טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentEdison-Tkach Natalia
SubjectIdentification of ARTS Interacting Proteins
Emphasis on BCl-2 Family Members
DepartmentDepartment of Medicine
Supervisor Ms. Sarit Larisch
Full Thesis textFull thesis text - English Version


Abstract

ARTS (Sept4_i2) is a mitochondrial pro-apoptotic protein, which promotes caspase activation through binding and degradation of X-linked Inhibitor of Apoptosis (XIAP) protein. The anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein protects against cell death.

We have found that ARTS and Bcl-2 reside at the mitochondrial outer membrane (MOM) and bind to each other directly. Upon apoptotic induction, ARTS, Bcl-2 and XIAP form a complex, with ARTS acting as a scaffold to bridge between Bcl-2 and XIAP. This induced proximity allows XIAP to function as an E3-ligase promoting degradation of Bcl-2 and initiating apoptosis. Bcl-2 degradation is ARTS-dependent as ubiquitination of Bcl-2 is significantly inhibited in ARTS knock-down (ARTS KD) cells, and levels of Bcl-2 remain high upon STS and Etoposide treatment in these cells.

Following apoptotic induction, ARTS translocates to the cytosol prior to MOM permeabilization (MOMP) and the release of cytochrome c and Smac/Diablo from mitochondria, and is required for that. ARTS-induced caspase activation leads to a cleavage of the pro-apoptotic Bcl-2 family protein Bid, known to promote MOMP. We propose that translocation of ARTS initiates a first wave of caspase activation, leading to the subsequent release of additional mitochondrial factors, which amplifies the caspase cascade and results in final cell death.

XIAP regulates the levels of ARTS by serving as its ubiquitin-ligase, thereby providing a potential feedback mechanism to protect against unwanted apoptosis. We showed that ARTS is directly ubiquitinated by XIAP, and identified the Lysine residue at position 3 in ARTS to be essential for its ubiquitination by XIAP.

Unlike other IAP-antagonists, ARTS does not contain a canonical IAP-Binding-Motif (IBM). Instead, ARTS binds to XIAP via a unique sequence termed ARTS-IBM (AIBM). AIBM alone can bind to BIR3 domain in XIAP and promote apoptosis. AIBM-derived peptides of nine residues can penetrate cancer cells without addition of any penetration sequences. These peptides are sufficient for binding and inducing XIAP degradation. Moreover, these peptides could promote caspase activation and apoptosis in various cancer cells.

Bcl-2 and XIAP are frequently over-expressed in tumors and have become promising targets for developing anti-cancer drugs. We have found that ARTS acts as an XIAP- as well as a new, non-BH3, Bcl-2-antagonist. This provides a proof-of-concept for the feasibility of developing novel ARTS-based anti-cancer therapeutics against cancers with high levels of XIAP and/or Bcl-2.