Noxa/Bcl-2 Protein Interactions Contribute to Bortezomib Resistance in Human Lymphoid Cells

Haiming Dai,Ingo Schmitz,Scott H. Kaufmann,Cristina Correia,Alyson J. Smith,Rie Takahashi,Sun-Hee Lee

doi:10.1074/jbc.m110.189092

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.

Highlights

Regression of sensitive tumors such as lymphomas after effective chemotherapy is thought to reflect the induction of apoptosis [1]
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
In this study we demonstrate that the anti-apoptotic protein Bcl-2 and the proapoptotic BH3-only protein Noxa interact under cell-free conditions, identify the domains responsible for this interaction, and show that lymphoma-associated Bcl-2 sequence variants have up to 20-fold higher affinity for Noxa

Summary

Introduction

Regression of sensitive tumors such as lymphomas after effective chemotherapy is thought to reflect the induction of apoptosis [1]. 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. To assess whether Noxa was bound to Bcl-2 during bortezomib treatment, S peptide-tagged Bcl-2 was affinity-purified after transient expression in Jurkat cells.

Results

Conclusion