Resources > MitoNews > Archives > Volume 01, Issue 04 - April, 2005

Volume 01, Issue 04 - April, 2005




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MitoNews
Mitochondrial Research Bulletin

Published by:
MitoSciences
Advancing Vital Discoveries in Mitochondrial Research
http://www.mitosciences.com

Edited & Moderated by:
Dr. Roderick Capaldi
rcapaldi@mitosciences.com

Volume 01, Issue 04 - April 19, 2005
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In This Issue...


RECENT RESEARCH
1. Mitofilin is involved in control of mitochondrial cristae
morphology.

2. Stress the organ and cytochrome c oxidase levels go up?

3. Deconstructing the PTP.

4. A fourth isoform of human ANT is described.

5. Ulcer-causing bacteria attack the mitochondrion and
induce apoptosis.

6. CREB protein is present in mitochondria and may regulate
mitochondrial gene expression.

7. Mitochondria have their own ferritin.

8. Supercomplexes of the OXPHOS complexes; real
or artifactual?

9. Identification of a novel mitochondrial protein involved
in type 2 diabetes.



....................................................................................................
RECENT RESEARCH
....................................................................................................


1. Mitofilin is involved in control of mitochondrial cristae
morphology.

The presence of mitofilin in mitochondria has been known for
some time but its function has proved elusive. In the paper
below strong evidence is presented that this protein is an
important organizer of the cristae that pack the interior of the
organelle. Mitofilin is found as a large multimeric complex in
the space between the inner boundary and outer membrane.
(Note..more recent models of mitochondria distinguish 
between the so called inner boundary membrane and the
cristae membrane, while older models called the two collectively
the inner membrane. The two are connected only at discrete
points called cristae junctions). Down regulation of mitofilin
in HeLa cells using siRNA was found to cause decreased cell
growth rates and increased apoptosis. This was not due to
altered fission and fusion of mitochondrial units but instead
to a grossly altered arrangement of the cristae membranes.
With lowered levels of mitofilin, the typical tubular cristae were
missing and replaced by closely packed stacks of membrane
sheets and an absence of cristae junctions. The altered
mitochondria showed relatively normal oxidative
phosphorylation but there was increased membrane potential
attributable to increased metabolic flux and an increased
generation of reactive oxygen species.

JOHN. GB, SHANG. Y, LI.L, RENKEN.C, MANNELLA. CA,
SELKER. JM, RANGELL. L, BENNETT. MJ & ZHA.
J. MOL.BIOL.CELL 16. 1543-54 (2005)



2. Stress the organ and cytochrome c oxidase levels go up?

The two recent papers below measure the levels of cytochrome
c oxidase by activity, mRNA, and/or protein levels when tissue
is stressed by activity. Nikonova et al. examine COX activity
and mRNA levels of subunits I and IV (one mt and one nuclear
gene) of rats after sleep, wakefulness and sleep deprivation.
They found significant increases in COX activity and in mRNA
levels for both subunits I and IV in awake and sleep -deprived
brains compared to the sleep group and this was brain-region
related. Both subunits I and IV mRNA levels were increased
similarly. In the paper by Parise et al older (depending of your
reference point) individuals i.e. average 68 years of age were
subjected to resistance exercise for 14 weeks before muscle
biopsies were taken and analyzed for metabolic changes. 
Most protein contents were unaltered, including the levels of
mitochondrial complexes I,II and III, but COX activity and levels
were significantly higher (activity by around 25%). These
authors suggest that newly added COX could act as an
antioxidant and delay aging.

NIKONOVA. EV, VIJAYASARATHY.C, ZHANG.L, CATER.JR,
GALANTE. RJ, WARD. SE, AVADHANI. NG. & PACK. AI.
SLEEP 28. 21-7 (2005)

PARISE. G, BROSE. AN & TARNOPOLSKY. MA. EXP.
GERONTOL. 40. 173-80 (2005)



3. Deconstructing the PTP.

It may be time to change my lecture notes on apoptosis. 
I show the mitochondrial permeability transition pore (PTP)
as a super-complex containing cyclophilin d on the matrix side,
attached to the adenine nucleotide translocase in the inner
membrane, which interacts with outer membrane porin via the
adenylate kinase, while porin is associated directly with cytosolic
hexokinase. I also have the peripheral benzodiazipine receptor
in there and bax bound transiently. Iıve seen this model in
papers and read the evidence, at least I think I did. I talk about
a nucleotide channel being converted into a channel for other
ions and solutes, and if I say so myself, I sound like I know what
I am talking about. Oh dear, the 5 papers referenced below
suggest itıs time to update.

First some background. It has been known for more than 25
years, and accepted by most for the last 10 years, that cells
can undergo a sudden increase in mitochondrial inner
membrane permeability to ions and solutes. This disequilibrium
causes a dissipation of the membrane potential, loss of ion
homeostasis, and impairment of ATP synthesis, which results
in passive swelling of the organelle, release of cytochrome
c and cellular apoptosis, or in dire circumstances, necrotic
cell death.

While not everyone adhered to the model above, there did
seem concensus that at a minimum the PTP contains porin,
ANT and cyclophilin d. Then in early 2004 Kokoszka et al
provided strong evidence that ANT is not essential for the
PTP. These authors genetically inactivated the two forms
of ANT in mouse liver and analyzed PTP functioning. They
found that genetic knockout of ANT had no significant effect
on the permeability transition and release of cytochrome c
except that ANT ligands no longer affected the process. 

In the last month 3 papers have appeared in which the gene
for cyclophilin d has been inactivated in mice. They each
confirm the importance of cyclophilin d in the permeability
transition but one suggests that the PTP can form in its
absence. Thus both Baines et al. and Nakagawa et al found
that mice lacking cyclophilin d developed normally but that
these mice showed no cyclosporin A sensitivity of the
permeability transition (cyclosporin A binds selectively to
cyclophilin d to inhibit apoptosis). They report that cells
died normally in response to various apoptotic stimuli
including ones that use the mitochondrial apoptotic pathway,
except for cell death induced by Ca2+ overload or by reactive
oxygen species. Both studies showed that animals without
cyclophilin d were resistant to ischemia reperfusion induced
cardiac injury.

Bisso et al also report studies generating cyclophilin d-
knockout mice but different from the above, these authors
focused on biochemical properties of the resulting PTP. 
They conclude that the PTP can form and open in the absence
of cyclophilin d and that the pore remains sensitive to Ca2+
but not to the proton chemical gradient or oxidative stress.
Where does this leave us. 

In a paper reviewing the work of Kokoszka et al, Haalstrap,
a strong proponent of the ANT-cyclophilin d connection in
PTP raises several interesting issues and proposals. One
suggestion is that a less abundant member of the carrier
family than ANT with less sensitivity to Ca2+ or oxidative
stress can replace ANT in the pore when this protein is
absent i.e a compensation mechanism.

O.K. Iıll redraw my figure of the PTP as a black box with
porin attachedŠbut wait...what is the evidence that porin
is involved?

KOKOSZKA. JE, WAYMIRE. KG, LEVY. SE, SLIGH. JE, CAI.
J, JONES. DP, MACGREGOR. GR & WALLACE. DC.
NATURE 427. 461-65 (2004)

BAINES. CP, KAISER. RA, PURCELL.NH, BLAIR. NS, OSINSKA.
H, HAMBLETON. MA, BRUNSKILL. EW, SAYAN. MR, GOTTLIEB.
RA, DORN. GW, ROBBINS. J & MOLKENTIN. JD 
NATURE 434. 658-62 (2005)

NAKAGAWA. T, SHIMIZU.S, WATANABE.T, YAMAGUCHI.O,
OTSU.K, YAMAGATA. H, INOHARA.H, KUBO. T &
TSUJIMOTO. Y. 
NATURE 434. 652-58 (2005)

BASSO. E, FANTE. L, FOWLKES. J, PETRONILLI.V , FORTE.
ME & BERNARDI. P.
J BIOL CHEM MAR25 (2005)

HALESTRAP. AP.
NATURE BRIEF COMMUNICATION 26 AUG (2004)



4. A fourth isoform of human ANT is described.

ANT is more than a PTP component (or not). The predominant
function of this protein in mitochondria is the exchange of
cytosolic ADP for matrix ATP, and in reverse, exchange of
glycolytically-generated ATP for ADP when necessary, e.g.
in rho0 cells (devoid of mtDNA). It had long been thought
that humans have three isoforms of this protein. However in
the paper referenced below, a fourth isoform is described that
maps to chromosome 4q28.1. This isoform is 66-68% identical
to the 3 other isoforms. It is sensitive to the usual ANT
inhibitors carboxyatractyloside and bonkrekic acid. Transcripts
of this novel ANT are found exclusively in liver, testis and brain.

DOLCE. V, SCARCIA. P, IACOPETTA. D & PALMIERI. F. FEBS
Lett. 579. 633-7 (2005)



5. Ulcer-causing bacteria attack the mitochondrion and
induce apoptosis.

The gastric pathogen Heliobacter pylori produces a
pro-apoptotic toxin called Vac A. The review referenced
below presents the evidence that this polypeptide localizes
to mitochondria and acts to trigger apoptosis by forming a
permeability channel, releasing cytochrome c , with a resulting
cell death.

BLANKE. SR. TRENDS IN MICROBIOL 13. 64-71 (2005)



6. CREB protein is present in mitochondria and may regulate
mitochondrial gene expression.

Nuclear gene transcription is regulated by a calcium/cAMP
responsive element binding protein (CREB) via calcium
dependent kinases and phosphatases. The question is,
do mitochondria contain the same set of proteins. To test
this possibility, Schub et al used antibodies to detect CREB
and phosphorylated (p)CREB in mitochondria and mitoplasts
from rat brain. They found that energized mitochondria contain
pCREB, which is converted to the non-phosphorylated form in
the presence of increasing concentrations of Ca2+. In the
absence of this cation, CREB phosphorylation could be elevated
by the addition of ATP. The authors conclude that CREB is
located in the mitochondrial matrix or inner membrane and
that its phosphorylation state is regulated by a mitochondrial
calcium dependent kinase and phosphatase.

SCHUH. RA, KRISTIAN. T & FISKUM. G. 
J. NEUROCHEM. 92. 388-94 (2005)



7. Mitochondria have their own ferritin.

Ferritins play an important role in iron homeostasis by acting
as storage proteins for the metal ion. The paper listed below
characterizes a novel human mitochondrial ferritin. This
mitochondrial form shows a high degree of sequence homology
with the better understood, cytosolic H chain ferritin, but there
are several interesting differences in properties. It is a
homopolymer of 24 subunits. While the two forms have similar
diFe ferroxidase centers, the mitochondrial form does not
regenerate its ferroxidase activity after oxidation of its initial
load of Fe(II). Moreover, only one half of the ferroxidase
centers (one per subunit) appear to be active. Clear kinetic
differences in the forms of the enzyme are reported.

BOU-ABDALLAH. F, SANTAMBROGIO. P, LEVI.S, AROSIO.P
& CHASTEEN. ND. 
J. MOL BIOL 347. 543-54 (2005)



8. Supercomplexes of the OXPHOS complexes; real
or artifactual?

There have been several recent studies reporting resolution
of supercomplexes of the OXPHOS complexes including ones
between complexes I and III, III and IV, and the so called ATP
synthesome, a supercomplex between complex V ( the ATP
synthase), with the phosphate carrier and ANT. In essence
these supercomplexes are obtained by dissociating the
mitochondrial inner membrane (cristae membrane) with
detergents to the point where proteins are most solubilized
without breaking relatively weak interactions, or (possibly)
removing all of the lipids that bridge complexes together. 
The technique of choice for identifying these supercomplexes
has been BN-PAGE. Now a supercomplex containing one
copy of complex I and a dimer of complex III has been
obtained in solution and examined by single particle EM. 
A model is presented for the spatial organization of the
component complexes.

DUDKINA. NV, EUBEL.H, KEEGSTRA. W, BOEKEMA.EJ &
BRAUN. HP. 
PROC NATL ACAD SCI USA. 102. 3225-9 (2005)



9. Identification of a novel mitochondrial protein involved
in type 2 diabetes.

In the study below, mitochondrial proteins involved in type
2 diabetes were identified by differential gene expression
in skeletal muscle of rats with diabetes and insulin resistance
when compared to controls. One protein in particular was
significantly altered; it is a mitochondrial intramembrane
protease PSARL (presenilins associated rhomboid-like protein).
  Expression of this protein was reduced in the diabetic state
but restored after exercise training to successfully treat the
diabetes. PSARL gene expression in human skeletal muscle
was correlated with insulin sensitivity, as shown by glucose
disposal during hyperinsulinaemic clamp. Moreover a
mutation of this gene (Leu262Val) was found to be associated
with increased plasma insulin concentrations in humans, a
key risk for type 2 diabetes. The authors conclude that variation
in PSARL may be an important risk factor for type 2 diabetes.

WALDER.K, KERR-BAYLES.L, CIVATARESE. A, JOWETT.J,
CURRAN. J, ELLIOTT.K, TREVASKIS.J, BISHARA.N, ZIMMET.P,
MANDARINO.L, RAVUSSIN.E, BLANGERO.J, KISSEBAH.A &
COLLIER.GR.
DIABETOLGIA 48. 459-68 (2005)



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