title: Noxa/Bcl-2 protein interactions contribute to bortezomib resistance in human lymphoid cells.
abstract: Previous studies have suggested that the BH3 domain of the proapoptotic Bcl-2 family member Noxa only interacts with the anti-apoptotic proteins Mcl-1 and A1 but not Bcl-2. In view of the similarity of the BH3 binding domains of these anti-apoptotic proteins as well as recent evidence that studies of isolated BH3 domains can potentially underestimate the binding between full-length Bcl-2 family members, we examined the interaction of full-length human Noxa with anti-apoptotic human Bcl-2 family members. Surface plasmon resonance using bacterially expressed proteins demonstrated that Noxa binds with mean dissociation constants (K(D)) of 3.4 nm for Mcl-1, 70 nm for Bcl-x(L), and 250 nm for wild type human Bcl-2, demonstrating selectivity but not absolute specificity of Noxa for Mcl-1. Further analysis showed that the Noxa/Bcl-2 interaction reflected binding between the Noxa BH3 domain and the Bcl-2 BH3 binding groove. Analysis of proteins expressed in vivo demonstrated that Noxa and Bcl-2 can be pulled down together from a variety of cells. Moreover, when compared with wild type Bcl-2, certain lymphoma-derived Bcl-2 mutants bound Noxa up to 20-fold more tightly in vitro, pulled down more Noxa from cells, and protected cells against killing by transfected Noxa to a greater extent. When killing by bortezomib (an agent whose cytotoxicity in Jurkat T-cell leukemia cells is dependent on Noxa) was examined, apoptosis was enhanced by the Bcl-2/Bcl-x(L) antagonist ABT-737 or by Bcl-2 down-regulation and diminished by Bcl-2 overexpression. Collectively, these observations not only establish the ability of Noxa and Bcl-2 to interact but also identify Bcl-2 overexpression as a potential mechanism of bortezomib resistance.
authors: Department of Molecular Pharmacology, Mayo Clinic, Rochester, Minnesota 55905, USA.
authors: Alyson J
authors: AJ
authors: Smith
authors: Haiming
authors: H
authors: Dai
authors: Cristina
authors: C
authors: Correia
authors: Rie
authors: R
authors: Takahashi
authors: Sun-Hee
authors: SH
authors: Lee
authors: Ingo
authors: I
authors: Schmitz
authors: Scott H
authors: SH
authors: Kaufmann
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D019337:Hematologic Neoplasms / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008214:Lymphocytes / Q000378:metabolism*; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D011719:Pyrazines / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D001897:Boronic Acids; D064549:Myeloid Cell Leukemia Sequence 1 Protein; C498702:PMAIP1 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D011719:Pyrazines; D000069286:Bortezomib