Necrosis

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• 2002-12-17

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• Necrosis
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• Programmed Cell Death
• Apoptosis

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Fig.1. Schematic summary of biochemical
mechanisms of apoptosis.
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Mitochondria and Commitment to Cell Death

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Mitochondria and Commitment to Cell Death

• the effectors of apoptosis are represented by a
  family of intracellular cysteine proteases known
  as caspases.
• Inhibiting caspases, however, does not always
  inhibit cell death induced by proapoptotic
  stimuli. Although caspase inhibitors block some
  or all of the apoptotic morphology induced by
  growth factor withdrawal, etoposide, actinomycin
  D, ultraviolet (UV) radiation, staurosporine,
  enforced c-Myc expression, or glucocorticoids,
  they do not necessarily maintain replicative or
  clonogenic potential ultimately, the cells die
  despite inactivation of caspases by way of a
  slower, nonapoptotic cell death (6-9).

• In contrast, antiapoptotic proteins such as
  Bcl-2, Bcl-xL, and oncogenic Abl can maintain
  survival and clonogenicity in the face of these
  treatments. Conversely, some proapoptotic
  proteins such as Bax, a mammalian cell death
  protein that targets mitochondrial membranes, can
  induce mitochondrial damage and cell death even
  when caspases are inactivated (10). Such
  experimental observations argue that a
  caspase-independent mechanism for commitment to
  death exists. This mechanism is likely to involve
  mitochondria, as we will see.

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Mitochondrial Pathways in physiological cell
death

• the release of caspase activators (such as
  cytochrome c),
• changes in electron transport,
• loss of mitochondrial transmembrane potential,
• altered cellular oxidation-reduction,
• participation of pro- and antiapoptotic Bcl-2
  family proteins.

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Mitochondrial Pathways in physiological cell
death

• If mitochondria are pivotal in controlling cell
  life and death, then how do these organelles
  kill? At least three general mechanisms are
  known, and their effects may be interrelated,
  including

• (i) disruption of electron transport,
  oxidative phosphorylation, and adenosine
  triphosphate (ATP) production
• (ii) release of proteins that trigger
  activation of caspase family proteases and
• (iii) alteration of cellular reduction-oxidation
  (redox) potential

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Disruption of electron transport and energy
metabolism

• disruption of electron transport has been
  recognized as an early feature of cell death.
• ?-Irradiation induces apoptosis in thymocytes
  and a disruption in the electron transport chain,
  probably at the cytochrome b-c1/cytochrome c
  (cyto c) step.
• Ceramide (a "second messenger" implicated in
  apoptosis signaling) disrupts electron transport
  at the same step in cells as well as in isolated
  mitochondria.
• Ligation of Fas also leads to a disruption in
  cyto c function in electron transport.

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Disruption of electron transport and energy
metabolism

• One consequence of the loss of electron transport
  should be a drop in ATP production. Although such
  a drop has been observed during apoptosis, it
  often occurs relatively late in the process (14).
  
• Indeed, ATP appears to be required for downstream
  events in apoptosis (15).
• Thus, although loss of mitochondrial ATP
  production can kill a cell, it is unlikely that
  this is a mechanism for induction of apoptosis.

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Disruption of electron transport and energy
metabolism

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Release of caspase-activating proteins

• The importance of mitochondria in apoptosis was
  suggested by studies with a cell-free system in
  which spontaneous,
• Bcl-2-inhibitable nuclear condensation and DNA
  fragmentation were found to be dependent on the
  presence of mitochondria (16).
• Subsequently, studies in another cell-free system
  showed that induction of caspase activation by
  addition of deoxyadenosine triphosphate depended
  on the presence of cyto c released from
  mitochondria during extract preparation (17).
  During apoptosis (in vitro and in vivo) cyto c is
  released from mitochondria and this is inhibited
  by the presence of Bcl-2 on these organelles (18,
  19).
• Cytosolic cyto c forms an essential part of the
  vertebrate "apoptosome," which is composed of
  cyto c, Apaf-1, and procaspase-9 (20). The result
  is activation of caspase-9, which then processes
  and activates other caspases to orchestrate the
  biochemical execution of cells.

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Release of caspase-activating proteins

• Significantly, caspase inhibitors do not prevent
  cyto c release induced by several apoptogenic
  agents, including UV irradiation, staurosporine,
  and overexpression of Bax (14, 21, 22). An
  exception is cyto c release from mitochondria
  induced by the tumor necrosis factor receptor
  family member Fas, in which cyto c release is
  prevented by inhibition of caspases (primarily
  caspase-8) recruited to the cytosolic domain of
  ligated Fas (21). Nevertheless, cyto c release
  can sometimes contribute to Fas-mediated
  apoptosis by amplifying the effects of caspase-8
  on activation of downstream caspases (23).
• The emergent view is that once cyto c is
  released, this commits the cell to die by either
  a rapid apoptotic mechanism involving
  Apaf-1-mediated caspase activation or a slower
  necrotic process due to collapse of electron
  transport, which occurs when cyto c is depleted
  from mitochondria, resulting in a variety of
  deleterious sequelae including generation of
  oxygen free radicals and decreased production of
  ATP

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Reactive oxygen species and cellular redox.

• Mitochondria are the major source of superoxide
  anion production in cells.
• During transfer of electrons to molecular oxygen,
  an estimated 1 to 5 of electrons in the
  respiratory chain lose their way, most
  participating in formation of O2. Anything that
  decreases the coupling efficiency of electron
  chain transport can therefore increase production
  of superoxides.

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Reactive oxygen species and cellular redox.

• Superoxides and lipid peroxidation are increased
  during apoptosis induced by myriad stimuli (28).
• However, generation of ROS may be a relatively
  late event, occurring after cells have embarked
  on a process of caspase activation.
• In this regard, attempts to study apoptosis under
  conditions of anoxia have demonstrated that at
  least some proapoptotic stimuli function in the
  absence or near absence of oxygen, which implies
  that ROSs are not the sine qua non of apoptosis
  (29, 30).
• However, ROSs can be generated under conditions
  of virtual anaerobiosis (31), and thus their role
  in apoptosis cannot be excluded solely on this
  basis.

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Figure 2. Model for caspase activation by
mitochondria.
the other envisions opening of channels in the
outer membrane thus releasing cyto c from the
intermembrane space of mitochondria into the
cytosol.
osmotic disequilibrium leading to an expansion of
the matrix space, organellar swelling, and
subsequent rupture of the outer membrane
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PT Pore

• In many apoptosis scenarios, the mitochondrial
  inner transmembrane potential (m) collapses (32),
  indicating the opening of a large conductance
  channel known as the mitochondrial PT pore (33)
  (Fig. 2).
• its constituents include both inner membrane
  proteins, such as the adenine nucleotide
  translocator (ANT), and outer membrane proteins,
  such as porin (voltage-dependent anion channel
  VDAC), which operate in concert, presumably at
  inner and outer membrane contact sites, and
  create a channel through which molecules 1.5 kD
  pass (32, 34).

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PT Pore

• Opening of this nonselective channel in the inner
  membrane allows for an equilibration of ions
  within the matrix and intermembrane space of
  mitochondria, thus dissipating the H gradient
  across the inner membrane and uncoupling the
  respiratory chain.
• Perhaps more importantly, PT pore opening results
  in a volume dysregulation of mitochondria due to
  the hyperosmolality of the matrix, which causes
  the matrix space to expand.
• Because the inner membrane with its folded
  cristae possesses a larger surface area than the
  outer membrane, this matrix volume expansion can
  eventually cause outer membrane rupture,
  releasing caspase-activating proteins located
  within the intermembrane space into the cytosol
  (Fig. 1).

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Fig. 3. The mitochondrial permeability transition.
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PT Pore ???

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PT Pore ??????????

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  ???????(N-benzyloxycarbonyl-Val-Ala-Asp-fluorometh
  ylketone)????
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  acid)???PT??????
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Figure 2. The mitochondrial permeability
transition.

• A speculative model showing some of the
  components of the permeability transition pore.
  The roles of porin and the benzodiazepine
  receptor remain circumstantial. In the open
  configuration, water and solutes enter the
  matrix, causing matrix swelling and outer
  membrane disruption (see Fig. 1), leading to
  release of cyto c and other proteins.

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Fig. 4 The cytochrome c-induced caspase
activation pathway.
Oligomerization
Recruit
Cleave
Activiate
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Figure 1.   The cytochrome c-induced caspase
activation pathway.

• Apoptotic stimuli exert their effects on
  mitochondria to cause the release of cytochrome
  c. Cytochrome c in turn binds to Apaf-1, a
  cytosolic protein that normally exists as an
  inactive monomer. The binding of cytochrome c
  induces a conformational change in Apaf-1,
  allowing it to bind the nucleotide dATP or ATP.
  The nucleotide binding to the Apaf-1-cytochrome c
  complex triggers its oligomerization to form the
  apoptosome, which recruits procaspase-9. The
  binding of procaspase-9 to the apoptosome forms
  the caspase-9 holoenzyme that cleaves and
  activates the downstream caspases, such as
  caspase-3.

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Fig. 5. Displacement of IAPs from caspases by
Smac/Diablo.
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Figure 2.   Displacement of IAPs from caspases by
Smac/Diablo.

• The precursor of Smac/Diablo is synthesized in
  the cytosol and transported to the mitochondria.
  After mitochondrial entry, the mitochondrial
  targeting sequence of Smac (dark purple
  rectangle) is cleaved, exposing the four amino
  acid residues Ala-Val-Pro-Ile through which Smac
  binds to the BIR domain of IAPs. The mature Smac
  (aqua rectangle) is normally located in the
  mitochondrial intermembrane space. During
  apoptosis, cytochrome c (light purple circles)
  and Smac are released from the mitochondria.
  Cytochrome c triggers the activation of caspase-9
  (green rectangles) and caspase-3 (red
  rectangles). The IAP molecules bind and inhibit
  active caspase-9 and caspase-3 via their BIR
  domains (yellow boxes). The IAP inhibition is
  relieved by Smac that competitively binds to the
  BIR domains of IAP molecules and excludes them
  from binding caspases.

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Fig. 6. Multiple apoptotic pathways emanate from
the mitochondria.
chromatin condensation and fragmentation
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Figure 3.   Multiple apoptotic pathways emanate
from the mitochondria.

• Apoptotic stimuli are transduced to mitochondria
  by the BH3-only proteins and possibly by
  additional pathways. The signal from the BH3-only
  protein can be either neutralized by the
  antiapoptotic protein, such as Bcl-2 or Bcl-xL,
  or further transduced to mitochondria by the
  proapoptotic protein such as Bax or Bak. The
  mitochondrial damage caused by apoptotic stimuli
  triggers the release of apoptogenic proteins
  including cytochrome c, Smac, AIF, and EndoG.
  Cytochrome c triggers caspase activation through
  Apaf-1, and Smac relieves IAP inhibition of
  caspases. AIF and EndoG cause chromatin
  condensation and fragmentation in a
  caspase-independent manner. The mitochondrial
  damage may also passively lead to cell death due
  to loss of mitochondrial function.

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Thanks!

• 2002-12-17