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MitoNews
Mitochondrial Research Bulletin
Published by:
MitoSciences
Advancing Vital Discoveries in Mitochondrial Research
Ê http://www.mitosciences.com
Written by:
Dr. Roderick Capaldi
[email protected]
Volume 02, Number 01 - April 19, 2006
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Past Issues of MitoNews can be found at:
http://www.mitosciences.com/mitonews_archives.html
CYTOCHROME c OXIDASE: BOTH CAUSATIVE AND
A BIOMARKER OF DISEASE
In this issue....
1. A brief update on structure.
2. Cytochrome c oxidase is regulated by phosphorylation/
dephosphorylation, as well as nucleotide binding.
3. Regulation of cytochrome c oxidase activity by NO.
4. Cytochrome c oxidase is a target of cellular oxidative stress.
5. Cytochrome c oxidase dysfunction may be a part of and is at
least a biomarker for Alzheimers Disease.
6. Cytochrome c oxidase levels as a marker of cancerous tissue.
7. Cytochrome c oxidase as a biomarker for adverse effects in
HAART therapy for HIV infection.
8. New Product Announcement:
- Introduction of 96 well plates for measuring COX activity
and amount without isolating mitochondria.
- A special offer for MitoNews subscribers.
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1. A Brief Update on Structure
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One of the most exciting times of this reviewer's scientific career
was the day that 3D structure of the mammalian cytochrome c
oxidase (COX) was reported in Science by Yoshikawa and
colleagues. The doctrine at that time was Òthe enzyme complex
is too big for a structure solution by X-ray, and it is a membrane
protein and virtually impossible to crystallizeÓ With this assurance
of a long career in the field, my lab and many others toiled away
to understand the enzyme structure by a combination of bio-
chemical and biophysical approaches. Then one morning there
it was, in color, all 13 subunits!
TSUKIHARA et al. SCIENCE 272. 1136-1144 (1996)
More details have been added since, including exactly what
holds the physiologically relevant dimer together (LEE et al. ACTA
CRYST. D57 941-47, 2001), and the arrangement and function of
phospholipids in the molecule (YOSHIKAWA. S. BIOCHEM SOC
TRANS 33 934-7, 2005).
The issue of whether there are tightly bound phospholipids
with functional importance for cytochrome c oxidase has been
a contentious one. My group (Steve Vik and Neil Robinson)
provided data that there were 2-3 key cardiolipin molecules
associated with the COX dimer as long ago as 1981 (VIK et al
PROC. NATL. ACAD SCI. USA . 78. 1456-60, 1981).
The crystal structure data and a recent careful biochemical
study using arylazidocardiolipin probes by Robinson and
colleagues (SELDACK et al. BIOCHEMISTRY 45. 746 -54,
2006 ) has finally put this issue to rest (hopefully). Thanks largely
to the work of Kadenbach and colleagues, and against some
criticism at the time, it became clear that the mammalian COX
is present in tissue specific forms. In mammals, three subunits
VIa, VIIa and VIII have different isoforms. Recently a third
isoform of subunit VIII was reported (HUTTERMANN et al.
GENE 312. 95-102, 2003)
2. Cytochrome c oxidase is regulated by phosphorylation/
dephosphorylation, as well as nucleotide binding.
-----------------------------------------------------------------
Largely due to the work of Kadenbach and colleagues, it
became clear in the 1990s that COX is subject to allosteric
regulation by nucleotides both from the matrix side and inter-
membrane space-side of the mitochondrial inner membrane.
More recently, evidence has been presented that the enzyme
is subject to phosphorylation/dephosphorylation. (LEE et.al.
J.BIOL.CHEM 280. 6094-6100, 2005 & PRABU et al. J. BIOL.
CHEM 281. 2061-70, 2006) via protein kinase A. Phosphorylation,
which is stimulated by a cAMP-dependent pathway, inactivates
the enzyme by modification of Tyr304 of subunit I. In their study
Prabu et. al. claim hyper-phosphorylation (?) at subunits I, IV,
and Vb in hypoxia and ischemia reperfusion with a loss of activity,
and surprisingly, an increase in ROS production from the enzyme
complex.
3. Regulation of cytochrome c oxidase activity by NO.
-----------------------------------------------------------------
There is a considerable literature on the regulation of COX by
nitric oxide. (for a recent review, see SHIVA et. al. FREE
RADICAL BIOLOGY & MEDICINE 38. 297-306, 2005).
Two recent papers provide new insight into this process.
Persichini et al reported that mitochondrial-type nitric oxide
synthase physically interacts with COX based on co-
immunoprecipitation. (PERSICHINI et. al NEUROSCIENCE
LETTERS 384. 254-259, 2005). Also Poyton and colleagues
have reported the surprising observation that COX can
produce NO under hypoxic/anoxic conditions (CATELLO et.
al. CELL METAB. 3 277-87, 2006) This NO production is
nitrite dependent.
4. Cytochrome c oxidase is a target of cellular oxidative stress.
------------------------------------------------------------------------
Both COX and its tightly bound cardiolipin component are subject
to post-translational modification from oxidative stress, resulting
in enzyme inhibition. The lipid molecules are subject to
peroxidation, generating 4-hydroxynonenal, which reacts with
COX predominantly at subunits VIIc and VIII. (MUSATOV et. al.
BIOCHEMISTRY 41. 8212-20, 2002).
There is modification of Tryp in subunits IV and VIIc (MUSATOV
et al/. BIOCHEMISTRY 43. 1003-9 2004; TAYLOR ET AL. J.BIOL.
CHEM. 278. 19587-90. 2003), nitration and carbonylation (CHOSKI
et al. BIOCHIM. BIOPHYS ACTA. 1688. 95-101. 2004) and
glutathionylation at subunit Vb (LOPEZ et. al. PROTEOMICS 3.
1154-1161. 2003).
The above modifications, as well as hyperphosphorylation,
cause not only inactivation, but also dissociation of subunits
from the complex.
5. Cytochrome c oxidase dysfunction may be a part of and is at
least a biomarker for Alzheimers Disease.
-----------------------------------------------------------------------
The evidence that COX deficiency in the brain cortex correlates
with Alzheimers disease has been reviewed recently. (BEAL. ANN.
NEUROL. 58. 495-505. 2005). Two recent papers place amyloid-
beta 1-42 at or close to cytochrome c oxidase (in mitochondria)
and lead to the proposal that the amyloid protein presence leads
directly or indirectly to COX inhibition. In their paper Crouch et al.
report direct biochemical studies of the interaction of (dimeric)
amyloid-beta with COX in a copper Ðdependent manner (CROUCH
et. al. J. NEUROSCI. 25. 672-79. 2005). Manczak et. al. report
accumulation of amyloid-beta into the Alzheimers disease neurons
in transgenic mice with concomitant inhibition of cytochrome c
oxidase activity (MANCZAK et. al. HUMAN MOLECULAR GENETICS
ADVANCED PUBLICATION 2006)
6. Cytochrome c oxidase levels as a marker of cancerous tissue.
-----------------------------------------------------------------------
Many studies have established that cancerous cells grow
preferably by glycosis and down regulate OXPHOS (e.g.
ROSSIGNOL et. al CANCER RES. 64. 985-93.2004).
More recently it has been shown that there is a more profound
down regulation of mitochondrial transcripts (COX subunits I,II
and III) than nuclear transcripts and several laboratories are
using the ratio-ing of COX I to COX IV by Western blotting and/or
immunohistochemistry to detect cancer boundaries. See KIM
et. al. CLIN CANCER RES 10. 8512-5 2004 for a study of neck
lesions; PAYNE et. al CURRENT EPIDEMIOLOGY BIOMARKERS
& PEVENTION 14. 2066-75. 2005 for a study of colon cancer.
A more general study using several cancer cell lines has been
reported by Kreig et. al. (KREIG et. al. PROTEOMICS 4. 2789-
2795. 2004).
7. Cytochrome c oxidase as a biomarker for adverse effects in
HAART therapy for HIV infection.
------------------------------------------------------------------------
Reverse transcriptases used as antiviral therapy and several
antibiotics such as erythromycin alter mtDNA replication or
mitochondrial protein synthesis, and as a result, alter the levels
of OXPHOS proteins and the ratio of mt-encoded to nuclear
encoded subunits of the complexes. In an exciting new longitudinal
study of HAART therapy Lopez et al. used COX activity as a
monitor of adverse effects and showed that the enzyme
measurement accurately represented the mtDNA loss (but is
easier to conduct; editors note). LOPEZ et.al. AIDS RES.HUM.
RETROVIRUSES 1. 33-39 2006).
8. New Product Announcement:
Introduction of 96 well plates for measuring COX activity
and amount without isolating mitochondria.
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(* Special Offer for MitoNews Subscribers - read below)
As studies focus more on the role of COX in disease and
involve cell based and animal studies as well as patient analysis,
it becomes important to have the tools with which to analyze
the enzyme from very small amounts of material.
This month, MitoSciences will introduce 3 plate-based assay
kits in which COX can be immunocaptured from as little as
10,000 cells and less than a mg of tissue without having to
isolate mitochondria.
In one kit (MS400A) COX activity is measured, while in the
other (MS400Q), the amount of the complex is determined
in a sandwich immunoassay. The two tests can be multiplexed
using MS400MULTI and a specific activity of COX in a sample
obtained.
Moreover, the plates can be used to capture the enzyme,
measure either or both of the above parameters and then
used to determine post-translational modifications where there
is a sensitive chemical reaction (e.g. carbonyls) or good antibodies
(nitration, HNE or phosphorylation) for detection of the specific
modification.
We think these tests will have widespread utility as the review
here indicates.
For further information please visit:
http://www.mitosciences.com/complex_iv_microplates.html
Or email us at:
[email protected]
Or phone:
1-541-284-1802
1-800-910-6486 (toll free)
* As a special offer for MitoNews subscribers, mention that you
are a subcriber and you will receive 10% off your first order of
one of these new kits.
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