Currently, research in my lab is focused on 4
main areas. The following diagram depicts how these various
projects and directions are organized and relate to each other:
1) Mitochondrial free radical interactions: Mitochondria are
both a source of free radicals (e.g. superoxide, O2·-),
and a target for them (e.g. inhibition of respiration by nitric
oxide, NO·). Collectively, these molecules are known
as reactive oxygen and nitrogen species (RONS). The complex
interplay between RONS and mitochondria is believed to play
a key role in the pathogenesis of many cardiovascular and neurodegenerative
diseases. However, the molecular events at the level of the
oganelle are very poorly understood. We are investigating such
interactions using spectrophotometry, polarographic electrodes
to measure NO and oxygen, and other basic biochemical techniques,
both in isolated mitochondria, and cardiomyocytes. In addition,
we are using proteomics-based techniques (see below) to investigate
post-translational modifications of mitocondrial proteins that
are mediated by reactive oxygen and nitrogen species. We are
also interested in complex cell-signaling events mediated by
the mitochondrion.
2) Cardiac ischemia-reperfusion injury: In cardiac ischemia
and reperfusion (I-R), mitochondrial damage is thought to play
an important role. Ca2+ overload, and permeability transition
/ cytochrome c release are key events. We are currently investigating
strategies to minimize mitochondrial damage during I-R, including
antioxidants and K+ATP channel reagents. In particular, we are
interested in the inhibition of mitochondrial complex I that
occurs following I-R. The mechanism for this inhibition is currently
unknown, and the complexity of complex I (~900kDa, >45 subunits,
>12 prosthetic electron transfer groups) makes the application
of proteomic techniques (see below) necessary. We are also interested
in therapies for I-R injury, and the famous "French Paradox"
- i.e. the ability of red wines to offer cardioprotection. To
this end, we recently showed that the red wine polyphenol quercetin
is able to protect against mitochondrial dysfunction in a model
of cardiac I-R.
3) Mitochondrial proton leak: Mitochondrial use the chemical
energy released from oxidation of substrates, to generate a
trans-membrane proton gradient. This gradient (like the potential
difference in a battery) is then used to generate ATP. However
the membrane is not a perfect seal and some protons can leak
across, short-circuiting the system (termed "uncoupling").
This inefficiency has a potential role in the regulation of
energy metabolism, with implications for obesity and weight
loss/gain, as well as in thermoregulation. In addition, mitochondrial
uncoupling can potentially regulate the generation of reactive
oxygen species (ROS) by the organelle (see above). Overall we
are interested in the molecular mechanisms of H+ leak, including
the recently defined "uncoupling proteins" (UCPs).
In addition, mitochondrial H+ leak is increased in cardiac I-R
injury, and while this might initially be thought injurious
(by affecting ability to generate ATP), it may also offer protection,
by decreasing ROS levels. The mechanisms of elevated H+ leak
in cardiac I-R are currently under investigation.
4) Mitochondrial Proteomics: The mitochondrion is an ideal sub-proteome,
since it contains about 2000 proteins, is readily isolated from
the rest of the cell, and is involved in numerous pathologies
(see above). However, proteomic analysis of mitochondria by
2D gels has proven difficult, since so many of their proteins
are hydrophobic membrane proteins that precipitate at the basic
pole during iso-electroc focusing. To circumvent this, we have
adopted the method of 2D Blue-Native electrophoresis (BN-PAGE),
which separates the respiratory complexes and other proteins
in their intact, multi-subunit native form. We have greatly
streamlined these techniques for high throughput, and used them
to examine changes in mitochondrial proteins under a variety
of pathological conditions including cardiac I-R. We are also
using these techniques to examine RONS-mediated protein modifications.
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Davies JE, Digerness SB, Goldberg SP, Killingsworth CR, Katholi CR, Brookes PS, Holman WL. (2003) Intra-myocyte ion homeostasis during ischemia-reperfusion injury: effects of pharmacologic preconditioning and controlled reperfusion. Ann. Thorac. Surg. 76:1252-1258
Digerness SB, Brookes PS, Goldberg SP, Katholi CR, Holman WL (2003) Modulation of mitochondrial adenosine triphosphate-sensitive potassium channels and sodium-hydrogen exchange provide additive protection from severe ischemia-reperfusion injury. J. Thorac. Cardiovasc. Surg. 125:863-871.
Brookes PS (2004) Mitochondrial H+ Leak and ROS Generation: An Odd Couple. Free Rad. Biol. Med. 38:12-23.
Brookes PS (2004) Mitochondrial nitric oxide synthase. Mitochondrion 3:187-204.
Brookes PS, Darley-Usmar VM (2004) Role of calcium and superoxide dismutase in sensitizing mitochondria to peroxynitrite-induced permeability transition. Am. J. Physiol. 286:H39-H46.
Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS (2004) Ca2+, ATP & ROS - a mitochondrial love/hate triangle. Am. J. Physiol. 287:817-833.
Shiva S, Crawford JH, Ramachandran A, Ceaser EK, Hillson T, Brookes PS, Patel R, Darley-Usmar VM (2004) Mechanisms of the interaction of nitroxyl with mitochondria. Biochem. J. 379:357-366.
Shiva S, Moellering D, Ramachandran A, Levonen AL, Landar A, Venkatraman A, Ceaser E, Ulasova E, Crawford JH, Brookes PS, Patel RP, Darley-Usmar VM. (2004) Redox signalling: from nitric oxide to oxidized lipids. Biochem. Soc. Symp. 71:107-120.
Brand MD, Pakay JL, Ocloo A, Kokoszka J, Wallace DC, Brookes PS, Cornwall EJ. (2005) The basal proton conductance of mitochondria depends on adenine nucleotide translocase content. Biochem J. 392: 353-362.
Itoh S, Lemay S, Osawa M, Che W, Berdirian A, Tompkins A, Brookes PS, Yan C, Abe J-I. (2005) Mitochondrial Dok-4 recruits Src kinase and regulates mitochondria-derived reactive oxygen species (ROS) and subsequent NF-kB activation in endothelial cells. J. Biol. Chem. 280:26383-26396.
Tompkins AJ, Burwell LS, Digerness SB, Holman WL, Brookes PS (2005) Mitochondrial dysfunction in cardiac ischemia-reperfusion injury: ROS from complex I, without inhibition. Biochim. Biophys. Acta. 1762: 223-231.
Burwell LS, Nadtochiy SM, Tompkins AJ, Young S, Brookes PS (2006) Direct evidence for S-nitrosation of mitochondrial complex I. Biochem. J. (in press).
Nadtochiy SN, Tompkins AJ, Brookes PS (2006) Different mechanisms of mitochondrial proton leak in ischemia-reperfusion injury and preconditioning: implications for cardioprotection and pathology. Biochem. J. (in press).
PubMed Publication List
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