Molecular Mechanism Of EBV-LMP1 Inhibition Of ANT1 Conformational Changes And Cisplatin Resistance

Mitochondria are important intracellular organelles that,in addition to being involved in the production of ATP,fatty acids,amino acids,heme and other substances,are also closely involved in the maintenance of calcium homeostasis within the matrix and the formation of signaling molecules.The imbalance of mitochondrial homeostasis leading to the disruption of mitochondrial function is one of the important mechanisms for the development of diseases.Various intrinsic and extrinsic stress signals promote the degradation of damaged mitochondria and mitochondrial biogenesis,which regulates the synthesis of mitochondrial DNA,proteins and lipids to maintain the homeostatic balance of mitochondria.When mitochondrial dysfunction exceeds the capacity of mitochondria,mitochondrial outer membrane permeability ruptures and mitochondrial membrane permeability transition pore(m PTP)opening is enhanced,resulting in m PTP-driven programmed death,thus m PTP opening is the terminal mitochondrial homeostatic imbalance event.Adenine nucleotide translocases(ANTs)are mitochondrial inner membrane proteins that are primarily responsible for ADP/ATP transport in mitochondria and are the major components of m PTP.ANTs include four different(ANT1,ANT2,ANT3,and ANT4)isoforms that control the balance of oxidative phosphorylation and glycolysis,provide the basis for cellular metabolism,and govern cell death mitochondrial inner membrane proteins,ANTs have been shown to play a key role in tumorigenesis.A variety of viral proteins(including DNA and RNA viruses)can regulate m PTP by altering mitochondrial membrane potential,ROS levels,and Ca2+ in different ways,thereby regulating host cell metabolism or mitochondrial function,making m PTP an important target for tumorigenic viruses.Epstein-Barr virus(EBV)was the first DNA virus identified to be tumorigenic in humans and is a driver of nasopharyngeal carcinoma(NPC)development.Studies have demonstrated that EBV encodes a key oncogenic protein,latent membrane protein 1(LMP1),that regulates NPC mitochondrial function and cell fate by altering multiple signaling axes leading to activation of host cell signaling networks.In the first part of this paper,we use m PTP as the entry point and nasopharyngeal carcinoma cells as the main study model to explore whether EBV-LMP1 may regulate mitochondrial function by affecting m PTP? Through which components of m PTP might EBV-LMP1 affect mitochondrial membrane potential? In which way does EBV-LMP1 regulate the relevant components of m PTP? We utilized two different mitochondrial membrane potential probes,tetramethylrhodamine methyl ester(TMRM)and JC-1,and found a significant increase in fluorescence intensity in CNE1-LMP1 and HK1-LMP1 cells compared to CNE1 and HK1 parental cells by flowmetry and laser confocal microscopy,confirming a substantial increase in mitochondrial membrane potential in LMP1-positive nasopharyngeal carcinoma cells.In addition,we further verified that LMP1 could increase the mitochondrial membrane potential by inhibiting m PTP opening through mitochondrial swelling assay and m PTP opening assay.Then,we constructed truncated bodies of ANT1 and LMP1,respectively,and found that EBV-LMP1 localized to the inner mitochondrial membrane,bound directly to the binding domain 2(domain2)of ANT1,and reduced m PTP opening by inhibiting the formation of ANT1-VDAC1 complex,leading to the mitochondrial increase in membrane potential.We conclude from the above study that EBV-LMP1 is localized in mitochondria and interacts with domain 2 of ANT1,a key protein of m PTP,on the mitochondrial matrix side through its transmembrane domain,increasing mitochondrial membrane potential and inhibiting m PTP opening,thus reducing mitochondrial swelling rate.It is suggested that ANT1 is an important target of viral protein LMP1.ANT1 is a key transducer of mitochondrial energy metabolism,which can regulate oxidative phosphorylation and glycolytic homeostasis in the cell through periodic conformational changes.ANT1 has two different conformations.When ANT1 faces the mitochondrial matrix,it transports ATP to release ADP and is in the m-state,while when ANT1 faces the cytoplasmic side,it transports ADP to release ATP and is in the c-state;ANT1 achieves mitochondrial energy conversion through cyclic state transition.The protein complex of ANT1 and VDAC1 plays an important role in maintaining the mitochondrial membrane potential and permeability.The c-state of ANT1 has been found to be an important condition for m PTP opening,indicating that the transition between conformations of ANT1 is not only necessary for the synthesis of ATP in mitochondria,but also an important mechanism for maintaining cellular life activities.In the second part of this paper,we use the mitochondrial protein ANT1 as the entry point and nasopharyngeal carcinoma cells as the main study model to explore whether EBV-LMP1 may regulate the pathophysiological process of tumor cells by directly affecting the conformation of ANT1 to inhibit the opening of m PTP? Is the binding of EBV-LMP1 to ANT1 necessary for the regulation of ANT1 conformation?Does this conformational change affect the conversion of energy metabolism and thus regulate intracellular levels of oxidative phosphorylation? Can ANT1 inhibitors interfere with the effect of LMP1 on ANT1 and thus control the cellular membrane potential and tumor cell fate? We used the conformational inhibitors of ANT1,carboxyatractyloside(CATR)and bongkrekic acid(BKA),and liquid-phase 19F-NMR experiments to confirm that LMP1 enhances cell viability by inhibiting the opening of m PTP,and that the binding of LMP1 to ANT1 is required for this process.Next,we examined the mitochondrial membrane potential and viability of CNE1/CNE1-LMP1 and HK1/HK1-LMP1 cells after treating the cells with CATR(inhibition of ANT1 at c-state)/BKA(inhibition of ANT1 at m-state),respectively,and the results showed that EBV-LMP1 can inhibit the m-state of ANT1 by maintaining formation of the VDAC1-ANT1 complex,increasing mitochondrial potential and promoting NPC cell viability.Furthermore,mitochondrial ADP/ATP exchange was found to be restricted in EBV-LMP1 cells using hippocampal XF mitochondrial stress analysis,and cellular oxidative phosphorylation levels were reduced,indicating that LMP1 reduces ATP production via mitochondria.In this chapter we conclude that EBV-LMP1 increases mitochondrial potential by directly binding to ANT1 to maintain it in the m-state conformation,decreasing the level of mitochondrial oxidative phosphorylation and promoting NPC cell viability.Cisplatin(CDDP)is the first-line chemotherapeutic agent for the treatment of nasopharyngeal carcinoma.CDDP resistance is an inherent feature of head and neck squamous carcinoma.EBV infection is an important reason why nasopharyngeal carcinoma is chemoresistant,and EBV-LMP1 regulates tumor cell resistance to CDDP through multiple signaling pathways.Studies have shown that mitochondria are important targets for tumor chemotherapy and ANT1 may be an important factor in LMP1 triggering tumor resistance.Chapter 3 of this paper explores whether EBV-LMP1 resists CDDP by affecting ANT1 conformational changes using NPC cells and animal models? Does CATR,a conformational inhibitor of ANT1,exert CDDP sensitization by targeting the ANT1 conformation? We confirmed that CATR increased the chemosensitivity of nasopharyngeal carcinoma cells to CDDP by in vitro and in vivo experiments.In in vitro experiments,we found that IC5 was significantly higher in CDDPLMP1-positive cells than in LMP1-negative cells.We divided the cells into control group,CATRtreated group,CDDP-treated group,and combined CDDP and CATRtreated group.The results of the experiments by CCK-8,Annexin V(PE)and 7-AAD double staining showed that CATR combined with CDDP significantly reduced mitochondrial membrane potential and viability of LMP1-positive cells and increased CDDP-induced cell death compared to CDDP alone,indicating that conformational changes in ANT1 mediated the resistance to CDDP.Furthermore,in an in vivo nude mouse tumorigenesis assay,we found that ANT1 was a potential molecular target for reducing NPC cell proliferation and survival by detecting Ki67,TUNEL,and caspase-3 and caspase-9,and that interference with ANT1 increased the chemosensitivity of tumor cells to CDDP.In this chapter we conclude that: EBV-LMP1 maintains ANT1 in the m-state to increase CDDP resistance;and the conformational inhibitor CATR promotes m PTP opening and enhances the sensitivity of NPC cells to CDDP.In summary,this study investigated the molecular mechanisms by which the EBV-encoded oncogenic protein LMP1 regulates the conformational changes of mitochondrial ANT1 and its potential as a target site.It was found that EBV-LMP1 localizes to the mitochondria and ANT1’s domain 2 directly on the matrix side to inhibit m PTP opening,increase mitochondrial membrane potential,and decrease mitochondrial swelling rate.In addition,it was confirmed that EBV-LMP1 fixes ANT1 conformation in the m-state,decreases the mitochondrial ADP/ATP exchange rate and oxidative phosphorylation level,and promotes the viability of nasopharyngeal carcinoma cells.In contrast,the ANT1 conformation inhibitor CATR promoted m PTP opening,decreased mitochondrial membrane potential,and enhanced the sensitivity of nasopharyngeal carcinoma cells to CDDP.This study identified a novel mechanism by which EBV-LMP1 regulates ANT1 conformational changes to affect mitochondrial function,and presented experimental evidence that EBV-LMP1 causes CDDP resistance from a novel perspective,suggesting that conformational changes of target molecules are new targets for chemoresistance in nasopharyngeal carcinoma.