As shown above, a comprehensive new theory of cellular ATP production has emerged through the
proposal of Nath’s torsional mechanism of energy transduction and ATP synthesis using original
systems biology/engineering approaches. In addition to providing the detailed molecular mechanism of
ATP synthesis by F1FO-ATP synthase, the torsional mechanism has explicitly emphasized and
quantitatively analyzed the key role of the transport steps through the adenine nucleotide translocase
(ANT), the Pi/OH- antiporter and FO preceding ATP synthesis by F1, and has specified their exact
sequential order as a law [1, 2, 11, 13]. The mechanism visualized the nature of these events as
localized, coordinated and ordered in time and space by the (local) electrical potentials induced by
these transport steps in their real molecular setting in the inner mitochondrial membrane. Recently, the isolation and characterization of the mitochondrial ATP synthasome as a 1:1:1 supercomplex of ANT,
Pi/OH- and F1FO located near one another in the membrane has been accomplished [48]. This finding
of co-localization contradicts the classical view that regards these components as separate entities in
the energy-transducing membrane and among all candidate mechanisms only the torsional mechanism
is in accord with these important new data on membrane association of these entities to form the ATP
synthasome supercomplex.
We now ask the question why apoptotic cell death by the intrinsic pathway is initiated in the
mitochondria of cells [81-87]. This has generally been considered as a paradox, i.e. why organelles
that are central to cell life are also involved in apoptosis and cell death. Either apoptosis is an ATP
requiring process (but then this must be in the cytoplasm, e.g. in the apoptosome), or there is a need to
block/inhibit and consequently shut down the energy transduction processes in the mitochondrion and
this is a central and primary process in the intrinsic apoptotic pathway [81-84] or in the action of viral
accessory proteins such as Vpr (Viral protein R) from Human Immunodeficiency Virus HIV-1 [85] or
PB1-F2 from Influenza A Virus IAV [86, 87]. Otherwise there is no logical reason why the
mitochondrion should be involved. Currently there is a substantial body of experimental evidence that
implicates a direct and specific interaction of pro-apoptotic viral peptides such as Vpr from HIV-1 and
PB1F2 from IAV with the ANT in mitochondria [81-87]. We propose here that pro-apoptotic agents
achieve their effect by inhibiting mitochondrial ATP production via the ATP synthasome. The modes
of inhibition can be further divided into classes of apoptotic agents/viral proteins that inhibit ANT,
Pi/OH-, F1FO or form pores in the membrane. Since according to the torsional mechanism, the
availability of ADP is the rate-limiting step of ATP synthesis, it is further proposed that inhibition via
ANT, either by blocking exit of ATP from it or entry of ADP through it, i.e. by blocking nucleotide
exchange through ANT is the most economical way of achieving the postulated reduction in
mitochondrial ATP generation, and thereby reduction of cellular ATP levels, leading to an overall
bioenergetic deficit in the entire cell and apoptosis through subsequent downstream events. It is also
proposed here that intrinsically/in vivo, Vpr and PB1-F2 have an anti-apoptotic function and act by
sequestering the amphipatic α-helix of BH3 regions of pro-apoptotic protein members of the Bcl-2
family such as Bax and/or Bak. However, in the absence of these pro-apoptotic members, Vpr and
PB1-F2 lose this anti-apoptotic function, especially at the relatively higher concentrations in various
experiments [85-87], and act as mimics of the pro-apoptotic members of the Bcl-2 family and
themselves cause apoptosis. The oligomerization properties of these viral peptides may be vital for
this function.
But the question arises why Vpr from HIV-1 and PB1-F2 from IAV should promote apoptosis in
the in vivo situation? In a stressed condition, i.e. the pro-apoptotic state, the Bax/Bak proteins are
activated through the absence or inability of the anti-apoptotic Bcl-2/Bcl-xL family of proteins to
maintain the pro-survival state of the cell. Therefore there is no natural anti-apoptotic agent available
that can antagonize and stop the natural apoptotic response, which leads to cell death. However, the
virus requires the host machinery to continue functioning, thereby allowing further viral production.
As a consequence, we surmise that in the above context, the viral proteins function in an anti-apoptotic
capacity by interaction and binding with Bax through neutralization of its BH3 domain, by analogy
with the known interactions of the anti-apoptotic Bcl-2 family of proteins with the BH3 domains of
Bax/Bak-like proteins. Thus, the Bcl-2 or its homolog Bcl-xL family of proteins achieves its anti-apoptotic effect by maintaining Bax in an inactivated state and preventing Bax from exerting its
apoptotic role.
What then could be the exact mechanism by which Bax induces apoptosis under conditions when it
is freed from its direct physical interaction with Bcl-2/Bcl-xL, which was responsible for its
inactivation, as discussed above? In our systems model of apoptosis, we propose a subtle and specific
direct interaction of Bax with ANT, which is responsible for inhibiting the enzymatic function of
ADP3-/ATP4- exchange of the transporter, and is in agreement with a previous proposal [83]. In our
view, this action of Bax on ANT is an essential primary event that is sufficient to cause and propagate
the later events in apoptosis.
Why is it necessary for the natural apoptotic program or for various pro-apoptotic agents to inhibit
ATP production and what consequences does it have for apoptosis by the intrinsic pathway? To the
best of our knowledge, there is no experimental evidence in the literature that either the natural
apoptotic program or pro-apoptotic agents directly target the electron transport or respiratory chain in
mitochondria. In our view, the only natural way (i.e. without the experimentalist’s intervention) to
inhibit the redox side, especially since electrons can readily tunnel through quantum mechanically, is
to inhibit the process to which it is coupled i.e., ATP synthesis and especially ion translocation (H+ and
A-) (Section 5). If this flow of ions is inhibited then the redox activity (which would have actively
transported these ions) is consequently inhibited too. Once the electron transport chain is itself
inhibited, we can expect the water-soluble cytochrome c to accumulate in the intermembrane space. If
the outer mitochondrial membrane is ruptured or if access channels in the outer membrane are open,
then cytochrome c can be readily released along its concentration gradient from the intermembrane
space to the cytoplasm, which then is the signal for initiating further downstream apoptotic
events/cascades such as caspase activation and the various processes in the apoptosome.
Finally, on the basis of the torsional mechanism and the unified theory, diverse experimental data in
the apoptosis/necrosis field can be naturally explained, including the role of oligomycin as a powerful
inhibitor of apoptosis, the lack of any significant effect of azide on apoptosis, the effects of
atractyloside, the local hyperpolarization/decrease of local depolarization in the vicinity of the the ATP
synthasome induced by certain ester anions and other agents etc. If the above hypothesis of the
involvement and role of ATP inhibition in apoptotic cell death via the intrinsic mitochondrial pathway
is correct, then the occurrence of fundamental cell life as well as cell death processes in the same
organelle is not “paradoxical” (as often stated), but is in fact a logical requirement for the desired
function in both cases. Cell life and cell death can then be viewed as two sides of the same coin and
studies of one side can synergize, impose further constraints, and truly help understand the other side,
with manifold applications for both health and disease. Furthermore, failure of the apoptosis program
will lead to irreversible disease, including cancer, via altered (e.g. glycolytic) pathways, and
mechanisms for the resistance and lowered apoptotic potential of tumor cells, and reversal of the ATP
synthase that is known to occur in such diseases and in necrosis can then also be further developed and
conceptually understood based on the detailed ATP hydrolysis mechanisms that have already been
postulated in the new framework of the unified theory and shown to be a microscopic reversal of the
torsional mechanism of ATP synthesis. The cellular mitochondrial vs. glycolytic potentials may then
provide a bioenergetic index for a logical assessment of carcinogenesis and the progression of
carcinoma. In summary, a systems-level biological understanding of the torsional mechanism of energy transduction and ATP synthesis and the unified theory in cell life suggests a bioenergetic basis
for mitochondrial apoptotic cell death and disease, and a large number of applications of our
fundamental work in cell life are possible in cell death and disease also.
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