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Development of Cell Penetrating Bax Inhibiting Peptides (BIP)

Gomez, Jose A.
http://rave.ohiolink.edu/etdc/view?acc_num=case1252025677

Abstract Details

2010, Doctor of Philosophy, Case Western Reserve University, Pharmacology.
Bax is a pro-apoptotic protein that mediates intrinsic cell-death signaling. Using a yeast-based functional screening approach, interferon gamma receptor beta chain (IFNγR2) and the DNA repair protein Ku70 were identified as Bax suppressors. IFNγR2 is a component of the IFNγ receptor complex along with the IFNγR alpha chain (IFNγR1). Upon IFNγ binding, a conformational change in the receptor complex occurs that activates the Jak2/STAT1 signaling cascade. The Bax binding domain is located in the C-terminal region IFNγR2296-337. This portion does not contain the Jak2-binding domain; therefore, the antiapoptotic function of IFNγR2 is independent of JAK/STAT signaling. IFNγR2296-337 rescued human cells from apoptosis induced by Bax overexpression but not Bak. IFNγR2 inhibited apoptosis induced by the BH3-only protein Bim-EL, suggesting that IFNγR2 inhibits Bax activation induced by a BH3-only protein. Bax and IFNγR2 were co-immunoprecipitated from cell lysates prepared from HEK293 and DAMI cells. Addition of recombinant Bcl-2 protein to cell lysates significantly reduced the interaction of IFNγR2 and Bax, suggesting that Bcl-2 and IFNγR2 bind a similar domain of Bax. The C-terminal fragment (cytoplasmic domain) of IFNγR2 is expressed in human cancer cell lines of megakaryocytic cancer (DAMI), breast cancer (MDA-MD-468), and prostate cancer (PC3 cells), this fragment may confer cancer cells resistance to apoptotic stresses. From the Bax binding domain of Ku70 and IFNγR2 a series of cell penetrating cytoprotective penta-peptides were developed, these peptide are known as Bax Inhibiting Peptides (BIPs), e.g. VPMLK, VPTLK, and VPALR. Another cell penetrating peptide KLPVM that does not inhibit Bax-mediated apoptosis was also developed as a control. All the five amino acids cell permeable peptides (whether they inhibit Bax or not) were named: cell penetrating penta-peptides (CPP5s). The CPP5s VPTLK and KLVPM penetrated in mammalian cells in a dose dependent manner, and did not show any significant toxicity. One of the promising applications of cell penetrating peptides with low cytotoxic effects is the ability to transduce proteins into mammalian cells; both VPTLK and KLPVM delivered the site-specific DNA recombinase Cre into mammalian cells, indicating that CPP5s may be an effective method for therapeutic delivery in human cells.
Shigemi Matsuyma, PhD (Advisor)
Anthony Berdis, PhD (Committee Chair)
Yu-Chung Yang, PhD (Committee Member)
George Stark, PhD (Committee Member)
189 p.
Biomedical Research; Cellular Biology; Pharmacology
Interferon gamma receptor beta chain (IFN&947; R2); Bcl-2 associated protein X (Bax), Apoptosis; Cytosolic IFN&947; R2; antiapoptotic protein; Bcl-2 family of proteins; Interferon gamma (IFN&947; ); Cell Penetrating Pepta-Peptides (CPP5)

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Citations

  • Gomez, J. A. (2010). Development of Cell Penetrating Bax Inhibiting Peptides (BIP) [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1252025677

    APA Style (7th edition)

  • Gomez, Jose. Development of Cell Penetrating Bax Inhibiting Peptides (BIP). 2010. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1252025677.

    MLA Style (8th edition)

  • Gomez, Jose. "Development of Cell Penetrating Bax Inhibiting Peptides (BIP)." Doctoral dissertation, Case Western Reserve University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1252025677

    Chicago Manual of Style (17th edition)

case1252025677
490
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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.