Resources > MitoNews > Archives > Volume 01, Issue 08 - August, 2005

Volume 01, Issue 08 - August, 2005




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MitoNews
Mitochondrial Research Bulletin

Published by:
MitoSciences
Advancing Vital Discoveries in Mitochondrial Research
http://www.mitosciences.com

Written by:
Dr.Sashi Nadanaciva
snadanaciva@mitosciences.com

Volume 01, Issue 08 - August 31, 2005
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Past Issues of MitoNews can be found at:
http://www.mitosciences.com/mitonews_archives.html


In this Issue:

Special Focus: Mitochondrial Proteins in Apoptosis

1. Introduction

2. Cytochrome c and caspase-dependent apoptosis
A. The PTP or not the PTP
B. The Bcl-2 proteins
C. Targeting Bcl-2 proteins in cancer therapy
D. The Apoptosome activates caspases

3. AIF and caspase-independent apoptosis

4. A mitochondrial connection for p53 in apoptosis

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1. Introduction

This issue of MitoNews is timed to coincide with the release,
by MitoSciences, of two antibody-based kits for detecting
apoptosis: one an immunocytochemistry kit, the other a
Western blotting kit. More details on these kits are available
at our website, http://www.mitosciences.com.

Apoptosis or programmed cell death is a highly regulated
natural process by which unwanted, damaged, or infected
cells are eliminated from the body during embryonic
development, tissue homeostasis and normal cell turnover.
Suppression of apoptosis is associated with disease states
such as cancer and autoimmune diseases.

Apoptosis occurs by at least 2 pathways, one of them
involving the mitochondrion. This issue of MitoNews deals
with some of the mitochondrial proteins involved in apoptosis.

General Reviews:

CYTOCHROME C-MEDIATED APOPTOSIS.
Jiang X., and Wang, X.
Annu.Rev.Biochem. 73: 87-106 (2004)

APOPTOTIC PATHWAYS: 10 MINUTES TO DEAD.
Green, D.R
Cell 121 (5): 671-674 (2005)

MITOCHONDRIAL EFFECTORS IN CASPASE-INDEPENDENT
CELL DEATH.
Lorenzo, H.K., and Susin S.A.
FEBS Lett. 557 (1-3): 14-20. (2004)

TRANSCRIPTION, APOPTOSIS AND p53: CATCH-22.
Schuler, M., and Green D.R.
Trends in Genetics, 21 (3): 182-187. (2005)



2. Cytochrome c and caspase-dependent apoptosis

A key player in the mitochondria-dependent pathway of
apoptosis is cytochrome c, a mitochondrial intermembrane
space protein. Cytochrome c's primary role is in mitochondrial
respiration, where it acts as an electron carrier between 2
oxidative phosphorylation complexes, the cytochrome bc1
complex (Complex III) and cytochrome c oxidase (Complex IV).
Under a variety of apoptotic stimuli, cytochrome c is released
from mitochondria into the cytosol where it binds to a cytosolic
protein, Apaf-1, leading to the activation of caspases (cysteine
containing proteases that cleave proteins at aspartate residues)
which cleave and destroy proteins. The resulting fragments of
the cell - apoptotic bodies - are then engulfed by macrophages
and dendritic cells. Release of cytochrome c into the cytosol is
an early event in apoptosis.



A. The PTP or not the PTP

How is cytochrome c released from the mitochondrial
intermembrane space into the cytosol? There seem to be two
prevailing views, one involving rupture of the outer mitochondrial
membrane and the other involving formation of pores in the
outer membrane without disruption of this membrane. According
to the former view, an assembly of proteins known as the
permeability transition pore (PTP) opens during apoptosis,
causing water to rush into the mitochondrial matrix. This leads
to swelling of the matrix, stretching of the inner mitochondrial
membrane, rupture of the outer mitochondrial membrane and
release of intermembrane space proteins. The composition
and structure of the PTP is still not resolved but it is thought
that Porin (Voltage Dependent Anion Channel), a mitochondrial
outer membrane protein, ANT (adenine nucleotide translocase),
an inner membrane protein, and cyclophilin D, a matrix protein,
are major components of the PTP (the April issue of MitoNews
has a discussion of what constitutes the PTP).

Two recent papers cast doubt on the importance of the PTP
in apoptosis and cytochrome c release. Cyclophilin D-knockout
mice show no developmental abnormalities (indicating that
apoptosis occurs in these animals), and cells from these mice
undergo apoptosis, showing cytochrome c release, in the
presence of various apoptotic stimuli. Interestingly, these
knockout mice do show resistance to necrosis ("accidental"
cell death), thus implicating the PTP in necrosis.

CYCLOPHILIN D-DEPENDENT MITOCHONDRIAL
PERMEABILITY TRANSITION REGULATES SOME NECROTIC
BUT NOT APOPTOTIC CELL DEATH.
Nakagawa, T., Shimizu, S., Watanabe, T., Yamaguchi, O.,
Otsu, K., Yamagata, H., Inohara, H., Kubo, T., and Tsujimoto,Y.
Nature 434: 652-658. (2005)

LOSS OF CYCLOPHILIN D REVEALS A CRITICAL ROLE FOR
MITOCHONDRIAL PERMEABILITY TRANSITION IN CELL DEATH.
Baines, C.P., Kaiser, R.A., Purcell, N.H., Blair, N.S., Osinska, H.,
Hambleton M.A., Brunskill, E.W., Sayen, M.R., Gottlieb, R.A.,
Dorn, G.W., Robbins, J., Molkentin, J.D.
Nature, 434:658-62. (2005)



B. The Bcl-2 proteins

If the PTP is not involved in cytochrome c release, what is?
Enter, the family of Bcl-2 proteins. These proteins which share
a conserved a-helical Bcl-2 homology domain (BH3), consist
of pro-apoptotic proteins (e.g. Bax, Bak, Bid and Bim) and
anti-apoptotic proteins (Bcl-2, Bcl-XL and Mcl-1). A delicate
balance occurs between the pro-apoptotic and anti-apoptotic
members of this family. Two small pro-apoptotic proteins,
Bid and Bim, promote two larger pro-apoptotic proteins, Bax
and Bak, to form pores in the mitochondrial outer membrane
under apoptotic conditions but are held in check by anti-apoptotic
proteins, in healthy cells. Unlike in the PTP model, cytochrome c
release through the pores made by oligomers of Bax or Bak
leaves the mitochondrial outer membrane intact. Newmeyer's
group has shown that tBid (a truncated version of Bid) promotes
oligomerization of Bax and membrane permeabilization in intact
mitochondria. It was also shown that the pores formed by Bax
were large enough to release fluorescein dextrans (2000kDa)
which had been pre-loaded into mitochondrial outer membrane
vesicles and protein-free liposomes. This process was dependent
on cardiolipin (a mitochondria-specific lipid) and was inhibited
by the anti-apoptotic protein, Bcl-XL.

BID, BAX, AND LIPIDS COOPERATE TO FORM
SUPRAMOLECULAR OPENINGS IN THE OUTER
MITOCHONDRIAL MEMBRANE.
Kuwana T., Mackey M.R., Perkins G., Ellisman M.H., Latterich M.,
Schneiter R., Green D.R., and Newmeyer D.D.
Cell 111(3):331-42. (2002)

BH3 DOMAINS OF BH3-ONLY PROTEINS DIFFERENTIALLY
REGULATE BAX-MEDIATED MITOCHONDRIAL MEMBRANE
PERMEABILIZATION BOTH DIRECTLY AND INDIRECTLY.
Kuwana T., Bouchier-Hayes L., Chipuk J.E., Bonzon C.,
Sullivan B.A., Green D.R., and Newmeyer D.D.
Mol Cell. 17(4):525-35. (2005)



C. Targeting Bcl-2 proteins in cancer therapy

Increased expression of anti-apoptotic proteins such as Bcl-2
and Bcl-XL and decreased expression of pro-apoptotic proteins
such as Bax and Bak have been found in several cancers. Not
surprisingly, these proteins are the target of cancer therapies.

A recent paper by Oltersdorf et al (2005) describes a compound
(developed from high-throughput NMR screening of a chemical
library and parallel chemical synthesis) which binds to a
hydrophobic groove in the anti-apoptotic proteins Bcl-XL and
Bcl-2 thus inhibiting their anti-apoptotic action. The inhibitor
shrunk tumors in mouse models of small cell lung cancer by
inducing apoptosis as evidenced by increased caspase activity.
Using a different approach, Walensky et al (2004) constructed a
synthetic peptide of 23 amino acids mimicking a region of the a
helical BH3 domain of the pro-apoptotic protein, Bid. In order to
maintain the a helical nature of the peptide and decrease its
susceptibility to proteases in the cell, the researchers used a
technique of hydrocarbon-stapling in which hydrocarbon side
chains attached to non-natural amino acids were "stapled" in
a ruthenium catalyzed reaction. The stabilized a helical peptide
induced cytochrome c release in mouse mitochondria and
inhibited growth of leukemic cell lines.

AN INHIBITOR OF BCL-2 FAMILY PROTEINS INDUCES
REGRESSION OF SOLID TUMORS
Oltersdoft T and 32 other authors
Nature. 435 (7042):677-81. (2005)

An audio broadcast about the development of this inhibitor,
ABT-737, and its effects ("New research helps cancer cells die")
can be heard at:
http://www.visualwebcaster.com/event.asp?id=29324

ACTIVATION OF APOPTOSIS IN VIVO BY A
HYDROCARBON-STAPLED BH3 HELIX.
Walensky L.D., Kung A.L., Escher I., Malia T.J., Barbuto S.,
Wright, R.D., Wagner, G., Verdine, G.L., and Korsmeyer S.J.
Science 305: 1466-1470. (2004)



D.The Apoptosome activates caspases

Cytochrome c, once released into the cytosol, binds to Apoptotic
protease-activating factor 1 (Apaf-1) in the presence of dATP or
ATP to form an apoptosome which activates procaspase 9.
Caspase 9 then triggers activation of caspase 3 and an ensuing
caspase cascade causes destruction of certain proteins.

A recent crystal structure of Apaf-1 containing bound ADP,
resolved at 2.2 shows that Apaf-1, an ATPase, has 3 domains -
a caspase recruitment domain, a nucleotide binding domain,
and a WD40 domain - which are locked in a conformation preventing
binding of procaspase 9 and dATP. According to modeling studies
and cryoelectron microscopy of an apoptosome resolved to 27,
the apoptosome looks like a seven-spoked wheel. It has been
suggested that when cytochrome c binds to the WD40 domain in
Apaf-1, a conformational change occurs leading to binding of dATP
at the nucleotide binding domain. This in turn causes another
conformational change in Apaf-1 resulting in oligomerization of
7 Apaf-1 molecules and 7 (or more) cytochrome c molecules) to
form an apoptosome. The caspase recruitment domain of each
Apaf-1 is now able to bind procaspase 9 molecules and activate
them.

STRUCTURE OF THE APOPTOTIC PROTEASE-ACTIVATING
FACTOR 1 BOUND TO ADP.
Riedl, S.J., Li, W., Chao, Y., Schwarzenbacher R., and Shi Y
Nature 434: 926-933. (2005)

THREE-DIMENSIONAL STRUCTURE OF THE APOPTOSOME:
IMPLICATIONS FOR ASSEMBLY, PROCASPASE 9 BINDING,
AND ACTIVATION.
Acehan D, Jiang X, Morgan DG, Heuser JE, Wang X, Akey CW.
Mol Cell. 9(2):423-32. (2002)



3. AIF and a caspase-independent pathway of apoptosis

Cytochrome c does not seem to be the only mitochondrial
protein which plays a dual role in the life and death of a cell.
The Apoptosis-inducing factor, AIF, is a 57kDa mitochondrial
intermembrane space protein which has been known for
some years to be important in apoptosis. Involved in a caspase-
independent pathway of apoptosis, AIF is released from the
mitochondrial intermembrane space to the cytosol and then
to the nucleus where it binds to DNA and is involved in
chromatin condensation and DNA breakdown.

Unlike the function of cytochrome c in mitochondria, the
physiological importance of AIF in its mitochondrial location is
not yet clear. Crystal structures of AIF show that it has 3
domains: a FAD-binding domain, an NADH-binding domain
and a C-terminal domain. One paper (Miramar et al, 2002)
demonstrates that AIF shows NADH oxidase activity in vitro
and produces free radicals, whereas another paper (Klein
et al, 2002) suggests that AIF is a scavenger of free radicals
in mutant mice having low levels of this protein. Remarkably,
a recent paper (Vahsen et al, 2005) describes loss of Complex I
subunits such as NDUFB6, NDUFS7, NDUFA9 and Grim 19 in
AIF-deficient embryonic stem cells and in the retina and brain
of mice showing reduced expression of AIF. The authors show
that although AIF itself is not part of Complex I, it seems to be
involved in the assembly of this large multisubunit assembly.

THE CRYSTAL STRUCTURE OF THE MOUSE APOPTOSIS-
INDUCING FACTOR AIF.
Mate M.J., Ortiz-Lombardia M., Boitel B., Haouz A., Tello D.,
Susin S.A., Penninger J., Kroemer G., Alzari P.M.
Nat Struct Biol. 9(6):442-6. (2002)

DNA BINDING IS REQUIRED FOR THE APOPTOGENIC
ACTION OF APOPTOTIS INDUCING FACTOR.
Ye, H. Cande, C., Stephanou N.C., Jiang, S., Gurbuxani, S.,
Larochette, N., Daugas, E., Garrido, C., Kroemer G., and Wu, H.
Nat.Struct.Biol.9, 680-684. (2002)

NADH OXIDASE ACTIVITY OF MITOCHONDRIAL
APOPTOSIS-INDUCING FACTOR. Miramar, M.D.,
Costantini, P., Ravagnan, L., Saraiva, L.M., Haouzi, D.,
Brothers, G., Penninger, J.M., Peleato, M.L., Kroemer, G.,
and Susin, S.A.
J.Biol.Chem. 276: 16391-16398. (2001)

THE HARLEQUIN MOUSE MUTATION DOWNREGULATES
APOPTOSIS-INDUCING FACTOR.
Klein, J.A., Longo-Guess, C.M., Rossman, M.P., Seburn,
K.L., Hurd, R.E., Frankel, W.N., Bronson, R.T, and
Ackerman, S.L.
Nature 419: 367-374. (2002)

AIF DEFICIENCY COMPROMISES OXIDATIVE
PHOSPHORYLATION
Vahsen N., Cande C., Briere J..J, Benit P., Joza N.,
Larochette N., Mastroberardino P.G., Pequignot M.O.,
Casares N., Lazar V., Feraud O., Debili N., Wissing S.,
Engelhardt S., Madeo F., Piacentini M., Penninger J.M.,
Schagger H., Rustin P., and Kroemer G.
EMBO J. 23(23):4679-89. (2004)



4. A mitochondrial connection for p53 in apoptosis

p53, a tumour suppressor protein and transcription factor,
has been studied for a decade with much interest since it
plays critical roles in cell cycle arrest and apoptosis.
Mutations of p53 are found in approximately 50% of solid
tumours and the protein has been intensely studied as a
target for cancer therapy. While it has been known for a
while that p53 promotes transcription of genes encoding
pro-apoptotic proteins, it now appears that p53 can also
translocate to mitochondria where it has a transcription-
independent pro-apoptotic effect. Mihara et al (2003) have
shown that p53 moves to the mitochondria under certain
conditions of apoptosis and interacts with the anti-apoptotic
proteins Bcl-2 and Bcl-XL. This results in disruption of the
interactions between the anti-apoptotic proteins and the
pro-apoptotic Bak, leading to oligomerization of Bak and
release of cytochrome c.

Another group (Leu et al, 2004)
has shown that p53 interacts with Bak by disrupting its
interaction with the anti-apoptotic protein, Mcl-1, causing
oligomerization of Bak and release of cytochrome c from
mitochondria. The same group also suggests that a polymorphic
variant of p53 containing Arg72 has a greater propensity than
the other variant, Pro72, to induce apoptosis since it is more
likely to be transported from the nucleus to mitochondria. Douglas
Green's group, using an in vitro system of liposomes, has
demonstrated that p53 promotes Bax oligomerization and
membrane permeabilization of liposomes. While the binding
partners of p53 seem to differ in each of these reports, the
unifying theme seems to be that p53 is able to interact with
members of the Bcl-2 family (either anti-apoptotic proteins
or pro-apoptotic proteins or both types) to promote apoptosis
in damaged cells.

p53 HAS A DIRECT APOPTOGENIC ROLE AT THE
MITOCHONDRIA.
Mihara, M., Ernster, S., Zaika A., Petrenko O., Chittenden T.,
Pancoska P., and Moll U.M
Molecular Cell, 11, 577-590. (2003)

MITOCHONDRIAL p53 ACTIVATES BAK AND CAUSES
DISRUPTION OF A BAK-MCL-1 COMPLEX.
Leu J.I, Dumont P., Hafey M., Murphy M.E., and George D.L.
Nat Cell Biol. 6:443-50. (2004)

THE CODON 72 POLYMORPHIC VARIANTS OF p53
HAVE MARKEDLY DIFFERENT APOPTOTIC POTENTIAL.
Dumont, P., Leu, J.I., Della Pietra AC 3rd, George D.L., and
Murphy M.
Nat Genet. 33(3):357-65. (2003)

DIRECT ACTIVATION OF BAX BY p53 MEDIATES
MITOCHONDRIAL MEMBRANE PERMEABILIZATION AND
APOPTOSIS.
Chipuk J.E., Kuwana T., Bouchier-Hayes L., Droin N.M.,
Newmeyer D.D., Schuler M., Green D.R
Science 303 (5660):1010-4. (2004)



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