Dataset Information

The Role of Microtubules in the Trafficking of Ectopic ATP Synthase

ABSTRACT: Adenosine triphosphate (ATP) synthase is located on the inner membrane of mitochondria which generate ATP for various cellular processes. Our previous studies showed that ATP synthases existed on plasma membrane of several types of cancer cells, which was defined as ectopic ATP synthase. However, the trafficking pathway of ectopic ATP synthase is still unclear. To investigate how ATP synthase subunits are involved in the trafficking process, we performed shot-gun proteomic analysis to reveal the plasma membrane proteins of A549 lung cancer cells and identified four subunits of ATP synthases, ATP5A1, ATP5B, ATP5H, and MT-ATP6. With the results of gene set enrichment analysis, we inferred that ectopic ATP synthase subunits are assembled as complex consistently and then translocated to cell surface through the trafficking of mitochondria. On the other hand, microtubules are involved in many intracellular transport. During protein trafficking, mitochondria and vesicles are transported to their final destination along microtubules by motor proteins such as kinesins and dyneins. To explore the function of microtubules in the trafficking of ectopic ATP synthase, we treated MCF-7 breast and A549 lung cancer cells with nocodazole, a microtubule depolymerization agent, and found that the expression level of ectopic ATP synthase decreased using both immunocytochemistry and flow cytometry. Besides, since kinesin family member 5B (KIF5B) is an important motor proteins involved in the transport of mitochondria, we further performed the immunoprecipitation of KIF5B followed by liquid chromatography−tandem mass spectrometry (LC−MS/MS). The result revealed that one of the mitochondrial outer membrane proteins, dynamin-1 like proteins (Drp1), was able to associate to KIF5B. From our recent research results and other reports show that Drp1 is a mediator of mitochondrial dynamic through regulation of mitochondrial fission. Taken together, our findings suggested that Drp1-KIF5B complex-mediated mitochondrial trafficking along microtubules may play a critical role in the transport of ectopic ATP synthase

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Ting-Yu Huang

LAB HEAD: Hsueh-Fen Juan

PROVIDER: PXD006791 | Pride | 2023-05-10

REPOSITORIES: Pride

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Publications

Spatial and temporal dynamics of ATP synthase from mitochondria toward the cell surface.

Chang Yi-Wen YW Tony Yang T T Chen Min-Chun MC Liaw Y-Geh YG Yin Chieh-Fan CF Lin-Yan Xiu-Qi XQ Huang Ting-Yu TY Hou Jen-Tzu JT Hung Yi-Hsuan YH Hsu Chia-Lang CL Huang Hsuan-Cheng HC Juan Hsueh-Fen HF

Communications biology 20230418 1

Ectopic ATP synthase complex (eATP synthase), located on cancer cell surface, has been reported to possess catalytic activity that facilitates the generation of ATP in the extracellular environment to establish a suitable microenvironment and to be a potential target for cancer therapy. However, the mechanism of intracellular ATP synthase complex transport remains unclear. Using a combination of spatial proteomics, interaction proteomics, and transcriptomics analyses, we find ATP synthase comple ...[more]

PMID: 37072500

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Project description:Ectopic adenosine triphosphate synthase (eATP synthase) was found on the cell surface to generate extracellular ATP in various cancer cells. Recently using proteomics approach, several reports identified ATP synthase in extracellular vesicles (EVs) which are important in cell-cell communications. However, the functions of ATP synthase in EVs are still unclear. Here we increased the expression of eATP synthase on the plasma membrane of lung cancer A549 cells via 0.1% FBS starvation and collected EVs including microvesicles and exosomes from the culture medium using serial centrifugation. Both of them were assuredly isolated and confirmed by transmission electron microscopy, nano tracking analysis, and specific EV markers. We also used western blot to demonstrate the presence of ATP synthase in EVs. Besides, dot blot experiment revealed that the F1 portion faced towards extracellular space using anti-ATP beta subunit antibody. Under starvation, the expression of eATP synthase was enhanced on cell surface and exosomes as well as increased EV secretion. To further investigate the functions, we first elucidated whether eATP synthase on EV membrane produced ATP by ATP luciferase analysis. Our data showed that there was no significant ATP production. Moreover, we performed proteomic analysis using dimethyl labeling and LC-MS/MS to compare the difference between EVs released by A549 control and starvation. We identified a total of 1504 and 869 proteins in microvesicles and exosomes, respectively. Using 1.5- and 0.67-fold change as the thresholds, there were 171 and 46 differential expressed proteins in microvesicles and exosomes, respectively. Seven ATP synthase subunits in microvesicles and five in exosomes were identified. The proteomic data were further analyzed using gene set enrichment analysis (GSEA) within the C2 (curated gene sets) subclasses and setting 0.25 as FDR cutoff to find out biological functions related to eATP synthase. Among these up-regulated terms, metabolism, cell cycle and apoptosis were enriched, which indicating that starvation influences cell behavior. Additionally, the immune response is upregulated strikingly in exosomes within C2 gene set. One of these immune response pathways, MHC-1 antigen presentation, was previously proved to interact with eATP synthase which could be recognized by several immune cells.

Project description:Purpose:to verify Drp1-mediated biological pathways under physiological and ischemia conditions.Methods:C57BL/6 mice and Drp1 knockdown mice were used for ischemia treatment. Right femoral arteries were catheterized with polyethylene catheters for bleeding 50% of total blood volume (≈7% of weight). The ischemia time started to calculate after the model was established. A laparotomy was then carried out to obtain superior mesenteric artery tissues (SMAs) for transcriptome analysis. Series-Cluster analysis was performed to identify the global trends of mitochondrial DEGs after Drp1 knockdown. Gene ontology (GO) analysis was performed to facilitate elucidating the biological process (BP), molecular function (MF) and cellular component (CC) of unique genes in the significant or representative profiles. Results:In addition to its traditional roles in GTPase regulation and mitochondrial fission, Drp1 may also regulate actin cytoskeleton and the movement of microtubules. Besides, Drp1 may participate in the regulation of morphology and functions of other organelles, including endoplasmic reticulum, peroxisome, phagolysosome, etc. As for general organ functions, Drp1 may regulate vascular constriction and dilation. These consequences indicated the important role of Drp1 in ischemia-induced cellular regulation. Conclusions: Drp1 deficiency proves the important role of Drp1 in ischemia-induced biomedical processes through multiple potential fission-independent manners. Methods:C57BL/6 mice and Drp1 knockdown mice were used for ischemia treatment. Right femoral arteries were catheterized with polyethylene catheters for bleeding 50% of total blood volume (≈7% of weight). The ischemia time started to calculate after the model was established. A laparotomy was then carried out to obtain superior mesenteric artery tissues (SMAs) for transcriptome analysis. Series-Cluster analysis was performed to identify the global trends of mitochondrial DEGs after Drp1 knockdown. Gene ontology (GO) analysis was performed to facilitate elucidating the biological process (BP), molecular function (MF) and cellular component (CC) of unique genes in the significant or representative profiles. Results:In addition to its traditional roles in GTPase regulation and mitochondrial fission, Drp1 may also regulate actin cytoskeleton and the movement of microtubules. Besides, Drp1 may participate in the regulation of morphology and functions of other organelles, including endoplasmic reticulum, peroxisome, phagolysosome, etc. As for general organ functions, Drp1 may regulate vascular constriction and dilation. These consequences indicated the important role of Drp1 in ischemia-induced cellular regulation. Conclusions: Drp1 deficiency proves the important role of Drp1 in ischemia-induced biomedical processes through multiple potential fission-independent manners.

Project description:Non-small cell lung cancer is the leading cause of cancer death worldwide. Gefitinib, epidermal growth factor receptor tyrosine kinase inhibitor, is the first-line treatment of NSCLC, however, many patients eventually become resistant and experience progressive disease. Therefore, development of efficient therapeutic agents to overcome resistance is urgent. We previously found that citreoviridin, one of toxic mycotoxins derived from fungal species, can suppress lung cancer cell growth by inhibiting the activity of ectopic ATP synthase, but has limited effect on normal cells. Citreoviridin suppresses mitogen-activated protein kinase/extracellular signal-regulated kinase signaling by site-specific dephosphorylation of HSP90AB1 on Serine 255 in gefitinib non-resistant lung cancer CL1-0 cells and xenograft model. We are curious whether signaling pathways underlying citreoviridin-treated gefitinib-acquired resistant lung cancer cells are different. In this study, we showed that citreoviridin inhibited cell proliferation and anchorage-dependent growth of gefitinib-acquired resistance NCI-H1975 cells with EGFR T790M mutation. Furthermore, we explored the dynamic molecular response by temporal phosphoproteomic approach. We identified 1476 phosphopeptides corresponding to 738 phosphoproteins and quantified 1901 phosphorylation sites. There were 274 phosphosites corresponding to 174 phosphorylated proteins significantly differential expressed. Functional enrichment analysis demonstrated that citreoviridin treatment affected chromatin organization, cell cycle and apoptosis. Interestingly, we found that citreovirdin suppressed cell proliferation by site-specific phosphorylation of topoisomerase on serine 1106. Citreovirdin induced double strands breaks, and then leaded to DNA damage response. The DNA lesions triggered cells to cell cycle arrest at S phase for repairing or apoptosis for cell death. The results indicated that citreoviridin could potentially be a therapeutic agent against gefitinib-resistant NSCLC.

Project description:Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system with diverse clinical behaviors. Even with multimodal therapies, high-risk neuroblastoma has an unfavorable outcome irrespective of MYCN amplification, a well-established oncogenic driver in neuroblastoma pathogenesis, and its genetic heterogeneity has largely impeded efforts to correlate molecular targets with biological consequences for more effective treatment strategies. Here, using a gene expression-based approach, we identified the FDA-approved anthelmintic niclosamide as a potential anti-neuroblastoma drug. By combining the gene expression signature associated with high-risk neuroblastoma and the recurrent drug−transcript relationships inferred from up to one million perturbational gene expression profiles, our algorithm predicted effective therapeutic candidates by evaluating the extent to which a given compound or their combinations could ‘reverse’ the high-risk signature. Furthermore, we performed quantitative polymerase chain reaction (qPCR) to validate top five candidate reverse genes which are involved in DNA replication, including cyclin A2 (CCNA2), minichromosome maintenance 10 replication initiation factor (MCM10), ERCC excision repair 6 like, spindle assembly checkpoint helicase (ERCC6L), kinesin family member 20A (KIF20A), and RuvB like AAA ATPase 1 (RUVBL1). Indeed, those five genes were downregulated in niclosamide-treated cells, indicating niclosamide suppressed DNA replication and then inhibited cell proliferation. Using cell proliferation and clonogenic assays as well as flow cytometry, we determined the cytotoxic effects of niclosamide in MYCN-amplified SK-N-DZ and non-amplified SK-N-AS cells. The results showed that niclosamide could effectively reduce not only cell proliferation and colony formation but also trigger cell cycle arrest and apoptosis. Moreover, we conducted human tumor xenografts in a nude mice model to evaluate the in vivo efficacy of niclosamide and found that it significantly suppressed tumor growth and prolonged survival rate, but doesn’t cause organ damage and change body weight. To explore the molecular mechanism of niclosamide, stable-isotope dimethyl labeling strategy for quantitative proteomics was performed on both cell-based or xenograft-based MYCN-amplified SK-N-DZ and MYCN-nonamplified SK-N-AS models. We confirmed niclosamide not only mediated the function of mitochondrial electron transport chain but also the other functions in high risk neuroblastoma cell lines and xenografts. The results suggest that our developed expression-based strategy is useful for drug discovery and provides the possibility of repurposing the anthelminthic drug niclosamide for treating high-risk neuroblastoma therapy.

Dataset Information

The Role of Microtubules in the Trafficking of Ectopic ATP Synthase

Publications

Spatial and temporal dynamics of ATP synthase from mitochondria toward the cell surface.

Similar Datasets

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