechains allows you to make predictions about protein structure.
- Hydrophobic – transmembrane region
- Polar/charged/hydrophilic-extramembrane - ligand binding etc.
68
How is x-ray crystallography used to determine ion channel structure?
- Standard technique
- Sample must be crystallised in a solid frozen structure.
- Any size macromolecules
- Atomic resolution but crystallisation may take years and damage protein structure.
69
What do we know about ion channel structure from X-ray crystallography?
- Transmembrane structure
- Large proteins – growing and producing in e. coli can be tricky.
- Multiple subunits/conformations
- Dynamic and disordered.
- Not very soluble
70
How is cryo-electron microscopy used to determine ion channel structure?
- Sample is frozen in its native state.
- Any size macromolecule.
- Near-atomic resolution, fast sample preparation.
71
Name 2 types of channel inactivation.
1. N-type inactivation
- Amino acids at N-terminus occlude the intracellular side of the channel pore.
- Leads to rapid inactivation.
2. C-type inactivation – hinged lid
- Conformational change at the selectivity filter or at the extracellular entrance to the channel
- Slow inactivation
72
Name 4 major types of animal tissues.
1. Epithelial
2. Muscle
3. Nervous
4. Connective
73
Why is connective tissue different to the others?
Reduced cellular content, increased ECM content, cell-cell contact is rare.
74
What do epithelial, muscle, nervous tissues have in common.
Similar structure with high frequency of cell-cell interactions. 4 types of junctions. Also interact with basal lamina
75
What is ECM?
Main stress-bearing component of connective tissue and forms an indirect means of cell-cell contact.
76
Functions of the ECM.
1. Support and strength - basal lamina, bone cartilage
2. Cell migration, polarity and shape - embryonic development, angiogenesis, wound repair, tumour development
3. Cellular communication - hormones, growth factors, cytokines
77
What is the basal lamina?
- Very thin layer of ECM produced by cells above and below.
- Evolutionary conserved
- Essential for maintaining epithelial tissues.
- Composed of laminin, type IV, XVIII collagen, nidogen, perlecan fibronectin
78
Name some indigenous cells.
1. Primitive mesenchymal cells: undifferentiated cells that can lead to the generation of other connective tissue cells, fat cells (adipocytes), mast cells (release histamine)
2. Fibroblasts: found in many connective tissues and synthesize most of the molecules Found in the ECM.
3. Specialized cells (adipocytes, mast cells, chondrocytes, osteoblasts): found in specialized connective tissues (e.g., cartilage, bone)
79
Name an immigrant cell
Immune cells
80
What are integrins?
- Key receptors that bind ECM components.
- Allow ECM to interact with cytoskeleton providing strength.
- Different integrin alpha and beta chain combinations allow an array of different ECM ligands to be bound.
- Mediate signalling - recruitment of focal adhesion kinase (FAK) = altered gene expression.
81
How many integrins are there? Describe the structure.
- 24 different integrins
- Alpha and beta chain with large N terminal domain.
- Short intracellular domain that binds adapters like those seen with cadherins.
82
Why do integrins need an inactive conformation?
- Many cells in connective tissue are not stationary and migrate through ECM.
- Use integrins to “pull” themselves through the ECM.
- Cell-ECM contacts therefore need to be made and broken.
83
What does activation of integrins require?
Intracellular signalling - provides physical link to cytoskeleton for application of force.
84
What makes up the composition of connective tissue ECM?
1. High MW, highly charged polysaccharides (glycosaminoglycans (GAGs)) covalently attached to proteins (proteoglycans) bind lots of water
2. Fibrous proteins
3. Glycoproteins e.g., elastins fibronectins
85
What are GAGs?
- Most anionic molecules produced by animals)
- Consist of repeating sulphated disaccharide units
- Often linked to a core protein to form proteoglycans (PG)
- PG can be relatively simple or complex, that can also self-aggregate.
86
What are fibrous proteins?
- Most abundant proteins in mammals
- Long, stiff, triple stranded helical structure provides tensile (pulling) strength of tissues.
- Members of collagen family.
87
What is the collagen family of proteins?
- Synthesized by indigenous ECM cells (e.g., fibroblasts)
- 40 different types encoded by different genes.
- 3 polypeptides (alpha chains; rich in proline/glycine) form a coil, which can then self-aggregate into fibrils and then fibers.
- Different alpha chain combinations lead to different types of collagens (Type I-XVIII) found in differing connective tissues. e.g., type II, IX found in cartilage, type XVIII basal lamina.
- Procollagen prevents aggregation inside cells.
88
What are elastins?
- Provide elasticity to connective tissues (similar in structure to collagen)
- Dominant component of ECM found in arteries.
89
What are fibronectins?
- Bind other matrix/cell membrane proteins.
- Organise matrix and provide cell-matrix link.
90
What are the different reasons for division?
- Cloning cells of a given type to make tissues.
- Making cells of different types (differentiation) – can involve asymmetric divisions.
- Making cells with half normal DNA content – eggs and sperm (meiosis).
91
Purpose of the cell cycle
To allow a cell to reproduce
92
Importance of the cell cycle
- Required for growth, development, and procreation.
- High fidelity required to ensure stable inheritance of cell and organism characteristics.
- Must be controlled to allow development and prevent disease
93
What needs to happen for the cell cycle to be carried out?
- Chromosomes need to be duplicated.
- Other organelles need to be copied.
- Cells need to grow.
- Chromosomes need to be segregated accurately.
- Cell needs to physically divide.
94
Stages of the basic cell cycle.
1. G1: Gap 1
- Deciding if conditions are right for a full cell cycle.
- Growing and preparing for DNA synthesis
2. S: Synthesis
- Replicating DNA and centrosomes
3. G2: Gap 2
- Deciding if conditions are right for mitosis.
4. M: Mitosis
- Chromosomes segregation and cytokinesis
5. G0: Resting State
- Cells not in the cell cycle
- Terminally differentiated cells.
- Quiescent cells
- Senescent cells
95
What are cyclin-dependent kinases (Cdks)?
- Drive the cell cycle
- Protein kinases that transfer a phosphate onto their substrates
- Act as ‘master regulators’
- Have multiple target proteins to control numerous processes in the cell cycle.
- Have little activity by themselves but are activated by cyclin proteins.
96
Name some additional Cdk regulators
* Upstream kinases and phosphatases.
* Cdk inhibitory proteins (CKIs)
97
What is the APC/C?
- The APC/C signals degradation of M-Cyclin to end mitosis and initiate cell division.
- The APC/C is a ubiquitin ligase.
- It covalently attaches the small protein Ubiquitin to client proteins such as M-Cyclin.
- Ubiquitinylation is a tag for protein degradation.
98
Why do cyclin levels oscillate?
- Cyclin synthesis is crucial to drive the cell cycle: mechanisms controlling synthesis include changes in transcription and translation rate, which vary depending on cell type.
- Control of cyclin destruction is also vital: important example – degradation of M-Cyclin is triggered by the APC/C
99
What is SCF?
- SCF signals degradation of CKIs to promote G1-S transition.
- SCF is a ubiquitin ligase.
- It covalently attaches the small protein Ubiquitin to client proteins such as CKIs (Cdk inhibitor proteins).
100
How is cell cycle fidelity maintained?
- Cyclin oscillations provide timing for the successive phases of the cell cycle.
- If something goes wrong, most cell types have cell cycle checkpoints.
- These are monitoring systems that check if conditions are right before allowing the next phase to occur.
- They act by promoting Cdk activation or inactivation.
101
What does the checkpoint ask in the G1-S transition?
- Are nutritional conditions suitable?
- Is the cell receiving proliferation signals?
- Has any DNA damage been repaired?
- Was the previous mitosis too long?
Once passed, the cell is committed to the entire cell cycle.
102
What does the checkpoint ask in the G2-M transition?
- Is DNA replication complete?
- Has and DNA damage been repaired?
- Is the cell big enough (yeast)?
103
What does the checkpoint ask in the metaphase-anaphase transition?
Are chromosomes attached to the spindle?
-Once checkpoint is satisfied:
* The APC/C is activated to degrade M-Cyclin
* The cells exit metaphase into anaphase.
104
What happens if a checkpoint cannot be satisfied?
- Cells will resume the cell cycle if errors or damage can be fixed.
- In some cases, things cannot be corrected in a timely way, e.g., extensive DNA damage or trial error correction of chromosome attachments takes too long
- Then, cells can withdraw from the cell cycle or can undergo apoptosis
105
What happens if a checkpoint is defective?
- Checkpoints that don’t operate properly can result in disease:
- Aberrant mitogen signalling can inappropriately drive cells through the G1 checkpoint into the cell cycle.
- Defects in G2 checkpoint can allow proliferating cells to accumulate DNA damage.
- Defects in the mitotic checkpoint can cause aneuploidy (wrong number of chromosomes)
106
How is a healthy ECM maintained?
- Cells within ECM secrete the components of the ECM.
- The same cells also secrete enzymes which digest/breakdown these components to remove damaged matrix, i.e., ‘remodel’ matrix.
107
What happens if there is too much matrix synthesis?
tissues scarring, fibrosis, cancer (alteration of function)
108
What happens if there is too much matrix breakdown?
developmental/induced deficiencies/breakdown, arthritis/metastasis (loss of function)
109
Name some intracellular proteinases
* Aspartic proteinases
* Cysteine proteinases
* Threonine proteinases
110
Name some extracellular proteinases
* Serine proteinases
* Metallo-proteinases
111
What is the degradome?
- 570 genes in human genome that encode proteinases.
- Collectively called the ‘degradome’.
112
What domains characterise the metalloproteinase family of enzymes?
1. MMP
2. ADAM
3. ADAMTS
113
When is ECM synthesis/breakdown essential?
During embryonic development, wound healing, prevention of tumour development
114
What are channelopathies?
- Pathology/disease arising from ion channel dysfunction.
- Usually arise from ion channel subunits or proteins that regulate ion channels.
115
What is ion channel dysfunction and where does it affect?
- Ion channel dysfunction is a hallmark of many disease phenotypes.
- Brain, pancreas, muscle, heart (occupied by many excitable cells), but also affect other tissues, e.g., kidneys and lungs.
116
Define and give examples of a phenotype.
- An observable physical property of an organism.
* Classic phenotypes:
Height
BMI
Eye colour
* Disease
Diabetes
Alzheimer’s
* Others
Financial income
- Phenotype is an interplay between genetics and environment.
117
What is a monogenic, idiopathic and acquired trait?
Monogenic - influenced by genetics
Idiopathic - influenced by both
Acquired - influenced by environment
118
Describe characteristics of CF.
- Monogenic – arises from mutations in the CFTR gene.
- Dysfunction in the influx of chloride ions (and water).
- Leads to mucus accumulation and inflammation.
- Subtypes functionally classified on the effect of the mutation on the channel.
119
What are the classes of CF?
* Class 1 – mutation in gene causing failure in translational process.
* Class 2 – mutation in resulting protein and can’t go anywhere.
* Class 3 – mutation affecting gating of ion channel.
* Class 4 – mutation forming faulty protein.
* Class 5 – mutation affecting amount of protein produced.
* Class 6 – not anchored to plasma membrane.
120
What is SCN9A?
- Sodium voltage gated channel gene
- Encodes NaV subtype: 1.7
121
What does SCN9A do?
- Contributes to the rising phase and amplifies subthreshold stimuli.
* Low activation threshold (compared to other NaV channels)
* Fast kinetics
* Responsive to TTX
- Highly expressed by sensory neurnos of dorsal root ganglia
122
SCN9A mutations and pain disorders
* Paroxysmal extreme pain disorder (PEPD)
* (primary) inherited erythromelalgia (IEM)
* Small-fibre neuropathy
123
What is IEM?
- Inherited Erythromelalgia
- Mutation in SCN9A (autosomal dominant)
- Bilateral episodes of burning pain in feet &
hands
- Attacks triggered by exercise and/or heat.
- Increased ion channel functions
124
What is paroxysmal extreme pain disorder?
- Mutation in SCN9A (autosomal dominant)
- Severe pain in the rectal, ocular, and mandibular areas
- Attacks triggered by chewing and/or heat.
125
Gain of function mutations for PEDP?
* Lowered threshold for channel activation - the channel opens more easily in response to small depolarizations.
* Delayed channel inactivation - the channel stays open longer than usual.
* Enhanced response to repetitive stimulations - the channel recovers more quickly between activations.
* Altered voltage-dependence - the relationship between membrane voltage and channel opening is shifted.
126
What is complete insensitivity to pain (CIP)?
- Mutation in SCN9A (autosomal recessive)
- Loss of all pain sensations
- Tend to be non-sense mutations (within domains I & II) leading to a truncated protein.
- CIP patients often suffer major injuries as a result.
- Characterised by loss of function
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4 types of cell junctions in epithelial cells
1. Adherens junctions - cadherin (calcium dependent). Binding is homophilic. Connects actin filament bundles.
2. Desmosomes - similar to adherens junctions but contain specialised cadherins that connect with intermediate filaments.
3. Tight junctions - seals gap between epithelial cells
4. Gap junctions - channels made from connexins and innexins. Allows passage of small water-soluble molecules.
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Shapes epithelial cells can be.
* Columnar – long and thin
* Cuboidal
* Squamous – squashed.
* Stratified – more stretched as they get further away from basal lamina.
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3 types of cartilage.
Hyaline, fibro, elastic.
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Where is hyaline cartilage found?
Ribs, nose, larynx, trachea, articular joints
Pre-cursor for bone
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Where is fibro cartilage found?
Joint capsules, ligaments
Least flexible, fewest cells, highest collagen
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Where is elastic cartilage found?
Ear, epiglottis, larynx
Most flexible due to increased elastin
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Characteristics of articular joints
- Stops bones rubbing together.
- Defined by the presence of the indigenous chondrocyte.
- Collagen (type 2) and aggrecan are the two key components found in articular cartilage, important for strength and support.
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What is collagen?
* Synthesised in rough endoplasmic reticulum.
* Processed in Golgi.
* Pro peptides removed so can auto aggregate.
* Collagen mutations cause hyperextensible joints.
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What is Ehlers Danlos syndrome?
* Defective deposition of collagen
* Hyperextensible joints
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What is aggrecan?
- Aggregates with other aggrecans by binding with other high mw GAG.
- PG are highly charged due to GAG content.
- Attract water and form a hydrated gel:
* Provides resistance to compression.
* Results in swelling pressure (turgor)
* Provides strength and support.
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What do chondrocytes make?
Makes PTHrP - parathyroid hormone-related protein - after you've got Sox-9. Basically makes the EMC that forms cartilage.
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What does secreted hedgehog do?
Prevents Ci being cleaved, if Ci isn't cleaved it can't interact with the repressor and therefore hedgehog responsive genes are turned off.
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What is osteoarthritis?
- Usually slow but progressive loss of ECM and the 'chondrogenic phenotype' in articular cartilage due to mechanical or degradative damage without obvious cause (primary OA)
- Results in limitation in joint movement that reduces quality of life.
- Often associated with formation of new bone in wrong place
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Current therapies for OA.
1. Surgery
2. Non-steroidal anti-inflammatories - don't stop disease progression
3. Identify and target key proteinases
4. Genetic screening
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What is Rheumatoid arthritis/inflammatory arthritis
- Progressive loss of chondrogenic phenotype in articular cartilage due to immune cell-mediated damage
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What is cell transduction?
Process by which an extracellular signal molecule activates a membrane receptor that in turn alters intracellular molecules to be transduced via a certain pathway to activate a cellular response.
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Name some first messengers
- Chemicals that can serve as extracellular signalling molecules:
* Amines – epinephrine, adrenaline
* Peptides and proteins – angiotensin II, insulin
* Steroids – hormones
* Other small molecules – amino acids, ions (calcium), gases (nitric oxide).
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Where are receptors for peptide hormones usually found?
Tend to be cell surface receptors built into the plasma membrane of cells and are thus referred to as trans membrane receptors, e.g., insulin.
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Where are receptors for steroid hormones usually found?
Within the cytoplasm and are referred to as intracellular or nuclear receptors, e.g., testosterone.
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4 main types of receptors
1. Ligand-gated ion channels - ionotropic receptors, metabotropic receptors
2. G-protein coupled receptors
3. Enzyme-linked receptors
4. Nuclear receptors
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What is PKA?
A tetrameric protein with 2 types of polypeptide chains - catalytic and regulatory
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What is Diabetes Mellitus?
- Inability to regulate blood glucose.
- High or low glucose both cause acute symptoms.
* Chronic high (glucose) damages almost all tissues.
* Untreated type 1 diabetes leads to body wasting.
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Pathogenesis of type 1 diabetes
* Caused by a failure of insulin secretion.
* Characterised by low/absent insulin and high glucose.
* Sudden onset
* Usually develops early in life.
* Relatively rare (~5% of diabetes)
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Pathogenesis of type 2 diabetes
* Caused by insulin resistance in tissues.
* Insulin present in circulation but glucose remains elevated.
* Gradual onset
* Usually develops later in life.
* Most common form of diabetes, strongly associated with obesity.
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How is type 1 diabetes developed.
- Caused by destruction of B cells.
- Involves an autoimmune mechanism (CD8 cytotoxic T cells mediated)
- Total failure of insulin secretion
- Evidence of hereditary tendency
- But can develop spontaneously in absence of family history or environmental trigger.
* Insulin dependent diabetes mellitus:
- B-cells of pancreas destroyed by cytotoxic CD8 T cells reactive against peptides of insulin and of other specific proteins which are complexed with MHC molecules and recognized by cytotoxic T lymphocytes (CTL)
- HLA-DR3 and DR4 – type 1 diabetes
- DR4-DQ8 haplotype for Caucasians
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Symptoms of type 1 diabetes.
- Lack of insulin (hyperglycaemia):
*Tissues cannot accumulate and store glucose.
*Tissues cannot use glucose as metabolic fuel.
*Body cannot store excess energy as fat.
*Reduced synthesis of protein
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Symptoms from hyperglycaemia
- High [Glucose] enters glomerular filtrate and overwhelms glucose absorbing capacity of proximal convoluted tubule =
- Increased fluid osmolarity in tubules =
- More water is secreted from cells into the PCT =
- Causes increased urine flow – diuresis. Water reabsorption is reduced =
- Dehydration, excessive urine production and thirst.
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What is insulin therapy.
- Aim is to artificially regulate blood glucose.
- Patient monitors their own blood sugar levels with a pump and regulates the amount of carbohydrate in their diet.
- Develop a protocol to match insulin injection/food consumption and thus obtain control over blood glucose levels.
- However, in practise good control over [glucose] is hard to achieve.
- Most patients with diabetes eventually develop long term complications.
- Glycosylated haemoglobin can be used to predict glucose values of the past 6-8 weeks and to monitor the long-term control.
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Problems with repeatedly injecting insulin.
- Exogenous insulin into general circulation – natural insulin portal circulation.
- A major effect of insulin is to promote the deposition of fat.
- Cells close to site of insulin injection exposed to high insulin.
- If same site used again and again, will promote deposition of fat around injection site (lipohypertrophy).
- Clinically important as leads to unpredictable rate of insulin absorption.
- Could lead to poor glycaemic control and patients could experience hyper/hypoglycaemic events. Important to change site frequently to avoid this.
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Forms of insulin used for therapy.
1. Animal insulin (porcine/bovine)
2. Human insulin
3. Human insulin analogue
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Types of human insulin
- Soluble insulin: Rapid, short-lived, intravenous emergency treatment of hyperglycaemic emergencies only
- Isophane insulin: Forms precipitates, intermediate acting
- Insulin zinc suspension: forms precipitates, long acting.
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Types of human insulin analogue
- Insulin Lispro: modified insulin analogue obtained by switching a Lys28 and Pro29, very rapid, very short lived, taken before a meal.
- Glargine: modified insulin obtained by mutating Asn21 in Gly and adding 2 Arg at the end of the B chain, long acting.
- Detemir: modified inulin obtained by mutating Thr30 (deletion), long acting. Taken before a meal in combination with a short-acting form, forms a micro precipitate at the physiological pH of subcutaneous tissue, slowly absorbed.
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What causes type 2 diabetes?
Genetic and environmental predisposition
* Lifestyle
* Bad dietary habits
* Obesity
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How is type 2 diabetes developed over time?
- Insulin resistance
* ↓ glucose uptake
* Insulin pathway defects
- Hyperinsulinemia
* B cells try to compensate for peripheral resistance
* Normal glucose levels can be maintained for years.
- B cells failure and hyperinsulinemia
* B cells become exhausted and cannot keep up with the peripheral demand of insulin
* Insulin secretion decrease
- Diabetes
* Hyperglycaemia develops.
* Total failure of insulin secretion
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What are free fatty acids?
- Lead to insulin resistance in muscle and liver.
- When in excess they are transformed in second messenger DAG
- DAG activate PKC, which phosphorylate IRS-1 on Ser residues.
- This attenuates Insulin Receptor signalling pathway.
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What are adipokines?
- Released by adipocytes.
- Pro-hyperglycaemic or anti-hyperglycaemic adipokines
- Adiponectin is anti-hyperglycaemic, because improves insulin sensitivity by activating AMPK, enzyme promoting lipolysis in liver and muscle.
- Adiponectin expression is reduced in obesity.
- Also activates IRS1/2 improving insulin signalling and GLUT4 improving glucose uptake.
- AMPK activators can be used in therapy (metformin)
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What is PPARy
- Nuclear receptor involved in adipocyte differentiation.
- Also expressed in liver and muscle
- Promotes secretion of anti-hyperglycaemic adipokines
- Mutations/Post-translational modifications associated with diabetes (e.g., Ser273 phosphorylation)
- Agonists used in therapy.
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Therapy at the beginning of type 2 diabetes
Weight loss/exercise can reverse the development of normal sensitivity to insulin.
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Effects of AGEs on blood vessels
- AGEs crosslink with collagen
- The basal membrane of the endothelium thickens.
- The thickened endothelium traps LDL and IgGs.
- Oxidation, complement activation and inflammation.
- Blood vessel damage.
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Long term complications of type 2 diabetes
Macrovascular disease/ microvascular disease = damage to blood vessels
Caused by ROS generation and AGEs generation.
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Main sites of glucose storage
Muscle and liver
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Glucose homeostasis
- Glucose absorbed from GI tract
- Enters circulation
- Used to fuel metabolism in many tissues
- Brain can only use glucose
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What happens to glucose after a meal?
- Increased glucose absorption after meal
- Would increase [glucose] in circulation
- May stimulate metabolism
- Increase O2 demand
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Glucose in between meals
Between meals glucose absorption minimal
Would lower [glucose] in circulation
May limit metabolism
Reduce O2 demand
Mammals maintain metabolism between meals
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What does insulin promote
Glucose storage as glycogen
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What does glucagon promote?
Glucose release from stored glycogen
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Structure of insulin
2 polypeptide chains held together by disulphide bridges.
Synthesised within pancreatic islet B cells as polypeptide
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Glucose transport into B cell
- B cells express a type 2 glucose transport system (GLUT2)
- Hormone-insensitive so always active
- In B cells, glucose is phosphorylated to glucose-6-P by glucokinase and metabolised by glycolysis and mitochondrial oxidation to generate ATP
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What happens when B cell is exposed to low glucose?
Normal glucose - normal internal ATP - K+ channels are open - Vm is hyperpolarised - Ca2+ channels are closed - B cell does not secrete insulin
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What happens when B cell is exposed to high glucose?
High glucose - high internal ATP - K+ channels close - Vm depolarised - Ca2+ channels open - B cells secrete insulin
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3 domains of p53
N-terminal domain, core domain, c-terminal domain
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How do mitogens drive progression into S phase?
Mitogen binds to cell surface receptor tyrosine kinase
Ras-Raf-MAPK kinase signalling pathway is triggered
“Immediate early” genes including Myc are expressed
Myc is a transcription factor which upregulates genes including Cyclin D
Cyclin D, together with Cdk4 or 6, forms G1-Cdk
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How does G1-Cdk drive progression into S?
A key target of G1-Cdk is Retinoblastoma protein, Rb
In an early G1 cell, Rb binds to and inactivates the transcription factor E2F
Phosphorylation of Rb by G1-Cdk inactivates Rb
E2F is now free to upregulate expression of genes including Cyclin E and Cyclin A
Cyclins E and A associate with Cdk2 to form G1/S-Cdk and S-Cdk
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What can prevent G1-Cdk driven progression into S?
p53 is a central regulator of checkpoint responses to stress
p53 is normally maintained at low levels in cells by Mdm2-mediated degradation
DNA damage activates kinase signalling through ATM/ATR and Chk1/Chk2
Phosphorylation of p53 displaces Mdm2 and p53 is stabilised
p53 acts as a transcription factor to turn on expression of CKIs such as p21 which inhibits G1/S-Cdk activity
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How is the end of linear DNA replicated?
Telomeres - GGGTTA repeated.
Extend the 3' end of the chromosome so it can be back filled by lagging strand synthesis without losing genetic information.
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3 DNA damage repair pathways
1. Detection of nucleotide mis-incorporation during replication
2. Detection of damaged nucleotides or bases
3. Detection of DNA breaks
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What is M-Cdk
'Master regulator' of mitosis
1. directly phosphorylates key substrate proteins
2. regulates downstream mitotic kinases (eg Aurora and Polo kinases) which then phosphorylate additional substrates
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Kinetochore assembly (prophase)
The kinetochore:
- the microtubule binding site on a chromosome
- a large macromolecular complex that assembles on the centromere
M-Cdk (Cyclin B-Cdk1) and Aurora B kinases are required to recruit kinetochore proteins in early mitosis
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Prophase summary
Interphase chromosome structure is lost
Chromosomes condense
Kinetochore assembly begins
Microtubule dynamics change so that the spindle starts to form
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Prometaphase summary
Nuclear envelope breaks down - allows access of microtubules to chromosomes
Spindle assembles
Microtubules attach to chromosomes
Microtubule adapter proteins and motor proteins become active - allows chromosomes to be moved on the spindle
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What does kinase Aurora B do?
Detects bi-orientation
Localises to centromeres, detects tension
Phosphorylates Ndc80 to remove microtubules from kinetochores
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Anaphase A
Chromosomes move towards the spindle poles
Driven largely by microtubule depolymerization at the plus ends of kinetochore microtubules (in human cells)
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Anaphase B
Spindle poles move apart
Driven largely by microtubule motors eg kinesin-5
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Telophase
Chromosomes decondense (Condensins lost, Cohesin recruited); interphase chromosome structure re-established
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Cytokinesis
Contractile ring causes cleavage furrow formation and cell is divided into two daughters
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What happens when a cell is forced into the cell cycle?
ABL protoncogene activation
Operates in the G1-S transition in response to mitogens
ABL is a tyrosine kinase that becomes aberrantly activated as a result of a reciprocal translocation
BCR-ABL chimeric protein is the cause of Chronic Myeloid Leukemia (CML)
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What happens when a cell cant exit the cell cycle?
Rb tumour-suppressor inhibition
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