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BIOC2120 Lectures 2010: Dr. J.A. Illingworth

Integration & Compartmentation of Metabolism

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Lecture 2 topics:

Why do organisms have compartments?

Eukaryotic cells are large and divided into numerous compartments, in contrast to the smaller prokaryotes which have only one intracellular space. These lectures consider the distribution of low molecular weight compounds between the mitochondrial and cytosolic compartments, but it should be remembered that macromolecules are also confined in these compartments, and that there are other subcellular spaces: nucleus, endoplasmic reticulum, lysosomes, Golgi, peroxisomes, chloroplasts; each of which has its own unique metabolism.

Multi-cellular organisms also show metabolic specialisation between their differentiated tissues. Such adaptations must confer some selective advantages, and it is instructive to consider what these might be:

Self-directed learning:

[Complete the lecture before answering the questions.]

1.

Compartmentation raises metabolite concentrations: allows enzymes to work faster with lower substrate affinities and higher product dissociation rates. Why is there emphasis on the dissociation rates?

2.

Compartmentation reduces diffusion barriers by bringing enzymes and substrates into physical proximity. Why is diffusion such a problem?

3.

Compartmentation allows eukaryotic cells to maintain different environments in different sections of each cell. Name three specific examples:

4.

Compartmentation assists with metabolic control by keeping enzymes, substrates and regulators in separate locations with only intermittent access between them. Give three specific examples:

5.

Compartmentation is in any case required to accommodate the pH and potential gradients necessary for oxidative phosphorylation. How big are these gradients, and how might they have evolved?

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Cooperation between compartments:

Many pathways extend across several compartments, so it is often necessary to move metabolites in bulk from one compartment to another. The cytosol joins all the other compartments together. It is normally the largest compartment in most eukaryotic cells (and it is the ONLY compartment in red blood cells) with mitochondria in second place (up to 40% of the cell volume in cardiac muscle). Other significant subcellular spaces are the lumen of the endoplasmic reticulum, the nucleus, the Golgi apparatus, various secretory and storage vesicles, lysosomes and peroxisomes.

Electron microscope image of plant cell organelles

EM picture

Photo: Dr Alison Baker, University of Leeds. The picture shows part of a cell from Arabidopsis thaliana, with a chloroplast, peroxisome and mitochondrion in close juxtaposition.

Fat metabolism nicely illustrates the metabolic specialisation and cooperation between compartments. Fat and steroid biosynthesis takes place mainly in the cytosol, using acetyl-CoA derived from citrate cleavage, although subsequent steroid and bile acid processing occurs in the mitochondrial matrix space. Lipid catabolism is split at least three ways. Xenobiotics, drugs and really awkward molecules get a preliminary free radical assault from the cytochrome p450 system anchored in the endoplasmic reticulum, which introduces additional hydroxyl groups and opens up new lines of chemical attack. The dicarboxylic products of these reactions, together with branched chain fatty acids and very long chain linear fatty acids (>C20) transfer to the peroxisomes, where they are converted to coenzyme A derivatives which undergo a modified type of β-oxidation to reduce them to a manageable size.

Although the peroxisomal fat oxidation "spiral" looks similar to the mitochondrial pathway, it is not connected to the respiratory chain and it does not directly generate any ATP. The first enzyme, acyl-CoA oxidase, is a flavoprotein oxidase that converts oxygen to hydrogen peroxide. The peroxide is detoxified by catalase, which has very high activity in peroxisomes. Hydration and dehydrogenation of the enoyl-CoA are catalysed by a single enzyme, which produces NADH. Peroxisomes lack a respiratory chain, and they are impermeable to pyridine nucleotides so the only available method to recycle the NADH back to NAD is through lactate dehydrogenase, as shown in the diagram below. This is a simple example of a shuttle system (see below) which are widely used to balance up metabolic pathways in different compartments.

diagram illustrating the mitochondrial and peroxisomal lipid oxidation spirals

Peroxisomal oxidation stops at 8 carbon atoms, so the octanoyl-CoA and acetyl-CoA products of peroxisomal lipid breakdown are converted to the corresponding carnitine derivatives and shuttled across to the mitochondria to complete their conversion to carbon dioxide and water.

Most of the cellular lipids contain linear C16 and C18 fatty acids which are activated in the cytosol, and then transfered to the mitochondria as acyl-carnitine derivatives. Fat oxidation within the mitochondria is coupled to the synthesis of ATP. Long chain fatty acids (>C12) are processed by a system in the mitochondrial inner membrane, but when the chain length has been reduced to about 12 carbon atoms the partially degraded lipid joins the stream from the peroxisomes and is processed by a soluble enzyme system located in the mitochondrial matrix. Acetyl-CoA from all three sources is finally oxidised via the Krebs cycle within the mitochondrial matrix space.

diagram illustrating the role of carnitine in mitochondrial lipid uptake

There has recently been great research interest in the various fat oxidation pathways, and in the control of gene expression by the peroxisome proliferator activated receptors (PPARs). This system is intimately connected with the development of obesity and type 2 diabetes, which are major medical problems in the modern world. There are numerous lengthy reviews, for example Reddy & Hashimoto (2001). We will return to these topics in lectures 3 and 4.

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Cardiac Metabolite Contents:

There must be selection pressure for high metabolic rates (at least in times of plenty) because organisms that metabolise quickly can find food faster, escape faster, grow faster and reproduce faster than their competitors. Only when the food supplies are erratic is there any pressure to slow things down.

Cells might speed up metabolism by cramming more in: more enzymes and more metabolites per gram live weight. There must be a downside to this strategy, because very few organisms have used it. Most animals are about 75% water, and their internal osmotic pressure is close to 300 milliosmoles per litre (equivalent to 150mM NaCl) even for many ocean dwelling species where their external environment contains 540mM salt. However, the available solute capacity is very efficiently used, and it would be difficult to squeeze any more metabolites within the constraints imposed by the 300 milliosmole limit.

The values given below are a general guide for cardiac muscle oxidising glucose at moderate work outputs. They vary with metabolic status and from one tissue to another, but a similar pattern is often observed. A large proportion of the total ADP is actin-bound in all tissues, and plays little role in metabolism. It is clear that the negative charges on the principal metabolites, taken together, account for most of the total cellular cation content (about 450mEq/g dry). It would be difficult to accommodate any more metabolites, bearing in mind that the proteins and nucleic acids also have a significant negative charge.

Subcellular compartmentation helps to solve this problem, by confining key enzymes and their substrates within the same section of the cell, so that enzymes can work efficiently at high metabolite concentrations, with modest Km's and high turnover numbers.

metabolitechargescontent
(mmol/g dry)
commentscations
mEq/l
anions
mEq/l

creatine phosphate

1+, 3-

35

entirely cytosolic

11.735.0

creatine

1+, 1-

25

entirely cytosolic

8.38.3

ATP

4-

20

mostly cytosolic

 

26.7

ADP

3-

4

mitochondrial / actin bound

 

4.0

AMP

2-

0.4

almost all mitochondrial

 

0.3

inorganic phosphate

2-

15

all compartments

 

10.0

bicarbonate

1-

45

all compartments

 

15.0

NAD+

1+, 2-

4

all compartments

1.32.7

NADH

2-

0.25

almost all mitochondrial

 

0.2

glutamate

1+, 2-

20

all compartments

6.713.3

aspartate

1+, 2-

15

almost all cytosolic

5.010.0

alanine

1+, 1-

10

all compartments

3.33.3

citrate

3-

1

almost all mitochondrial

 

1.0

oxoglutarate

2-

0.2

mostly mitochondrial

 

0.1

malate

2-

0.5

mostly mitochondrial

 

0.3

oxaloacetate

2-

0.005

mostly cytosolic

 

 

acetyl CoA

2-

0.015

 

 

 

CoASH

2-

0.35

 

 

0.2

acetyl carnitine

1+, 1-

0.15

 

0.10.1

carnitine

1+, 1-

2.3

 

0.80.8

 

 

 

TOTALS37.2131.3

[These values are expressed as mmol/g dry weight. Do not attempt to learn them: they are here to illustrate a point. Most tissues contain about 75% water in vivo, and almost all the solids are proteins. The extracellular space is about 20% of the total volume. Discounting the extracellular contributions to both water and solids, a typical heart has 3ml intracellular water per gram dry weight, of which approximately 0.75ml is in the mitochondrial matrix space.]

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Mitochondrial Metabolite Transport:

Proton pumping by the respiratory chain complexes creates pH and electrical gradients across the inner mitochondrial membrane:

DpH = 0.5 pH units, inside ALKALINE; H+out /H+in = 3

DE = 150mV, inside NEGATIVE; bilayer thickness = 5 nanometres

Potential gradient = 0.15 volts / 5E-9 metres = 30,000,000 volts / metre

In 1966 Peter Mitchell correctly predicted that the existence of large mitochondrial transmembrane gradients would require highly specific transport proteins to control the movement of small ions across the mitochondrial inner membrane, and prevent the dissipation of the various gradients. At that time no mitochondrial membrane transport proteins had yet been isolated. Mitchell's paper was published privately because because he wasn't working in a "recognised" university laboratory. Forty six members of this "solute carrier family 25" have subsequently been identified in the human genome:

Mitochondrial Solute Carrier Proteins listed at NCBI

OMIMfunctiongene

190315

citrate transporter

SLC25A1

608157

ornithine transporter 2

SLC25A2

600370

phosphate carrier

SLC25A3

103220

adenine nucleotide translocator ANT1 (4q35 )

SLC25A4

300150

adenine nucleotide translocator ANT2 (Xq24-q26)

SLC25A5

300151

adenine nucleotide translocator ANT3 (X & Y chromosomes)

SLC25A6

113730

UCP1 uncoupling protein 1, thermogenin (brown fat)

SLC25A7

601693

UCP2

SLC25A8

602044

UCP3

SLC25A9

606794

dicarboxylate transporter

SLC25A10

604165

oxoglutarate carrier

SLC25A11

603667

aspartate / glutamate carrier 1 [binds Ca++], "aralar"

SLC25A12

603859

aspartate / glutamate carrier 2 [binds Ca++], "citrin"

SLC25A13

300242

UCP5 (brain)

SLC25A14

603861

ornithine transporter 1

SLC25A15

139080

Graves disease autoantigen

SLC25A16

606795

peroxisomal membrane protein

SLC25A17

609303

H+ glutamate carrier 2

SLC25A18

606521

mitochondrial thiamine pyrophosphate carrier

SLC25A19

212138

carnitine / acylcarnitine translocase

SLC25A20

607571

oxodicarboxylate carrier [lysine, tryptophan 2-oxoadipate]

SLC25A21

609302

H+ glutamate carrier 1

SLC25A22

608746

phosphate carrier

SLC25A23

608744

phosphate carrier

SLC25A24

608745

phosphate carrier

SLC25A25

611037

phosphate carrier

SLC25A26

 

UCP4

SLC25A27

609767

mitoferrin-2

SLC25A28

610793

kidney mitochondrial carrier protein-1

SLC25A30

610796

adenine nucleotide translocator ANT4 (4q28.1 )

SLC25A31

610815

folate carrier

SLC25A32

610816

not known

SLC25A33

610817

not known

SLC25A34

610818

not known

SLC25A35

 

not known

SLC25A36

610387

mitochondrial iron carrier

SLC25A37

610819

not known

SLC25A38

610820

not known

SLC25A39

610821

not known

SLC25A40

610822

not known

SLC25A41

610823

not known

SLC25A42

300641

not known

SLC25A43

610824

not known

SLC25A44

610825

not known

SLC25A45

610826

not known

SLC25A46

Students are not expected to learn the table above! These genes are still being annotated, and the functions of several remain to be elucidated. Note the presence of tissue-specific isoenzymes for several of these solute carriers, reflecting the tissue-specific metabolic pathways in which they participate.

The principal types of transport system are listed in the table below, which also shows some inhibitors.

Mitochondrial carrier system

Stoichiometry

Inhibitors & other features

phosphate:hydroxyl

electroneutral

N-ethyl maleimide, mersalyl

dicarboxylate carrier
(dibasic phosphate or malate or succinate)

electroneutral antiport

n-butyl malonate

tricarboxylate carrier
(citrate or isocitrate or malate + hydroxyl)

electroneutral antiport

benzene 1,2,3 tricarboxylate
mainly located in liver and adipocytes (for lipogenesis)

malate:oxoglutarate

electroneutral antiport

used for malate - aspartate cycle

adenine nucleotides

electrical antiport

atractyloside, bongkrekic acid

glutamate:aspartate

electrical antiport

used for malate - aspartate cycle

glutamate:hydroxyl

electroneutral

mainly in liver (for urea cycle)

ornithine:citrulline + proton

electroneutral antiport

mainly in liver (for urea cycle)

calcium ions

electrical uniport

calcium uptake in most tissues

calcium:sodium

electroneutral antiport

calcium export in heart

calcium:proton

electroneutral

calcium export in liver

sodium:proton

electroneutral

most tissues

[Note: using current techniques it is impossible to distinguish experimentally between a proton symport and a hydroxyl antiport if no other ion is involved in the exchange, so in these cases no indication is given in the table above.]

It makes a huge difference to the outcome whether or not the total electrical charges on all the ligands "balance out" over the whole catalytic cycle when the transporter is operating. If the charges balance then the carrier cannot feel the membrane potential, and it is described as "electroneutral". If the charges do not balance, the carrier is described as "electrical". Such carriers experience the full force of the membrane potential, and the ligand distribution is radically affected.

The operation of the electroneutral anion carriers leads to a modest accumulation of polybasic acids (e.g. citrate) within the matrix space. In general, at equilibrium:

The carriers may not reach equilibrium if the ligand flux is high, and the pH differential may vary under physiological conditions, but as an approximate guide we would expect the following pattern:

anion

pyruvate

phosphate

malate

citrate

charge at pH 7.2

-1-1.5-2-3

ratio [in]/[out]

35927

This is generally helpful for the operation of the citric acid cycle, and also has implications for the regulation of glycolysis and lipogenesis by phosphofructokinase and acetyl-CoA carboxylase, where citrate is an allosteric effector.

The case of phosphate is interesting, since this anion has a pK near neutrality and a fractional charge at physiological pH values. The pH is different on the two sides of the mitochondrial inner membrane, so the ratio between singly and doubly charged phosphates will differ in the two compartments. Try to convince yourself that it makes absolutely no difference to the outcome whether phosphate is taken up as H3PO4 in exchange for nothing (i.e. a proton symport), H2PO4- in exchange for one OH- or as HPO42- in exchange for two OH- followed by appropriate re-ionisations on the inside.

All electroneutral mechanisms lead to the same thermodynamic result. However, it is absolutely vital that the carriers are specific: if the carrier doesn't care about the anion charge, so that it sometimes operates electrically and sometimes not, then it would completely uncouple oxidative phosphorylation and normal mitochondrial functions would cease.

Some simple sums...

All such carriers must be pernickety about charges and stoichiometry. Citrate, for example, is actually transported by an electroneutral system, but it is possible to calculate the effects of an electrical leak...

How many electron volts (eV) would be wasted for each citrate ion that escaped through an electrical uniport?

If 1 eV=1.6E-19 Joules, what would be the energy cost per mole of citrate ions that escaped?

How much ATP would be wasted per citrate ion lost? [ATP is worth ~50kJ/mole]

The electrical porters:

The charge imbalance associated with the adenine nucleotide carrier leads to a large difference in the ATP/ADP ratio between matrix space and cytosol. Mitochondria scavenge almost all the ADP from the cytosol, and exploit the huge membrane potential to expel their ATP against a concentration gradient. ATP:ADP is high in the cytosol and low in the mitochondria. ATP is effectively "worth more" in the cytosol, because the ATP:ADP couple is maintained further away from equilibrium.

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The Gibbs equation

The mathematical relationship between DG and the distance from equilibrium was first described by the 19th century chemist J. Willard Gibbs. It applies to all chemical reactions (in fact, to all physical processes) and in essence is little more than common sense. If you begin with large amounts of reactants and no products then the reaction will proceed to equilibrium with formation of products, but conversely, starting with all products and no reactants the reaction will proceed in the opposite direction to the same final equilibrium position, where DG is zero.

Standard DG0 values are measured with everything at 1 molar concentration, but the actual concentrations in living cells will differ from this, and the effective DG values may vary in different parts of the cell. The precise relationship is a logarithmic one, as shown in the figure below. For different chemical reactions each graph will be displaced along the horizontal axis, but it will always have the same general shape.

Eukaryotic cells do not, however, get something for nothing by pumping ATP into the cytosol. The transport of ATP, ADP and phosphate across the inner mitochondrial membrane costs 33% additional energy, over the minimum required for the synthesis of ATP within the mitochondrial compartment. This extra energy must be supplied by the respiratory chain.

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Myokinase

The high cytosolic ATP/ADP implies a very low cytosolic AMP concentration, as a result of the myokinase equilibrium:

This enables 5' AMP to serve as an emergency signal, which indicates a threat to the ATP supply. Several key enzymes (notably glycogen phosphorylase and phosphofructokinase) are strongly activated by 5'AMP. This nucleotide also gives rise to adenosine which stimulates blood flow to active tissues, and it regulates its own protein kinase, AMPK. Note that 5'AMP differs from 3'5' cyclic AMP produced by adenyl cyclase, and that AMPK is NOT the same as protein kinase A.

Self directed learning: log into Web of Knowledge Run a 'words in title' search with the topic 'AMPK'. You will get over a thousand hits, so click in 'Document Types' and restrict the output to Reviews. In early 2010 there were about 40 of these. Pick an interesting 'mainstream' topic, download and read one of them as a PDF file. [Most of these articles are available free of charge via the University Library.]

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Creatine kinase

Muscle contraction apparently requires a very high ATP/ADP ratio, and it is difficult to maintain a low myofibrillar ADP because it will not diffuse quickly enough back to the mitochondria at low ADP levels. (Diffusion rate is directly proportional to concentration.) To overcome this problem, active muscles contain creatine phosphokinase, and large amounts of creatine and creatine phosphate. These compounds equilibrate with the adenine nucleotide pools, and provide high concentrations of two highly diffusible energy carriers to increase the energy transport rate:

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Shuttle systems

One of the most important asymmetric transporters is the glutamate:aspartate carrier, which plays a key role in the re-oxidation of glycolytic NADH by the malate-aspartate cycle.

Aspartate- swops for glutamate plus a proton, so the full proton motive force is applied to the exchanger. The effect is to maintain the cytosolic compartment highly oxidising (with a low NADH/NAD ratio) while the mitochondrial compartment is kept correspondingly reduced. This suppresses lactate formation during aerobic glycolysis.

The system above is widespread in mammals, and is probably present in all vertebrates, but other kingdoms and phyla have their own solutions to this problem. Plant mitochondria have an outward facing NADH dehydrogenase which reacts with cytosolic NADH, in addition to the normal inward facing enzyme that processes NADH from the matrix space. The outward facing enzyme does not pump protons as it donates electrons to ubiquinone, so it achieves a lower overall P:O ratio than the inward facing route. This keeps the cytosol more highly oxidised than the mitochondria, which seems to be a desirable outcome. Insects use a glycerol phosphate shuttle to achieve the same result: cytosolic NADH reduces dihydroxyacetone phosphate (DHAP) to glycerol phosphate. The glycerol phosphate is recycled to DHAP by an outwardly directed glycerol phosphate oxidase in the mitochondrial inner membrane, which donates electrons to ubiquinone. Once again their is no proton pumping, so the result once again is a highly oxidised cytosol when compared with the mitochondrial matrix space. Intriguingly, humans also have this enzyme system, which is activated by thyroid hormone, but the activity is normally low.

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Cation transport

The pH and potential gradients also provide the energy for cation transport across the mitochondrial inner membrane, but the roles are reversed when compared with anion transport. The electrical gradient drives cation uptake, while the pH gradient powers cation export. This is neatly illustrated by the systems that regulate intramitochondrial calcium, and transmit this important cellular activation signal from the cytosol to the mitochondrial matrix space.

Calcium uptake is mediated by an electrical uniport which is restricted under physiological conditions by the very low cytosolic calcium ion concentration in the resting state. This carrier has a sigmoid saturation curve for calcium, so that when the cell is activated and the cytosolic calcium concentration starts to rise, a powerful jolt of calcium ions is delivered to the mitochondrial matrix space. Left to its own devices, the large transmembrane potential would lead to a very high matrix calcium concentration if equilibrium were ever attained. Fortunately, a separate electroneutral export system bails out the surplus calcium, driven directly by the pH gradient in liver, and indirectly via sodium ions in cardiac muscle.

Calcium signalling is important, because it is one of the few cytosolic second messengers that has access to the intramitochondrial compartment. Phosphoinositol and nucleotides like cAMP are unable to cross the mitochondrial inner membrane, but calcium ions can switch on the main energy-yielding pathways whenever cells are called upon to perform some particularly arduous task.

Mitochondrial enzymes activated by calcium ions include pyruvate dehydrogenase, NAD-linked isocitrate dehydrogenase and oxoglutarate dehydrogenase.

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Apoptosis

Apoptosis or programmed cell death is an ancient strategy for killing defective or unwanted cells which is probably present in all multicellular organisms, both animals and plants. It is employed extensively within the immune system to select the most useful clones. Apoptosis also plays a major role in embryology, pathology and in cancer chemotherapy. (These references are included for general interest, to illustrate the current direction of research activity. You don't need all this detail for BIOC2120!) Apotosis is a major anti-cancer and anti-viral mechanism in humans, and it is also involved in growth and differentiation, heart attacks and stroke.

There are at least four independent trigger mechanisms:

Apoptosis is opposed by the Bcl-2 oncogene [Bcl = B cell lymphoma] and promoted by Bax.

Major events on the mitochondrial pathway

Intracellular apoptotic signalling exploits amplifying cascades of cysteine proteases called caspases that activate their protein substrates by cleaving them after aspartate residues. A major event in some of these signalling pathways is the mitochondrial permeability transition, which involves a bizarre change in mitochondrial compartmentation. The adenine nucleotide carrier and other proteins are incorporated into new transmembrane pores. This creates holes in both mitochondrial membranes, collapsing the inner membrane potential, preventing ATP synthesis and releasing cytochrome c from the inter-membrane space into the cytosol. Once released, the cytochrome c associates with another protein called Apaf-1 and initiates a proteolytic cascade. The ultimate result is fragmentation of the DNA and the orderly dissolution of the cell.

Greatly simplified diagram of the apoptotic signalling cascades

PARP stands for poly(ADP-ribose) polymerase, an enzyme which ADP-ribosylates a wide variety of nuclear proteins, using NAD as the ADP-ribosyl donor. (You will encounter this curious process in other parts of the cell when you study the actions of cholera and diphtheria toxins on mammalian cells.) Nuclear ADP-ribosylation is strongly induced by the presence of DNA strand breaks. It plays a role in DNA repair and the recovery of cells from moderate DNA damage. Inactivation of PARP implies that the cell has "given up" on DNA repair, and the cellular machinery now regards death as inevitable.

Ow et al (2008) Cytochrome c: functions beyond respiration. Nature Reviews Molecular Cell Biology 9, 532-542.

Riedl & Salveston (2007) The apoptosome: signalling platform of cell death. Nature Reviews Molecular Cell Biology 8, 405-413.

The life or death decision depends on a continuously shifting balance between pro- and anti-apoptotic factors. Radiation and cytotoxic drugs are effective against susceptible tumours because these cells are already teetering on the brink of self-destruction, and the slightest push will tilt the balance. The proto-oncogene Bcl-2 is an anti-apoptotic gene whose protein product inhibits the mitochondrial permeability transition. It is named after B cell lymphomas, which are tumours of the MALT tissue in the small intestine where redundant B cells fail to die at the appropriate time.

There is cross-talk between the different pathways, so (for example) granzymes and caspase 8 can both activate the mitochondrial route, and are consequently subject to modulation by Bcl-2. Marsden et al (2002) have shown that Bcl-2 over-expression inhibits apoptosis, even in the absence of Apaf-1 or caspase 9. Once triggered, apoptosis is auto-catalytic because activated caspase 3 can itself activate caspase 8, which can in turn initiate the mitochondrial permeability transition and the release of cytochrome c. The cross-talk and positive feedback normally ensures a clear decision on whether each cell will live or die.

Don't forget to try the self-assesment tests!

introduction
metabolic map
lecture 1
lecture 2
lecture 3
lecture 4
self-assessment


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