Conformational change of adenine nucleotide translocase‐1 mediates cisplatin resistance induced by EBV‐LMP1

Article9 November 2021Open Access Source DataTransparent process Conformational change of adenine nucleotide translocase-1 mediates cisplatin resistance induced by EBV-LMP1 Lin Zhao Lin Zhao Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Xiangying Deng Xiangying Deng Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Yueshuo Li Yueshuo Li orcid.org/0000-0002-1680-915X Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Jianmin Hu Jianmin Hu Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Longlong Xie Longlong Xie Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Feng Shi Feng Shi Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Min Tang Min Tang orcid.org/0000-0002-3381-7207 Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Ann M Bode Ann M Bode The Hormel Institute, University of Minnesota, Austin, MN, USA Search for more papers by this author Xin Zhang Xin Zhang Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Weihua Liao Weihua Liao Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Ya Cao Corresponding Author Ya Cao [email protected] orcid.org/0000-0002-3558-3336 Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Molecular Imaging Research Center of Central, South University, Changsha, China Research Center for Technologies of Nucleic Acid-Based Diagnostics and Therapeutics Hunan Province, Changsha, China National Joint Engineering Research Center for Genetic Diagnostics of Infectious Diseases and Cancer, Changsha, China Search for more papers by this author Lin Zhao Lin Zhao Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Xiangying Deng Xiangying Deng Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Yueshuo Li Yueshuo Li orcid.org/0000-0002-1680-915X Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Jianmin Hu Jianmin Hu Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Longlong Xie Longlong Xie Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Feng Shi Feng Shi Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Min Tang Min Tang orcid.org/0000-0002-3381-7207 Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Search for more papers by this author Ann M Bode Ann M Bode The Hormel Institute, University of Minnesota, Austin, MN, USA Search for more papers by this author Xin Zhang Xin Zhang Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Weihua Liao Weihua Liao Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Ya Cao Corresponding Author Ya Cao [email protected] orcid.org/0000-0002-3558-3336 Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China Molecular Imaging Research Center of Central, South University, Changsha, China Research Center for Technologies of Nucleic Acid-Based Diagnostics and Therapeutics Hunan Province, Changsha, China National Joint Engineering Research Center for Genetic Diagnostics of Infectious Diseases and Cancer, Changsha, China Search for more papers by this author Author Information Lin Zhao1,2,3, Xiangying Deng1,2,3, Yueshuo Li1,2,3, Jianmin Hu1,2,3, Longlong Xie1,2,3, Feng Shi1,2,3, Min Tang1,2,3, Ann M Bode7, Xin Zhang8, Weihua Liao9 and Ya Cao *,1,2,3,4,5,6 1Key Laboratory of Carcinogenesis and Cancer Invasion, Chinese Ministry of Education, Department of Radiology, Xiangya Hospital, Central South University, Changsha, China 2Cancer Research Institute and School of Basic Medical Science, Xiangya School of Medicine, Central South University, Changsha, China 3Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, China 4Molecular Imaging Research Center of Central, South University, Changsha, China 5Research Center for Technologies of Nucleic Acid-Based Diagnostics and Therapeutics Hunan Province, Changsha, China 6National Joint Engineering Research Center for Genetic Diagnostics of Infectious Diseases and Cancer, Changsha, China 7The Hormel Institute, University of Minnesota, Austin, MN, USA 8Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha, China 9Department of Radiology, Xiangya Hospital, Central South University, Changsha, China *Corresponding author. Tel: +86 0731 84805448; E-mail: [email protected] EMBO Mol Med (2021)13:e14072https://doi.org/10.15252/emmm.202114072 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Adenine nucleotide translocase-1 (ANT1) is an ADP/ATP transporter protein located in the inner mitochondrial membrane. ANT1 is involved not only in the processes of ADP/ATP exchange but also in the composition of the mitochondrial membrane permeability transition pore (mPTP); and the function of ANT1 is closely related to its own conformational changes. Notably, various viral proteins can interact directly with ANT1 to influence mitochondrial membrane potential by regulating the opening of mPTP, thereby affecting tumor cell fate. The Epstein–Barr virus (EBV) encodes the key tumorigenic protein, latent membrane protein 1 (LMP1), which plays a pivotal role in promoting therapeutic resistance in related tumors. In our study, we identified a novel mechanism for EBV-LMP1-induced alteration of ANT1 conformation in cisplatin resistance in nasopharyngeal carcinoma. Here, we found that EBV-LMP1 localizes to the inner mitochondrial membrane and inhibits the opening of mPTP by binding to ANT1, thereby favoring tumor cell survival and drug resistance. The ANT1 conformational inhibitor carboxyatractyloside (CATR) in combination with cisplatin improved the chemosensitivity of EBV-LMP1-positive cells. This finding confirms that ANT1 is a novel therapeutic target for overcoming cisplatin resistance in the future. SYNOPSIS EBV-encoded tumorigenic protein LMP1 regulates conformational changes of mitochondrial protein adenine nucleotide translocase-1 (ANT1), and thereby chemoresistance of tumor cells. Direct interaction of the EBV-encoded tumorigenic protein LMP1 with ANT1 in the mitochondria induced its conformational change. EBV-LMP1 fixed ANT1 conformation in the m-state, increased mitochondrial membrane potential, inhibited mitochondrial ADP/ATP exchange rate and decreased oxidative phosphorylation levels, which led to increased NPC cell viability. Carboxyatractyloside (CATR) targeted conformational changes of ANT1 and enhanced the sensitivity of tumor cells to cisplatin. The paper explained Problem The Epstein–Barr virus (EBV) encodes the key oncogenic protein LMP1, which plays an important role in promoting resistance to therapy. In this study, we explored novel targets and potential mechanisms by which EBV-LMP1 regulates resistance to cisplatin in nasopharyngeal carcinoma (NPC). Results We found that EBV-LMP1 can localize to the mitochondria to bind directly to adenine nucleotide translocase-1 (ANT1), fixing the ANT1 conformation in the m-state, thereby increasing the mitochondrial membrane potential and promoting the viability of NPC cells. Carboxyatractyloside (CATR), a conformational inhibitor of ANT1, which contributes to mPTP opening and cell death, enhanced the sensitivity of tumor cells to cisplatin. Impact Our study links for the first time ANT1 conformational changes to cisplatin chemosensitivity, highlighting the importance of protein conformational changes in tumor chemotherapy. Introduction Adenine nucleotide translocase-1 (ANT1) is a mitochondrial inner membrane protein located in the mitochondria and is responsible for mitochondrial ADP/ATP transport (Parodi-Rullaán et al, 2019; Ruprecht & Kunji, 2020). A recent study confirms that ANT1 is a key protein driving mitochondrial autophagy (Hoshino et al, 2019), while ANT is involved in proton transport in the presence of fatty acids (Bertholet et al, 2019). Adenine nucleotide translocase-1 is also a major component of the mitochondrial membrane permeability transition pore (mPTP) (Bround et al, 2020). Protein complexes of ANT with VDAC and cyclophilin D play a crucial role in maintaining the mitochondrial membrane potential (Δψm) and permeability (Bertholet et al, 2019; Ruprecht et al, 2019). Despite extensive work, the molecular composition of mPTP is currently not fully understood and remains an area of debate, with ANT and F0F1-ATP synthase being the main contenders for its components (Brustovetsky, 2020). ANT has two different conformations: When ANT1 is facing the mitochondrial matrix, it transports ATP to release ADP, which is in the m-state, and when ANT1 is facing the cytoplasmic side, it transports ADP to release ATP, which is in the c-state; ANT achieves mitochondrial energy conversion through a cyclic state transition, the inhibitors CATR and bongkrekic acid (BKA) fix ANT1 in the c-state and m-state, respectively (Bertholet et al, 2019; Ruprecht et al, 2019; Ruprecht & Kunji, 2020). The c-state of ANT has been found to be an important condition for mPTP opening, suggesting that transitions between conformations of ANT are not only critical for mitochondrial synthesis of ATP but also key mechanisms for maintaining the fate of cancer cells (Novgorodov et al, 1992; Halestrap & Brenner, 2003; Ruprecht et al, 2019; Zhao et al, 2021). Several viral proteins have been shown to interact directly with ANT1 to regulate mPTP function. For example, the cytomegalovirus protein (vMIA) can inhibit the opening of mPTP by interacting with ANTs, and the human immunodeficiency virus-1 (HIV-1) encoding viral protein R (Vpr) can bind to ANTs to induce mPTP-driven apoptosis (Jacotot et al, 2001; Vieira et al, 2001; Sabbah et al, 2006; Tanaka et al, 2008; Green et al, 2011; Wang et al, 2017). The Epstein–Barr virus (EBV) is the first DNA virus determined to be tumorigenic and persistently infectious in humans, resulting in approximately 95% of the world's asymptomatic infections. It is associated with multiple cancers, including nasopharyngeal carcinoma (NPC) (Chou & Talalay, 1983; Lieberman, 2014; Young et al, 2016; Chen et al, 2019). EBV encodes the key oncogenic protein, latent membrane protein 1 (LMP1), which plays a central role in promoting therapeutic resistance in related tumors (Shair et al, 2018; Shi et al, 2019; Xie et al, 2020). Cisplatin is the most important and classic first-line synchronous chemotherapeutic agent for the treatment of nasopharyngeal carcinoma; and EBV-LMP1 regulates cellular cisplatin resistance through multiple signaling pathways (Tang et al, 2018). Inhibition of AKT after LMP1 knockdown enhances tumor cell sensitivity to cisplatin; and LMP1 regulates mitochondrial dynamic protein-related protein 1 (Drp1) to promote NPC cell survival and cisplatin resistance (Mei et al, 2007; Xie et al, 2020). In addition, the pivotal mitochondrial protein ANT1 showed increased sensitivity to cisplatin after knockdown in non-small cell lung cancer (Tajeddine et al, 2008). These findings suggest that ANT1 may be an important cause of LMP1-triggered tumor drug resistance, which is not yet fully understood. Our findings provide novel insights into viral protein regulation of ANT1 conformational changes affecting mitochondrial function and tumor drug resistance. Here, we show for the first time that EBV-LMP1 localizes to the inner mitochondrial membrane to directly interact with ANT1, resulting in a block of the mPTP opening and elevated mitochondrial membrane potential, thereby increasing tumor cell viability. The specific mechanism involves an inhibition of the conformational change of ANT1 by EBV-LMP1, which decreases the transport activity of ANT1 and attenuates the formation of the ANT1-VDAC1 complex. In addition, the ANT1 conformation inhibitor, CATR, reversed this process, resulting in a significant increase in the sensitivity of NPC cells to cisplatin. Thus, a molecular link between LMP1 and cisplatin resistance was established in which the inhibition of ANT1 conformation by EBV-LMP1 was a determinant of reduced cisplatin sensitivity in tumor cells. Results EBV-LMP1 inhibits mPTP opening to increase the mitochondrial membrane potential (Δψm) To understand the differences in membrane potential in LMP1-negative or LMP1-positive NPC cells, two different Δψm sensitive probes, TMRM (tetramethyl rhodamine methyl ester) and JC-1, were used. Two sets of NPC cells, CNE1/CNE1-LMP1 and HK1/HK1-LMP1, were stained with TMRM and then observed by laser confocal microscopy to show that the fluorescence intensity of CNE1-LMP1 and HK1-LMP1 cells was significantly increased compared with CNE1 and HK1 parental cells (Fig 1A). The JC-1 assessment further confirmed that the mitochondrial membrane potential of LMP1-positive NPC cells increased substantially; and the difference was statistically significant (Fig 1B). Figure 1. EBV-LMP1 inhibits mPTP opening and increases Δψm Confocal observation of mitochondrial potential after TMRM staining of LMP1-negative or LMP1-positive NPC cells. Red fluorescence, which indicates normal mitochondrial potential, was converted into green fluorescence after a reduction in mitochondrial potential. Images were analyzed using ImageJ software (scale bar, 10 μm). Data are presented as means ± SEM (paired t-test, n = 6, biological replicates per group, *P < 0.05, **P < 0.01). The mitochondrial potential was detected by JC-1 staining. Data are presented as means ± SEM (paired t-test, n = 3, biological replicates per group, **P < 0.01). Extent of Ca2+-mediated mitochondrial swelling in NPC cells. Data are presented as means ± SEM (paired t-test, n = 6, biological replicates per group, **P < 0.01). The extent of mPTP opening in nasopharyngeal carcinoma cells (scale bar, 20 μm). Calcimycin (calcium ionophore) served as a positive control. Images were analyzed using ImageJ software. Data are presented as means ± SEM (paired t-test, n = 6, biological replicates per group, **P < 0.01). Download figure Download PowerPoint Under normal physiological conditions, the mPTP allows free passage of solutes ≤ 1.5 KD; but in some pathological conditions the mPTP opens, mitochondria undergo swelling, and the Δψm decreases, eventually leading to cell death (Bonora et al, 2015; Fricker et al, 2018). Therefore, an analysis of the degree of mitochondrial swelling could be important in examining changes in the mitochondrial membrane potential. Next, we investigated the effect of EBV-LMP1 on Ca2+-mediated mitochondrial swelling by extracting mitochondria from the two sets of NPC cells separately. The mitochondrial swelling rates of CNE-LMP1 (7%) and HK1-LMP1 (6%) cells were significantly lower than those of CNE1 (27%) and HK1 (23%) cells, suggesting that EBV-LMP1 decreases Ca2+-mediated mitochondrial swelling and increases the mitochondrial membrane potential (Fig 1C). We further used the calcein-AM probe to detect the opening of mPTP in the two sets of NPC cells. The reduced fluorescence compared to the initial fluorescence amount represents the degree of mPTP openness (Fig 1D). In these two sets of NPC cells, the openness of mPTP in LMP1-positive NPC cells was significantly reduced compared to the control group. The above results suggest that EBV-LMP1 increases the mitochondrial potential by inhibiting mPTP opening. The main components of mPTP consist of VDAC1/2 at the outer membrane, ANT1/2/3 at the inner membrane, and CypD at the matrix (Wang et al, 2014). We first examined the efficiency of VDAC1/2, ANT1/2, and CypD knockdown (Fig EV1A). Subsequently, we compared the differences in cell membrane potential changes: after VDAC2/ANT2/3 and CypD knockdown, the membrane potential of CNE1-LMP1 and HK1-LMP1 cells was significantly higher than that of CNE1 and HK1 (Fig EV1B). In contrast, no statistically significant difference was observed between VDAC1 and ANT1 knockdown (Fig EV1B). These results suggest that VDAC1 and ANT1 affect the regulation of mitochondrial membrane potential by LMP1, which in turn affects mPTP opening. We confirmed the specificity of ANT1 antibody by both prokaryotic and eukaryotic methods: First, we overexpressed and knocked down ANT1 in HK1 cells and then detected ANT1 expression by WB (Fig EV1C); second, we expressed HK1 cell-derived ANT1 in prokaryotic cells, purified it, and detected it by ANT1 antibody (Fig EV1D). Click here to expand this figure. Figure EV1. EBV-LMP1 interacting with ANT1 localizes to mitochondria A. Knockdown effect of mPTP complex components. B. Effect of knockdown of each mPTP component on the membrane potential of EBV-LMP1-negative or EBV-LMP1-positive nasopharyngeal carcinoma cells. Data are presented as means ± SEM (paired t-test, n = 5, biological replicates per group, *P < 0.05, **P < 0.01). C, D. The specificity of ANT1 antibody was verified in this experiment using: knockdown or overexpression of ANT1 in HK1 cells, respectively (C); expression of HK1-derived ANT1 in prokaryotes and validation after purification (D). E. LMP1 is present in the cytoplasm and mitochondria. EBV-LMP1-positive nasopharyngeal carcinoma cells were isolated from cytoplasm and mitochondria and western blot assayed for LMP1 expression. F. Co-IP detection of the interaction of EBV-LMP1 and ANT1 in CNE1-LMP1 cells. G. Laser confocal analysis of CNE1-LMP1 cells revealed the presence of co-localization of LMP1 with ANT1 (scale bar, 5 μm). The quantified graph on the lower right shows the percentage of yellow fluorescence (merge) to red fluorescence (ANT1) in CNE1-LMP1 cells. Data are presented as means ± SEM (n = 6, biological replicates per group). H. PLA detects the presence of direct binding of LMP1 to ANT1, but not VDAC1, in CNE1-LMP1 cells (scale bar, 10 μm). Download figure Download PowerPoint EBV-LMP1 localizes mitochondrial endosomes to interact with ANT1 The previous section showed that the difference in mPTP opening leads to different membrane potentials in LMP1-positive cells. The question is why would this difference be caused in LMP1-positive tumor cells and does LMP1 cause this change by directly regulating ANT1 or VDAC1. To answer these questions, we first needed to determine whether LMP1 is present in mitochondria. We isolated cytosolic and mitochondrial proteins and Western blot results confirmed that LMP1 is present in both cytoplasm and mitochondria (Fig EV1E). Using proteinase K to remove the outer mitochondrial membrane (OMM) protein prevented LMP1 from remaining on the OMM (Fig 2A). Further separation of the mitochondrial membrane and matrix revealed that LMP1 was mainly present on the inner mitochondrial membrane (Fig 2A). Figure 2. LMP1 localizes to the mitochondrial inner membrane and interacts with ANT1 in NPC cells LMP1 is localized to the inner mitochondrial membrane. Isolated mitochondria were incubated with proteinase K (40 μM) for 30 min before LMP1 expression was detected (left); the mitochondria were further subjected to submitochondrial fractionation. VDAC1, TIM23, and HSP60 were used to represent MOM, MIM, and mitochondrial matrix protein, respectively (right). The interaction of LMP1 and endogenous ANT1 was analyzed by an immunofluorescence confocal assay. HK1-LMP1 cells were immunostained with anti-LMP1 (green) and anti-ANT1 (red) antibodies and then subjected to confocal microscopy, and use ImageJ to calculate the co-location level of both. The nuclei were stained with DAPI (scale bar, 5 μm). The quantified graph on the top right shows the percentage of yellow fluorescence (merge) to red fluorescence (ANT1) in HK1-LMP1 cells. Data are presented as means ± SEM (n = 6, biological replicates per group). IP/IB analysis was used to detect the interaction of LMP1 with endogenous ANT1 in CNE1-LMP1-expressing cells. Detection of the LMP1-ANT1 interaction using a proximity ligation assay. Red fluorescence corresponds to the PLA-positive signal and blue fluorescence corresponds to nuclei (DAPI staining; scale bar, 10 μm). LMP1 interaction with ANT1 in vitro. GST-ANT1 expressed in bacteria was incubated with a Flag-LMP1 protein and the proteins were subjected to Western blot analysis. ANT1 interaction with LMP1 is dependent on its domain 2 in 293T cells. ANT1 and LMP1 domain architecture and schematic representation of its respective deletion constructs used in this study in 293T cells. IP/IB was used to detect the interaction between Flag-tagged LMP1 and Myc-tagged ANT1 mutants. IP of cell lysates using a Flag or Myc antibody. LMP1 binds directly to ANT1 dependent on its transmembrane domain in 293T cells. IP/IB was used to detect the interaction between Flag-tagged LMP1 mutants and Myc-tagged ANT1. IP of cell lysates using a Flag or Myc antibody. Download figure Download PowerPoint We further determined whether LMP1 localizes to the inner mitochondrial membrane and interacts directly with ANT1. We observed the presence of co-localization between LMP1 and ANT1 in HK1-LMP1 and CNE1-LMP1cells (Figs 2B and EV1G). EBV-LMP1 binding to ANT1 was further confirmed by immunoprecipitation (Figs 2C and EV1F). In addition, the existence of direct binding of EBV-LMP1 to ANT1 was confirmed by an in vitro pull-down assay and prokaryotic expression of GST-ANT1 (Fig 2E). We next performed an in situ proximity ligation assay (PLA) in LMP1-positive NPC cells transiently co-express Flag-LMP1 and Myc-ANT1 or Myc-VDAC1. Similar to the above results, there was a positive fluorescent signal observed in cells co-expressing Flag-LMP1 and Myc-ANT1, while such a signal was not observed in cells co-expressing Flag-LMP1 and Myc-VDAC1 (Figs 2D and EV1H), indicating a specific interaction between LMP1 and ANT1 in this cell compartment. The EBV-LMP1 protein consists of 386 amino acid residues and includes the N-terminal, six transmembrane domains, and the C-terminal activation domain, localized to the plasma membrane and endoplasmic reticulum (Lee & Sugden, 2008; Liu et al, 2018). Hence, to further examine the interaction domain of LMP1 responsible for binding to ANT1, we constructed recombinant plasmids encoding a truncated form of Flag-LMP1 (Fig 2F). In immunoprecipitation assays, we found that only the protein expressed by the LMP1 truncator that retained the transmembrane region was able to bind to ANT1 (Fig 2F), indicating that the transmembrane region is essential for the interaction between LMP1 and ANT1. The functional unit of ANT1 is a homodimer consisting of two 32 kD proteins, containing six hydrophobic transmembrane sheet layers and three homology domains (Brenner et al, 2011; Liu & Chen, 2013). To identify the interaction domain of ANT1 responsible for LMP1 binding, we generated a Myc-tagged ANT1 deletion construct (Fig 2G). Co-immunoprecipitation experiments showed that only the constructs of ANT1 retaining the domain2 were able to bind with LMP1 (Fig 2G). EBV-LMP1 induces an ANT1 conformational change Studies have confirmed that the function of ANT1 is mostly associated with its conformational changes (Ruprecht & Kunji, 2020; Zhao et al, 2021); and we confirmed that EBV-LMP1 and ANT1 are co-localized in the inner mitochondrial membrane. We next used two specific conformational inhibitors, CATR and BKA, to examine whether the LMP1-induced changes of mitochondrial membrane potential are associated with ANT1 conformation. We found that CATR inhibited ANT1 at the c-state, which is one of the necessary conditions for mPTP opening (Novgorodov et al, 1992; Ruprecht et al, 2019), and BKA maintained ANT1 at m-state in NPC cells (Fig EV2A). We considered whether EBV-LMP1 could inhibit ANT1 to maintain it in the m-state. To test this hypothesis, HK1 cells were treated with different concentrations of BKA or CATR for 24 h, and immunoprecipitation results demonstr