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Necrosis

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Ligation of Fas also leads to a disruption in cyto c function in electron transport. ... (primarily caspase-8) recruited to the cytosolic domain of ligated Fas (21) ... – PowerPoint PPT presentation

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Title: Necrosis


1
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  • ??? ?? ???
  • 2002-12-17

2
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  • ???????

3
????
  • ?????
  • Necrosis
  • ??????????
  • Programmed Cell Death
  • Apoptosis

4
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  • ??????,???????????????????????,???????????,??????.
    ??????.
  • ????
  • ?????, ??????DNA?????,???????DNA??,?180-200DP
    ?????????DNA??.

5
Fig.1. Schematic summary of biochemical
mechanisms of apoptosis.
6
Mitochondria and Commitment to Cell Death
  • ??????????????,???????ATP??????
  • ???????????????????????NADH?NADH?????????????
  • ????,?????????????????/??????
  • ???????ATP???????????????ATP?
  • ???ATP???????ADP/ATP???????ADP???????,????????????

7
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.

8
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.

9
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

10
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.

11
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.

12
Disruption of electron transport and energy
metabolism
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  • ????????????????????????????,???????????????????
    ??????????,????????????????????????????????,??????
    ???????????,?????mClCCP???????????????????????????
    ?????

13
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.

14
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

15
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.

16
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.

17
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
18
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).

19
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).

20
Fig. 3. The mitochondrial permeability transition.
21
PT Pore ???
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  • ?????????????????????,?????????
  • PT??????????????????????????????
  • PT??????????????(??????ATP?ADP?NAD???m?pH??????),?
    ?????????????????????
  • PT???????ADP-ATP????????????,??ADP-ATP????????????
    ??????,????????????????,PT??????????????????
  • PT?????????????
  • PT????m??,???? mClCCP???m??????PT???PT???????m
    ??,????????????PT?????PT?????, ???????????????????
    ,???????m???,?????????????????????????????????

22
PT Pore ??????????
  • PT???????????
  • ?PT??????????????????(AIF)?
  • AIF??????????,?????????,??????????N-????-???-???-?
    ???????(N-benzyloxycarbonyl-Val-Ala-Asp-fluorometh
    ylketone)????
  • ?????PT?????
  • ??????ADP-ATP?????????
  • ?????????????C?????????????
  • ?PT????????????
  • ?PT???????A(cyclosporin A)?SH,?????(bongkrek
    acid)???PT??????
  • ??????ADP-ATP????????????

23
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.

24
Fig. 4 The cytochrome c-induced caspase
activation pathway.
Oligomerization
Recruit
Cleave
Activiate
25
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.

26
Fig. 5. Displacement of IAPs from caspases by
Smac/Diablo.
27
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.

28
Fig. 6. Multiple apoptotic pathways emanate from
the mitochondria.
chromatin condensation and fragmentation
29
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.

30
Thanks!
  • 2002-12-17
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