The Bcl-2 protein family comprises both pro- and antiapoptotic members that control the permeabilization of the mitochondrial outer membrane, a crucial step in the modulation of apoptosis. Recent research has demonstrated that the carboxyl-terminal transmembrane domain (TMD) of some Bcl-2 protein family members can modulate apoptosis; however, the transmembrane interactome of the antiapoptotic protein Mcl-1 remains largely unexplored. Here, we demonstrate that the Mcl-1 TMD forms homooligomers in the mitochondrial membrane, competes with full-length Mcl-1 protein with regards to its antiapoptotic function, and induces cell death in a Bok-dependent manner. While the Bok TMD oligomers locate preferentially to the endoplasmic reticulum (ER), heterooligomerization between the TMDs of Mcl-1 and Bok predominantly takes place at the mitochondrial membrane. Strikingly, the coexpression of Mcl-1 and Bok TMDs produces an increase in ER mitochondrial-associated membranes, suggesting an active role of Mcl-1 in the induced mitochondrial targeting of Bok. Finally, the introduction of Mcl-1 TMD somatic mutations detected in cancer patients alters the TMD interaction pattern to provide the Mcl-1 protein with enhanced antiapoptotic activity, thereby highlighting the clinical relevance of Mcl-1 TMD interactions.

Biochemie Zentrum Heidelberg University of Heidelberg 69120 Heidelberg Germany

Departament de Bioquímica i Biologia Molecular Estructura de Recerca Interdisciplinar en Biotecnologia i Biomedicina Universitat de València E 46100 Burjassot Spain

Department of Physics University of Helsinki FI 00014 Helsinki Finland

Laboratorio de Péptidos y Proteínas Centro de Investigación Príncipe Felipe 46012 Valencia Spain

Laboratorio de Péptidos y Proteínas Centro de Investigación Príncipe Felipe 46012 Valencia Spain;

Molecular Modeling Laboratory Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 166 10 Prague 6 Czech Republic

Zobrazit více v PubMed

Kluck R. M., Bossy-Wetzel E., Green D. R., Newmeyer D. D., The release of cytochrome c from mitochondria: A primary site for Bcl-2 regulation of apoptosis. Science 275, 1132–1136 (1997). PubMed

Birkinshaw R. W., Czabotar P. E., The BCL-2 family of proteins and mitochondrial outer membrane permeabilisation. Semin. Cell Dev. Biol. 72, 152–162 (2017). PubMed

Kale J., Osterlund E. J., Andrews D. W., BCL-2 family proteins: Changing partners in the dance towards death. Cell Death Differ. 25, 65–80 (2018). PubMed PMC

Edlich F., et al. , Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol. Cell 145, 104–116 (2011). PubMed PMC

Ausman J., et al. , Ceramide-induced BOK promotes mitochondrial fission in preeclampsia. Cell Death Dis. 9, 298 (2018). PubMed PMC

Morciano G., et al. , Intersection of mitochondrial fission and fusion machinery with apoptotic pathways: Role of Mcl-1. Biol. Cell 108, 279–293 (2016). PubMed

Morciano G., et al. , Mcl-1 involvement in mitochondrial dynamics is associated with apoptotic cell death. Mol. Biol. Cell 27, 20–34 (2016). PubMed PMC

Schulman J. J., et al. , Bok regulates mitochondrial fusion and morphology. Cell Death Differ. 26, 2682–2694 (2019). PubMed PMC

Williams A., et al. , The non-apoptotic action of Bcl-xL: Regulating Ca(2+) signaling and bioenergetics at the ER-mitochondrion interface. J. Bioenerg. Biomembr. 48, 211–225 (2016). PubMed PMC

Andreu-Fernández V., et al. , Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes. Proc. Natl. Acad. Sci. U.S.A. 114, 310–315 (2017). PubMed PMC

Chang T., Glazko A. J., Effect of an adenosine deaminase inhibitor on the uptake and metabolism of arabinosyl adenine (Vidarabine) by intact human erythrocytes. Res. Commun. Chem. Pathol. Pharmacol. 14, 127–140 (1976). PubMed

Chen L., et al. , Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol. Cell 17, 393–403 (2005). PubMed

Stehle D., et al. , Contribution of BH3-domain and transmembrane-domain to the activity and interaction of the pore-forming Bcl-2 proteins Bok, Bak, and Bax. Sci. Rep. 8, 12434 (2018). PubMed PMC

Fernández-Marrero Y., Spinner S., Kaufmann T., Jost P. J., Survival control of malignant lymphocytes by anti-apoptotic MCL-1. Leukemia 30, 2152–2159 (2016). PubMed

Lin J., Fu D., Dai Y., Lin J., Xu T., Mcl-1 inhibitor suppresses tumor growth of esophageal squamous cell carcinoma in a mouse model. Oncotarget 8, 114457–114462 (2017). PubMed PMC

Sieghart W., et al. , Mcl-1 overexpression in hepatocellular carcinoma: A potential target for antisense therapy. J. Hepatol. 44, 151–157 (2006). PubMed

Campbell K. J., et al. , MCL-1 is a prognostic indicator and drug target in breast cancer. Cell Death Dis. 9, 19 (2018). PubMed PMC

Feng C., Yang F., Wang J., FBXO4 inhibits lung cancer cell survival by targeting Mcl-1 for degradation. Cancer Gene Ther. 24, 342–347 (2017). PubMed

Lee E. F., et al. , BCL-XL and MCL-1 are the key BCL-2 family proteins in melanoma cell survival. Cell Death Dis. 10, 342 (2019). PubMed PMC

Fletcher S., MCL-1 inhibitors–Where are we now (2019)? Expert Opin. Ther. Pat. 29, 909–919 (2019). PubMed

Akgul C., Moulding D. A., White M. R., Edwards S. W., In vivo localisation and stability of human Mcl-1 using green fluorescent protein (GFP) fusion proteins. FEBS Lett. 478, 72–76 (2000). PubMed

Andreu-Fernández V., et al. , Peptides derived from the transmembrane domain of Bcl-2 proteins as potential mitochondrial priming tools. ACS Chem. Biol. 9, 1799–1811 (2014). PubMed

Grau B., et al. , The role of hydrophobic matching on transmembrane helix packing in cells. Cell Stress 1, 90–106 (2017). PubMed PMC

Wassenaar T. A., et al. , High-throughput simulations of dimer and trimer assembly of membrane proteins. The DAFT approach. J. Chem. Theory Comput. 11, 2278–2291 (2015). PubMed

Lelimousin M., Limongelli V., Sansom M. S., Conformational changes in the epidermal growth factor receptor: Role of the transmembrane domain investigated by coarse-grained metaDynamics free energy calculations. J. Am. Chem. Soc. 138, 10611–10622 (2016). PubMed PMC

Lemmon M. A., et al. , Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-helices. J. Biol. Chem. 267, 7683–7689 (1992). PubMed

Mineev K. S., et al. , Dimeric structure of the transmembrane domain of glycophorin a in lipidic and detergent environments. Acta Naturae 3, 90–98 (2011). PubMed PMC

Lemmon M. A., Flanagan J. M., Treutlein H. R., Zhang J., Engelman D. M., Sequence specificity in the dimerization of transmembrane alpha-helices. Biochemistry 31, 12719–12725 (1992). PubMed

García-Sáez A. J., et al. , Peptides derived from apoptotic Bax and Bid reproduce the poration activity of the parent full-length proteins. Biophys. J. 88, 3976–3990 (2005). PubMed PMC

Berger B. W., et al. , Consensus motif for integrin transmembrane helix association. Proc. Natl. Acad. Sci. U.S.A. 107, 703–708 (2010). PubMed PMC

Gotoh T., Terada K., Oyadomari S., Mori M., hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria. Cell Death Differ. 11, 390–402 (2004). PubMed

Kerppola T. K., Bimolecular fluorescence complementation: Visualization of molecular interactions in living cells. Methods Cell Biol. 85, 431–470 (2008). PubMed PMC

Javadpour M. M., Eilers M., Groesbeek M., Smith S. O., Helix packing in polytopic membrane proteins: Role of glycine in transmembrane helix association. Biophys. J. 77, 1609–1618 (1999). PubMed PMC

Duarte J. M., Biyani N., Baskaran K., Capitani G., An analysis of oligomerization interfaces in transmembrane proteins. BMC Struct. Biol. 13, 21 (2013). PubMed PMC

Teese M. G., Langosch D., Role of GxxxG motifs in transmembrane domain interactions. Biochemistry 54, 5125–5135 (2015). PubMed

Orzáez M., Lukovic D., Abad C., Pérez-Payá E., Mingarro I., Influence of hydrophobic matching on association of model transmembrane fragments containing a minimised glycophorin A dimerisation motif. FEBS Lett. 579, 1633–1638 (2005). PubMed

Arbely E., Granot Z., Kass I., Orly J., Arkin I. T., A trimerizing GxxxG motif is uniquely inserted in the severe acute respiratory syndrome (SARS) coronavirus spike protein transmembrane domain. Biochemistry 45, 11349–11356 (2006). PubMed

Bi G., et al. , SOBIR1 requires the GxxxG dimerization motif in its transmembrane domain to form constitutive complexes with receptor-like proteins. Mol. Plant Pathol. 17, 96–107 (2016). PubMed PMC

Faingold O., Cohen T., Shai Y., A GxxxG-like motif within HIV-1 fusion peptide is critical to its immunosuppressant activity, structure, and interaction with the transmembrane domain of the T-cell receptor. J. Biol. Chem. 287, 33503–33511 (2012). PubMed PMC

Yano Y., et al. , GXXXG-mediated parallel and antiparallel dimerization of transmembrane helices and its inhibition by cholesterol: Single-pair FRET and 2D IR studies. Angew. Chem. Int. Ed. Engl. 56, 1756–1759 (2017). PubMed PMC

Pan L., et al. , Higher-order clustering of the transmembrane anchor of DR5 drives signaling. Cell 176, 1477–1489.e14 (2019). PubMed PMC

Kwon B., Lee M., Waring A. J., Hong M., Oligomeric structure and three-dimensional fold of the HIV gp41 membrane-proximal external region and transmembrane domain in phospholipid bilayers. J. Am. Chem. Soc. 140, 8246–8259 (2018). PubMed PMC

Willis S. N., et al. , Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev. 19, 1294–1305 (2005). PubMed PMC

Germain M., Duronio V., The N terminus of the anti-apoptotic BCL-2 homologue MCL-1 regulates its localization and function. J. Biol. Chem. 282, 32233–32242 (2007). PubMed

Hockings C., et al. , Mcl-1 and Bcl-xL sequestration of Bak confers differential resistance to BH3-only proteins. Cell Death Differ. 25, 721–734 (2018). PubMed PMC

Llambi F., et al. , BOK is a non-canonical BCL-2 family effector of apoptosis regulated by ER-associated degradation. Cell 165, 421–433 (2016). PubMed PMC

Javanainen M., Equilibration simulations of TM domains of Bcl-2 proteins Mcl-1, Bok, and Bax. Zenodo. 10.5281/zenodo.4012224. Deposited 2 September 2020. DOI

Javanainen M., Universal method for embedding proteins into complex lipid bilayers for molecular dynamics simulations. J. Chem. Theory Comput. 10, 2577–2582 (2014). PubMed

Marrink S. J., Tieleman D. P., Perspective on the martini model. Chem. Soc. Rev. 42, 6801–6822 (2013). PubMed

Javanainen M., Martinez-Seara H., Vattulainen I., Excessive aggregation of membrane proteins in the Martini model. PLoS One 12, e0187936 (2017). PubMed PMC

Lee J., et al. , CHARMM-GUI input generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM simulations using the CHARMM36 additive force field. J. Chem. Theory Comput. 12, 405–413 (2016). PubMed PMC

Lolicato F., Kulig W., Dimerization simulations of TM domains of Bcl-2 proteins: Mcl-1, Bok, and Bax. Zenodo. 10.5281/zenodo.4022900. Deposited 10 September 2020. DOI

Ren Z., Ren P. X., Balusu R., Yang X., Transmembrane helices tilt, bend, slide, torque, and unwind between functional states of rhodopsin. Sci. Rep. 6, 34129 (2016). PubMed PMC

Haschka M. D., Villunger A., There is something about BOK we just don’t get yet. FEBS J. 284, 708–710 (2017). PubMed

Echeverry N., et al. , Intracellular localization of the BCL-2 family member BOK and functional implications. Cell Death Differ. 20, 785–799 (2013). PubMed PMC

Einsele-Scholz S., et al. , Bok is a genuine multi-BH-domain protein that triggers apoptosis in the absence of Bax and Bak. J. Cell Sci. 129, 2213–2223 (2016). PubMed

Hsu S. Y., Kaipia A., McGee E., Lomeli M., Hsueh A. J., Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members. Proc. Natl. Acad. Sci. U.S.A. 94, 12401–12406 (1997). PubMed PMC

D’Orsi B., et al. , Bok is not pro-apoptotic but suppresses poly ADP-ribose polymerase-dependent cell death pathways and protects against excitotoxic and seizure-induced neuronal injury. J. Neurosci. 36, 4564–4578 (2016). PubMed PMC

Onyeagucha B., et al. , Novel post-transcriptional and post-translational regulation of pro-apoptotic protein BOK and anti-apoptotic protein Mcl-1 determine the fate of breast cancer cells to survive or die. Oncotarget 8, 85984–85996 (2017). PubMed PMC

Alam M. S., Proximity ligation assay (PLA). Curr. Protoc. Immunol. 123, e58 (2018). PubMed PMC

Tate J. G., et al. , COSMIC: The catalogue of somatic mutations in cancer. Nucleic Acids Res. 47, D941–D947 (2019). PubMed PMC

Shihab H. A., et al. , An integrative approach to predicting the functional effects of non-coding and coding sequence variation. Bioinformatics 31, 1536–1543 (2015). PubMed PMC

Hellmuth S., Stemmann O., Separase-triggered apoptosis enforces minimal length of mitosis. Nature 580, 542–547 (2020). PubMed

Beroukhim R., et al. , The landscape of somatic copy-number alteration across human cancers. Nature 463, 899–905 (2010). PubMed PMC

Shah K., Huang H., Bradbury N. A., Li C., White C., Mcl-1 promotes lung cancer cell migration by directly interacting with VDAC to increase mitochondrial Ca2+ uptake and reactive oxygen species generation. Cell Death Dis. 5, e1482 (2017). PubMed PMC

Song K.-A., et al. , Increased synthesis of MCL-1 protein underlies initial survival of EGFR-mutant lung cancer to EGFR inhibitors and provides a novel drug target. Clin. Cancer Res. 24, 5658–5672 (2018). PubMed

Wen Q., et al. , Elevated expression of mcl-1 inhibits apoptosis and predicts poor prognosis in patients with surgically resected non-small cell lung cancer. Diagn. Pathol. 14, 108 (2019). PubMed PMC

Hird A. W., Tron A. E., Recent advances in the development of Mcl-1 inhibitors for cancer therapy. Pharmacol. Ther. 198, 59–67 (2019). PubMed