Apoptosis

 

For excellent general reviews on apoptosis click here. and here.

 

The most heavily investigated PCD death-mechanism.

Derived from a Greek term describing autumn leaf-fall.

 

Animal cells use cell-death : 

 

(1) Developmentally e.g. finger separation during  

       embryogenesis  

 

(2) control organ development and size e.g. skin.

 

      inappropriate activation leads to e.g. AIDS

      inappropriate inhibition leads to cancer.

 

(3) destruction of infected cells

 

BUT cell contents are inflammatory so cannot simply rupture!

 

Click for apoptosis movies : Movie 1 and Movie 2

 

………...and compare with necrosis

 

Hence apoptosis involves –

 

(i)      nuclear  and cytoplasmic condensation margination of

chromatin along nuclear envelope

 

(ii)   membrane blebbing compartmentalisation of nuclear and

cytoplasmic material → apoptotic bodies

 

(iii)  DNA ladder formation

 

DNA fragmentation into 200bp increments corresponding

to internucleosomal spacing.

 

              

 

                          Used as a diagnostic marker for apoptosis.

 

 

Caspases : The workhorses of apoptosis

 

CYSTEINE-PROTEINASES a. k. a Caspases. named as activity is dependent on cysteine in active site

 

Proteinases which will cleave at aspartate residues within proteins

 

             

 

 

Translated as inactive precursor polypeptide separated by   aspartate residues

 

             

 

represents a positive feedback mechanism

 

SUBSTRATES

 

(A) AFFECTING CELL SHAPE

 

(i)   Microfilaments : gelsolin, actin and intermediate filaments.     

       Alters cell-shape and movement during apoptosis

(ii)  Lamin :  cleavage unpins nuclear envelope

 

(B)  AFFECTING DNA

 

(iii) Caspase Activated DNase (CAD)

 

           

 

(iv)    poly-(ADP-ribose) polymerase- (PARP) used in DNA

repair . Contains asp-glu-val-asp sites "unhooks" DNA repair from damage

 

          A suppressors of apoptosis

 

Bcl-2          onco-gene giving rise to B-cell lymphoma

                   an intracellular membrane protein. 

 

Bcl-2 also has a asp-glu-val-asp site--> therefore a caspase substrate!

 

Digestion acts to (i) inhibits caspase function

(ii) breakdown product - Bax, stimulates which forms pores in mitochondrial membrane.- this releases cytochrome c.

 

        

 

 

The regulator – the apoptosome

 

 

How can Bcl-2 suppress caspases and be a caspase substrate?

 

Revolves around another complex: the apoptosome

 

This is a complex formed between Bcl-2/caspase 9/ and Apaf-1

 

Apaf-1  (i) essential for cell-death but is not a caspase.

            (ii) processes caspase into an active form.

            (iii)          dependent on ATP hydrolysis -> conformational

change but is absolutely dependent on cytochrome c released from the mitochondria.

 

Bcl-2 binding prevents ATP  hydrolysis (and Bcl-2 prevents cytochrome release)

 

 

1

 

 

 

TNF, IL-1 and NF-kB and the regulation of apoptosis

 

 

TNF and the FAS pathway

 

TNF-a has well-characterised apoptotic function by interaction with the FAS-FADD pathway.

 

 

 

The Fas receptor (CD95) is part of the TNF-receptor superfamily.

 

FAS signalling is not suppressed by Bcl-2 – thus does not use the apoptosome mechanism.

 

FAS-Pathway

 

 

A. Death receptor Fas with intracellular death domain (DD)

B. Fas Ligand binds to Fas. Receptors cluster and the Fas death domain associates with

 the death domain on the adaptor protein FADD

C. This death-inducing signalling complex (DISC) recruits pro-caspase 8 (aka FLICE)

        molecules via the death effector domain (DED) on the FAD

 

TNF Pro-apoptotic pathway No 1

 

   Click here for larger image

 

 

 

LI-1 Pro-apoptotic pathway No 2

 

click here for larger image

 

It is possible for IL-1 to initiate apoptosis via the adaptor protein MyD88. This is a has both  a TIR domain and death domain –

 

Apoptosis can be intiated via FADD- Caspase 8 mechanism.

 

Pro and anti-apoptosis (Pathway No 3)

 

 

p53

 

Functional p53 is thought to provide a protective effect against tumorigenesis,

and indeed, mutations of p53 have been found in nearly all tumor types and are

 estimated to contribute to around 50% of all cancers.

 

Structural and functional aspects of p53

There are four conserved domains in p53:
1. The N-terminal domain is required for transcriptional transactivation
2. A sequence-specific DNA binding domain
3. A tetramerization domain near the C-terminal end
4. The C-terminal domain interacts directly with single stranded DNA.

Activation

Stress signals (e.g. hypoxia, radiation, DNA damage or chemotherapeutic drugs ...) activate

 

·       p53 activation,

·       ubiquitin-dependent degradation of the p53 protein is blocked.

 

The resulting increase in p53-dependent gene transcription leads to

 

·       the p53-mediated induction of programmed cell death

·       and/or cell cycle arrest.

 

 

p53 Target Genes

·       Wild-type p53 binds to specific genomic sites with a consensus binding site

5'-PuPuPuC(A/T)(T/A)GPyPyPy-3'.

·       p53 binds as a tetramer and stimulates expression of downstream genes that

negatively control growth and/or invasion or are mediators of apoptosis.

·       It was predicted that the expression of about 200-300 genes might be controlled

by p53 transactivation. 


 

p53 and apoptosis.

 

 

p53 induces

 

·       the expression of proteins that target both the mitochondrial- and the

death-receptor-induced apoptotic pathways,

·        represses transcription from several death-inhibiting genes.

·       They include the ability of p53 to drive relocalization of death receptors

such as Fas/CD95 from the Golgi to the cell surface

·        Murine double minute 2 (Mdm2), is a transcriptional target of p53.

Mdm2 binds to p53 and targets p53 for ubiquitin/proteasome-dependent

degradation. Ubiquitination (Ub) of p53 by Mdm2 probably also enhances

the export of p53 from the nucleus to the cytoplasm, where degradation takes place.


pp90rsk

 

·       forms part of MEK1/2-MAPK (ERK1/2) signaling pathway-

·       originally isolated as an Insulin associated kinase

 

 

·       Schematic diagram showing steps in the signaling pathway by which

P. aeruginosa up-regulates human MUC2 mucin gene transcription.

 

·       P. aeruginosa releases LPS, thereby activating a c-Src-Ras-Raf-1-

MEK1/2-MAPK (ERK1/2)-pp90rsk pathway leading to the activation

of NF-kappa B –mediated MUC2 transcription.

 

·        LBP, LPS-binding protein. The overproduced mucin, in concert with

abnormal airway lining fluid secondary to CFTR mutation, leads to airway

mucus obstruction and lung failure.

 

 Click here for larger image

 

 

NF-kB – mediated suppression of apoptosis

 

Two main functions 

 

(a)           Inducing the expression of superoxide dismutase (SOD)

(b)          Inducing the expression of IAPs – inhibitor of apoptosis.

 

Deveraux and Reed (1999) Gene and Development 13, 239-252.

 

IAP proteins characterised by having BIR domains –

 

BIR = Baculoviral IAP repeat

 

Conserved spacing of cysteine residues

 

(Cx2, Cx6, Wx5, Dx6, C)

 

 

 

CARD = Caspase recrutiment domain

 

IAPs bind to an inhibit a series of CASPASES by bind to the BIR domains