department of pharmacology

Shigemi Matsuyama, Ph.D

matsuyama

Associate Professor, Medicine

WRB3-353
Phone: (216) 368-5832
Fax: (216) 368-8919
E-mail: shigemi.matsuyama@case.edu
Office/Laboratory: Wolstein Research Building 3 F.

Research

Cancer Cell Biology, Cell Death Regulation, Cell Penetrating Peptide.

My laboratory has two research projects as explained below:

(1) How does the stressed cell decide its fate to live or die?

Several types of stresses, including DNA damage, protein misfolding, reactive oxygen species generation, viral infection and tropic factor deprivation are known to activate apoptosis pathways controlled by evolutionarily conserved Bcl-2 family proteins (Reviewed in 1-3). There are two types of Bcl-2 family proteins: anti-apoptotic and pro-apoptotic proteins. Bcl-2, Bcl-X, and Mcl-1 are anti-apoptotic proteins that inhibit cell death through several types of stress. Bax, Bak, Bid, and Bim are the well-known members in pro-apoptotic Bcl-2 family proteins, and these proteins trigger apoptotic signal cascades that lead the stressed cell to undergo apoptosis.

Cellular stresses activate pro-apoptotic Bcl-2 family proteins and these pro-apoptotic proteins initiate the release of apoptogenic factors (e.g. cytochrome c) from mitochondria. Contrarily, anti-apoptotic Bcl-2 family proteins work as antagonists to inhibit the release of apoptogenic factors from mitochondria. Therefore, the balance of pro- and anti-apoptotic Bcl-2 family proteins is one important factor in deciding whether a cell dies or survives.

One of the fundamental unanswered questions in apoptosis regulation is the mechanism controlling how pro-apoptotic Bcl-2 family proteins (such as Bax) are activated by stresses. In other words, how do different types of stresses activate a single Bax molecule to induce apoptosis? Is there any specific mediator for each stress, and/or is there any common upstream stress sensor mediating the degree of stresses to Bax?

Recently, my laboratory found that Ku70 is one of the Bax-inhibiting factors that keeps Bax inactive in the cytosol (summarized in 4, 5). Importantly, Ku70 is a ubiquitously expressed and evolutionarily conserved protein that has been known to play an important role in DNA double-strand break repair 6. We have found that the cytosolic Ku70 has a novel function as a Bax inhibitor, independent from Ku70’s previously known function in DNA repair in the nucleus. We hypothesize that Ku70 is a stress sensor protein that controls the early phase of Bax activation, and that the Ku70 modification influencing the Ku70-Bax interaction sets the sensitivity of the cells to determine whether to initiate apoptosis.

To test our hypothesis, our research currently has the following aims:

Aim 1: To determine the mechanism of post-translational control of Ku70 levels and functions in normal and stressed cells.

Our laboratory has found that ubiquitin-dependent modification of Ku70 is one of the mechanisms to decrease Ku70 levels in apoptotic cells 7. In addition, the acetylation of Ku70 has also been shown to induce the dissociation of Ku70 from Bax by other groups 8, 9. We are currently studying the impact of these post-translational modifications on the half-life, subcellular localization, affinities to binding proteins (Bax and Ku80) and biological activities which regulate cell death and DNA repair. We are also searching for the Ku70 modifying enzymes that mediate cellular stresses to this sensor protein.

Aim 2: To determine the molecular mechanism behind the Ku70-dependent inhibition of Bax activation.

We are currently studying how the binding of Ku70 inhibits the activation process of Bax, such as the conformational change of Bax and the interaction with Bax activators such as BH3 only proteins that are the members of pro-apoptotic Bcl-2 family proteins.

Aim 3: To determine the biological significance of Ku70-Bax interaction to control cell death sensitivity using mouse genetics.

Ku70-/- mice have been generated by other groups 10, 11 and these mutant mice show the phenotypes of growth retardation, accelerated replicative senescence of cultured fibroblasts, increased cell death in gastrointestinal tissue and T-cell lymphoma development. We hypothesize that the disease phenotypes of Ku70-/- mice may be, at least in part, due to the increased Bax-mediated cell death caused by the absence of Ku70. Bax-/- mice have been established by other group 12. By crossing Ku70-deficient and Bax-deficient mice, we are generating Ku70-/-Bax-/- mice and will analyze the phenotype of the Ku70-/-Bax-/- mice to determine whether Bax deletion corrects some of the disease phenotypes of a Ku70-/- mouse.

(2) Development of Cytoprotective Cell-Penetrating Penta-Peptides

The cellular membrane has a strict selectivity for molecular transport to maintain cellular life. Because of this selectivity, delivering potentially effective drugs into damaged cells often become difficult. Therefore, there is a strong need to develop the technologies for molecular transport across the cellular membrane. My laboratory has discovered specific penta-peptides (5-amino-acid peptides) that penetrate the cellular membrane (please see the next paragraph for details) 4, 5. These penta-peptides are categorized as the shortest cell-penetrating peptides13, and the mechanism of how these peptides penetrate the cellular membrane is now being investigated in my laboratory. By elucidating the cell penetrating mechanism of these penta-peptides, we hope to develop the technology to deliver effective drugs into the cell.

Development of Bax-Inhibiting Peptide

Previously, we found that Ku70 binds Bax and inhibits Bax-mediated cell death 7. We identified the Bax binding domain of Ku70 and the penta-peptide derived from this domain was sufficient to bind and inhibit Bax4. Interestingly, these penta-peptides entered the cells when the peptides were added to the culture medium, indicating that these peptides are cell permeable. These peptides have been named Bax-Inhibiting Peptides (BIPs). The discovery of BIPs brought us two different types of research opportunities: (a) Utilization of BIPs to rescue damaged cells, and (b) Elucidation of the mechanism of membrane penetration of BIPs.

(a) Utilization of BIPs to rescue damaged cells
The BIP has been shown to suppress drug-induced apoptosis in cell culture 4, 14. Importantly, the BIP rescued retinal ganglion cells from optic nerve injury-induced apoptosis and also increased the survival rate of the hepatocytes after transplantation in mice15, 16. Currently, we are examining whether BIP can be utilized to protect damaged tissue by ischemia-reperfusion treatment in a mouse model. We are also developing more effective cytoprotective, cell-permeable peptides by introducing mutations in BIPs.

(b) Elucidation of the mechanism of membrane penetration of BIPs and BIP-derived cell-penetrating peptides.

In addition to BIPs, we have also developed new cell-permeable peptides by mutating the sequence of BIPs. These mutant peptides do not bind Bax and thus do not inhibit cell death. BIP and these mutant peptides have been labeled “BIP-derived Cell Penetrating Penta-Peptides (BCP)” 5.

BCPs belong to the growing family called “Cell Penetrating Peptides” that include the Human Immunodeficiency Virus (HIV) tat peptide13. The cell entry mechanism of cell-penetrating peptides is not yet known, and it is possible that there are different mechanisms depending on the type of cell-penetrating peptide. My laboratory is focusing on the cell entry mechanism of BCPs. We are currently examining the following questions: (1) Do BCPs use energy-dependent and/or energy-independent mechanism(s) to enter the cell? (2) Is there any unidentified receptor molecule(s) on the cell surface that is required for the cell entry of BCPs? (3) Can we design new penta-peptides that have improved cell entry activity? Our study will advance the understanding of the protein traffic in the plasma membrane, and we hope that our study will contribute the development of new drug delivery technology into the cell.

(References)

1.         Reed, J. C. Double identity for proteins of the Bcl-2 family. Nature 387, 773-6. (1997).
2.         Korsmeyer, S. J. BCL-2 gene family and the regulation of programmed cell death. Cancer Res 59, 1693s-1700s. (1999).
3.         Green, D. R. At the gates of death. Cancer Cell 9, 328-30 (2006).
4.         Yoshida, T. et al. Bax-inhibiting peptide derived from mouse and rat Ku70. Biochem Biophys Res Commun 321, 961-6 (2004).
5.         Gomez, J. A., Gama, V., Matsuyama, S. Cell-permeable penta-peptides derived from Bax-inhibiting peptide. Cell Penetrating Peptide, 2nd edition., 469-481 (2006).
6.         Downs, J. A. & Jackson, S. P. A means to a DNA end: the many roles of Ku. Nat Rev Mol Cell Biol 5, 367-78 (2004).
7.         Gama, V. et al. Involvement of the ubiquitin pathway in decreasing Ku70 levels in response to drug-induced apoptosis. Exp Cell Res 312, 488-99 (2006).
8.         Cohen, H. Y. et al. Acetylation of the C terminus of Ku70 by CBP and PCAF controls Bax-mediated apoptosis. Mol Cell 13, 627-38 (2004).
9.         Subramanian, C., Opipari, A. W., Jr., Bian, X., Castle, V. P. & Kwok, R. P. Ku70 acetylation mediates neuroblastoma cell death induced by histone deacetylase inhibitors. Proc Natl Acad Sci U S A 102, 4842-7 (2005).
10.       Gu, Y. et al. Growth retardation and leaky SCID phenotype of Ku70-deficient mice. Immunity 7, 653-65 (1997).
11.       Li, G. C. et al. Ku70: a candidate tumor suppressor gene for murine T cell lymphoma. Mol Cell 2, 1-8 (1998).
12.       Knudson, C. M., Tung, K. S., Tourtellotte, W. G., Brown, G. A. & Korsmeyer, S. J. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science 270, 96-9. (1995).
13.       Joliot, A. & Prochiantz, A. Transduction peptides: from technology to physiology. Nat Cell Biol 6, 189-96 (2004).
14.       Zi, X. & Simoneau, A. R. Flavokawain A, a novel chalcone from kava extract, induces apoptosis in bladder cancer cells by involvement of Bax protein-dependent and mitochondria-dependent apoptotic pathway and suppresses tumor growth in mice. Cancer Res 65, 3479-86 (2005).
15.       Qin, Q., Patil, K. & Sharma, S. C. The role of Bax-inhibiting peptide in retinal ganglion cell apoptosis after optic nerve transection. Neurosci Lett 372, 17-21 (2004).
16.       Tanaka, K. et al. Prolonged survival of mice with acute liver failure with transplantation of monkey hepatocytes cultured with an antiapoptotic pentapeptide V5. Transplantation 81, 427-37 (2006).

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Publication by Shigemi Matsuyama (1997-) and Matsuyama laboratory (from 2003-).

1.    Shendel SL, Xie Z, Montal MO, Matsuyama S, Montal M, Reed JC (1997) Channel formation by anti-apoptotic protein, Bcl-2.  Proc Natl Acad Sci USA 94, 5113-5118.
2.    Matsuyama S, Xu Q, Velrous J, Reed JC (1998) F0F1-proton-pump ATPase is required for the function of a pro-apoptotic protein, Bax, both in yeast and mammalian cells. Molecular Cell 1: 327-336.
3.    Xie Z, Schendel SL, Matsuyama S, Reed JC (1998) Acidic pH promotes dimerization of Bcl-2 family proteins. Biochemistry 37:6410-6418.
4.    Matsuyama S (1998) Molecular mechanism of Bax-induced cell death. Cell Technology 17:891-898 (Japanese article).
5.    Xu Q, Jurgensmeier JM, Zha H, Matsuyama S, Reed JC (1998) Exploiting yeast for the investigation of mammalian proteins that regulate programmed cell death. Apoptosis Detection and Assay Methods, Edited by Li Zhu and Jerald Chun, BioTechniques Books, Natick, MA. Page 93-115.
6.    Reed JC, Jurgensmeir J, Matsuyama S (1998) Bcl-2 family proteins and mitochondria (Review). Biochemica et Biophisica Acta, 1366, 127-137.
7.    Marzo I, Brenner C, Zamzami N, Jurgensmeier JM, Susin SA, Vieira HLA, Prevost MC, Xie Z, Matsuyama S, Reed JC, Kroemer G. (1998) Bax and adenine nucleotide translocator cooporate in the mitochondrial control of apoptosis. Science, 281. 2027-2031.
8.    Matsuyama S, Schendel SL, Xie Z, Reed JC (1998) Cytoprotection by Bcl-2 requires the pore-forming a5 and a6 helicies. Journal of Biological Chemistry 273. 30995-31001.
9.    Matsuyama S (1999) Mitochondria dependent cell death pathway. In "Apoptosis and the digestive organ-diseases" (Edited by H. Ishi), p.p. 31-46 (Japanese article)
10.  Matsuyama S, Nouraini S, Reed JC (1999) Yeast as a tool for apoptosis research. Current Opinion in Microbiology 2:618-623.
11.  Xu Q, Matsuyama S , Reed JC (2000) Utilization of yeast genetics to explore mammalian cell death mechanism. Methods in Enzymology 322, 283-296.
12.  Matsuyama S (1999) Advantages of the utilization of yeast genetics to study apoptosis mechanism. Experimental Medicine (Japanese article). 17, 2607-2608.
13. Matsuyama S, Llipos J, Devraux Q, Tsien R, Reed JC (2000) Changes in intramitochondrial and cytosolic pH:early events that modulate caspase activation during apoptosis. Nature Cell Biology, 2, 318-325.
14.  Nuoraini S, Six E, Matsuyama S, Krajewski S, Reed JC (2000). The putative pore forming-domain of Bax regulates mitochondrial localization and interaction with Bcl-XL. Mol Cell Biol,  20, 1604-1615.
15.  Zhang H, Huang Q, Matsuyama S, Ke N, Godzik A, and Reed JC (2000). Drosophila Pro-Apoptotic Bcl-2/Bax Homologue Reveals Evolutionary Conservation of Cell Death Mechanisms.
J Biol Chem, 275:27303-6.
16.  Matsuyama S and Reed JC (2000). Mitochondria-dependent Apoptosis and Cellular pH Regulation. (Review article) Cell Death and Differentiation, 7:1155-1165.
17.  Gu C, Sun W, Sawada M, Matsuyama S, Newman P (2003). PECAM-1 suppresses Bax-mediated apoptosis. Blood, 102, 169-179.
18. Yoshida T, Sawada M, Okuno M, Gama V, Hayes P, Matsuyama S (2004). New Bax-inhibiting peptides (BIPs) derived from mouse and rat Ku70. Biochemical and Biophysical Research Communications, 321, 961-966.
19. Gohil V, Hayes P, Matsuyama S, Schägger H, Schlame M, Greenberg M (2004). Cardiolipin biosynthesis and  mitochondrial respiratory chain function are interdependent. Journal of Biological Chemistry, 279: 42612-8.
20. Martinez JJ, Seveau S, Veiga-Chacon E, Matsuyama S, Cossart P (2005). Identification of Ku70, a component  of the DNA-dependent protein kinase, as a receptor involved in Rickettsia conorii invasion of mammalian cells. Cell, 123. 1013-1023.
21. Gama V, Yoshida T, Gomez J, Mayo L, Haas A, Matsuyama S. (2006) Involvement of ubiquitin-pathway in Ku70 decrease in response to apoptosis. Experimental Cell Research, 312: 488-99.
22. Gomez J, Gama V, Matsuyama S. Cell permeable pentapeptides derived from Bax inhibiting domain of Ku70 (2006).  “Cell Permeable Peptides” (2nd edition, edited by Ulo Langel), CRC Press, ,p.p. 469-481.
23. Sun W, Hattori N, Matsuyama S,hiota K (2006), “Proliferation related acidic leucine rich protein PAL31 functions as a caspase-3 inhibitor” Biochemical and Biophysical Research Communications, 342(3):817-23.
24. Chitamber C, Wereley JP, Kotamraju S, Matsuyama S. (2006) Induction of
Apoptosis by Galium Nitrate in Lymphoma Cells:  Involvement of Bax, mitochondria, and Proteasome Pathway. Molecular Cancer Therapeuitic, 5; 2834-43.
25. Bergom C, Gao C, Matsuyama S, Newman PJ. (2006) The Cell Surface Molecule PECAM-1 Mediates Resistance Against Chemotherapy-Induced Apoptosis., Cancer Biology and Therapy 5: 1699-707
26. Chen YN, Yamada H, Mao W, Matsuyama S, Aihara M, Araie M. (2007).
Hypoxia-Induced Retinal Ganglion Cell Death and the Neuroprotective
Effects of Beta-adrenergic Antagonists. Brain Research, in press.

Li Y, Ykota T, Yoshida T, Gama V, Sawada M, Ishikawa K, Cohen HY, Sinclair DA, Mizushima H, Matsuyama S.  Bax-inhibiting Peptide (BIP) suppresses poly-glutamine-induced cell death that induces Ku70 acetylation. submitted.

Gomez J, Yoshida T, Stapleton M, Gama V, Paddock C, Newman P, Wilcox  D, Matsuyama S. Bax inhibiting peptides derived from Ku70 suppress chemotherapy-induced cell death of megakaryocytes. submitted.