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MitoNews
Mitochondrial Research Bulletin
Published by:
MitoSciences
Advancing Vital Discoveries in Mitochondrial Research
http://www.mitosciences.com
Edited by:
Dr. Roderick Capaldi
[email protected]
Volume 02, Number 02 - August, 2006
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Past Issues of MitoNews can be found at:
http://www.mitosciences.com/mitonews_archives.html
In this Issue:
Pyruvate Dehydrogenase: Big, Complicated, and Fascinating
1. Overview
2. Reviews of Recent Research:
-- Anoxia affects PDH activity through HIF1 mediated
expression changes.
-- Hypoxia, PDH regulation and cancer.
-- The PDH complex is oxidatively damaged during reperfusion after
cerebral ischemia.
-- Novel genetic mutations causing PDH deficiency
continue to appear.
-- Studies of dichloroacetate, a potential treatment for
PDH deficiency.
-- A New Approach to Assaying PDH Activity: No more radioacitivity,
no more isolating mitochondria - Microplate Assay
Kits from MitoSciences (advertisement)
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1. Overview
If you believe that big is better, that there is beauty in complexity,
and you want to study a protein at the center of many important
human diseases, then you should consider working on pyruvate
dehydrogenase.
PDH is a large complex of close to 9 million MW composed of
three different enzymes: pyruvate dehydrogenase (E1alpha
and E1beta), dihydrolipoamide transacetylase (E2), and
dihydrolipoamide dehydrogenase (E3). Also present in the
complex is a fifth different polypeptide, the E3 binding protein
(E3bp). These different proteins are arranged in an elegant
structure (reviewed in MILNE et al. J. BIOL CHEM 281. 4364
(2006)), with sixty copies of E2 forming a core, and with the
other proteins organized regularly around its periphery.
Functioning involves sensuous movement of arms by which
intermediates of one enzyme are passed physically to become
substrate for the next in a reaction sequence that is a prototype
substrate channeling mechanism.
PDH catalyzes the physiologically irreversible oxidative
decarboxylation of pyruvate to acetyl CoA. Thus it links and
regulates the flow of energy in cells by determining when pyruvate
should be used for oxidative phosphorylation versus "neutralized"
to lactic acid to allow continued glycolysis. At the same time the
control of acetyl CoA directly influences fatty acid oxidation and
production of ketone bodies.
PDH shows a beautifully complex and comprehensive regulation
(reviewed in PATEL.M.S & KOROTCHKINA.LG BIOCHEM. SOC
TRANS 34. 217. 2006), which includes substrate activation and
product inhibition. Also it is inactivated by phosphorylation at three
different sites by a set of four differently expressed kinases
(PDK1-4) whose expression are differently regulated by factors
such as starvation, hypoxia and utilization of glucose and fatty acids
in various tissues. All the best transcription regulators are involved,
including PGC-1alpha, retinoic acid, and the glucocorticoid receptor.
Oh bliss!
Through its regulation of glucose metabolism, and by different
regulation in heart and skeletal tissue compared to liver, PDH
is at the center of physiological response to energy needs.
Not surprisingly then, the complex is important and much studied
for its involvement in diabetes (reviewed in SUGDEN M.C &
HOLNESS MJ AM J. PHYSIOL.ENDOCRINOL. METAB. 284
E855 2003; HARRIS.RA, HUANG.B & WU.P. ADV. ENZYME
REGUL. 41. 269 (2001)).
Alterations in the enzyme are also reputed to be involved in
Alzheimer's disease (possibly oxidative modifications altering
functioning), and PDH deficiency is the most common cause
of mental retardation in children, mostly through mutation of
the E1alpha gene which is on the X chromosome (reviewed in
CEMERON ET AL. AM J MED GENET A. 131(1):59-56 (2004)).
2. Reviews of Recent Research
-- Anoxia affects PDH activity through HIF1 mediated
expression changes.
Two articles back to back in Cell Metabolism show that when
glycolytic enzymes are activated by anoxia through HIF1, at
the same time the transcription factor upregulates PDK 1,
one of the four PD kinases, thereby suppressing metabolism
through the OXPHOS system and Krebs cycle. Prior to this
work, most studies of regulation had "fingered" PDK 4 involvement.
"HIF-1 Mediated expression of PD kinase; a metabolic switch
required for cellular adaption to hypoxia."
KIM.J, TCHERNYSHYOV I, SEMENZA. G.L & DANG.C.V.
"HIF1 mediates adaption to hypoxia by actively down regulating
mitochondrial oxygen consumption."
PAPANDREOU.I, CAIRNS.RA. FONTANA.L, LIM.AL & DENKO.NC.
CELL METAB 3 187 2006.
-- Hypoxia, PDH regulation and cancer.
In an interesting extension of the issues of PDH regulation in
hypoxia, Denko and colleagues recently discussed the link
between this regulation and tumor biology, the old Warburg
effect being revisited not for the first time recently.
"Overcoming physiologic barriers to cancer treatment by
molecularly targeting the tumor microenvironment."
CAIRNS.R, PAPANDREOU.I & DENKO. N.
MOL CANCER. RES. 4. 61 (2006).
-- The PDH complex is oxidatively damaged during reperfusion after
cerebral ischemia.
In another interesting study Fiskum and associates evaluated the
possibility of altered functioning of the PDH complex during
reperfusion after cerebral ischemia. Using a canine ventricular
fibrillation cardiac arrest model and reperfusion under hyperoxic
conditions they found reduced PDH activity and elevated 3
nitrotyrosine levels in hippocambal but not cortex tissue. In vivo
exposure of PDH to the likely culprit, peroxynitrite, caused
reduced activity and increased oxidative modification of the enzyme.
"Postischemic hyperoxia reduces hippocambal pyruvate
dehydrogenase activity."
RICHARDS.EM, ROSENTHAL.RE, KRISTIAN.T & FISKUM.G.
FREE RADIC.BIOL.MED 40. 1960 (2006)
-- Novel genetic mutations causing PDH deficiency continue
to appear.
A recent report describes a novel mutation in the E3bp, the
second most common site for PDH defects. In this case there
was a 3913bp deletion in the gene for the protein.
"Leigh's disease due to a new mutation in the PDHX gene."
SCHIFF et al. ANN.
NEUROL. 59. 709 (2006)
Kerr and colleagues have reported a novel male patient mosaic
for a PDH E1alpha mutation. Mosaicism is expected and
common in females as the E1alpha gene is on the X chromosome,
but a male mosaic! In this patient two populations of the E1alpha
gene were identified, the normal, and another lacking exon 6.
There were different ratios of these forms in different tissues.
"Somatic mosaicism in a male with an exon skipping mutation in
PDHA1 of the pyruvate dehydrogenas complex results in a milder
phenotype."
OKAJIMA.K, WARMAN.ML, BYRNE. LC & KERR.DS.
MOL. GENET. METAB. 87 162.(2006)
In a third important paper on PDH genetic diseases, Robinson
and colleagues describe mutations in the PDH phosphatase
genes which cause severe exercise intolerance and mild
developmental delay. They point out that such mutations are
often overlooked in diagnosis.
"Pyruvate dehydrogenase phosphatase deficiency; orphan
disease or an under-diagnosed condition?"
MAJ.MC, CAMERON.JM & ROBINSON.BH.
MOLECULAR & CELLULAR ENDOCRINOLOGY 249. 1 2006
-- Studies of dichloroacetate, a potential treatment for
PDH deficiency.
It has been suggested based on in vitro experiments that DCA
may be effective in treating patients with congenital lactic acidosis,
including ones with PDH deficiency and defects in OXPHOS, all
of which present with overlapping symptomology, including LA.
In this first report or the effect of oral DCA treatment with 43
patients, it was found that DCA significantly decreased rises
in blood lactate caused by carbohydrate feeding. The drug
was well tolerated but there were no significant differences in
Global Assessment of Treatment Efficiency scores or severity
of intercurrent illnesses.
"Controlled clinical trial of dichloroacetate for treatment of
congenital lactic acidosis in children."
STACKPOOLE AND 19 OTHERS.
PEDIATRICS 117. 1519 2006.
- Advertisement -
A NEW APPROACH TO ASSAYING PDH ACTIVITY
At MitoSciences we decided that with all of the activity in
research on PDH that it would be useful if there was a
relatively simple way of assaying the enzyme.
Presently almost all researchers feed 14C labeled pyruvate
and measure the kinetics of radioactive CO2 production.
We are now introducing a new approach to assaying the
enzyme. This involves immunocapturing the PDH complex
away from other NAD and NADH-using enzymes so that
the assay can be done spectrophotometrically.
The amount of enzyme captured can be measured by our
quantitation assay, activity can then be measured, with
both assays using a 96 well plate format . Taken together
the data provide specific activities from very small amounts
of material without isolating mitochondria.
Overview of Assay Technology:
http://www.mitosciences.com/pdh_microplates.html
The Products:
PDH Activity Microplate Assay Kit
http://www.mitosciences.com/msp00a.html
PDH Quantity Microplate Assay Kit
http://www.mitosciences.com/msp00q.html
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