Shigemi Matsuyama, PhD

Associate Professor
Department of Medicine
School of Medicine
Associate Professor
Department of Pathology
School of Medicine
Assistant Professor
Department of Pharmacology
School of Medicine
Molecular Oncology Program
Case Comprehensive Cancer Center

Research Information

Research Interests

  • Cancer Cell Biology
  • Cell Death Regulation
  • Cell Penetrating Peptide

Research Projects

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.