Immunity in Health and Disease (BS963) 2009- 2010 Dr Minnie O’Farrell APOPTOSIS

Apoptosis in model systems. Molecular mechanisms of apoptosis in mammalian cells, mitochondrial pathway (intrinsic) and cell death receptor pathway (extrinsic). bcl-2 gene family. Caspases. Apoptosis in the development of the immune system and in pathologies.

General text books/monographs:

Alberts et al. The Molecular Biology of the Cell 5 th ed Lodish

et al.

Molecular Cell Biology 6 th ed.

Weinberg The Biology of Cancer Normal white blood cell Apoptotic white blood cell Lodish 6 th Fig 1-19 During apoptosis (programmed cell death) cells bleb and eventually break apart

without

releasing contents.

Alberts et al. Fig 18-3 Apoptosis during the metamorphosis of a tadpole into a frog.

The cells in the tadpole tail are induced to undergo apoptosis stimulated by the increases in thyroid hormone that occurs during metamorphosis The nematode

Caenorhabditis elegans

has also been a very important model system for studying apoptosis in development.

Weinberg Fig 9.19

Apoptosis and normal morphogenesis.

TUNEL assay (immunodetection) detects breaks in DNA as cells undergo apoptosis + 1 day Types of cells that undergo apoptosis Apoptosis in the embryonic mouse paw.

Apoptosis occurs between 12.5 to 14.5 days in embryogenesis Cell Biology

Pollard and Earnshaw

Apoptosis is also important in the development of the nervous system

Healthy Two lymphocytes Staurosporin-treated HeLa cells Apoptosis Apoptosis Apoptotic

Weinberg Fig 9.18 Different parts of the apoptotic programme. Markers for the process.

Fragmentation of DNA

Pyknotic nuclear fragments Apoptotic cell

Fragmentation of Golgi bodies Phosphorylation of Histone 2B Phagocytosis of apoptotic bodies

Necrosis

Comparison between two forms of cell death, apoptosis and necrosis

Apoptosis

Apoptosis is a highly regulated process requiring gene expression.

Weinberg Table 9.3 Apoptosis v necrosis

Apoptosis is a very “clean” process.

Phagocytic macrophage removing remains of apoptotic cell.

Also a highly regulated process involving cell-cell interaction and signalling.

Cell and Molecular Biology Karp

This slide and the next……… Two useful tables

Regulators of apoptosis

An important model system in which the molecular basis of apoptosis has been studied

Lodish et al. 6 th Fig 1.25

Caenorhabditis elegans

Nomarski interference microscopy Lodish et al. 5 th Fig 22.8

Lineage of all somatic cells, from fertilized egg to mature worm has been traced.

1030 cells generated but 131 cells die

C. elegans

Cell fate data

Genetic screens:

Ced-3, Ced-4 and Egl1

required for cell deaths

Ced-9

represses cell death programme Mutations in the

ced-3

gene block apoptosis Lodish et al.6

th Fig 21.36

ced-1

mutant (defective in engulfment so dead cells visible (refractile) and can identify apoptosed cells (yellow arrows)

ced-1- ced-3

double mutant. There are no refractile cells in these double mutants indicating that no cell deaths occurred (yellow arrowheads)

Lodish et al. 6 th Figure 21.37 Evolutionary conservation of apoptotic pathways

There are two major pathways of apoptosis intrinsic pathway extrinsic pathway Maniati

et al.

2008 Fig 1 The molecular basis of apoptosis in mammals. Simplified overview

A family of apoptosis proteins has been discovered in mammalian cells . The first member of the Bcl-2 family was identified during a study of B cell lymphoma.

The oncogenic version is formed through a reciprocal chromosomal translocation in which parts of the chromosome 14 and chromosome 18 are exchanged.

The translocated

bcl-2

gene is now under the control of an active immunoglobulin promoter that drives high levels of constitutive expression.

Bcl-2 is pro-survival (anti-apoptotic) and is homologous to CED-9 in

Caenorhabditis elegans

.

Quite early in the study of Bcl-2 it was found to localise to the outer membrane of the mitochondria.

We now know that there are at least 24 Bcl-2-related proteins, 6 are anti apoptotic and 18 are pro-apoptotic. Lets examine these in a little more detail.

Introducing the BCL-2 family of proteins important in apoptotic pathways

= anti-apoptotic Weinberg Fig 9.25

Bcl-2 and related proteins part A

The structure of many of these proteins has been determined and interactions between them investigated.

Weinberg Fig 9.25

Bcl-2 and related proteins The anti-apoptotic protein, BCL-X L , is inhibited by binding of the pro-apoptotic BH3 only protein (orange) in the groove between BH1 and BH3

BH3 only protein binding specificity for BCL-2 homologues

BIM and PUMA bind to all BCL-2 family members tested; by contrast NOXA only binds to A1 and MCL1. These binding specificities recapitulate the ability of these proteins to activate apoptosis e.g. BIM et al can induce apoptosis alone whereas a combination of NOXA and BAD is required.

Youle and Strasser (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology,

9

, 47-59

Conformational changes in BCL-2 family members during apoptosis.

BAX undergoes extensive conformational changes during the mitochondrial translocation process. The protein changes from a soluble cytoplasmic protein in healthy cells to one that appears to have at least 3 helices inserted in the mitochondrial membrane in apoptotic cells.

Youle and Strasser (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology,

9

, 47-59

Alberts et al.

Fig 18-7

Release of cytochrome c from mitochondria during apoptosis GFP = Green Fluorescent protein Control UV-treated to induce apoptosis

To summarize…..

BCL-2 family of proteins have opposing apoptotic activities

1

st

set (e.g. Bcl-2 itself) inhibit apoptosis.

2

nd

set (e.g. BAX) promotes apoptosis.

3

rd

set (e.g. the BH3 only proteins) bind and regulate the anti apoptotic BCL-2 proteins to promote apoptosis.

Extrinsic or death receptor pathway

Maniati

et al.

2008 Fig 1 The molecular basis of apoptosis. Simplified overview

Death receptors Weinberg Fig 9.31 The extrinsic apoptotic pathway

Opferman (2008) Fig 1 Death receptor mediated apoptosis

Maniati

et al.

2008 Fig 1 The molecular basis of apoptosis. Simplified overview

Weinberg Fig 9.29 The intrinsic apoptotic pathway

Heptamer ( 7 chains) The blue helices in the middle bind and activate procaspase 9 to caspase 9 Caspase 9 activates procaspases 3, 6 and 7 Weinberg Fig 9.28 The APOPTOSOME is assembled in the cytoplasm when cytochrome c is released from the mitochondria and binds to Apaf-1

Weinberg Fig 9.32 Convergence of intrinsic and extrinsic apoptotic pathways

Caspases are proteolytic enzymes activated by both extrinsic and intrinsic pathways

Caspase family, 12-13 members Two classes: Initiators Effectors All caspases have a similar domain structure Not all mammalian caspases participate in apoptosis. For example Caspases 1, 4, 5, and 12 are activated during innate immune responses and are involved in the regulation of the inflammatory reponse

Effector caspases (such as caspase-3, -6 and -7 in mammals) function to breakdown cell structures through cleavage of specific substrates.

Actin cytoskeleton Lamins Golgi Translation apparatus Taylor

et al.

(2008)

Cory and Adams (2002) Nature Reviews Cancer, 2, 647-656

Phagocytic macrophage removing remains of apoptotic cell.

This is a highly regulated process involving recognition and signalling between the apoptotic cell and macrophage.

Cell and Molecular Biology Karp

Stages of engulfment of apoptotic cells can be divided into 4 stages

Binding Recognition Phagocytosis Internalization

Kinchen and Ravichandran (2007) Fig1

Lauber et al.(2004) Fig 2 Lack of “don’t eat me” signals on the surface of apoptotic cells

Taylor

et al

. (2008)

Lauber et al.(2004) Fig 1 The engulfment synapse

Lauber

et al.

(2004) Fig 6 The three steps of apoptotic cell removal.

Alberts

et al

. Fig 25-13 Mechanisms of immunological tolerance to self antigens

25-64

Alberts Fig 25-47 Two strategies by which effector cytotoxic T cells kill their target cells

Following are three supplementary slides for overview and glossary of terms.

A useful diagramatic overview with a short review. Willis et al. (2003) J. Cell Science,

116

, 4053-4057

Cory and Adams (2002) Nature Reviews Cancer, 2, 647-656

Cory and Adams (2002) Nature Reviews Cancer, 2, 647-656