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Protein Traffic in Apoptosis

April 8, 2009

Edited by Roderick Capaldi, D.Phil.
Past issues are available for review in the archives.

Table of Contents

I. INTRODUCTION

II. ALPHA B-CRYSTALLIN DURING EX VIVO ISCHEMIA

III. POSSIBLE MECHANISM OF ANTIMYCIN

IV. BAXBETA IN HUMAN CELLS

V. BRCA ISOFORMS REPRESS ELK-1

VI. CTMP A PKB INHIBITOR

VII. PRESENILIN AND APOTOSIS

IIX. BINDING CAPSAICIN

IX. hNOA INTERACTION AND REGULATION

X. GSK3BETA ACTIVITY

XI. THE BAX BINDING DILEMMA

I. INTRODUCTION
Regular readers of MitoNews will be aware of the significant pleomorphy of mitochondria, with fragmented and reticular forms, whose presence is linked to functioning of the organelle in energy metabolism as well as mitochondrial replication. Another and more recently appreciated aspect of mitochondrial structure is the compositional variation of the organelle. There is regular movement of a significant number of proteins including kinases and transcription factors into and out of mitochondria as part of co-ordination of organelle biogenesis. Moreover there is trafficking of a number of pro- and anti-apoptotic proteins between the cytosol, mitochondria and nucleus as part of programmed cell death. The number of the proteins identified as being transiently present on or in the mitochondrion as part of the apoptotic process, continues to increase. Further, novel mitochondrial proteins with pro-apoptotic or anti-apoptotic function continue to be discovered. Finally, unexpected interactions of proteins previously known to be present but not thought to participate in apoptosis, are still being described. Some of the recent work is described below.



II. ALPHA B-CRYSTALLIN DURING EX VIVO ISCHEMIA
Alpha B crystallin, a small heat shock protein present in high amount in heart, is phosphorylated by p38MAP kinase and this helps protect the heart from ischemia/reperfusion injury. In this study the authors show that the protein moves to mitochondria from the cytosol as a response to ischemia, and that phophorylation follows soon after, with the net result that mitochondrial damage is reduced upon reperfusion.

Whittaker R, Glassy MS, Gude N, Sussman MA, Gottlieb RA, Glembotski CC. Am J Pysiol. Heart Circ. Physiol (2209) available on line.
Kinetics of the Translocation and Phosphorylation of {alpha}B-crystallin in Mouse Heart Mitochondria during Ex Vivo Ischemia.


III. POSSIBLE MECHANISM OF ANTIMYCIN
This article provides a possible mechanism of antimycin induced apoptosis which involves/requires NO and is inhibited when nitric oxide synthase inhibitors are present. It is concluded that antimycin induced cell death is cytochrome c independent, does not require the caspase cascade, and instead involves AIF translocation to the nucleus.

Ogita M, Ogita A, Usuki Y, Fujita K, Tanaka T. J. Antibiot (Tokyo) (2209) available on line.
Antimycin A-induced cell death depends on AIF translocation through NO production and PARP activation and is not involved in ROS generation, cytochrome c release and caspase-3 activation in HL-60 cells.


IV. BAXBETA IN HUMAN CELLS
This study shows that a second form of bax called baxbeta is present in all human cells with activity regulated by proteasomal degredation (cf. HIF 1alpha) Baxbeta spontaneously integrates into the mitochondrial outer membrane and potently releases cytochrome c with resulting apoptosis. The authors show that baxbeta is upregulated by apoptotic stimuli via inhibition of its ubiquitinylation. They also find that baxbeta associates with and promotes baxalpha activation.

Fu NY, Sukumaran SK, Kerk SY, Yu VC. Mol. Cell 33. 15-29 (2009)
Baxbeta: a constitutively active human Bax isoform that is under tight regulatory control by the proteasomal degradation mechanism.

V. BRCA ISOFORMS REPRESS ELK-1
BRCA isoforms are tumor suppressors that are mutated in families with breast cancer. In this study the authors show that BRCA-1, 1a and 1b are all localized in mitochondria and that together they repress ELK-1 transcriptional activity. They conclude that " since mitochondrial dysfunction is a hallmark of cancer, the organellar location may be functionally significant in regulating mitochondrial DNA damage".

Maniccia AW et al. J Cell Physiol (2009) available on line.
Mitochondrial localization, ELK-1 transcriptional regulation and growth inhibitory functions of BRCA1, BRCA1a, and BRCA1b proteins.

VI. CTMP A PKB INHIBITOR
CTMP (Carboxy-Terminal Modulator Protein) has been identified as a PKB (Akt/protein Kinase B) inhibitor and the authors show that this protein exists in mitochondria in two pools, one in the inter-membrane space and one a membrane-bound pool. Further, they show that this protein is released from mitochondria during apoptosis. CTMP over-expression led to increased mitochondrial membrane potential and both caspase 3 and PARP cleavage.

Parcellier A et al. Cell Signal 21. 639-50 (2009)
Carboxy-Terminal Modulator Protein (CTMP) is a mitochondrial protein that sensitizes cells to apoptosis.

VII. PRESENILIN AND APOTOSIS
Presenilin 1-associated protein/mitochondria carrier homolog 1 (who thinks up these names!) is a pro-apoptotic mitochondrial outer membrane protein. In this study the authors show that targeting the protein to the organelle is sufficient to induce apoptosis. This cell death does not depend on bax or bak and the authors propose that the mechanism of action is linked to PTP.

Lamarca V, Marzo I, Sanz-Clemente A, Carrodeguas JA. Eur. J. Cell Biol. 87. 325-34 (2008)
Exposure of any of two proapoptotic domains of presenilin 1-associated protein/mitochondrial carrier homolog 1 on the surface of mitochondria is sufficient for induction of apoptosis in a Bax/Bak-independent manner.

IIX. BINDING CAPSAICIN
Capsaicin is a candidate anti-cancer compound because of its ability to inhibit apoptosis in cells. In the present study the authors show that capsaicin binds to prohibitin 2, a protein normally localized in the mitochondrial inner membrane and causes its translocation to the nucleus.

Kuramori C et.al. Biochem. Biophys. Res. Commun. 379. 519-25 (2009)
Capsaicin binds to prohibitin 2 and displaces it from the mitochondria to the nucleus.

IX. HNOA INTERACTION AND REGULATION
hNOA1 is a large mitochondrial GTPase first identified in Arabidopsis. This protein has been shown to localize to mitochondria and be peripherally associated with the inner membrane. The authors show that overexpression or knockdown of this protein is linked to morphology changes of the organelle. Immunoprecipitation studies show that hNOA1 is associated with Complex I and with DAP3 (death associated protein 3) a positive regulator of apoptosis. Knockdown of the protein renders cells more resistant to apoptotic stimuli including by staurosporine.

Tang T et.al. J Biol Chem 284. 5414-24 (2009)
hNOA1 interacts with complex I and DAP3 and regulates mitochondrial respiration and apoptosis.

X. GSK3BETA ACTIVITY
This study expresses GSK3beta in mitochondria and showed that it enhanced MPP+ and rotenone induced apoptosis signaling via complex I. Complex I activity as well as ATP production were also reduced, there was increased ROS, and a perturbation of mitochondrial morphology.

King TD, Clodfelder-Miller B, Barksdale KA, Bijur GN. Nerotox Res. 14. 367-82 (2008)
Unregulated mitochondrial GSK3beta activity results in NADH: ubiquinone oxidoreductase deficiency.

XI. THE BAX BINDING DILEMMA
There have been several models to explain the binding and resulting mitochondrial outer membrane permeabilization and cytochrome c release by bcl2 pro-apoptotic proteins including bax and bid. In one model these proteins form a pore by themselves. Other studies have concluded that these interact with the PTP. Now several recent studies have implicated the Tom complex in bax-induced apoptosis. Three interesting new papers in this regard are;

Colin J, Garibal J, Mignotte B, Guénal I. Biochem. Biophys. Res Commun. 379. 939-43 (2009)
The mitochondrial TOM complex modulates bax-induced apoptosis in Drosophila.

Cartron PF et al. FEBS Lett 582. 3045-51 (2008)
Bax inserts into the mitochondrial outer membrane by different mechanisms.

Bellot G. et al. Cell Death Differ. 14. 785-94 (2007)
TOM22, a core component of the mitochondria outer membrane protein translocation pore, is a mitochondrial receptor for the proapoptotic protein Bax.


Products for Apoptosis:
Cell Fractionation Kit HT (MS862)
Provides a highly novel method and reagents for a simple and rapid preparation of cytosolic, mitochondrial and nuclear fractions from adherent cells grown in 96-well plates.

ApoTrack™ Cytochrome c WB Antibody Cocktail (MSA12)
A 4-plex Western blot antibody cocktail that allows for the detection of cytochrome c in cytoplasmic and mitochondria-containing fractions for determining the proportion of released cytochrome c from mitochondria to the cytoplasm from apoptosis. Includes a set of control markers allows for the monitoring and/or optimization of the permeabilization conditions

Cytochrome c Protein Quantity Microplate Assay Kit (MSA41)
A 96-well microplate assay that is used to determine the amount of cytochrome c in a human, mouse, rat or bovine sample.

Apoptosis-Inducing Factor Protein Quantity Dipstick Assay Kit (MSA31)
This kit is used to quantitate the amount of AIF in a human sample using the rapid, simple and quantitative dipstick technology.

Apoptosis-Inducing Factor Protein Quantity microplate Assay Kit (MSA42)
This kit is used to quantitate the amount of AIF in a human sample using a high-throughput 96-well plate.

ApoTrack™ Cytochrome c ICC Antibody Kit (MSA07)
Apoptotic cells which have released mitochondrial cytochrome c into the cytosol can be differentiated from non apoptotic cells which still retain cytochrome c in their mitochondria by fluorescence microscopy with this 2-plex ICC kit.
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