Simmen (Ph.D. University of Lausanne)
Department of Cell Biology
5-65 Medical Sciences Building
Phone: (780) 492-1546
Fax: (780) 492-0450 (fax)
Redox-based signaling between the endoplasmic reticulum (ER) and mitochondria as a critical determinant in cancer
Malignant tumors develop when cells proliferate uncontrollably or fail to undergo cell death upon various cues. Current research shows that countless mutations on all chromosomes contribute to the tumor phenotype, with no particular area or gene standing out as a particularly detrimental contributor. Moreover, this myriad of mutations leads to even more drastic changes in the protein composition of the tumor versus healthy tissue. Therefore, it is the task of today’s cancer researchers to identify and characterize signaling mechanisms that govern cellular life span and tumor growth.
In our research, we have identified a central player in the form of an intracellular signaling hub termed the mitochondria-associated membrane (MAM). The mitochondria-associated membrane, part of the endoplasmic reticulum, is closely apposed to mitochondria, thus forming a signaling axis. On this structure, calcium and protein-based signaling mechanisms determine the onset of apoptosis (programmed cell death originating within mitochondria) and tumor growth (through the production of growth factors in the endoplasmic reticulum that is contiguous with the mitochondria-associated membrane) in an on/off switch manner, often dependent on the cellular redox state. Not surprisingly, the mitochondria-associated membrane houses numerous oncoproteins and tumor suppressors, which are all postulated to regulate the signaling of the mitochondria-associated membrane either towards its pro-death or pro-life readout.
Our laboratory focuses on the identification of these signaling proteins on the mitochondria-associated membrane and on the characterization of their function. We expect our findings to have profound impact on future approaches to fight cancer, since they will allow researchers to precisely tweak signaling on the mitochondria-associated membrane towards the desired direction. We chose to study redox-sensitive proteins and chaperones found here, because this group of proteins sits at the crossroads between growth factor secretion, oxidative stress and cell death. In particular, we focus for now on the mechanisms that regulate their enrichment to the mitochondria-associated membrane. For this task, we have developed a subcellular fractionation protocol that isolates the MAM from other domains of the ER, such as the rough ER (Figure 1).
Figure 1: MAM isolation. HeLa cells were fractionated on a discontinuous Optiprep gradient. Marker proteins indicate cell surface (biotinylated proteins), Golgi (b-COP), ERGIC (ERGIC53), rER (Ribophorin I), mitochondria-associated membrane (ACAT1), and mitochondria (mitochondrial complex II). The chaperone calnexin (CNX) was identified as a mitochondria-associated membrane component (peak in fractions 5 and 6), whose distribution depends on the presence of the cytosolic protein PACS-2, since its distribution was changed in the absence of PACS-2 (siPACS-2).
Figure 2: MAM immunofluorescence. A portion of the chaperone calnexin (CNX) colocalizes with PACS-2 in the vicinity of mitochondria, indicative of a localization to the mitochondria-associated membrane. Bottom row, magnified area, indicated with the white frame on the overlay. White arrows, PACS-2/mitochondria overlap; red arrowheads, triple overlap. Scale bar, 25 µm.
We also detect mitochondria-associated membrane localization of ER proteins by assaying for immunofluorescence overlap with mitochondria (Figure 2). With our biochemical isolation protocol and our immunofluorescence technique, we were able to demonstrate that Phospho-Furin Acidic Cluster Sorting protein 2 (PACS-2) contributes to mitochondria-associated membrane enrichment of calnexin, an ER chaperone (Myhill et al., 2008) and characterized the intracellular targeting of the redox-sensitive enzyme TMX4 (Roth et al., 2010).
Key research areas:
- We aim to identify novel proteins, which act in parallel or together with PACS-2 to regulate ER-mitochondria contacts and apoptosis.
- We want to elucidate the role of redox-sensitive, mitochondria-associated membrane-targeted proteins in ER-stress and hypoxia-derived apoptosis and in the regulation of pro-angiogenic growth factor secretion.
- Among these proteins and in collaboration with Alberta clinicians, we aim to identify candidate mitochondria-associated membrane proteins that could serve as tumor biomarkers or future targets of intervention in cancer.
Bui, M., Gilady, S.Y., Fitzsimmons, R.E.B., Benson, M.D., Lynes, E.M., Gesson, K., Alto, N.M., Strack, S., Scott, J.D., and Simmen, T. (2010) Rab32 modulates apoptosis onset and mitochondria-associated membrane (MAM) properties. J Biol Chem, in press.
Simmen, T., Lynes E. M., Gesson K., and Thomas G. (2010) Oxidative protein folding in the endoplasmic reticulum: Tight links to the mitochondria-associated membrane (MAM). BBA-Biomembranes,1798, pp. 1465-1473
Gilady, S. Y., Bui, M., Lynes, E. M., Benson, M. D., Watts, R., Vance, J. E., and Simmen, T. (2010) Ero1a Requires Oxidizing Conditions and Oxygen Supply to Localize to the Mitochondria-Associated Membrane (MAM). Cell Stress and Chaperones, MS ID #CSAC-253, in press
Roth, D., Lynes, E., Riemer, J., Hansen, H. G., Althaus, N., Simmen, T. and Ellgaard, L. (2010). A di-arginine motif contributes to the ER-localization of the type I transmembrane ER oxidoreductase TMX4. Biochem J. 425(1):195-205
Myhill, N., E.M. Lynes, J.A. Nanji, A.D. Blagoveshchenskaya, H. Fei, K.C. Simmen, T.J. Cooper, G. Thomas, and T. Simmen. (2008) The Subcellular Distribution of Calnexin is Mediated by PACS-2. Mol Biol Cell 19(7), 2777-2788.
Simmen, T., J. E. Aslan, A. D. Blagoveshchenskaya, L. Thomas, L. Wan, Y. Xiang, S. F. Feliciangeli, C.-H. Hung, C. M. Crump and G. Thomas (2005). "PACS-2 controls ER-mitochondria communication and Bid-mediated apoptosis." Embo J, 24, 717-729.
Bertoli, G., T. Simmen, T. Anelli, S. N. Molteni, R. Fesce and R. Sitia (2004). "Two conserved cysteine triads in human Ero1alpha cooperate for efficient disulfide bond formation in the endoplasmic reticulum." J Biol Chem 279(29): 30047-52.
Simmen, T., S. Höning, A. Icking, R. Tikkanen and W. Hunziker (2002). "AP-4 binds basolateral signals and participates in basolateral sorting in MDCK cells." Nature Cell Biology, 4(2): 154-159.
Carolina Ortiz Sandoval
Dr. Boyin Liu
Dr. Samira Samtleben