TOPIC 7 - membranes, lipids and signalling Flashcards
How many membranes do gram positive bacteria have?
No outer membrane and a thick peptidoglycan layer
How many membranes do gram negative bacteria have?
Outer and inner membrane and a thinner peptidoglycan layer
Where are membranes in eukaryotic cells?
- nucleolus
- mitochondria
- lysosomes
- endoplasmic reticulum
- golgi
- vesicles
- chloroplasts (in plants)
What are the functions of membranes?
- functional barrier
- provide cells with energy
- organise and regulate enzyme activities
- signal transduction
- substrates for biosynthesis and signalling molecules
- protein recruitment platform
What are membranes composed of?
Proteins and lipids
What are the three types of lipids?
- glycerophospholipids
- sphingolipids
- sterols
How are phospholipids amphiphatic?
They have a polar head and non polar tail so can from bilayer structures
What group is on the end of a fatty acid chain?
A terminal carboxylic acid
How does a lipid bilayer form?
Lipids spontaneously aggregate to bury their hydrophobic tails in the interior and expose their hydrophilic heads to water
How do you name fatty acids?
Fatty acids vary in chain length, double bond number, double bond position and hydroxylation
XX:Y n-y
XX = number of carbons in the chain
Y = number of double bonds
n-y = position of first double bonded carbon
What are the shapes of saturated and unsaturated lipid tails?
Saturated - no double bonds, straight
Unsaturated - one or more double bond, can be straight (trans bond) or have a 30º kink (cis bond)
Can a phospholipid have tails of different lengths
Yes, it influences how the phospholipids pack against one another
- straight chains= thicker membranes
What do glycerophospholipids consist of?
- 2 fatty acid tails
- a glycerol backbone
- a head group
Are the glycerophospholipid tails usually saturated or unsaturated?
sn-1 is usually saturated or monounsaturated
sn-2 is more often monounsaturated or polyunsaturated
Which glycerophospholipids have a net negative charge/anionic phospholipids ?
PS - Serine
PI - Inositol
PG - Glycerol
CL - Cardiolipin
What glycerophospholipids have zero charge/ Zwitterionic phospholipids ?
PE - ethanolamine
PC - Choline
What glycerophospholipids contain amines that can form H bonds?
PS - serine
PE - ethanolamine
How many tails does cardiolipin have?
4 - makes it bulky and so affects its packing
How many PIP species?
7 - PI(4,5)P2 is the most abundant
What leaflet of the membrane are PIPs found?
Inner (cytoplasmic)
What do sphingolipids consist of?
A sphyngoid base, N-acyl chain and head group
-both tails likely to have no double bonds = saturated
What bond is there between the acyl chains and glycerol backbone in glycerophospholipids?
Ester
What bond is there between the acyl chain and sphingoid base backbone in sphingolipids?
Amide linkage - the amide group allows it to form hydrogen bonds and so can interact with cholesterol or polar parts of proteins
What is the most common sphingolipid?
Sphingomyelin (SM) has a phosphocholine headgroup
Are the acyl chains of sphingolipids or glycerophospholipids longer?
Sphingolipid acyl chains tend to be longer and more saturated
What are glycolipids?
Glycosphingolipids
- have different oligosaccharides as head groups (composed of mainly sugars)
What leaflet of the membrane are glycolipids found?
Exclusively on the outer leaflet (only make up about 5% of the membrane)
What role do glycolipids have?
Interaction of the cell with its surroundings (cell to cell adhesion) and allow membranes to act as recognition sites (sugar site exposed to the outside)
How many sugars do ganglioside GM1, GM2 and GM3 have?
GM1 - 4
GM2 - 3
GM3 - 2
-If these lipids found on outer membrane= tend to aggregate= have these suagrs and sugras can interact with outer sugars = clustering
What do sterols consist of?
Hydroxyl group and a hydrocarbon tail
What is the most common sterol in animals?
Cholesterol
What effect does cholesterol have on membranes?
- increases thickness
- increases packing
- increases compressibility
- decreases mobility of lipids and proteins
What type of tail does cholesterol interact more tightly with?
Straight saturated tails - more packing due to its shape
What affects membrane curvature?
The relative size of the head group and hydrophobic tails of lipids affect the shape of the lipid and the spontaneous curvature of the membrane
What are the two types of lipid diversity?
Chemical/structural - defines specific properties of lipids
Compositional between tissues, organelles and leaflets - affects collective behaviour of lipids
Why is lipid asymmetry functionally important?
Change in membrane leaflet composition could act as a signal - eg phosphatidylserine in animal cells translocate to the extracellular monolayer when cell is dying - acts as a signal to neighbouring cells
What enzyme aids the movement of PS lipids?
Scramblases
What is lipid interdigitation?
When lipids from separate leaflets overlap due to lipid length asymmetry. This couples the two leaflets together and decreases thickness
What is rotational movement of lipids?
Spinning of lipids around their axes, does not alter their position but affects interactions with neighbouring molecules
What is lateral movement of lipids?
Neighbouring lipids exchange places within a bilayer leaflet
What is transverse movement of lipids?
Exchange of lipids between leaflets - can move spontaneously by transverse diffusion or it can be mediated by proteins
What are the three phases of lipids when in the lamellar (bilayer) phase?
Lamellar liquid crystalline - membrane more fluid due to loose packing
Solid gel - long unsaturated lipid tails pack more tightly
Liquid-ordered - presence of cholesterol
What is the fluid mosaic model?
The bulk of the lipids forms the bilayer providing the solvent for embedded proteins.
The bilayer is fluid – lateral mobility of lipids and of some proteins. It is mosaic in the sense that proteins are scattered across it.
Most proteins are integral and some are peripheral.
This model emphasises the fluidity of the bulk lipids allowing random diffusion
What is the concept of membrane domains/rafts?
Suggests that in a eukaryotic plasma membranes there will be different areas ‘domains’ with different composition of cholesterol and sphingomyelin .
Proteins are either excluded or included in the raft regions
What are lipid droplets?
Storage organelles that help to maintain the lipid and energy homeostasis.
Hydrophobic core of neutral lipids enclosed by a phospholipid monlayer.
originate from ER
Where do lipid droplets originate from?
The endoplasmic reticulum and are initiated when neutral lipids are produced
How do neutral lipids form?
Result from the esterification of a fatty acid to a triacylglycerol or a sterol to a sterol ester
Where are neutral lipids normally and what happens if their storage is impaired?
Normally dispersed in the leaflets of ER bilayer, as their concentration increases neutral lipids accumulate (demixing)
If fatty acid storage in lipid droplets is impaired can result in diseases such as type 2 diabetes and fatty liver disease
What is a monotopic protein?
an integral membrane protein that inserts into the membrane but does not span it
what is a bitopic protein?
integral membrane protein with one helix that spans the bilayer once
type 1 - N-terminal is outside
type 2 - N-terminal is inside
what is a polytopic protein?
integral membrane protein with segments spanning the membrane, connected by loops
what is a oligomeric protein?
integral membrane protein formed when multiple bitopic proteins oligomerize (join together, but not connected by loops)
Give the 5 functions of integral proteins
transport - move molecules across the membrane by changing shape
enzymatic activity - participate in electron transport and metabolism of phospholipids and sterols
signal transduction - transfer information in response to the binding of a ligand or molecule
cell-cell interactions - glycoproteins are recognised by other cells
attachment to cytoskeleton/extracellular matrix - can be non-covalently bound to cytoskeleton, regulates cell shape and stabilises protein position in membrane. also facilitate cell-cell adhesion
what is the transmembrane region of integral proteins made up of?
amino acids with hydrophobic side chains
how is glucose transported into cells?
glucose transporters (GLUTs) - made up of 12 ⍺-helices
- glucose binds to membrane outer surface, causing a conformational change
- at inner surface, glucose is released and protein returns to its original conformation
what can cause ion channels to open?
ligand biding, electric potential, pH, temperature, pressure or lipids
what do potassium channels do in the
- cardiovascular system?
- epithelial cells?
- regulate heartbeat
- regulate passage of salt and water
how many subunits do potassium channels have?
4
- each subunit has two transmembrane helices and a pore half helix
what do mechanosensitive channels respond to?
mechanical stress (touch, sound and gravity)
what are the critical roles of ß barrel proteins?
cell structure and morphology, nutrient acquisition, protection of bacteria against toxic threats
what are peripheral membrane proteins?
proteins that bind to the surface of the membrane or to an integral protein
how do peripheral proteins bind?
interact with anionic lipid head-groups or with charged groups on another protein
what are lipid binding molecules?
positively charged surfaces on a peripheral protein that recognise specific lipids in the membrane - means they bind in specific places
what type of membrane proteins are cytoskeleton?
peripheral membrane proteins
- form a scaffold on the cytosol side of membrane
- attach to integral proteins
- in RBC’s this is important as cytoskeleton allows cell to undergo stress without fragmentation
what is hereditary elliptocytosis?
condition of RBC’s which causes disruption of interactions with the cytoskeleton, forming elliptically shaped cells - causes anaemia
what is hereditary spherocytosis?
condition of RBC’s as a result of loss of cohesion between plasma membrane and cytoskeleton due to defective anchoring - cells become spherocyte
define dementia
an umbrella term for the serious deterioration in mental functions, such as memory, language, orientation and judgement
alzheimers is the most common cause
what are the clinical features of alzheimers?
- amnesia
- aphasia (language problems)
- agnosia (difficulty recognising and naming objects)
- apraxia (difficulties in complex tasks)
- visuospatial difficulties
- functional impairment
- mood disorders
- psychosis
- personality change
what causes alzheimers?
neurons malfunction causing the chemical and electrical signalling to go wrong - the nerve cells die and the connections deteriorate
- post mortems find senile plaques made of amyloid-beta peptide
what are amyloid-beta peptides?
short peptides derived from the larger membrane bound amyloid precursor protein (APP), goes onto form senile plaques which are abundant in alzheimer brains
how does cholesterol impact alzheimers?
- more amyloid plaques in those dying of heart disease
- in cholesterol rich areas of the membrane there is more cleavage of APP and so more amyloid-beta peptide is made
what is an O-linked glycoprotein?
a carbohydrate attached to the oxygen atom in the side chain of serine or threonine
- often 2-5 sugars
what is an N-linked glycoprotein?
carbohydrate attached to the amide nitrogen atom in the side chain of asparagine
- usually large and branched with up to 30-40 sugars
what sequence must the amino acids be for an asparagine residue to accept an oligosaccaride?
asn - X - ser
asn - X - thr
why do cells communicate with each other?
- regulate their development and organisation into tissues
- control their growth and division
- co-ordinate their functions
give an example of a disease where cell signal is lost/ no longer sent
Type 1 diabetes
- beta cells are damaged
- so no insulin produced
- insulin receptors do not mobilise glucose transporters
give an example of a disease where the target cell ignores the signal
type 2 diabetes
- insulin binds to receptors
- but receptors do not send signal to mobilise glucose transporters
give an example of a disease where the signal doesn’t reach its target
multiple sclerosis
- damage to myelin sheath
- signal from brain doesn’t reach extremities
give an example of a disease where there is too much signal
brain damage (ie stroke)
- blood vessels blocked so neurone cells die due to lack of O2
- glutamate released which causes neighbouring cells to die
give an example of a disease where there are multiple breakdowns
cancer
- hallmarks of cancer
- eg cell proliferation - too much cell signal so too much growth factor produced so too much cell division
what is autocrine signalling?
where a cell produces the signal and has the receptors
what is endocrine signalling?
signal is released from a cell into the blood stream and travels to a remote target cell
what is paracrine signalling?
signal is released to a nearby cell
what is juxtacrine signalling?
contact signalling by plasma membrane-bound molecules (eg T helper cells and antigen presenting cells)
what is gap junction signalling?
physical connection between cells via channels (eg cardiomyocytes)-allows movement of some cytoplasmic contents between cells
give 4 examples of extracellular signalling molecules (1st messengers)
- growth factors
- neurotransmitters
- hormones
- cytokines
what is the difference between exocrine, endocrine and paracrine hormones?
exocrine - secreted directly into the blood
endocrine - secreted into ducts first: distant cells
paracrine - diffuse through interstitial tissues to target cells - act on nearby cells
what are the four classes of receptor?
(cell surface) - ligand gated ion channel - G protein coupled receptor - receptor tyrosine kinase (intracellular) - nuclear hormone receptor
ligand-gated ion channels
- ionotropic receptors
- binding and opening is very fast - involved in fast synaptic transmission
- ligand binding site on extracellular side
- example - nicotinic acetylcholine receptor (increases Na+ and K+ permeability, Na+ in and K+ out - causes membrane depolarisation)
- also GABA, GlyR and 5-HT3R
G-protein-coupled receptors
- metabotropic (ie no pore but triggers signalling response) or heptahelical receptors
- couple to an intracellular effector system via a G-protein
- examples - muscarinic ACh receptor, adrenoceptors, angiotensin II receptors
what does the renin-angiotensin system control
blood pressure (negative feedback system)
outline RAS
- low blood pressure = renin released by kidneys
- renin converts angiotensinogen to (through cleaving) angiotensin I
- ACE 1 (from lungs and kidney) converts angiotensin I to agiotensin II
- agiotensin II has a variety of effects to increase blood pressure
what affects does angiotensin II have
- increase nervous activity (contraction of vessels)
- retention of water
- aldosterone secretion (retention of water)
- vasoconstriction
- ADH secretion (reabsorption of water in collecting duct)
why is understanding RAS important?
plays an important role in pathogenesis of heart failure (all work by reducing BP)
- can control bp by inhibiting renin release or stop trigger of RAS syestem
- ACE inhibitors
- AT1 receptor antagonists
- aldosterone receptor antagonists
- example of negative feedback
kinase-linked receptors
- single transmembrane helix with a large extracellular binding domain and an intracellular catalytic domain
- some receptors are enzymes themselves- catalytic receptors
- some act through cytoplasmic tyrosine kinases
- extracellular c terminus and intercellular n terminal
nuclear hormone receptors
- intracellular receptors in the cytosol or nucleus - ligand activated transcription factors
- Regulate gene transcription
- monomeric structure
- hormones diffuse across membrane Interact with cytosolic or nuclear receptors and form hormone-receptor complexes, bind to regions of DNA and affect gene transcription
- examples - steroid and thyroid hormones
what are the 5 neurotransmitters?
- acetylcholine (ACh)
- monoamines (noradrenaline, adrenaline, dopamine, histamine, serotonin)
- amino acids (glutamate, aspartate, glycine, GABA)
- peptides (endorphins, substance P, neurokinins, neurotensin)
- lipids (anandamide)
outline the lifecycle of a neurotransmitter
- synthesis
- storage - synaptic vesicles
- release
- receptor activation
- neurotransmitter inactivation and reuptake
what are 5 treatments for depression
- monoamine reuptake inhibitors (tricyclic antidepressants, selective seretonin reuptake inhibitors, seretonin/noradrenaline reuptake inhibitors)
- monoamine oxidase inhibitors
- atypical antidepressants
- electroconvulsive therapy
- mood-stabilising drugs
how do reuptake inhibitors work?
prevent neurotransmitters going back into presynaptic cleft so they stay in synapse longer and more stimulation of receptors
how do autoreceptor blockers work?
inhibit negative feedback, Ca2+ still released into presynaptic cleft so neurotransmitter still released into synapse
how do monoamine oxidase inhibitors work?
stop breakdown of serotonin, so more serotonin in vesicles so more released into synapse
what type of signalling do gasotransmitters take part in?
paracrine - pass readily across cell membranes
outline the enzymatic production of NO
L-arginine converts to NO + L-citrulline aided by eNOS, nNOS and iNOS
outline the enzymatic production of CO
haem + O2 = NADPH
HO-2 and HO-1 remove Fe2+ and bilirubin = CO
outline the enzymatic production of H2S
homocysteine with CBS = cystathionine
cystathionine with CSE = L-cysteine
L-cysteine with CSE, CBS, 3MST = H2S
where do we get cholesterol from?
25% from diet, 75% synthesised in the liver
how soluble is cholesterol?
insoluble in blood plasma so transported with a carrier - lipoprotein
give the 4 classifications of lipoproteins from biggest to smallest and their function
Chylomicrons, VLDL - large not v dense, made in intestines - transport dietray fats from intestines to tissues
VLDL - transport lipids made in liver to peripheral tissues
LDL - smaller, denser- main cholesterol carrier, provide cholesterol to peripheral tissues
HDL - transport cholesterol to liver from peripheral tissues “good cholesterol”
what is an apolipoprotein?
- single protein strand that runs around the outside of lipoprotein
- allows lipids to be transported to areas they are required in the body
- determine start and end points for cholesterol transport
- LIPID+APOLIPOPROTEIN = LIPOPROTEIN
where is cholesterol found in a lipoprotein?
free cholesterol is found in the external monolayer
cholesterol esters and triacylglycerols are in the hydrophobic particle core
what are the 4 major classes of apolipoprotein?
ApoA - present in HDL, mediates efflux from tissues and influx to liver
ApoB - facilitates LDL uptake into tissues
ApoC - activator of lipoprotein lipase
ApoE - stabilises LDL for cellular uptake
how is synthesis of apoplipoproteins regulated?
intestine - dietary fat intake= made according to demand
liver - hormones (insulin, glucagon) and drugs (statins, alcohol)
what are the main functions of apolipoproteins?
- regulate key enzymes in lipoprotein metabolism
- ligands for interaction with lipoprotein receptors, targeting lipoproteins to the correct tissues
what are the structural differences between LDL and HDL?
LDL has ApoB apolipoprotiens
HDL has Apo A-I and Apo A-II, so highly resistant to being oxidised = anti inflamatory properties
(LDL oxidation = chief culprit of CV disease in form artherosclerosis)
What are the different types of membrane transport?
- Passive transport (simple diffusion, facilitated diffusion)
- active transport (ATP-driven and ion-driven)
Describe simple diffusion.
- Type of passive transport
- Can be used by gases, hydrophobic molecules and small polar molecules
- No metabolic energy required
- No specificity
- Rate of diffusion proportional to conc. gradient
Describe facilitated diffusion.
- Type of passive transport
- Occurs down the conc. gradient
- No metabolic energy required
- Depends on integral membrane proteins (carriers, permeates, channels, transporters)
- Specific
- Effected by temperature and pH
- Able to be inhibited
What are ionophores?
- Example of facilitated diffusion
- Ion carrying proteins
- Carrier ionophores (carry molecule across) or channel-forming ionophores (molecule passes through channel)
What are ion channels?
- Example of facilitated diffusion
- Allow rapid and gated passage of ions
- Highly selective- stimulated by a specific stimulus that allows it to open
- Down conc. gradient
- Essential for maintaining osmotic balance, signal transduction and nerve impulses
How is glucose transported across the membrane?
- Integral membrane protein GLUT1
- Facilitated diffusion
- GLUT1 has 12 alpha helical transmembrane domains and a central pore where glucose binds
- Transporter undergoes conformational change when glucoses binds
- Important that glucose conc. is higher outside of the cell
How is a low conc. of glucose maintained within the cell?
Hexokinase forms G6P from glucose using ATP
What are aquaporins?
- Example of facilitated diffusion
- Water channel proteins required for the bulk flow of H2O across membranes
- 6 transmembrane alpha helices come together to form a pore
- Tetramer with four pores through which H2O can pass
- Abundant in erythrocytes and kidney cells
Describe ATP-driven active transport.
- Na+, K+, Ca+ and H+ transport is directly coupled to ATP hydrolysis
- There is a high conc. of K+ and low conc. of Na+ inside the cell = Na+/K+ gradient
- Facilitated by Na+/K+ ATPase
- Na+/K+ ATPase is a tetramer with two alpha and two beta subunits. It pumps 3 Na+ ions out and 2K+ ions into the cell which polarises the cell membrane
- ATP hydrolysis induces conformational changes, pumping Na+ and K+ against their conc. gradients
- coupled system: ATP is not hydrolysed unless Na and K+ are transported and visa versa
Describe ion-driven active transport.
- Coupled to the movement of an ion (Na+ or H+) down its conc. gradient
- Symports: both molecules move in the same direction, e.g. Na+/glucose transporter
- Antiports: both molecules move in opposite directions, e.g. Na+/Ca2+ exchanger
What are Cardiac Glycosides (Cardiotonic Steroids)?
- Used in congestive heart failure
- Naturally occurring plant steroids (e.g. Digitalin, Ouabain, Digoxin, Digitoxin) that inhibit Na+/K+ ATPases
- In healthy people digitalis causes heart failure but in lower doses it strengthens the heart beat of patients with congestive heart failure- used at treatment
- Inhibition of Na+/K+ ATPase leads to increased conc. of Na+ inside the cell as less pumped out and a decreased Na+ gradient across the membrane. This decreases Na+/Ca2+ exchange, so there is a greater conc. of Ca2+ inside the cell. Increased intracellular Ca2+ leads to enhanced strength of cardiac muscle contraction
How do intestinal epithelial cells transfer molecules from the intestinal lumen to the blood?
- Movement of glucose from the lumen into the cell is driven by movement of Na+ ions down the conc. gradient (Na+/glucose transporter transports both into cells)
- Glucose diffuses into the blood via GLUT1
- Low conc. of intracellular Na+ is maintained by Na+/K+ ATPase
- As glucose flows through cell, water follows by osmosis due to osmotic gradient
How does oral rehydration therapy work?
Uptake of glucose is dependant on Na+ so an oral solution of both glucose and Na+ is given. This increases osmotic pressure in epithelial cells, so H2O follows.
How are macromolecules moved across membranes?
Exoctyosis/endocytosis
What is exocytosis and what are its different types?
Exocytosis is the excretion/secretion of molecules. It can be constitutive or regulated.
What is constitutive exocytosis?
Constitutive exocytosis is used by all cells. Secreted proteins & plasma membrane. It is continuous or regulated.
What is regulated exocytosis?
Regulated exocytosis is used only by specialised cells such as neurones and pancreatic cells. It is Ca2+ dependent.
What is endocytosis and what are its different types?
Endocytosis is the ingestion/uptake of molecules. It can be phagocytosis, pinocytosis or receptor-mediated endocytosis
What is phagocytosis?
- Ingestion of large particles (bacteria/debris)
- Specialist cells - macrophages, neutrophils, dendritic cells
- Bacteria/debris is degraded by enzymes in the lysosomes and the remnants are displayed on the surface of the phagocyte to alert other immune cells to fight the infection
What is pinocytosis?
- Cell ‘drinking’
- Uptake of fluid
- All cells are able to do this
What is receptor-mediated endocytosis?
- Selective (receptor recognition)
- Uptake from extracellular fluid via Cathrin-coated vesicles and pits
- The ligand being taken up must first bind to a specific cell surface receptor
- Receptor-molecule complexes accumulate in a Cathrin-coated pit and are endocytose in a clathrin-coated vesicle
- fuse with endosome and lysosomes = lysosome degradation
- cell membrane recycled by cell
- Way of concentrating specific macromolecules that are present at low conc.
- E.g. cholesterol uptake via LDL receptor
- Receptor-mediated endocytosis may be exploited by viruses to gain entry to cells (eg. rhinovirus and covid-19)
what is signal transduction
also known as intracellular signalling
= converts extracellular signal into cellular response
what are the 3 main stages of signal transduction
1- reception
2- signal transduction
3- cellular response
what are the 4 Important features of signal transduction pathways
Hierarchy, amplification, specificity, complexity
what is hierarchry
things happen in a particular order to transmit signal from outside to inside cell
eg. 1st messenger, receptor, G protein, effector enzyme, 2nd messenger, protein kinase, target protein , cellular response
what is amplification
A single molecule of first messenger can induce many downstream signalling molecules and lead to a larger cellular response
what are the main points of amplification
1-G-protein activation - is activated by the receptor as long as the receptor remains in an activated state = single G-protein molecule can activate many molecules of effector enzyme
2-Effector enzyme -catalyse reactions without being used up. So a single effector enzyme molecule can catalyse the production of many molecules of second messenger.
3- ) Protein kinase - single protein kinase molecule can catalyse the phosphorylation of many molecules of protein substrate
what is the process in glycogen breakdown pathway
1- binding of epinephrine to G protein receptor 2- activates G protein 3- g protein activates adenylyl cyclase 4- which turns ATP to cAMP 5- a activates protein kinase A 6- activates phosphorylase kinase 7- activates glycogen phosphorylase 8- turns glycogen to glucose-1-phosphate
what is specificity
signal transduction pathways are highly specific
one stimulus can cause different cellular function depending on what receptor and where it binds
what hormone/1st messenger activates different processes on different cells
adrenaline
as it binds to different receptors and activates different signalling pathways
3 examples of different cellular functions regulated by Adreneline
- in muscle/liver = glycogen degradation
- in adipose = fatty acid production
- in cardiovascular = increases heart rate and blood pressure
what is complexity
Signal transduction pathways are highly COMPLEX
due to number of different types of receptor, G-proteins, effector enzymes, second messengers, protein kinases and downstream substrates
what is ‘cross talk’
when signalling pathways interact with one another
allows for fine tuning of cellular responses
what are Main components of signal transduction pathways
G proteins, effector enzymes, 2nd messengers, kinases
what are G proteins
Guanine nucleotide binding proteins
what does GTP do
high energy molecule that activates G proteins
what are G proteins also known as
GTPases
what do G proteins do to GTP
hydrolyse GTP to GDP
create lipid anchored membrane proteins to associate them with different parts of membrane via process prenylation
what are the 2 major groups of G proteins
- G prtoeins (receptor associated) :
heterotrimeric(3 subunits) (a,b,y subunits) - small GTPases:
monomeric (one subunit)- Rho and Ras
why are G proteins reffered to as molecular switches
- switched on by ligand binding to receptor
- switched off by intrinsic GTPase activity
how are G proteins switched off
removal of phosphate group by GTPase from GTP = GDP
off always bound to GDP
how are G proteins switched on
binding of ligand = confrontational change = allows G protein bind to receptor = GDP exchanged to GTP
on always bound to GTP
What is the Gs protein
trimeric G protein
combination of a, B, and gamma subunits
alpha subunit is the one that binds to GDP/GTP
step 1 - what happens when ligands bind to Gs protein
confrontational change in receptor
causes the G-protein to release GDP and swap it for GTP = ON
alpha unit separates from beta and gamma subunits
step 2- what happens in the activation of effector enzyme and 2nd messnegr
GTP-bound Gs-alpha subunit binds to and activates adenylyl cyclase which catalyses conversion of ATP to the second messenger cyclic AMP
step 3 - what happens in hydrolysis of GTP to GDP
The GTPase activity of the Gs-alpha subunit hydrolyses GTP to GDP (with release of inorganic phosphate, Pi)= G-protein “OFF” state
Step 4 - what happens in re-association
The GDP-bound alpha subunit then re-associates with the beta and gamma subunits
system resets itself
step 5- what happens to the 2nd messenger
loss of second messenger
Cyclic AMP is broken down to AMP by phosphodiesterases
what happens if the receptor is still active?
process will be repeated so the signal continues to be amplified
what enzyme and 2nd messenger does Gs effect?
- adenylate cyclase - stimulation
- increase in cAMP
what enzyme and 2nd messenger does Gai effect?
- adenylate cyclase - inhibition
- decrease in cAMP
what enzyme and 2nd messenger does Gaq effect?
- phospholipase C stimulation
- increase DAG, IP3
what 2 G proteins have opposing effects
Gs and Gi on cyclic AMP levels
what G protein does cholera toxin target
Gs
what G protein does pertussis toxin (whooping cough) target
Gi
what is the name of the bacteria causing cholera
Vibrio cholerae bacteria in contaminated water & food
what is the treatment for cholera
Oral rehydration therapy
water, salts, glucose
what is the mechanism for the cholera toxin on Gs
- Cholera toxin prevents GTPase activity of Gs =therefore GTP remains bound to Gs and it stays in the “ON” state
- over-stimulation of adenylate cyclase and accumulation of cyclic AMP
- intestinal epithelial cells, elevated cAMP increases loss of Cl- ions through chloride channels
- resultant osmotic gradient= water excreted into the intestinal lumen= diarrhoea and dehydration
what bacteria is whooping cough caused by
Bordetella pertussis
what is the virulence factor of whooping cough
Pertussis toxin
what is the virulence factor of cholera
Virulence factor = Cholera toxin
mechanism of pertussis toxin on Gi
inhibition of Gi G-protein
- Pertussis toxin prevents GDP/GTP exchange by Gi
- the Gi protein is locked in the “off”
- unable to inhibit adenylate cyclase, resulting in accumulation of cyclic AMP
- physiological effects = increased insulin secretion and increased sensitivity to histamine
what are second messengers
• Short-acting intracellular molecules that are rapidly formed or released as a result of receptor activation
Five common second messengers
• Cyclic AMP (cAMP) • Cyclic GMP (cGMP) • Diacylglycerol (DAG) • Inositol 1,4,5-trisphosphate (IP3) - intracellular calcium (Ca2+i)
production of cAMP?
cyclic AMP from ATP; a reaction catalysed by adenylate cyclase (also called adenylyl cyclase)
production of cGMP?
cyclic GMP from GTP; a reaction catalysed by guanylate cyclase (also called guanylyl cyclase
what breaks down cAMP and cGMP
Cyclases and Phosphodiesterases (PDEs)
- some Phosphodiesterases break down cAMP or anther one will specifically breakdown cGMP
- same cyclases can break both down
well known PDE inhibitors?
caffeine and Viagra
- Caffine inhibits breakdown cAMP
- Viagra inhibits breakdown of cGMP
step 1 of Gq activation?
- ligand binding GDP/GTP exchange,ON, due to confrontational change
- alpha subunit dissociates from beta/gamma subunits
step 2 of Gq activation?
activation of effector enzyme and production 2nd messenger
- alpah subunit binds Phospholipase C
- catalyses production of two different second messengers 1,2-diacyclycerol (DAG) and inositol 1,4,5-trisphosphate (IP3)
- release of stored Ca2+ ions into the cytosol due to interaction of ER with IP3
- DAG and stimulates protein kinase C (PKC) that phosphorylates target proteins leading to cellular response
- release of calcium also stimulates responses
why is Ca importnant 2nd messenger?
(NB: not “formed” but is “released” into the cytosol)
1-activate various molecules (e.g calcium-dependent kinases) to modulate cellular function
2- trigger opening of calcium channels in the plasma membrane = more Ca floods in
how is calcium removed from the cell
taken back up into the ER through a calcium ATPase in the ER membrane, or is pumped or exchanged out of the cell
what are protein kinases?
- enzymes that transfer Pi from ATP to a SPECIFIC AA residue: Ser, Thr,Thy on a SPECIFIC protein
- phosphorylation may activate or inhibit protein function
what are the 3 main groups of protein kinase
- serine/theronine kinases
- thyrosine kinases
- dual specificity kinases
function of serine/theronine kinases
- phosphorlyate Ser and or Thr residues
- eg. PKA,PKC,PKG
function of thyrosine kinases
-phosphorlyte only Tyr residues
function of dual specificity kinases
phosphorlyate Ser, Thr, Tyr resides
eg.MAP kinase kinases- MKKs
how is LDL receptor gene expression regulated (endocytosis of LDL)?
intracellular cholesterol concentration, binding of LDL to receptor (binds to apoB or apoE) stimulates endocytosis into hepatic or peripheral cells
- form a coated vesicle
- become uncoated and triskelions return to plasma membrane
- ph decreases and LDL -> amino acids, cholesterol, fatty acids
- receptors recycled to membrane
what does high intracellular levels of cholesterol do to LDL uptake and so why is excess LDL bad?
suppresses LDL receptor synthesis - prevents excess cholesterol uptake
as a result excess cholesterol remains in the blood as LDL
what does high levels of HDL correlate with?
low incidence of atherosclerosis as is resistant to oxidation through apoAand scavenges excess cholesterol, returns to liver and is excreted as bile
REVERSE CHOLESTEROL TRANSPORT
what is normal level of cholesterol in a healthy adult?
175 mg/100ml
what are the cardiac, cerebral and peripheral manifestations of atherosclerosis?
cardiac - chest pain, palpitations, hear tattack
cerebral - stroke, cerebral haemorrhage
peripheral - pain, ischaemia , ulceration and gangrene
what is the cholesterol synthetic pathway?
- HMG-CoA
- —> HMG-CoA reductase - mevalonate (key intermediate)
- IPP
- FPP
- Squalene (fatty precursor)
- cholesterol
what do statins do?
prevent cholesterol synthesis in the liver as inhibit HMG-CoA reductase —> less mevalonate —> less cholesterol —> more expression of LDL receptor —> more uptake of cholesterol from blood
what is pleiotropism?
extra unintended affects from a drug - could be good or bad
what are statins pleiotropic effects?
- improve endothelial dysfunction
- antioxidant
- inhibition of inflammatory response
- stabilise atherosclerotic plaques
what do kinases do
put phosphate groups onto molecules
- very specific action
what do phosphateses do
remove phosphate groups from AA
-not very specific
how can kinases modulate protein function
- Phosphorylation to induce change in protein conformation / function
- § Phosphorylation of a transcription factor that alters gene transcription and hence protein expression levels
Protein kinase inhibitors as therapeutic agents in…
Cancer Cardiovascular Disease HIV/AIDS Rheumatoid Arthritis Alzheimer’s Disease
how can disregulation in kinases be linked to cancer?
in solid tumours changes in protein kinase expression levels and activities + posttranslational modifications can contribute to cancer development
what are kinase therapies used for?
- as a cancer drug therapy
- Conventional chemotherapy can be ineffective and harmful- by combining chemotherapeutics and protein kinase inhibitors= more succesfull
functions of lipids?
- energy storage - as fat deposits
- major components of cell membranes
- signalling molecules
- required to solubilise fat soluble vitamins
- biosynthetic precursors (eg. steroid hormones from cholesterol)
function of lipoproteins?
-means of lipid (trigyceriol and cholesterol) transport in blood
LIPID+ APOLIPOPROTEIN=?
LIPOPROTEIN
similarities between apoA and apoB structure
- surface monolayer of phospholipids and free cholesterol
- hydrophobic core of mainly cholesterol esters and some triglycerides
How are lipids transported by lipoproteins?
- Triglycerides hydrolysed by lipoprotein lipase to fatty acids= taken up by target tissues for energy production (eg muscle) or stored (adipose tissue).
- this means Chylomicrons shrink, remnants transported back to liver.
- VLDL transport lipids to target tissues- same process as above
how to HDL work?
- HDL= opposite function to LDL and can remove cholesterol from the tissues.
- HDL synthesised in the blood and acquire their cholesterol by extracting it from cell membranes and transporting back to the liver.
- The liver is the only organ that can dispose of significant quantities of cholesterol, primarily in the form of bile salts.
what is the main function of lipoproteins
- Membrane-bound receptors to enable cholesterol entry to hepatic and peripheral cell
- Lipoprotein receptors=necessary for cholesterol uptake into cells.
what happens to VLDL remnants?
-VLDL remnants remain in the blood, become LDL that are then taken up by target cells by the LDL receptor, digested in the lysosome to release the cholesterol.
how is LDL taken into cells?
- apoB recognised by receptors in clathrin coated pit
- membranes takes this in by endocytosis
- clathrin released back onto surface
- receptor and cholesterol processed in vesicle then released to endosome
- receptors released from LDL receptor
- receptors recycled back to plasma membrane
- cholesterol processed by lysosome and degraded to fatty acidsf
how does reverse cholesterol transport work?
- nascent HDL extracts cholesterol form cell membrane of macrophage and swells
- turns to mature HDL
- recognisable by scavenger cells on liver
- cholesterol in membrane of HDL processed in liver and secreted in bile
mutations with LDL receptors are a common cause of?
FAMILIAL HYPERCHOLESTEROLAEMIA= cells lacking LDL receptors= amount LDL in blood increases
major consistent of arhteroscelroic plaque?
cholesterol-enriched LDL
how do arhterocelrtic plaques form?
1-fatty streak in lumen (at young ages - process takes decades)
2- fibrous plaque forms = reduces area blood flows
3- advanced plaque = complete occlusion - plaque rupture on top of blood clot = MF
what drugs control hypercholesterolaemia?
-Statins
Prevent cholesterol synthesis in the liver
-Cholesterol absorption inhibitors
Prevent uptake from the intestine
-Fibrates
Reduce triglycerides and increase HDL, less effective
how do statins work?
- inhibit HMG-CoA reductase
- decreases cholesterol synthesis
- increased expression of LDL receptor
- increase uptake cholesterol from blood
what are Ras and Rho?
small G proteins only function when prenylated
what prenylates Rho and Ras
FPP and GGPP = called isoprenoids
- required for a process known as PRENYLATION (sticking a lipid tail onto intracellular signalling molecules) = so associated with cell membrane and signal cascade
- Ras is farnesylated whilst Rho is geranylgeranylated
why is lipid tail G proteins important?
Without lipid tail G proteins floating in cytosol and cant signal
what is PCSK9?
- Protease enzyme expressed by liver & intestine
* Important role in lipid metabolism
why is PCSK9 important?
- Promotes intracellular degradation of LDL-R
- Prevents recycling of LDL-R to cell surface
- Reduces LDL-R population on cell membrane
- Link between PCSK9 mutations and coronary heart disease
- Implicated in familial hypercholesterolaemia
- Genetic variation in PCSK9 (gain/loss of function)- beneficial if loss of function? LDL receptors remain in tact?
how does PCSK9 work?
- PCSK9 binds LDL and internalised with LDL receptor
- degrades receptor and cannot be recycled to accept LDL at surface
- PCSK9 inhibitor stops this from happening
what are neutral lipids a result of?
-the esterification of a fatty acid to triacylglycerol or a sterol (such as cholesterol) to sterol ester
what is a sterol ester
- cholesteral with lipid tail attached to one side of cholesteral
- At low concentrations, neutral lipids are dispersed between the leaflets =no deformation
- As their concentration increases, neutral lipids accumulate (de-mixing- membranes start deforming ) = may threaten burst membrane
what are the 3 proteins that help lipids move between leaflets?
- P type fliptases – proteins which help lipids move from outside to inside of cells-require energy from ATTP
- ABC fliptase- help proteins move from inner to outer membrane – ATP
- Scrambalses- can move proteins in any directions- no ATP but Ca needed
what membrane curvature do cylindrical lipids from?
cylindrical shape so can pack next to each other tightly= flat membrane
what membrane curvature do conical shape lipids form?
negative curvature ‘U shape’
what membrane curvature do inverted conical lipids form?
only one tail= create a positive curvature ‘N shape’
why CO bad?
- highly toxic
- exposure is common
- chronic exposure leads long term neurological CV disorders
why is CO good?
- endogenous signalling molecule
-cardioprotective
neuroprotective
what are. gasotransmitters?
gaseous molecules synthesised in the body. They include nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S).
what other effects do kinase linked enzymes have?
•Act by indirectly regulating gene transcription •Roles in controlling: cell division tissue repair apoptosis -cellualr effects: cellular poliferation
what is angiotensin II made of?
- Octapeptide
- Angiotensin II receptors are GPCRs]
- only 8 AA
what do hormones regulate?
•body energy needs •protein and nucleic acid metabolism •mineral and electrolyte metabolism •synthesis and release of hormones Activity is regulated through negative or positive feedback mechanisms
how do cells communicate with one another?
- Remote signalling by secreted molecules
- Contact signalling by plasma membrane-bound molecules (‘Juxtacrine’ signalling)
- Contact signalling via gap junctions- channels that form between two cells and allow molecules to move across
which isomoer of glucose is the glucose transporter specific for?
D-isomer of glucose relative to the L-isomer or to other related sugars
what ions are directly transported into a cell by ATP hydrolysis?
Na+, K+, Ca2+ and H+ transport is directly coupled to ATP hydrolysis
e.g. Na+/K+ ATPase
§ Coupled system: ATP is not hydrolysed unless ions (eg.Na+ and K+) are transported and vice versa
what does the Na+/K+ gradient ensure?
- controls cell volume
- makes nerve and muscle cells electrically excitable
- facilitates ion-driven active transport of amino acids and sugars
what is the symport system?
both molecules travel in same direction
what do intestinal epithelial cells do?
•Line the lumen of small intestine
•Large surface area for absorption from villi and microvilli
•Absorb nutrients from digested food (sugars, amino acids, lipids, etc.)
•Transfer nutrients into the blood
•Similar cells found in kidney tubules
-cells are polarised - have 2 distinct sides: the apical (brush-border) membrane that faces the lumen of the gut and the basolateral surface that faces the bloodstream
