Death-Associated Protein 3 Triggers Intrinsic Apoptosis via Miro1 Upon Inducing Intracellular Calcium Changes

Abstract

Mitochondrial homeostasis is essential for cell survival and function, necessitating quality control mechanisms to ensure a healthy mitochondrial network. Death-associated protein 3 (DAP3) serves as a subunit of the mitochondrial ribosome, playing a pivotal role in the translation of mitochondrial-encoded proteins. Apart from its involvement in protein synthesis, DAP3 has been implicated in the process of cell death and mitochondrial dynamics. In this study, we demonstrate that DAP3 mediates cell death via intrinsic apoptosis by triggering excessive mitochondrial fragmentation, loss of mitochondrial membrane potential (ΔΨm), ATP decline, and oxidative stress. Notably, DAP3 induces mitochondrial fragmentation through the Mitochondrial Rho GTPase 1 (Miro1), independently of the canonical fusion/fission machinery. Mechanistically, DAP3 promotes mitochondrial calcium accumulation through the MCU complex, leading to decreased cytosolic Ca²⁺ levels. This reduction in cytosolic Ca²⁺ is sensed by Miro1, which subsequently drives mitochondrial fragmentation. Depletion of Miro1 or MCU alleviates mitochondrial fragmentation, oxidative stress, and cell death. Collectively, our findings reveal a novel function of the mitoribosomal protein DAP3 in regulating calcium signalling and maintaining mitochondrial homeostasis.

Keywords

calcium / cell death / death-associated protein 3 / mitochondrial dynamics / reactive oxygen species

References

[1] A. Misrani, S. Tabassum, and L. Yang, “Mitochondrial Dysfunction and Oxidative Stress in Alzheimer's Disease,” Frontiers in aging neuroscience 13 (2021): 617588.

[2] F. J. Bock and S. W. G. Tait, “Mitochondria as Multifaceted Regulators of Cell Death,” Nature Reviews Molecular Cell Biology 21, no. 2 (2020): 85-100.

[3] Q. Li and T. Hoppe, “Role of Amino Acid Metabolism in Mitochondrial Homeostasis,” Frontiers in Cell and Developmental Biology 11 (2023): 1127618.

[4] F. Wang, D. Zhang, D. Zhang, P. Li, and Y. Gao, “Mitochondrial Protein Translation: Emerging Roles and Clinical Significance in Disease,” Frontiers in Cell and Developmental Biology 9 (2021): 675465.

[5] A. Cheong, R. Lingutla, and J. Mager, “Expression Analysis of Mammalian Mitochondrial Ribosomal Protein Genes,” Gene Expression Patterns 38 (2020): 119147.

[6] J. Han, O. An, H. Hong, et al., “Suppression of Adenosine-to-inosine (A-to-I) RNA Editome by Death Associated Protein 3 (DAP3) Promotes Cancer Progression,” Science Advances 6, no. 25 (2020): eaba5136.

[7] J. Han, O. An, X. Ren, et al., “Multilayered Control of Splicing Regulatory Networks by DAP3 Leads to Widespread Alternative Splicing Changes in Cancer,” Nature Communications 13, no. 1 (2022): 1793.

[8] B. J. Greber and N. Ban, “Structure and Function of the Mitochondrial Ribosome,” Annual Review of Biochemistry 85 (2016): 103-132.

[9] Z. Wei, J. Jia, G. Heng, et al., “Sirtuin-1/Mitochondrial Ribosomal Protein S5 Axis Enhances the Metabolic Flexibility of Liver Cancer Stem Cells,” Hepatology 70, no. 4 (2019): 1197-1213.

[10] S. R. Chintharlapalli, M. Jasti, S. Malladi, K. V. Parsa, R. P. Ballestero, and M. Gonzalez-Garcia, “BMRP Is a Bcl-2 Binding Protein That Induces Apoptosis,” Journal of Cellular Biochemistry 94, no. 3 (2005): 611-626.

[11] L. Sun, Y. Liu, M. Fremont, et al., “A Novel 52 kDa Protein Induces Apoptosis and Concurrently Activates c-Jun N-terminal Kinase 1 (JNK1) in Mouse C3H10T1/2 Fibroblasts,” Gene 208, no. 2 (1998): 157-166.

[12] L. Liu, C. Luo, Y. Luo, et al., “MRPL33 and Its Splicing Regulator hnRNPK Are Required for Mitochondria Function and Implicated in Tumor Progression,” Oncogene 37, no. 1 (2018): 86-94.

[13] E. Cavdar Koc, A. Ranasinghe, W. Burkhart, et al., “A New Face on Apoptosis: Death-associated Protein 3 and PDCD9 Are Mitochondrial Ribosomal Proteins,” Febs Letters 492, no. 1-2 (2001): 166-170.

[14] C. Saveanu, M. Fromont-Racine, A. Harington, F. Ricard, A. Namane, and A. Jacquier, “Identification of 12 New Yeast Mitochondrial Ribosomal Proteins Including 6 That Have no Prokaryotic Homologues,” Journal of Biological Chemistry 276, no. 19 (2001): 15861-15867.

[15] T. Suzuki, M. Terasaki, C. Takemoto-Hori, et al., “Proteomic Analysis of the Mammalian Mitochondrial Ribosome. Identification of Protein Components in the 28 S Small Subunit,” Journal of Biological Chemistry 276, no. 35 (2001): 33181-33195.

[16] A. Amunts, A. Brown, J. Toots, S. H. W. Scheres, and V. Ramakrishnan, “Ribosome. The Structure of the human Mitochondrial Ribosome,” Science 348, no. 6230 (2015): 95-98.

[17] R. K. Koripella, M. R. Sharma, P. Risteff, P. Keshavan, and R. K. Agrawal, “Structural Insights Into Unique Features of the human Mitochondrial Ribosome Recycling,” PNAS 116, no. 17 (2019): 8283-8288.

[18] J. L. Kissil, L. P. Deiss, M. Bayewitch, T. Raveh, G. Khaspekov, and A. Kimchi, “Isolation of DAP3, a Novel Mediator of Interferon-gamma-induced Cell Death,” Journal of Biological Chemistry 270, no. 46 (1995): 27932-27936.

[19] J. L. Kissil, O. Cohen, T. Raveh, and A. Kimchi, “Structure-function Analysis of an Evolutionary Conserved Protein, DAP3, Which Mediates TNF-alpha- and Fas-induced Cell Death,” Embo Journal 18, no. 2 (1999): 353-362.

[20] T. Miyazaki and J. C. Reed, “A GTP-binding Adapter Protein Couples TRAIL Receptors to Apoptosis-inducing Proteins,” Nature Immunology 2, no. 6 (2001): 493-500.

[21] T. Miyazaki, M. Shen, D. Fujikura, et al., “Functional Role of Death-associated Protein 3 (DAP3) in Anoikis,” Journal of Biological Chemistry 279, no. 43 (2004): 44667-44672.

[22] F. Broecker, R. Horton, J. Heinrich, et al., “The Intron-enriched HERV-K(HML-10) family Suppresses Apoptosis, an Indicator of Malignant Transformation,” Mob DNA 7 (2016): 25.

[23] X. Liu, R. Xi, X. Du, et al., “DNA Methylation of microRNA-365-1 Induces Apoptosis of Hair Follicle Stem Cells by Targeting DAP3,” Noncoding RNA Res 9, no. 3 (2024): 901-912.

[24] Y. Jia, Z. Li, X. Cheng, et al., “Depletion of Death-associated Protein-3 Induces Chemoresistance in Gastric Cancer Cells Through the Beta-catenin/LGR5/Bcl-2 Axis,” Journal of Investigative Medicine 67, no. 5 (2019): 856-861.

[25] D. R. Green, “The Coming Decade of Cell Death Research: Five Riddles,” Cell 177, no. 5 (2019): 1094-1107.

[26] S. Elmore, “Apoptosis: A Review of Programmed Cell Death,” Toxicologic Pathology 35, no. 4 (2007): 495-516.

[27] S. J. Riedl and G. S. Salvesen, “The Apoptosome: Signalling Platform of Cell Death,” Nature Reviews Molecular Cell Biology 8, no. 5 (2007): 405-413.

[28] S. W. Tait and D. R. Green, “Mitochondria and Cell Death: Outer Membrane Permeabilization and Beyond,” Nature Reviews Molecular Cell Biology 11, no. 9 (2010): 621-632.

[29] Z. Mukamel and A. Kimchi, “Death-associated Protein 3 Localizes to the Mitochondria and Is Involved in the Process of Mitochondrial Fragmentation During Cell Death,” Journal of Biological Chemistry 279, no. 35 (2004): 36732-36738.

[30] T. Berger, M. Brigl, J. M. Herrmann, et al., “The Apoptosis Mediator mDAP-3 Is a Novel Member of a Conserved family of Mitochondrial Proteins,” Journal of Cell Science 113 Pt 20 (2000): 3603-3612.

[31] T. Berger and M. Kretzler, “Interaction of DAP3 and FADD Only After Cellular Disruption,” Nature Immunology 3, no. 1 (2002): 3-5.

[32] T. Berger and M. Kretzler, “TRAIL-induced Apoptosis Is Independent of the Mitochondrial Apoptosis Mediator DAP3,” Biochemical and Biophysical Research Communications 297, no. 4 (2002): 880-884.

[33] D. F. Suen, K. L. Norris, and R. J. Youle, “Mitochondrial Dynamics and Apoptosis,” Genes & Development 22, no. 12 (2008): 1577-1590.

[34] L. Xiao, H. Xian, K. Y. Lee, et al., “Death-associated Protein 3 Regulates Mitochondrial-encoded Protein Synthesis and Mitochondrial Dynamics,” Journal of Biological Chemistry 290, no. 41 (2015): 24961-24974.

[35] H.-R. Kim, H.-J. Chae, M. Thomas, et al., “Mammalian dap3 Is an Essential Gene Required for Mitochondrial Homeostasis in Vivo and Contributing to the Extrinsic Pathway for Apoptosis,” Faseb Journal 21, no. 1 (2007): 188-196.

[36] H. Imamura, K. P. Nhat, H. Togawa, et al., “Visualization of ATP Levels inside Single Living Cells With Fluorescence Resonance Energy Transfer-based Genetically Encoded Indicators,” PNAS 106, no. 37 (2009): 15651-15656.

[37] I. Kotera, T. Iwasaki, H. Imamura, H. Noji, and T. Nagai, “Reversible Dimerization of Aequorea Victoria Fluorescent Proteins Increases the Dynamic Range of FRET-based Indicators,” Acs Chemical Biology 5, no. 2 (2010): 215-222.

[38] Y. Chen, Z. Zhou, and W. Min, “Mitochondria, Oxidative Stress and Innate Immunity,” Frontiers in Physiology 9 (2018): 1487.

[39] G. B. Waypa, J. D. Marks, R. Guzy, et al., “Hypoxia Triggers Subcellular Compartmental Redox Signaling in Vascular Smooth Muscle Cells,” Circulation Research 106, no. 3 (2010): 526-535.

[40] T. Ozawa, Y. Natori, Y. Sako, H. Kuroiwa, T. Kuroiwa, and Y. Umezawa, “A Minimal Peptide Sequence That Targets Fluorescent and Functional Proteins Into the Mitochondrial Intermembrane Space,” Acs Chemical Biology 2, no. 3 (2007): 176-186.

[41] G. T. Hanson, R. Aggeler, D. Oglesbee, et al., “Investigating Mitochondrial Redox Potential With Redox-sensitive Green Fluorescent Protein Indicators,” Journal of Biological Chemistry 279, no. 13 (2004): 13044-13053.

[42] H. Flores-Romero, U. Ros, and A. J. Garcia-Saez, “Pore Formation in Regulated Cell Death,” Embo Journal 39, no. 23 (2020): e105753.

[43] A. Santel, S. Frank, B. Gaume, M. Herrler, R. J. Youle, and M. T. Fuller, “Mitofusin-1 Protein Is a Generally Expressed Mediator of Mitochondrial Fusion in Mammalian Cells,” Journal of Cell Science 116 Pt 13 (2003): 2763-2774.

[44] Y. Huo, W. Sun, T. Shi, S. Gao, and M. Zhuang, “The MFN1 and MFN2 Mitofusins Promote Clustering Between Mitochondria and Peroxisomes,” Communications Biology 5, no. 1 (2022): 423.

[45] P. Huang, T. Yu, and Y. Yoon, “Mitochondrial Clustering Induced by Overexpression of the Mitochondrial Fusion Protein Mfn2 Causes Mitochondrial Dysfunction and Cell Death,” European Journal of Cell Biology 86, no. 6 (2007): 289-302.

[46] J. Han, O. An, H. Hong, et al., “Suppression of Adenosine-to-inosine (A-to-I) RNA Editome by Death Associated Protein 3 (DAP3) Promotes Cancer Progression,” Science Advances 6, no. 25 (2020): eaba5136.

[47] K. Brickley and F. A. Stephenson, “Trafficking Kinesin Protein (TRAK)-mediated Transport of Mitochondria in Axons of Hippocampal Neurons,” Journal of Biological Chemistry 286, no. 20 (2011): 18079-18092.

[48] N. Nemani, E. Carvalho, D. Tomar, et al., “MIRO-1 Determines Mitochondrial Shape Transition Upon GPCR Activation and Ca(2+) Stress,” Cell Reports 23, no. 4 (2018): 1005-1019.

[49] H. Fu, H. Zhou, X. Yu, et al., “Wounding Triggers MIRO-1 Dependent Mitochondrial Fragmentation That Accelerates Epidermal Wound Closure Through Oxidative Signaling,” Nature Communications 11, no. 1 (2020): 1050.

[50] M. Yi, D. Weaver, and G. Hajnoczky, “Control of Mitochondrial Motility and Distribution by the Calcium Signal: A Homeostatic Circuit,” Journal of Cell Biology 167, no. 4 (2004): 661-672.

[51] E. L. Eberhardt, A. V. Ludlam, Z. Tan, and M. A. Cianfrocco, “Miro: A Molecular Switch at the Center of Mitochondrial Regulation,” Protein Science 29, no. 6 (2020): 1269-1284.

[52] M. Saotome, D. Safiulina, G. Szabadkai, et al., “Bidirectional Ca2+-dependent Control of Mitochondrial Dynamics by the Miro GTPase,” PNAS 105, no. 52 (2008): 20728-20733.

[53] X. Wang and T. L. Schwarz, “The Mechanism of Ca2+ -dependent Regulation of Kinesin-mediated Mitochondrial Motility,” Cell 136, no. 1 (2009): 163-174.

[54] S. Nagdas and D. F. Kashatus, “The Interplay Between Oncogenic Signaling Networks and Mitochondrial Dynamics,” Antioxidants 6, no. 2 (2017): 33.

[55] T. Yu, J. L. Robotham, and Y. Yoon, “Increased Production of Reactive Oxygen Species in Hyperglycemic Conditions Requires Dynamic Change of Mitochondrial Morphology,” PNAS 103, no. 8 (2006): 2653-2658.

[56] V. Brillo, L. Chieregato, L. Leanza, S. Muccioli, and R. Costa, “Mitochondrial Dynamics, ROS, and Cell Signaling: A Blended Overview,” Life (Basel) 11, no. 4 (2021): 332.

[57] H. Vakifahmetoglu-Norberg, A. T. Ouchida, and E. Norberg, “The Role of Mitochondria in Metabolism and Cell Death,” Biochemical and Biophysical Research Communications 482, no. 3 (2017): 426-431.

[58] Y. Zhao, S. Araki, J. Wu, et al., “An Expanded Palette of Genetically Encoded Ca(2)(+) Indicators,” Science 333, no. 6051 (2011): 1888-1891.

[59] H. Antonicka, Z. Y. Lin, A. Janer, et al., “A High-Density Human Mitochondrial Proximity Interaction Network,” Cell metabolism 32, no. 3 (2020): 479-497. e9.

[60] J. S. Graf, S. Schorn, K. Kitzinger, et al., “Anaerobic Endosymbiont Generates Energy for Ciliate Host by Denitrification,” Nature 591, no. 7850 (2021): 445-450.

[61] V. Scaltsoyiannes, N. Corre, F. Waltz, and P. Giege, “Types and Functions of Mitoribosome-Specific Ribosomal Proteins Across Eukaryotes,” International Journal of Molecular Sciences 23, no. 7 (2022): 3474.

[62] K. Boengler, G. Heusch, and R. Schulz, “Nuclear-encoded Mitochondrial Proteins and Their Role in Cardioprotection,” Biochimica Et Biophysica Acta 1813, no. 7 (2011): 1286-1294.

[63] J. F. Allen, “Why Chloroplasts and Mitochondria Retain Their Own Genomes and Genetic Systems: Colocation for Redox Regulation of Gene Expression,” PNAS 112, no. 33 (2015): 10231-10238.

[64] P. Bjorkholm, A. Harish, E. Hagstrom, A. M. Ernst, and S. G. Andersson, “Mitochondrial Genomes Are Retained by Selective Constraints on Protein Targeting,” PNAS 112, no. 33 (2015): 10154-10161.

[65] D. B. Zorov, M. Juhaszova, and S. J. Sollott, “Mitochondrial Reactive Oxygen Species (ROS) and ROS-induced ROS Release,” Physiological Reviews 94, no. 3 (2014): 909-950.

[66] H. Kalkavan and D. R. Green, “MOMP, Cell Suicide as a BCL-2 family Business,” Cell Death and Differentiation 25, no. 1 (2018): 46-55.

[67] Y. Zhang, S. S. Su, S. Zhao, et al., “RIP1 autophosphorylation Is Promoted by Mitochondrial ROS and Is Essential for RIP3 Recruitment Into Necrosome,” Nature Communications 8 (2017): 14329.

[68] E. Giampazolias, B. Zunino, S. Dhayade, et al., “Mitochondrial Permeabilization Engages NF-kappaB-dependent Anti-tumour Activity Under Caspase Deficiency,” Nature Cell Biology 19, no. 9 (2017): 1116-1129.

[69] P. Wang, S. Y. Zheng, R. L. Jiang, et al., “Necroptosis Signaling and Mitochondrial Dysfunction Cross-talking Facilitate Cell Death Mediated by Chelerythrine in Glioma,” Free Radical Biology and Medicine 202 (2023): 76-96.

[70] R. S. Whelan, K. Konstantinidis, A. C. Wei, et al., “Bax Regulates Primary Necrosis Through Mitochondrial Dynamics,” PNAS 109, no. 17 (2012): 6566-6571.

[71] X. Liu and G. Hajnoczky, “Ca2+-dependent Regulation of Mitochondrial Dynamics by the Miro-Milton Complex,” International Journal of Biochemistry & Cell Biology 41, no. 10 (2009): 1972-1976.

[72] A. F. Macaskill, J. E. Rinholm, A. E. Twelvetrees, et al., “Miro1 is a Calcium Sensor for Glutamate Receptor-dependent Localization of Mitochondria at Synapses,” Neuron 61, no. 4 (2009): 541-555.

[73] X. Liu, D. Weaver, O. Shirihai, and G. Hajnoczky, “Mitochondrial ‘Kiss-and-run’: Interplay Between Mitochondrial Motility and Fusion-fission Dynamics,” Embo Journal 28, no. 20 (2009): 3074-3089.

[74] S. Marchi and P. Pinton, “The Mitochondrial Calcium Uniporter Complex: Molecular Components, Structure and Physiopathological Implications,” The Journal of Physiology 592, no. 5 (2014): 829-839.

[75] D. De Stefani, M. Patron, and R. Rizzuto, “Structure and Function of the Mitochondrial Calcium Uniporter Complex,” Biochimica Et Biophysica Acta 1853, no. 9 (2015): 2006-2011.

[76] G. M. Cereghetti, A. Stangherlin, O. Martins de Brito, et al., “Dephosphorylation by Calcineurin Regulates Translocation of Drp1 to Mitochondria,” PNAS 105, no. 41 (2008): 15803-15808.

[77] D. Zhu, X. Li, and Y. Tian, “Mitochondrial-to-nuclear Communication in Aging: An Epigenetic Perspective,” Trends in Biochemical Sciences 47, no. 8 (2022): 645-659.

[78] C. Jacques, A. Chevrollier, D. Loiseau, et al., “mtDNA Controls Expression of the Death Associated Protein 3,” Experimental Cell Research 312, no. 6 (2006): 737-745.

[79] Y. Murata, T. Wakoh, N. Uekawa, et al., “Death-associated Protein 3 Regulates Cellular Senescence Through Oxidative Stress Response,” Febs Letters 580, no. 26 (2006): 6093-6099.

[80] G. Kak, M. Raza, and B. K. Tiwari, “Interferon-gamma (IFN-gamma): Exploring Its Implications in Infectious Diseases,” Biomolecular Concepts 9, no. 1 (2018): 64-79.

[81] Y. Liu, R. Xu, H. Gu, et al., “Metabolic Reprogramming in Macrophage Responses,” Biomarker Research 9, no. 1 (2021): 1.

[82] H. S. Prabhakaran, D. Hu, W. He, G. Luo, and Y. C. Liou, “Mitochondrial Dysfunction and Mitophagy: Crucial Players in Burn Trauma and Wound Healing,” Burns Trauma 11 (2023): tkad029.

[83] H. Xian, Q. Yang, L. Xiao, H.-M. Shen, and Y.-C. Liou, “STX17 dynamically Regulated by Fis1 Induces Mitophagy via Hierarchical Macroautophagic Mechanism,” Nature Communications 10, no. 1 (2019): 2059.

[84] A. Chaudhry, R. Shi, and D. S. Luciani, “A Pipeline for Multidimensional Confocal Analysis of Mitochondrial Morphology, Function, and Dynamics in Pancreatic Beta-cells,” American Journal of Physiology. Endocrinology and Metabolism 318, no. 2 (2020): E87-E101.

[85] X. Lin. Functional Studies of DAP3 In Mediating Mitochondrial Dynamics And Cell Death. 2013.

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