INTERNATIONAL VOLUNTEER PROGRAM
APPLICATION FORM
Name, address and telephone number of the person responsible for this proposal
Prof. Tamar Kleinberger
Dept. of Molecular Microbiology
Faculty of Medicine
Technion
1 Efron St., Bat Galim
Haifa 31096
ISRAEL
Tel. +972-4-8295257
Email: tamark@tx.technion.ac.il
Name of the laboratory and host institution
Laboratory of Molecular Virology
Dept. of Molecular Microbiology
Faculty of Medicine
Technion – Israel Institute of Technology
Title of the research project in which the volunteer would be involved
Induction of non-classical, cancer cell-specific cell death by the adenovirus E4orf4 protein
Have you already identified a candidate?
Name and email address of the candidate:
NO
Background, knowledge and level required from the applicant volunteer (including languages)
The applicant should know English. A background in Molecular Biology is an advantage. Experience in
Yeast genetics could help for part of the project.
Suitable date for
receiving the volunteer
Anytime
French Embassy in Israel
Office for Science & Technology
Rothschild Boulevard 7
Tel Aviv, 66881 Israel
Duration of the stay
Participation in the
monthly allowance
24 months
Tel: +972 (0)3 796 80 42
Fax: +972 (0)3 796 80 45
Web : http://www.fitscience.wordpress.com
Email : science@ambfr-il.org
750 euros
Description of the specific research work, which would be carried out
(Please enclose a two pages description of the project – Arial 10, Line spacing 1.5 point)
INTRODUCTION
The adenovirus E4orf4 protein induces a non-classical mode of apoptosis, which is p53- and caspaseindependent and was shown to be more efficient in cancer cells in tissue culture compared with normal cells.
Importantly, the E4orf4 cell death network is highly conserved in evolution, from yeast to mammals,
underscoring its importance to cellular regulation. Since the E4orf4-initiated pathway includes components that
respond differently to the E4orf4 signal in cancer and normal cells, and are not part of the widely studied
classical apoptotic pathways, exploiting components of this pathway as cancer drug targets could address
situations in which anti-cancer drugs currently in use are not efficient. The goals of our research are to
study the E4orf4-induced cell death network and to understand the reasons for its cancer cell
specificity, with the long-term goal of identifying novel drug targets for cancer therapy. Two subprojects are available, described below.
SUB-PROJECTS
1. Association of E4orf4 with the ATP-dependent chromatin remodeling factor ACF: characterization
and functional implications
We have recently shown that E4orf4 associates with the ATP-dependent chromatin remodeling
factor ACF that consists of a SNF2h ATPase and an Acf1 regulatory subunit. Furthermore, E4orf4 targets
protein phosphatase 2A (PP2A) to this complex and to chromatin. Obstruction of SNF2h activity inhibits
E4orf4-induced cell death, whereas knockdown of Acf1 results in enhanced E4orf4-induced toxicity in both
mammalian and yeast cells, and Acf1 overexpression inhibits E4orf4’s ability to downregulate early
adenovirus gene expression in the context of viral infection. Knockdown of the Acf1 homologue, WSTF,
inhibits E4orf4-induced cell death. Based on these results we suggest that the E4orf4-PP2A complex inhibits
ACF and facilitates enhanced chromatin remodeling activities of other SNF2h-containing complexes, such as
WSTF-SNF2h. The resulting switch in chromatin remodeling determines life vs. death decisions and
contributes to E4orf4 functions during adenovirus infection.
Brestovitsky, A., Sharf, R., Mittelman, K., and Kleinberger, T. The adenovirus E4orf4 protein targets PP2A to
the ACF chromatin-remodeling factor and induces cell death through regulation of SNF2h-containing
complexes. 2011. Nucleic Acids Res. 39, 6414-6427.
Continued investigation:
1A. We investigate E4orf4-induced changes in Acf1 and SNF2h protein complexes using proteomics
methods.
1B. We search for E4orf4-induced phosphorylation changes in the nucleus by proteomics methods.
1C. We search for E4orf4-induced changes in Acf1 and SNF2h association with chromatin by ChIP-Seq.
French Embassy in Israel
Office for Science & Technology
Rothschild Boulevard 7
Tel Aviv, 66881 Israel
Tel: +972 (0)3 796 80 42
Fax: +972 (0)3 796 80 45
Web : http://www.fitscience.wordpress.com
Email : science@ambfr-il.org
2. Association of E4orf4 with Ynd1 in yeast and mammalian cells: characterization and functional
implications
2.1. Yeast Ynd1. A genetic screen in yeast revealed that the Golgi apyrase Ynd1 associates with E4orf4 and
contributes to E4orf4-induced toxicity, independently of Ynd1 apyrase activity. Ynd1 and PP2A were shown
to contribute additively to E4orf4-induced toxicity in yeast, and to interact genetically and physically. A
mammalian orthologue of Ynd1 was shown to bind E4orf4 in mammalian cells, confirming the evolutionary
conservation of this interaction. Mutation analysis of yeast Ynd1 identified its cytosolic tail as the protein
domain required for mediation of the E4orf4 toxic signal and for the interaction with E4orf4. This cytosolic tail
is known to bind 10 membrane proteins involved in protein trafficking in the cell. We suggest that the Ynd1
cytosolic tail acts as a scaffold for protein complexes participating in protein trafficking which are targeted by
E4orf4.
Maoz, T., Koren, R., Ben-Ari, I., Kleinberger T. YND1 interacts with CDC55 and is a novel mediator of
E4orf4-induced toxicity. 2005. J Biol Chem. 280, 41270-41277.
Mittelman, K., Ziv, K., Maoz, T., and Kleinberger, T. The cytosolic tail of the Golgi apyrase Ynd1 mediates
E4orf4-induced toxicity in Saccharomyces cerevisiae. 2010. PLOS ONE, 2010 Nov 22;5(11):e15539.
2.2. Mammalian Ynd1 (Golgi UDPase). The contribution of human Ynd1 (Golgi UDPase) to E4orf4-induced
cell death has been studied. We found that E4orf4 associates with the UDPase and that overexpression of
UDPase significantly enhances E4orf4-induced cell death, but does not enhance cell death induced by an
E4orf4 mutant that does not bind PP2A. E4orf4 expression leads to increased UDPase levels in a PP2Adependent manner and dissociates high molecular weight protein complexes containing the UDPase in a
PP2A-independent manner. We conclude that the Golgi UDPase cooperates with E4orf4 to induce cell death
through PP2A-dependent and –independent mechanisms.
Avital-Shacham, M., and Kleinberger, T. The interactions between mammalian Ynd1, PP2A, and E4orf4
contribute to E4orf4-induced cell death. Manuscript in writing.
Continued investigation:
2A. We investigate the contribution of Ynd1-associating proteins in yeast to E4orf4-induced toxicity.
2B. We will use proteomics methods to identify proteins involved in the UDPase-containing high molecular
weight protein complex, which is disrupted by E4orf4.
French Embassy in Israel
Office for Science & Technology
Rothschild Boulevard 7
Tel Aviv, 66881 Israel
Tel: +972 (0)3 796 80 42
Fax: +972 (0)3 796 80 45
Web : http://www.fitscience.wordpress.com
Email : science@ambfr-il.org