metabolism + cell motility Flashcards
give an overview of glycolysis
Glycolysis overview;
Preparation phase -
Converts glucose, a 6C molecule, into two 3C molecules of glyceraldehyde-3-phosphate (G3P)
Consumes 2 ATP
Payoff phase -
Converts the two G3Ps into pyruvate
Produces 4 ATP and 2 NADH
Net products -
2 NADH, 2ATP
how is glycolysis regulated?
Pyruvate kinase activity is promoted by fructose 1,6-bisphosphate (product of step 3)
Pyruvate kinase is inhibited by ATP and acetyl CoA, products of glycolysis
briefly outline the process of the TCA or krebs cycle
Combines the 2C acetyl CoA with the 4C oxaloacetate to form a 6C molecule
Then removes electrons from the 6C molecule (as well as the 2 carbons) in order to reduce NAD+ and FAD+ for large ATP payoff in ox.phosphorylation
in terms of the Krebs cycle, what does one molecule of glucose get you?
One molecule of glucose = two cycles:
4x CO2
2x ATP (or GTP)
2x FADH2
6x NADH
why is losing weight difficult?
The only way to metabolise fats is by putting them into the krebs cycle and oxidative phosphorylation, they cannot be converted into glucose (glycerol can via gluconeogenesis but fatty acids cannot)
Fats are energy rich, one regular fatty acid provides around 4 acetyl-CoA molecules, which gives 8x FADH2 and 24x NADH, or 64 electrons
so don’t need loads of it for lots of energy
what molecules can we use as an energy source/to produce ATP?
amino acids - different ones can feed in to the Krebs cycle at different positions
glucose (sugars)
fats (fatty acids)
give an outline of the process of oxidative phosphorylation
Essentially electrons from FADH2 and NADH are transferred to oxygen to produce water
The process is about using the free energy provided by redox reactions to pump H+ from the matrix into the intermembrane space, so the H+ can travel back into the matrix via ATP synthase
Transport complexes 1, 3 and 4 pump H+ across into intermembrane space
ATP synthase then allows H+ to move down gradient, pushing it like a turbine to generate ATP
O2 + 4H2 —> 2H2O
what are uncoupling agents and why are they so dangerous?
bind protons in intermembrane space and transport them across membrane, dismantling the proton gradient before ATP can be produced
Drive oxidative phosphorylation harder and harder, literally generates heat and cells die - very dangerous
have previously been used (not officially) for weight loss
example = 2,4-dinitrophenol
aside from uncoupling agents, what kind of metabolism inhibitors are there?
e- transport inhibitor - can target the different complexes in the transport chain
examples = cyanide, CO, sodium azide (all target complex IV)
ATP synthase inhibitors
example = oligomycin
metabolism is C__________?
compartmentalised - glycolysis in cytosol, krebs and ox.phos in mitochondria
metabolism in the brain?
Normally, only uses glucose
In starved/fasted states, ketone bodies can be used but only after several days
Consumes 120g/day - 60-70% of your glucose production
metabolism in kidney?
Kidneys produce urine – ie secrete waste products
reabsorbs water and glucose in the process
during starvation the cortex of the kidneys are a major site of gluconeogenesis (1/2 blood glucose???)
muscle - what does it use to generate ATP?
Needs ATP, uses different fuels to get it
Mostly uses glucose, fatty acids and ketone bodies
muscles - what occurs during ‘burst’ exercise, and resting?
anaerobic respiration, uses glucose from glycogen stores in the muscle (¾ of all glycogen stores are in the muscles) or creatine …
Resting - aerobic respiration, typically uses fatty acids
how is creatine used?
In burst exercise, to give a chance for glycogen to be converted to glucose, phosphocreatine transfers its P to ADP, to form ATP, via the enzyme creatine kinase
what is the Cori Cycle?
(Glycogen stores in liver can be converted to G6P then) glucose (from the liver), goes into blood,
then into muscle for anaerobic respiration
Muscle produces lactate in glycolysis,
which goes back to - blood - liver - to be converted into G6P and glucose again (gluconeogenesis), back to the start, this glucose can go into the blood etc… forming a cycle
in a fed state, insulin rises and glucagon drops - what does this result in?
Insulin - Regulates the metabolism of carbohydrates, fats and protein
It promotes the absorption of glucose from the blood into liver, fat and skeletal muscle cells
Causes liver to turn glucose into fat
increases:
glucose uptake
glycogen synthesis
protein synthesis
fat synthesis
decreases:
gluconeogenesis
glycogen mobilisation
lipid mobilisation
protein degradation
(insulin alters gene expression to do some of these ^^^)
how does insulin work in terms of glucose uptake?
Insulin - causes insertion of glucose transporters (GLUT4) into plasma membranes
GLUT 4 needs insulin for it to be able to uptake glucose
Certain tissues have GLUT 1,2 and 3 which can take in glucose without insulin being presence - e.g. the brain has these
in a fasting state, glucagon rises and insulin drops - what does this up/downregulate?
upregulates:
gluconeogenesis
glycogen mobilisation
ketogenesis
protein degradation
uptake of amino acids
downregulates -
glycogen synthesis
protein synthesis
fat synthesis
in the muscles, what happens in a fasted state?
In a fasted state – GLUCOSE made from fats etc is used for muscle contraction
BUT proteins can be broken into amino acids and used to make energy in the liver
FATTY ACIDS can be used in muscle to make Acetyl-CoA for aerobic metabolism
There is a complex interaction between liver and muscle to keep things going
cell migration:
what are small GTPases?
a protein with:
guanine + ribose sugar (guanosine nucleotide) + three phosphates (GTP)
21 kDa proteins
change in conformation upon activation, swapping GTP for GDP
what is something not to get tripped up by when using terminology for GTPases?
GTP forms are ‘signalling active’
a GTPase that is ‘hydrolysis active’ is essentially inactivating itself tho
explain the structure of a small GTPase
main body, conserved across most GTPases
P-loop, or the phosphate coordinating loop - binds and stabilises the GTP nucleotide
Mg2+ - the positive charge is needed to bind the negatively charged nucleotide
Switch regions -
These are what have super subtle changes in conformation upon activation, and bind to downstream effectors
Switch 1
Switch 2
how are active small GTPases purified/isolated?
the change in structure when activated is so subtle its hard to identify, so the effector of the small GTPase is what is used to identify the active GTPase
how does the GTPase contribute to the GTP hydrolysis when deactivating
its all about the probability of getting water in position to form a nucleophilic attack and steal the phosphate
GTPases have a glutamine-61 residue that positions water in a favourable way for attacking the phosphate
If this glutamine is substituted or moved out of position by other mutations, hydrolysis won’t occur and the GTPase won’t be able to deactivate
how does the P-loop of the GTPase assist in hydrolysis?
P-loop has +ve residue like lysine, and forms some H-bonds in order to pull some -ve charge away from the phosphate, destabilising it’s bonds and catalysing hydrolysis
why is a GEF needed for GTPases and how effective are they?
GTPases are unstable when they are without a nucleotide, GTP or GDP (mid-switch)
GEFs (guanine nucleotide exchange factors) are needed to stabilise the GTPase for this exchange
Accelerates the exchange from 10 to 10^7 x faster
how do GAPs assist in GTP hydrolysis for small GTPases?
Several residues in the GTPase itself are restricting the freedom of the attacking water molecule = lowering the entropy barrier = making the reaction easier
The GAP - e.g. p50 - acts through its arginine residue -
The GAPs Arg stabilises the Glutamine residue that is already stabilising the water molecule
The +ve arginine is also pulling -ve charge away from the oxygen between the two phosphates, weakening the bond
how do GEFs work, give some examples of GEF groups?
they are all about stabilisation, accelerating GDP to GTP exchange, they also stabilise the nucleotide-free form, which is also Mg2+ free
you’ve got Dbl-homology domain GEFs (over 70 of these)
DOCK - family
Sec7 domain GEFs
what is the T17N mutation in a Rac (GTPase)?
causes the GTPase to bind so well to GEFs that it hoards them all, preventing activation of other GEFs in the cell
what are the sections/sub-families of the Rho family of GTPases?
Rac (protrusion in migration)
Cdc42 (polarity)
RhoA(contraction)
RhoG (trafficking)
GEFs are usually very specific, however…
VAVs, a group of GEFs - are quite promiscuous and will bind to all Rho family GTPases
give an example of a mutation in a GEF that alters it’s function
Tiam1: this GEF has 9 residues in its body bind to the switch 2 domain of Rac GTPases
Tiam1 - only effects Rac
another GEF, Intersectin 1 - only effects Cdc42
however, a single residue mutation can cause Tiam1 to go from being Rac sensitive to Cdc42 sensitive
what are the three stages in cell movement and what Rho GTPases are they governed by?
Filopodia - narrow projections at the front of the cell, formed in response to some kind of stimulus like a chemokine. Caused by Cdc42
Followed by formation of lamellipodia - narrow projections, then we get protrusion of the whole front of the cell, and the formation of new adhesions. The lamellipodia step = Rac1 drives this membrane protrusion
RhoA regulates the next step - formation and contraction of actin stress fibres, disassembly of adhesions at the back of the cell, to move the whole cell forward
what is the RhoA cascade involved in contraction of actin stress fibres?
RhoA - activated - binds to Rho kinase - phosphorylates myosin light chain - actomyosin contraction
what is important about the timing for Rac1 + Cdc42 vs RhoA?
the first two cause cell protrusion, RhoA causes cell contraction
these are antagonistic so must happen asynchronously
cell migration requires G…
GUIDANCE
a stimuli helps with going in the correct direction, but cells need some kind of track to follow in order to have a direction - this would be the ECM
without it migration is random, fast and cells are flat
restricting freedom = …
increasing efficiency
would increasing Rac1 activity speed up migration?
hard to say…
more protrusion, but you’d also need to increase RhoA if you want migration to be faster
You’d also need the signals to be in the correct location, e.g. Rac must be at the front of the cell/polarised to get directional movement, otherwise you get projections all over the place and movement is random, does not follow the ECM/track it is on
what are the four superfamilies or whatever of small GTPases?
Ras (cell proliferation)
Rho (migration)
Rab (endosomal trafficking)
Arf (membrane budding)
