BH3 Peptides Induce Mitochondrial Fission and Cell Death Independent of BAX/BAK Emelyn H. Shroff1, Colleen M. Snyder1, G. R. Scott Budinger1,3, Manu Jain1,3, Teng-Leong Chew2,3, Satya Khuon2,3, Harris Perlman1, Navdeep S. Chandel1,3* 1 Department of Medicine, Northwestern University Medical School, Chicago, Illinois, United States of America, 2 Cell imaging facility, Northwestern University Medical School, Chicago, Illinois, United States of America, 3 Department of Cell and Molecular Biology Northwestern University Medical School, Chicago, Illinois, United States of America Abstract BH3 only proteins trigger cell death by interacting with pro- and anti-apoptotic members of the BCL-2 family of proteins. Here we report that BH3 peptides corresponding to the death domain of BH3-only proteins, which bind all the pro-survival BCL-2 family proteins, induce cell death in the absence of BAX and BAK. The BH3 peptides did not cause the release of cytochrome c from isolated mitochondria or from mitochondria in cells. However, the BH3 peptides did cause a decrease in mitochondrial membrane potential but did not induce the opening of the mitochondrial permeability transition pore. Interestingly, the BH3 peptides induced mitochondria to undergo fission in the absence of BAX and BAK. The binding of BCL-XL with dynamin-related protein 1 (DRP1), a GTPase known to regulate mitochondrial fission, increased in the presence of BH3 peptides. These results suggest that pro-survival BCL-2 proteins regulate mitochondrial fission and cell death in the absence of BAX and BAK. Citation: Shroff EH, Snyder CM, Budinger GRS, Jain M, Chew T-L, et al. (2009) BH3 Peptides Induce Mitochondrial Fission and Cell Death Independent of BAX/ BAK. PLoS ONE 4(5): e5646. doi:10.1371/journal.pone.0005646 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received February 9, 2009; Accepted April 18, 2009; Published May 21, 2009 Copyright: 2009 Shroff et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is supported in part by National Institutes of Health Grants (HL071643-06A1) to NSC. E.S. is supported by a fellowship from the American Heart Association Grant 0815606G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nav@northwestern.edu Introduction The BCL-2 family of proteins are critical regulators in the process of cell death [1]. Characterized by their conserved BCL-2 Homology (BH) domains, these proteins are divided into groups that have pro-survival and pro-apoptotic functions. The pro- apoptotic group is divided into the single BH3 domain containing proteins and the multi BH3 domains proteins, BAX and BAK [2]. BH3 only proteins activate BAX and BAK to induce the permeabilization of the outer mitochondrial membrane (MOMP), and the subsequent release of cytochrome c [3]. In addition to the physical changes required for the release of cytochrome c, a remarkable alteration during the process of cell death is that the interconnected mitochondria network undergoes morphological changes from long tubular, to short punctiform structures [4]. The short punctated structures reflect fragmentation of the mitochon- dria, a process referred to as mitochondrial fission. The mammalian fission machinery to date consists of DRP1, FIS1 and OPA-1. FIS1 is distributed throughout the outer mitochondrial membrane and Dynamin related protein-1 (DRP- 1) is localized in the cytosol [5]. DRP1 and FIS1 control the fission of outer mitochondrial membrane while OPA1 regulates fusion of the inner membrane. DRP1 translocate and co-localizes with BAX on the surface of the outer mitochondrial membrane during early stages of apoptosis [6]. DRP1 inhibition delays or partially inhibits cell death suggesting a role for mitochondrial fission in cell death [7,8]. Furthermore, a small molecule that inhibits DRP-1 prevents BH3-only protein dependent cytochrome c release in isolated mitochondria [9]. In contrast other reports indicate that the downregulaton of hFIS1 or DRP1 prevents mitochondrial fission but not BAX/BAK dependent cell death during apoptosis [10]. The overexpression of hFIS1 does not induce death in cells devoid of BAX and BAK [11]. However, these cells still undergo mitochondrial fragmentation indicating that mitochondrial fission is distinct from BAX/BAK dependent cell death. Recent studies further support that fission and cytochrome c induced cell death are discrete events [12]. Nevertheless, OPA-1 mediated cristae remodeling is required for cytochrome c release [13]. Thus, the role of the fission machinery in cell death is complex [14]. Mitochondrial fission is an evolutionarily conserved process and has been suggested to regulate cell death in C. elegans [15]. Interestingly, in this model organism the multi BH3 proteins BAX and BAK are not present, and cytochrome c release does not play a role in initiating cell death [16]. Rather, binding of the BCL-2 like pro-survival protein CED-9, by a BH3-only protein EGL-1, is the key initiation event in the apoptotic pathway, and induces mitochondrial fission [15]. The overexpression of a dominant negative DRP-1 mutant increased survival of cells in this model. However, recent evidence suggests that the role of DRP1 in cell death is distinct from its role in mitochondrial fission in C. elegans [17]. Given the fact that BH3 proteins binding to prosurvival BCL-2 proteins can regulate both mitochondrial fission and cell death in C. elegans, here we have uncovered that negating the pro- survival proteins by BH3 peptides can also lead to mitochondrial fission and cell death in a mammalian system devoid of BAX and BAK. PLoS ONE | www.plosone.org 1 May 2009 | Volume 4 | Issue 5 | e5646