Molecular Mechanisms of Apoptosis

Molecular mechanisms of apoptosis
Cell death by apoptosis occurs when a specialised intracellular signalling pathway is activated
and kills the cell. Apoptosis is the most common way of cells to die in vivo but there are other
ways (necrosis has been defined as cell death that is not apoptosis; necrosis may in some
cases indeed be due to simple physical injuries but there also seems to be at least one
signalling pathway that causes necrosis; pyroptosis is when a cell dies as a consequence of the
activity of caspase-1, a protease involved in the maturation of cytokines; autophagy has also
been linked to cell death. Programmed cell death used to be a term for cell death especially
during development, where a cell has the predetermined fate to die. The term is now
commonly used to describe any cell death that is the result of intracellular signal transduction
(a program) and therefore especially encompasses apoptosis).
The pathways to apoptosis are incompletely but still fairly well understood. A simple
diagram is here:
Caspase-8-activation: death receptors and TRIF

Put simply, apoptosis probably always involves caspase-3, and caspase-3 is activated either
by caspase-8 or by caspase-9. The upstream mechanisms therefore activate either caspase-8 or
-9. Caspase-8 is typically activated by death receptors (Fas, TRAIL-receptors, TNFR) but it
can also be activated by the immune signalling adapter TRIF. In some situations the activation
of caspase-8 seems fairly straightforward, where it is recruited by death receptors and
activated by reaching a high local concentration. It turns out more and more however that the
signalling downstream of death receptors (in particular the TNF receptor) is amazingly
complex, with balancing apoptosis-inducing and non-apoptosis-inducing functions. Similarly,
the signalling molecule TRIF appears to be able to activate caspase-8. TRIF is better known
for its immune ability of inducing interferons and NF-B upon recognition of viral RNA but
TRIF can also recruit the TNF-receptor signalling machinery, including caspase-8.
One aspect we are looking at in this context is the role of the cellular inhibitor of
apoptosis proteins (cIAPs). cIAPs are ubiquitin ligases, and a fair bit about their molecular
function is still unclear. One function appears to be the ubiquitylation of RIP1, which at least
determines composition of the signalling complexes. One in part speculative model of how
signalling downstream of TRIF could work is shown here:
cIAPs appear to govern in part a decision of whether a TRIF-signal leads to activation of

caspase-8 or not. This is easily demonstrated by using a synthetic drug that is currently under
clinical evaluation (we use LBW242 from Novartis but other companies have similar
molecules). These drugs induce the loss of especially cIAP1 and sensitize e.g. tumour cells to
TRIF-induced apoptosis. How this works and what the function also in immune cells may be
is an intriguing question we are working on.
Mitochondrial apoptosis
It appears that mitochondrial apoptosis is much more common than caspase-8-dependent
apoptosis in vivo. Mitochondrial apoptosis is regulated through the Bcl-2-family of proteins:
within this family, the BH3-only proteins (triggers) often regulate the activation of the
effectors Bax and Bak; active Bax and Bak cause the release of cytochrome c from the
mitochondrial intermembrane space into the cytosol, where it binds to Apaf-1, inducing its
oligomerisation and thereby causing the activation of caspase-9.
Cytochrome c sits in between the two mitochondrial membranes and is released
through permeabilisation of the outer membrane (there may be something specific there as
cytochrome c is released more easily than other proteins). Permeabilisation is the result of
activation (detectable as conformational change) and oligomerisation of the effector proteins
Bax or Bak. Activation of Bax/Bak is probably in most situations achieved by BH3-only
proteins but this is a contentious area. The anti-apoptotic members of the Bcl-2-family block
apoptosis very likely by binding either BH3-only proteins or Bax/Bak. We have been looking
a lot at BH3-only proteins, their functions and their physiological role. A number of BH3-
only proteins are localised to mitochondria, where they have functions in activating Bax. The
insertion of one BH3-only protein (BimS) is shown here (if you look at higher resolution you
can see how Bim is inserted into the outer mitochondrial membrane):
This regulation of apoptosis is something that is still not particularly well understood, and this
is an area we are very actively investigating.
Apoptosis in host defence

This is really part of our immunology section. Apoptosis is used in host defence on a number
of levels. Youll see in our section Immunology how apoptosis regulates the termination of the
immune response. What we touch on here is the cell-autonomous defence. This has been best
defined in viral infections. Viral infections often kill the cell, and this is because the cell
recognises the virus and activates apoptosis. Viruses, on the other hand (in particular the
larger DNA viruses such as poxviruses and Herpes viruses) often carry genes whose products
inhibit apoptosis (especially Bcl-2-like genes/proteins are common). The understanding of
how viruses (and other micro-organisms) are detected (most often by so-called pattern
recognition receptors), how this leads to apoptosis, how these recognition pathways are linked
to and distinct from other defence functions such as interferon-induction, and how viruses try
to block this is a fascinating area where we are active. The figure shows an instance where a
poxvirus called MVA infects a cell (in blue host cell nuclei). Nothing much happens in terms
of apoptosis, but if we take away the MVA anti-apoptotic protein F1L (labelled F1L), then
the virus induces apoptosis (seen by condensation and fragmentation of the nuclei). Analysis
of these pathways, in part by rebuilding them synthetically, is one of our research areas.
Apoptosis in tumours
It is generally assumed that a defect in the ability to undergo apoptosis is one factor that may
drive or is otherwise involved in the emergence of tumours. Curiously, a defect in apoptosis is
often associated with tumour development but most tumour cells are actually more sensitive
than normal cells for apoptosis induced by irradiation or chemotherapy. There are a number of
changes known in the apoptosis system that determine this balance; for instance, most
tumours depend a lot more than normal cells on the continuous presence of anti-apoptotic
Bcl-2 proteins, and these proteins are a very promising target of tumour therapy (a drug
furthest in development is the Abbott compound ABT-737, which inhibits some Bcl-2-like
proteins very well and can help kill tumour cells). We are applying our knowledge from the
above approaches to understand apoptosis deregulation in some tumour cells.
Publications in this area:

Zall, H., Weber, A., Besch, R., Zantl, N. and Hcker, G. (2010) Chemotherapeutic drugs sensitize
human renal cell carcinoma cells to ABT-737 by a mechanism involving the Noxa-dependent
inactivation of Mcl-1 or A1. Molecular Cancer; 9(1):164
Grespi, F., Soratroi, C., Krumschnabel, G., Sohm, B, Ploner, C., Geley, S., Hengst, L., Hcker, G and
Villunger, A. BH3-only protein Bmf mediates apoptosis upon inhibition of CAP-dependent protein
synthesis. Cell Death Diff, 2010 Aug 13. [Epub ahead of print].
Weber, A., Kirejczyk, Z., Besch, R., Potthoff, S., Leverkus, M. and Hcker, G. (2010) Pro-apoptotic
signalling through Toll-like receptor 3 involves TRIF-dependent activation of caspase-8 and is under
the control of inhibitor of apoptosis proteins in melanoma cells. Cell Death Diff, 17(6):942-51.
Besch, R., Poeck, H., Hohenauer, T., Senft, D, Hcker, G., Berking, C., Hornung, V., Endres, S.,
Ruzicka, T., Rothenfusser, S and Hartmann, G. (2009) Proapoptotic signalling by RIG-I and MDA-5
results in tumor-specific apoptosis independent of type I interferons in melanoma. J Clin Invest.,
119(8):2399-411.
Poeck, H., Besch, R., Maihoefer, C:, Renn, M., Tormo, D., Morskaya, S.S., Kirschnek, S., Gaffal, E.,
Landsberg, J., Hellmuth, J., Schmidt, A., Anz, D., Bscheider, M., Schwerd, T., Berking, C., Bourquin,
C., Kalinke, U., Kremmer, E., Kato, H., Akira, S:, Meyers, R., Hcker, G., Neuenhahn, M., Busch,
D.H., Rothenfusser, S., Prinz, M., Hornung, V., Endres, S., Tting, T. and Hartmann, G. (2008) 5-
triphosphate si-RNA: turning gene silencing and RIG-I activation against melanoma. Nature Med,
14(11):1256-63.
Weber, A., Kirejczyk, Z., Potthoff, S., Ploner, C. and Hcker, G. (2009) Endogenous Noxa
determines the strong pro-apoptotic synergism of the BH3-mimetic ABT-737 with chemotherapeutic
agents in human melanoma cells. Transl. Oncology, 2(2):73-83.
Lohmann, C., Muschweckh, A., Kirschnek, S., Jennen, L., Wagner, H. and Hcker, G. (2009)
Induction of tumor cell apoptosis or necrosis by conditional expression of cell death proteins: analysis
of cell death pathways and in vitro immune stimulatory potential. J Immunol., 182(8):4538-46.
Zantl, N., Weirich, G., Hilpert, C., Seiffert, B., Fischer, S.F., Gillissen, B., Daniel, P. T. and Hcker,
G. (2007) Low expression of the BH3-only protein Bim is a determinant of apoptosis resistance in
renal cell carcinoma. Oncogene, (49):7038-48.
Weber, A., Paschen, S. A., Heger, K., Wilfling, F., Frankenberg, T., Bauerschmitt, H., Seiffert, B. M.,
Kirschnek, S., Wagner, H. and Hcker, G. BimS Induced Apoptosis Requires Mitochondrial
Localization but not Interaction with Anti-Apoptotic Bcl-2 Proteins. J Cell Biol. 177(4):625-36
(2007).
Hcker. H., Redecke, V., Blagoev, B., Kratchmarova, I., Wang, G., Kamps, M.P., Saha, S.K.,
Oganesyan, G., Raz, E., Wagner, H., Hcker, G., Mann, M., Cheng, G. and Karin, M. (2006)
Specificity in TLR Signaling: Activation of the Interferon Response Depends on TRAF3. Nature,
439:204-207.
Fischer, S.F., Rehm, M., Bauer, A., Hfling, F., Kirschnek, S., Rutz, M., Bauer, S., Wagner, H. and
Hcker, G. (2005) Toll-like receptor 9-signaling can sensitise fibroblasts for apoptosis. Immunol.
Letters, 97:115-122.
Ruckdeschel, K., Pfaffinger, G., Haase, R., Sing, A., Weighard, H., Hcker, G., Holzmann, H. and
Heesemann, J. (2004) Signaling of apoptosis through toll-like receptors critically involves Toll/IL-1
receptor domain containing adapter inducing IFN- but not myeloid differentiation factor 88 in
bacteria-infected murine macrophages. J. Immunol., 173:3320-3328.
Kreuz, S., Siegmund, D., Rumpf, J.J., Samel, D., Leverkus, M., Janssen, O., Hcker, G., Dittrich-
Breiholz, O., Kracht, M., Scheurich, P. and Wajant, H. (2004) NFB activation by Fas is mediated
through FADD, Caspase-8 and RIP and is inhibited by FLIP. J Cell Biol., 166:369-80.

Molecular Mechanisms of Apoptosis

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Molecular Mechanisms of Apoptosis

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Molecular mechanisms of apoptosis

Caspase-8-activation: death receptors and TRIF

cIAPs appear to govern in part a decision of whether a TRIF-signal leads to activation of

Apoptosis in host defence

Publications in this area:

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