Lecture 1 Flashcards
What is physiology?
Study of:
- purposeful interactions of matter energy and fields in a living system
- interactions are functional
- dynamic - movement in an organised way
- flow requires control and regulation
Example were ‘flow’ occurs in the living body
- Pulmonary system
- Cardiovascular system
- GI system (e.g. if too fast, can suffer from diahorrea, if too slow, can suffer from constipation)
- renal system
etc
Explain how flow is used in the pulmonary system? Why is flow needed? Does it overlap with other system? Where does flow occur?
o Enable Respiratory Gas Flow
o Supply of O2 /Removal CO2 matched to Physiological Demand
o Flows through Trachea - Bronchial Tree to Alveoli
o Transfer Matched to Cardiovascular Flow
o Increased Demand - Increased Flow
How to measure disease and disturbance to pulmonary flow? How is healthy flow determined?
o Lung Capacity (Volume)
o Peak Expiratory Flow Rate
o Airway Resistance
How can these indicate disease (flow - pulmonary system)
o Lung Capacity (Volume) decrease
o Peak Expiratory Flow Rate decrease
o Airway Resistance increase
What do drugs commonly want to improve?
Flow
Capacity
Explain how flow is used in the cardiovascular system
o Supply of O2 /Removal CO2
o Supply of Nutrients supporting Metabolism /Growth/Renewal - Removal of Waste Products
o Flow/Supply matched to Physiological Demand
o Flows Heart /Lungs - Arteries – Arterioles – Capillaries –Venules –Veins
o NB – Pulmonary Flow matched to Cardiovascular Flow
o Increased Demand - Increased Flow
How can you measure flow in cardiovascular system?
o Electrocardiogram (ECG) - Pump o Heart Rate x Stroke Volume = Cardiac Output o Blood Pressure o Blood Biochemistry (Cholesterol + Flow Factors)
Examples of disturbance to normal cardiovascular flow
Coronary heart disease - heart’s blood supply is blocked or interrupted by a build-up fatty substances in coronary arteries
What have of these systems got to do with cell physiology?
- Huge range of Membrane Transporters/Channels selectively regulating flow
- Nerve Action Potential precise spatio-temporal control of Na+ /K+ current - electrochemical flow
Connection between potential energy and kinetic energy
(blank)
Potential energy - what is this?
- Energy in ‘Stored Form’
- Can be Released and Harnessed to Perform ‘Work’ • It is called ‘Potential’ because it is not being used
- Body has huge reserves of PE re
Kinetic energy - what is this?
- Called ‘Kinetic’ as it is associated with Movement
- Energy made into movement of matter– considered as ‘Work’
- Body continually utilises and directs KE in a highly controlled way
Give some examples (some from this list) of potential energy in physiological systems
- PE stored in Chemical Bonds – Energy Released in Reaction
- PE in the ~Pi bond in ATP universally used as currency for delivery of Energy
- PE in Concentration Gradients across Cell Membranes
- PE in Electrochemical Gradients - 1 Generates Membrane Potential
- PE in Electrochemical Gradients - 2 The PE Source for 2o Active Transport
- PE in Electric Fields – ‘Action at a Distance’ on Voltage Sensitive Proteins
- ‘Elastic PE’ – Held in Molecular Structures For Release as Mechanical Energy
- The release of PE needs to match the demand for KE – Again Flow Control
Give some examples (some from this list) of Kinetic energy
- Chemical Bond -> Thermal Energy from exothermic reactions -> Random Brownian Motion (‘disorganised’ flow)
- Chemical Gradient -> Molecular Movement across Membrane
- Electrochemical Energy Gradient - 1 -> Current Flow across Membrane
- Electrochemical Energy - 2 -> Current Flow + Co-Transport secondary Active Transport
- Electrical Field -> Field Movement + Conformational Changes
- Elastic Energy -> Mechanical Energy
- > Conformational Changes -> Macromolecular Movement
Chemical bond PE can lead to KE due to heat being released
PE: Concentration gradient across membrane
KE: Diffusion of molecules across membrane
What is the correct name for the ‘sodium postassium pump’
Na+/K+ ATPase
How is the Na+/K+ ATPase involved in potential energy and kinetic energy…?
• Na+ /K+ ATPase is an enzyme coupled transporter at the heart of Cell Energetics.
• At rest uses 30-35% of Cellular ATP
• Uses Chemical Bond PE in ATP - enables conformational change in ATPase (Chemical Bond
-> Elastic Energy -> Mechanical Energy)
• Carries 3 Na+ out / 2K+ into the Cell
• This is Electrogenic - contributes to an Electrochemical Gradient across the Cell
• E. Chem. Gradients –V. important PE Source
• E.Chem. Gradients have an associated Electric Field – a further source of PE
• Electric Fields allow ‘Action at a Distance’
Electrochemical gradient PE and Electrochemical current flow KE
Electrical field PE -> Ions move KE
How can potential energy be added to a molecular structure?
‘Elastic’ Energy Stored in ‘Stressed Molecular’ Structure
• Proteins ability to switch between conformational shape underpins much function.
• Potential Energy added by phosphorylation ~ Pi to add ‘Elastic PE’ to attain new higher energy conformational state
• Elastic energy is released as Mechanical Movement when it ‘springs back’ to the lower energy conformation.
• Property underpins PE storage in proteins
Elastic energy (PE) -> Mechanical energy (KE)
How is physiology classified?
Characterised by the close control of the flow and interplay of work and information
How does pharmocology link to physiology?
Pharmacology studies defined effect of signalling molecules on physiological and biochemical work activity of cells right up through to the level of the person
Clinical pharmacology is based on proper understanding of endogenous signalling molecules and their cellular targets
Signalling molecule classification?
- Endogenous (within the body – our reference signalling molecules)
- Exogenous I – Natural (plant based – e.g. morphine, aspirin, antibiotics, anticancer)
- Exogenous II – Synthetic (Man made - many thousands of compounds)
Therapeutics definition
- the branch of medicine concerned with the treatment of disease and the action of remedial agents.
- a treatment, therapy, or drug.
What are the main extracellular signalling groups?
o Endocrine
o Paracrine
o Autocrine
• Characterised by distance/volume over which signalling molecules act
• Overlap between categories sites of action
All these processes work in….
…work in synchrony
Extracellular Signalling Molecules 1: Endocrine system (produced by… time they act… act where?… what must be the case for an action to occur… what is endocrine system involved in…)
• Endocrine system – glands produce hormones act as signalling molecules
• Typically act over long distances/throughout whole body ( upto +1m )
• More than 100 known endocrine hormones as signalling molecules in humans
• For an action cells need to express receptors/binding sites – many specific target types
• Major regulation of body function via neuroendocrine system
o Digestion
o Metabolism/Respiration
o Growth – Development
o Behaviours - Sexual - Stress Response
Extracellular Signalling Molecules 1: Endocrine system (where secreted… talk about potency… timescale of action… feedback…)
- Secreted into blood stream : ‘Global’ signal route - circulation to whole body
- Highly potent - picomolar to nanomolar range (10-12 M to 10-9 M)* More potent = the lower the conc of it is needed to have an effect
- Timescale of action ranges from seconds to months (molecule/receptor dependent)
- In health - synthesis release and degradation well controlled - subject to tight feedback control (see MEH Unit Semester 2)
- Close interaction synchrony and integration between endocrine signalling is key e.g. controlling ovulation or blood sugar (insulin/glucagon)
Endocrine system - What are the major types of signalling molecules
• Hydrophilic 1 - Amines
Amino acid derivatives (amino acids but modified groups) – small charged hydrophilic with Receptors in Plasma Membrane
Hydrophilic - can’t get through the membrane
e.g. Norepinephrine
• Hydrophilic 2 - Peptide hormone and Proteins hormones.
Peptide hormone:
Short chain of linked amino acids, e.g. Insulin 51 amino acid residues around 5.8 kDa (kDa - measure of weight) with Receptors in Plasma Membrane
Protein hormone:
Long chains of linked amino acids e.g Human Growth Hormone has 191 amino acid residues
Hydrophillic - can’t get through the membrane
• Lipophilic – Steroids
Common derivation from cholesterol Receptors are Intracellular
e.g. cholesterol and testosterone
Properties of the different types of endocrine signalling molecules (amines, peptides and proteins, steroids)
- solubility
- feedback regulating synthesis
- Plasma half-life
- receptor location
- mechanism of action
Extracellular signalling 2 - Paracrine signalling (scale of the signalling
Paracrine Signalling Molecules
• Signalling ‘extent’: Signalling coupled from cell to cell - or cells within nearby volume
• Scale of signalling: Communication scale is much lower than endocrine, 10-2 to 10-8 (1cm to ≈10 nm)
• Paracrine signalling molecules released into extracellular environment
• Induce changes in receptor cells - specific behavior / differentiation
Examples
• Very wide set of signalling molecules - ICPP cover specific examples
• Focus on Neurotransmitters - neurone to neuron - ICPP Session 08
Neurotransmitters are examples of _______ signalling
Paracrine signalling
Extracellular signalling 2: Paracrine Signalling Molecules
Neurotransmitters
- occurs over…
- direction…
- distance…
- trasmission time… (the amount of time from the beginning until the end of a message transmission)
- Tight coupling of signalling molecule transmission over synapse
- Synapse is highly specialised + controlled extracellular space
- One way transmission of signal
- Electrochemical– chemical signal conversion/coupling
- Chemical signal release is proportional to presynaptic electrical field
- Distance approximately 20 nm
- Transmission time is in msecs (very short)
How many types of neurotransmiiter singalling molecules are there?
About 100 types, different classifications of them
Paracrine signalling molecules - major groups
- Monoamines
- Amino acids
- Acetylcholine
Meaning of excitatory and inhibitory
- Excitatory - signal increase firing rate post synaptically
- Inhibitory - signal decrease firing rate post synaptically
3 main groups of neurotransmitters, what specific types of neurotransmitters are in these groups, what is there signalling function
Name of where drugs act…
Drug target (commonly at a receptor)
Endogenous signalling molecules…
- ‘Engineered’ via Evolution to carry and transfer signal
- ‘OptimaI Fit’ for the Job
- Endogenous Signallers are Agonists*
*There are few endogenous antagonists
These are the natural signalling molecules, e.g. like a baton in a relay race
Exogenous signalling molecules binding with its target (what is this… efficiency it works at…
not the ‘natural’ signalling molecule
e.g. banane in a relay race being used as a baton instead of the baton, the banae does work as a baton, but it is not quite as good as the natural endogenous version.
• Engineered via human design to carry ‘imposter’ signal
• Signal still carried although the fit may be ‘suboptimaI’ (as knowledge of drugs is improving and drugs get better - they are becoming more optimal)
• Many Exogenous molecules act as Antagonists and Partial Agonists – block or attenuate biological signal
• Antagonists very important in Therapeutics
• Side Effects Possible (as manufactured drug could affect other places of the body, that the endogenous neurotransmitter wouldn’t effect)
NOTE: Exogenous drugs can be manufactured version of endogenous molecules e.g. adrenaline, testoterone (just come through a different synthetic route compared to the natural form)
Overview of signalling molecule targets: Therapeutics
Majority of drug targets can be classified using the mnemonic: RITE
R Receptors
I Ion channels (includes ligand gated and voltage gated)
T Transporters
E Enzymes
Exception to ‘RITE’?
Important exceptions to RITE is chemotherapy. This targets structural protein or DNA
What does Receptors include? (Four drug target classes)
R from RITE
Receptor sub-group:
KING
NOTE - ALL RECEPTORS NEED A LIGAND
Overviw of signalling moelcule target 1: Receptors
KING
• Receptors all need a LIGAND
• A signalling molecule that activates them
In RITE - The ‘I’, stands for Ion channels (voltage gated)
Important to realise this is voltage gated - as it is not a receptor, as it does not have a ligand
Regulating voltage gated ion channels
- Voltage Gated Ion Channels - primary activity dependent of change in electric field density - (this first depends on LGIC current activation at synapse)
- Ion Channel activity can be facilitated or inhibited by phosphorylation
- Examples of the above seen in members of Na+ ; K+ ; Ca2+; Cl- families - including the VGICs
- Binding using exogenous channel blockers results in therapeutic outcome
- Examples VG Na+/Ca2+ Channel blockers for: epilepsy; chronic pain; migraine GABA Cl- channel agonists (agonist - work at seperate sites on the protein) for epilepsy
‘T’ in RITE (overview of signalling molecules)
T stands for Transporters/carriers
What do Transports/Carriers
- Transport of Ions/ small molecules - channels can use facilitated diffusion if there is gradient into/out of cell
- Active Transport needed if transported species
- going against gradient e.g. Na+ /K + ATPase
- Use ATP as 1o energy source or 2o pre-existing Electrochemical ion gradient
- Transport across many boundaries GI tract
- Renal Tubule Blood Brain Barrier
- E.g. SSRIs Inhibit Serotonin re-uptake in mood disorders
E in RITE (overview of ‘Enzymes’)
What do Enzymes Do?
• Signal Processing - Transformation - Synthesis - Degradation
• Phenomenal Range of Molecular Effectors > 5000 reaction types
Targeting Enzymes
• Competitive inhibition at active binding site with - substrate Aspirin binds to COX enzyme reduces prostaglandin synthesis
• ACE Inhibitors - Reduce levels of Angiotensin – decrease BP
General Functions of Biological Membranes (Cells and Organelles)
- Continuous, highly selective permeability barrier
- Control of the enclosed chemical environment
- Communication
- Recognition - signalling molecules:
i. adhesion proteins,
ii. immune surveillance - Signal generation in response to stimuli (electrical, chemical)
Different regions of plasma membrane may have different functions
For example
• Interaction with basement membrane
• Interaction with adjacent cells
• Absorption of body fluids
• Secretion
• Transport
• Synapses – nerve junctions
• Electrical signal conduction
• Changing shape may change the properties of a particular region
By dry weight, what is the most abundant component of membrane bilayers?
• Varies with source of membrane
• Generally membranes contain approximately (dry weight)
– 40 % lipid
– 60 % protein
– 1-10 % carbohydrate
NB. Membranes are hydrated structures, thus, 20 % of total weight is water
What word can be used to describe a lipid?
Amphipathic molecules
i.e. they contain both a hydrophilic and a hydrophobic moiety
Structure of a phospholipid - predominant lipid
What does a phospholipid molecule look like? Important part of there structure…
Cis double bond causing the kink
Phospholipid - two main bits of its structure
• Head groups: POLAR
– range of polar head groups
– e.g. choline, amines, amino acids, sugars
• Fatty acid chains (acyl groups): NON-POLAR
– Length between C14 and C24
– C16 and C18 most prevalent
– Cis double bond introduces a kink
Length of the acyl group affects the biophysical properties of the membrane. Electrical charge along these acyl chains is spread evenly, so described as non-polar
Phospholipid head groups - variety of these
Choline
Serine
Ethanolamine
Inositol
These phosphate groups exhibit some degree of electrical chage or polarity. The charge or polarity means they can easily interact non-covalently with the polar water molecule i.e. they are hydrophobic (water soluble)
Phosphatidylcholine
Phosphatidylcholines (PC) are a class of phospholipids that incorporate choline as a headgroup.
Overall, must be aware (regarding lipids in the membrane) about not just phospholipids in the membrane (traditionally phospholipids, phospholids with a head group and different lipids)
Must be aware that there are other structure of lipids, not just phospholipids in the membrane.
For example, sphingomyelin (still has a phosphate and choline head group, different tail structure), glycolipids (no phosphate, head group is a single monosaccharide or chain of monosaccharides)
Glycolipids - two types
No phosphate group
Cerebrosides: head group is a sugar monomer
Gangliosides: head group is oligosaccharide (sugar multimer i.e. joined monoaccharides)
Structures lipids can form
- Lipid micelle
- Lipid bilayer
Model showing the bilyaer-water interface
Liposomes - why are these really helpful?
Why are there many more water molecules associated with the membrane compared to lipid molecules?
Water has a low molecule weight of 18 Da
Other components have a large molecule weight e.g. 5000-200kDa
This means that there are many more water molecules associated with the cell membrane and that there are more lipid molecules than proteins
Top of the fatty acyl tails - key structural feature
Top end of the fatty acid chain residual, there is a C=O bond, which has strongly polar qualities. This group is the left over from the loss of the -OH group on the bonding of carboxylic acid. The C=O is very importnat. It enables hydrogen bonding with water and the polar -OH group in cholesterol.
Phospholipid motion
- Flexion: tails can ‘wobble’ about all single C-C bonds
- Rotation: whole lipid spinning around its axis
- Lateral diffusion: Lipids diffusing within the bilayer, random motion
- Flip flop (rare): Lipids flip between layers
How does the word ‘dynamic’ describe phospholipids?
Phospholipids are dynamic. This means they can move. (flexion, rotation, lateral diffusion and flip flop)
How does a cis bond in phospholipids affect the bilayer structure?
- Saturated straight hydrocarbon chains - This makes the packing of the phospholipids more ordered and more closely packed
- Unsaturated hydrocarbon chains with cis double bonds, reduce phospholipid packing - Reducing phospholipid packing, increases fluidity in the membrane meaning there is more movement. Also means that proteins can more easily change shape (conformational change)
Cholesterol - structure and what it affects
Cholesterol greatly affects stability and fluidity.
Central role in stabilisation and in formation of lipid rafts, helping proteins be siutated in an optimal position in the membrane
Cholesterol has a polar head group (important for it’s interacting with the -OH on the lipid) and a non-polar hydrocarbon tail.
How does cholesterol stabilise the lipid bilayer temperature properties?
Don’t need to learn this experiment, just need to understand this experiment
The experiment shows how cholesterol is stabilising temperature properties of the phospholipid bilayer:
- Pure DPPC (no cholesterol), increasing the temperature causes the membrane at around 320 degree K to change from a crystalline structure to a fluid liquid structure, in effect the membrane has ‘melted’ (shown by the large peak of the rate of heart flow)
- When more cholesterol is added, the change of state is less (doesn’t become as fluid)
- When 50mol % cholesterol is in the membrane, increasing the temp doesn’t change the state of the membrane at all. This shows how cholesterol is stabilising the phospholipid bilayer. The graph does show a decrease showing the membrane has become more fluid, but without the extent of the change in state seen above.
How is cholesterol inserted into the phospholipid bilyaer?
Hydroxyl group of the cholesterol forms a bond here with the fatty acid in the phospholipid. This locks the cholesterol onto the adjacent phospholipid.
What is the paradoxical effects of cholesterol in the phospholipid bilayer?
Two roles:
- Due to locking onto a phospholipid, it reduces fluidity
- However, by attaching to a phospholipid molecule, it prevents an adjacent phospholipid molecule from packing. This decreases packing and increases fluidity of the membrane. This is a paradoxical effect – as one way it reduces fluidity, another way it increases fluidity.
This paradoxical effect means it stops the membrane becoming too fluid and also too crystaline (too ‘solid’)
Showing the cis bond (double bond) in lipids
Lipid rafts
• Lipid rafts are dynamic, cholesterol-rich structures
• Comprised of
− sphingolipids with saturated fatty acid chains − tightly intercalated cholesterol (highly reduced fluidity)
• Can include or exclude membrane proteins
• Most proteins want to be out in the fluid part of the membrane to allow them to undergo conformational change (and therefore their cellular function). However, some proteins prefer to be in cholesterol ridge.
• Raft affinity for proteins modulated by intra- or extracellular stimuli (raft affinity may cause a receptor to be moved over into the raft)
• Proteins can move in and out (lipid rafts are dynamic - so cholesterols can move in and out of the lipid raft)
Lipid raft - function
• Scaffold proteins involved in signal transduction, e.g. receptors and signalling pathway components (there are a number of proteins that need to be located close together to get the message from outside to inside of the cell. The function of the raft is to act as a region within the membrane where these molecules can ‘scaffold together’ and make this transfer of information efficient. Therefore, often receptors and singalling pathway components are found on lipid rafts.
• Included proteins often in lipid rafts include:
− glycosylphosphatidylinositol (GPI)- anchored proteins
− doubly acylated (palmitoylated proteins, such as receptors with intrinsic kinase activity − α-subunits of heterotrimeric G proteins (transducing proteins)
• Crosslinking of signalling receptors increases their affinity for rafts.
• Partitioning changes their micro-environment, therefore being located close together in a lipid raft makes new interactions possible, affecting their signalling
• Raft clustering may amplify signalling by bringing signalling components together
Lipid raft - signalling scaffolds
Level of hydration of the lipid bilayer -
This is dependent on the whole body water content. Importantly, variation in body hydration either way by +/-10% of total body water will seriously affect normal membrane function
Evidence for proteins in membranes
Functional:
– Facilitated diffusion
– Ion gradients
– Specificity of cell responses
Biochemical:
– Membrane fractionation + gel electrophoresis
– Freeze fracture
SDS-PAGE gel electrophoresis e.g. of erythrocytes
This electrophoresis is a method of separating the proteins according to size.
- Coat the membrane sample with SDS, this is a detergent that coats all proteins with a negative charge (meaning all the proteins move in the same direction when put in electrophoresis)
- Sample applied at the top of the gel
- Electro gradient put across the cell, causing the negative proteins to migrate downwards
- Smaller the protein is = the further it will move, so you get banding patterns
Freeze fracture
Freeze fracture - more indepth
plane of fracture
2 faces
Mobility of proteins in the bilayer
Three modes of motion permitted:
- Conformational change
- Rotational
- Lateral
- NO FLIP- FLOP (proteins are locked in their orientation – energetically unfavourable to flip-flop)
Restriction of membrane protein mobility
- Aggregates - membrane protein associations within the membrane (AGGREGATES)
- *- Interactions of proteins with other molecules** and other proteins in and outside the cell with effect the proteins mobility, such as peripheral proteins… e.g. cytoskeleton (TETHERING)
- *- Interaction with other cells** – to form tissues (INTERACTION WITH OTHER CELLS)
- Lipid mediated effects, if proteins are separated by lipids in the membrane (e.g. not in lipid rafts). Also, if proteins tend to separate out into the fluid phase or cholesterol poor regions
Membrane Proteins - two types and what does this mean?
- *Peripheral**
- Bound to surface
- Electrostatic and hydrogen bond interactions holding proteins to the surface of the membrane
- These proteins can be removed by changes in pH or in ionic strength (e.g. increasing the salt conc)
- *Integral**
- Interact extensively with hydrophobic domains of the lipid bilayer
- Cannot be removed by manipulation of pH and ionic strength
- Are removed by agents that compete for non-polar interactions
e. g. detergents and organic solvents (these disrupt the membrane releasing the protein)
What are transmembrane polypeptides?
AA that are present are not normally acidic or basic, they are usually small or hydrophobic or polar and uncharged.
Often alpha helix. Usually 20 amino acids span across the protein.
What are hydrophathy plots?
As we know alpha helixes are common in transmembrane protiens and they are usually 20 aa, we can go to an unknown protein and work out if it is likely to be a transmembrane protein. Can use a hydropathy plot -
Summary - Association of proteins within the bilayer
What type of gel electrophoresis is used to investigate erythrocyte membrane?
SDS-PAGE
What does the bonding pattern look like in SDS-PAGE for erthyrocytes?
What happens when SDS-PAGE is put in salt water? What is left? What does this mean?
Salt wash only removes the peripheral proteins. Then spin it again (the only proteins present now in the second electrophoriisis will be integral proteins).
What is spectrins structure?
What does spectrin look like using EM?
Spectrin in the cytoskeleton
Erythrocyte cytoskeleton
Key points:
- Transmembrane protein anchors
- Spectrin lattice adhered through attachment proteins
Erythrocyte cytoskeleton is composed of spectrin dimers that are joined end to end to form the lattice structure of the cytoskeleton. This lattice (the cytoskeleton) is held in place and connected to the plasma membrane by integral proteins that are attached to peripheral proteins.
Forces exerted on Erythrocytes
Overview of Physical Forces acting on RBCs:
- The variety of forces that act on RBCs include: compression (forces pushing together; stretching forces pulling apart; bending (compression and stretching forces acting together at one point to cause bending; shearing forces in opposite direction acting on the surface); torsion (twisting forces). These are shown in the cartoon in Figure 15. These forces vary as RBCs go through different parts of the vascular tree. Fig. 15. Summary of the forces acting on RBCs. Most cell types may be subject to these forces stressful forces to differing extents.
- In the major arteries, there will be a mix of these as RBCs get buffeted due to turbulence near the artery walls. For RBCs at the wall, surrounding by adjacent faster moving RBCs just away from the wall, will experience shearing forces. This is where the whole RBC is experiencing forces in opposing directions and pushing the membrane in opposite directions.
- Within smaller capillaries with a diameter of about 3μm and RBCs about 7-8 μm you can see that there are multiple forces all acting on a single RBC. These then result in remarkable deformations of the RBC. Some of these shapes are illustrated in Fig. 16.
What is haemolytic anaemias?
Hemolytic anemia is a blood disorder that occurs when your red blood cells are destroyed faster than they can be replaced.
What is hereditary spherocytosis?
- Spectrin depleted by 40-50% (as one of the two alleles for spectrin is mutated so only half of spectrin is produced).
- As spectrin is a structural protein, it affects the structure of the cytoskeleton. This causes the erythrocytes to ‘round up’ (become spherical rather than concave)
- This leads to the RBC taking on a less efficient spherical shape, with a decrease in membrane surface area:volume. It can still carry adequate concentrations of O2 for modest levels of activity, however it is still less. However, the RBCs are much more fragile, unable to distribute and buffer the constant physical forces summarised in Fig.15.
- RBCs with cytoskeletal mutations are also osmotically fragile. The normal physiological excursions in water movement over the RBC lifetime also contribute to lysis. This fragility leads to RBCs having much shorter life cycle, typically of between 10-40 days (cf normal = 120 days).
- This deformability in these mutated RBCs leads to increased rates of splenic destruction which is the primary cause of haemolytic anaemia in this patient group. This process is illustrated in Fig.17. In less vulnerable cases, the anaemia can be offset by increased marrow erythropoiesis.
What is hereditary elliptocytosis?
- Defect in spectrin molecule - Unable to form heterotetramers – the end to end of the spectrin molecules are unable to form, this leads to fragile elliptoid cells.
- Fragile elliptoid cells – the erythrocytes are less resistant to shearing stress of the capillaries, so they lyse (burst), releasing haemoglobin, less oxygen carrying capacity (leading to aneamia).
Secreted protein biosynthesis:
- Learn how the process starts (for both non-transmembranal and integral proteins)
- For non-transmembranal proteins only
- For integral proteins
- More detail about the signal sequence
Secreted protein biosynthesis
1. Learn how the process starts (for both non-transmembranal and integral proteins)
- As normal, protein synthesis occurs with the ribosomes. The ribosomes attach to the 5 prime end of the RNA and reading down the RNA and the triplet code. Each triplet code, it brings in the required amino acid (translation). However, before the synthesis progresses very far, this translation then has to be stopped as the synthesis needs to be carried out in the ER membrane.
- The start of the translation makes a characteristic hydrophobic amino acid sequence of around 20 amino acids with a number of basic residues at the N-terminus of the new polypeptide (N-terminus, means the nitrogen in the amino acid is at the end of the sequence – rather than the carboxyl group of the amino acid residue). This short 20 amino acid sequence is termed the signal or leader sequence (it will eventually be cut off and doesn’t form part of the protein). This signal is recognised by a large protein/RNA complex called the signal recognition particle (SRP).
- The SRP then binds to the amino acid signal sequence. It also binds to the ribosome so the ribosome so the growing protein is physically held against the ribosome so the protein is unable to continue to translate more of the protein (this is called: the protein synthesis has been arrested)
- SRP is then recognised by a SRP receptor or docking protein on the ER. In this recognition, the SRP brings the ribosome and amino acid sequence down to the ER.
- In making the interaction with the docking protein, the SRP is released from the signal sequence of the newly forming polypeptide and ribosome removing the inhibitory constraint on further translation (therefore it can now continue to be translated).
- The signal sequence then binds with a signal sequence receptor (SSR) within a protein translocator complex (Sec61) (also called a pore complex) in the ER membrane, this directs further synthesis through the ER membrane.
- Now the ribosomes is attached to this pore complex, so the protein continues to be made but is extruded into the ER lumen (see next page for transmembranal proteins)
Secreted protein biosynthesis
2. For non-transmembranal protein only
- As the protein continues to be made, the signal sequence has done its job (see it diagram above), so it ‘cleaves’ the signal from the growing polypeptide chain. This signal sequence goes round to search for more ribosomes (cycle).
- Once the polypeptide chain is complete, it is fully extruded into the ER lumen (shown by the star over the polypeptide chain).
Secreted protein biosynthesis:
3. For integral proteins
(START from where the cloud is in the diagram)
- As the synthesis continues, there will be a region in the polypeptide of around 18-22 amino acids that is highly hydrophobic [not necessarily the first 20 amino acids made at the beginning, these 20 amino acids that will eventually make up the transmembrane protein across the membrane could be anywhere in this newly forming (nascent – another word for newly forming) polypeptide].
- At this point, i.e. when the group of roughly 20 highly hydrophobic amino acids (go back to electrophoresis slide above if don’t understand) is threading through the ER membrane, these amino acids on this protein would rather form an interaction with the membrane instead of passing through the membrane and into the lumen of the ER (as these amino acids are highly hydrophobic). Therefore, this sequence of amino acids act as a stop transfer sequence. This ‘locks’ this part of the protein into the membrane. This sequence forms the trans-membranous region of the protein.
- The ribosome can continue to make the protein. This means we end up with a transmembrane protein with the N-terminal (on the amino acid residue) in the lumen of the ER and the C-terminal in the cytoplasm.
- The mechanism explained above is sufficient to explain how proteins with an N-terminal signal sequence may be orientated with their N-terminus directed towards the ER lumen but does not explain how those with a lumen-directed C-terminal may be orientated. However, an understanding of this (when the protein has it’s C-terminal in the lumen of the ER) will not be assessed – so don’t worry!(START from where the cloud is in the diagram)
- As the synthesis continues, there will be a region in the polypeptide of around 18-22 amino acids that is highly hydrophobic [not necessarily the first 20 amino acids made at the beginning, these 20 amino acids that will eventually make up the transmembrane protein across the membrane could be anywhere in this newly forming (nascent – another word for newly forming) polypeptide].
- At this point, i.e. when the group of roughly 20 highly hydrophobic amino acids (go back to electrophoresis slide above if don’t understand) is threading through the ER membrane, these amino acids on this protein would rather form an interaction with the membrane instead of passing through the membrane and into the lumen of the ER (as these amino acids are highly hydrophobic). Therefore, this sequence of amino acids act as a stop transfer sequence. This ‘locks’ this part of the protein into the membrane. This sequence forms the trans-membranous region of the protein.
- The ribosome can continue to make the protein. This means we end up with a transmembrane protein with the N-terminal (on the amino acid residue) in the lumen of the ER and the C-terminal in the cytoplasm.
- The mechanism explained above is sufficient to explain how proteins with an N-terminal signal sequence may be orientated with their N-terminus directed towards the ER lumen but does not explain how those with a lumen-directed C-terminal may be orientated. However, an understanding of this (when the protein has it’s C-terminal in the lumen of the ER) will not be assessed – so don’t worry!
Secretory protein synthesis - N-terminal signal sequence folds into the membrane positioning positively charged resides on the cytoplasmic side…
I did just make it sound that the protein just ‘threads’ its way through the membrane, however, it can’t do this:
- Most of these starting signals (three dots at the beginning) have two or three positive charges in this sequence (very close to the N-terminal). These positive charges will resist passage across the membrane through the ‘translocating machinery’.
- These positive charges of the signal sequence bind to the signal sequence receptor in the membrane on the cytoplasmic side of the membrane.
- The signal peptidase cleavage site is still on the ER side (only the signal is now bound in the membrane – it is not free flowing in the lumen).
- The cleavage by the signal peptidase, releases the N-terminal of the forming polypeptide into the lumen of the ER.
- Synthesis just as explained above.
Membrane protein orientation
• How may membrane proteins contribute to the cytoskeleton?
-> integral proteins form a lattice structure with the cytoskeleton, cytoskeleton maintains cell shape
• How are membrane proteins inserted into membranes?
-> inserted into ER during synthesis…
• How is correct orientation of membrane proteins maintained
-> As they are inserted during their synthesis, this maintains the orientations of membrane proteins.
