Brand, Martin D.

Gerencser, Akos A.

Goncalves, Renata L.S.

Hey-Mogensen, Martin

Orr, Adam L.

Perevoshchikova, Irina V.

Quinlan, Casey L.

Treberg, Jason R.

Elsevier - Publisher Connector 

S6 – ROS and Aging
S6.L1
Characterization and suppression of mitochondrial
production of superoxide and hydrogen peroxide
under near-physiological conditions
Martin D. Brand, Casey L. Quinlan, Renata L.S. Goncalves,
Irina V. Perevoshchikova, Jason R. Treberg, Martin Hey-Mogensen,
Akos A. Gerencser, Adam L. Orr
Buck Institute for Research on Aging, USA
E-mail: mbrand@buckinstitute.org
Superoxide and H2O2 are important in redox signaling, disease and
aging. However, there are ten sites associated with the mitochondrial
respiratory chain that are known to produce these species at mea-
surable rates, making it hard to assess the contribution of particular
sites in cell physiology or pathology. We have deﬁned the maximum
capacities and many kinetic aspects of each site in skeletal muscle
mitochondria. In order of capacity they are sites IIIQo in complex III; IQ
and IIF in complexes I and II; OF, PF and BF in the 2-oxoglutarate,
pyruvate and branched chain 2-oxoacid dehydrogenase complexes; GQ
in mitochondrial glycerol phosphate dehydrogenase (mGPDH); IF in
complex I; EF in ETF/ETF:Q oxidoreductase and DQ in dihydroorotate
dehydrogenase. By screening libraries of small molecules against these
sites, we identiﬁed a novel suppressor of superoxide/H O production
at site IQ that does not affect oxidative phosphorylation (CN-POBS), and
a novel inhibitor of mGPDH (iGP-1). The rate of superoxide/H2O2
production at any site depends on its redox state, which can be
measured for sites IIIQo and IF. Therefore, we can assess the rates from
these sites in complex substrate mixes in different conditions. Using
speciﬁc inhibitors with correction for changes at sites IIIQo and IF,
and by measuring the suppression of signal by CN-POBS, the rates from
all signiﬁcant sites can be estimated. In this way we analyzed the rates
of superoxide/H2O2 production from different sites in mitochondria
oxidizing conventional substrates. The overall rates differed tenfold
and, importantly, the contribution of each site varied with substrate;
e.g. with succinate, most of the signal was from site IQ, with a little from
IF and IIIQo, whereas with glutamate +malate, it was shared between
sites IF, IIIQo and OF. In a complex mix of substrates mimicking resting
muscle cytosol, the major contributors were sites IQ and IIF, with
smaller contributions from IF, IIIQo and maybe EF and GQ. In a medium
mimicking contracting muscle at about 30% VO2max the overall rate
was ﬁvefold lower and site IF was dominant, with smaller contributions
from IQ, IIF, IIIQo and maybe EF, PF and GQ. These results highlight the
sites that may operate in intact muscle at rest and during exercise. This
new suite of approaches revolutionizes our ability to assess and control
production of superoxide and H2O2 from speciﬁc mitochondrial sites
that are important in physiology and pathology.
doi:10.1016/j.bbabio.2014.05.224
S6.L2
Mitochondria signal the oncogenic phenotype
Jose M. Cuezva
Centro de Biología Molecular, Universidad Autónoma de Madrid, Spain
E-mail: jmcuezva@cbm.csic.es
Cancer is a complex disease in which abnormal cells divide without
control and are able to invade other tissues. However, a most relevant
phenotypic trait of cancer cells is the reprogramming of cellular
metabolism to an enhanced aerobic glycolysis to satisfy the demand
of carbon skeletons and energy that is required to sustain proliferation.
Mitochondria are essential organelles in promoting rewiring of metab-
olism during proliferation. An additional aspect in which mitochondria
are also critical organelles is in deciding and executing cell death, a
trait that is also altered in cancer cells. Cell death is molecularly and
functionally integrated with the bioenergetic function of the organelle.
A critical hub in the reprogramming of energy metabolism and cell
fate is the mitochondrial H+-ATP synthase, the engine that synthesizes
most of the ATP that sustains cellular activity of the normal dif-
ferentiated cell and that also forms part of the molecular machinery
required to execute apoptosis. Two main mechanisms are used by
human carcinomas of the breast, colon and lung to silence the H+-ATP
synthase. One is the translation masking of the mRNA that encodes
the catalytic subunit of the H+-ATP synthase (β-F1-ATPase) which is
mediated by RNA binding proteins that upon binding onto the mRNA
impede its efﬁcient translation. A secondmechanism involves the over-
expression of the ATPase Inhibitory Factor 1 (IF1), a short-lived protein
that by inhibiting the H+-ATP synthase not only contributes to
metabolic rewiring but also retrograde signals to the nucleus of the
cancer cell features of the oncogenic phenotype. In this presentation
we will summarize these ﬁndings to further emphasize the tumor
suppressor role of mitochondrial oxidative phosphorylation, a promis-
ing new therapeutic target of cancer.
doi:10.1016/j.bbabio.2014.05.225
S6.L3
Allospeciﬁc expression of AOX in mice: Insights into pathology
and therapeutic potential
Howard T. Jacobs
BioMediTech, FI-33014 University of Tampere, Finland
E-mail: howard.t.jacobs@uta.ﬁ
In organisms where they are found, the alternative respiratory
chain enzymes provide a potential buffer of oxidative stress and
Biochimica et Biophysica Acta 1837 (2014) e57–e66
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
j ourna l homepage: www.e lsev ie r.com/ locate /bbab io


Characterization and suppression of mitochondrial production of superoxide and hydrogen peroxide under near-physiological conditions 

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Characterization and suppression of mitochondrial production of superoxide and hydrogen peroxide under near-physiological conditions

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