ABSTRACT
Mitochondria require the constant import of nuclear-encoded proteins for proper functioning. Impaired protein import not only depletes mitochondria of essential factors but also leads to toxic accumulation of un-imported proteins outside the organelle. Here, we investigate the consequences of impaired mitochondrial protein import in human cells. We demonstrate that un-imported proteins can clog the mitochondrial translocase of the outer membrane (TOM). ATAD1, a mitochondrial ATPase, removes clogged proteins from TOM to clear the entry gate into the mitochondria. ATAD1 interacts with both TOM and stalled proteins, and its knockout results in extensive accumulation of mitochondrial precursors as well as decreased protein import. Increased ATAD1 expression contributes to improved fitness of cells with inefficient mitochondrial protein import. Overall, we demonstrate the importance of the ATAD1 quality control pathway in surveilling protein import and its contribution to cellular health.
ABSTRACT
The association of circular RNAs (circRNAs) with non-small cell lung cancer (NSCLC) has been recognized extensively. In view of this, our study particularly surveyed the underlying mechanism of circ-ATAD1 in the disease. First, an analysis of the clinical expression of circ-ATPase family AAA domain containing 1 (ATAD1) was performed, followed by further evaluation of the relationship between circ-ATAD1 expression and prognosis. Then, A549 cells were treated with single transfection or combined transfection with the plasmid vectors that interfere with circ-ATAD1 or miR-191-5p. circ-ATAD1 and miR-191-5p levels were detected by reverse transcription quantitative polymerase chain reaction to verify the transfection success. Then, cell proliferation was checked by cell count kit-8 and clonal formation test. Cell apoptosis was analyzed by flow cytometry. Cell migration and invasion were examined by wound healing assay and Transwell. Finally, the targeting of miR-191-5p to circ-ATAD1 or Forkhead Box K1 (FOXK1) was verified by bioinformation website starBase analysis and dual-luciferase reporter assay. circ-ATAD1 was expressed abundantly in tumor tissues of NSCLC patients and had a predictive value in poor prognosis. circ-ATAD1 underexpression or miR-191-5p overexpression could obstruct A549 cells to behave aggressively, while circ-ATAD1 upregulation or miR-191-5p depletion resulted in the promotion of aggressiveness of A549 cells. Interestingly, circ-ATAD1 could decoy miR-191-5p. miR-191-5p negatively regulated FOXK1 expression, and downregulating miR-191-5p or upregulating FOXK1 rescued circ-ATAD1 downregulation-mediated influences on NSCLC cells. circ-ATAD1 accelerates NSCLC progression by absorbing miR-191-5p to upregulate FOXK1 expression.
ABSTRACT
Maintenance of desmin intermediate filaments (IF) is vital for muscle plasticity and function, and their perturbed integrity due to accelerated loss or aggregation causes atrophy and myopathies. Calpain-1-mediated disassembly of ubiquitinated desmin IF is a prerequisite for desmin loss, myofibril breakdown, and atrophy. Because calpain-1 does not harbor a bona fide ubiquitin-binding domain, the precise mechanism for desmin IF disassembly remains unknown. Here, we demonstrate that the AAA-ATPase, ATAD1, is required to facilitate disassembly and turnover of ubiquitinated desmin IF. We identified PLAA and UBXN4 as ATAD1's interacting partners, and their downregulation attenuated desmin loss upon denervation. The ATAD1-PLAA-UBXN4 complex binds desmin filaments and promotes a release of phosphorylated and ubiquitinated species into the cytosol, presenting ATAD1 as the only known AAA-ATPase that preferentially acts on phosphorylated substrates. Desmin filaments disassembly was accelerated by the coordinated functions of Atad1 and calpain-1, which interact in muscle. Thus, by extracting ubiquitinated desmin from the insoluble filament, ATAD1 may expose calpain-1 cleavage sites on desmin, consequently enhancing desmin solubilization and degradation in the cytosol.
Subject(s)
Intermediate Filaments , Muscles , ATPases Associated with Diverse Cellular ActivitiesABSTRACT
BACKGROUND: CircRNA circ-ATAD1 has been characterized as an oncogenic circRNA in gastric cancer, while its role in colorectal cancer (CRC) is unknown. This study was carried out to explore the role of circ-ATAD1 in CRC. METHODS: Paired CRC and adjacent non-tumor tissue samples collected from 64 CRC patients were subjected to RNA extractions and RT-qPCRs to analyze the expression of circ-ATAD1, premature miR-618, and mature miR-618 in CRC. The effects of circ-ATAD1 overexpression on miR-618 maturation were analyzed by transfecting circ-ATAD1 expression vector into CRC cells, followed by determining the expression of premature miR-618 and mature miR-618 using RT-qPCR. The subcellular location of circ-ATAD1 was analyzed by nuclear fractionation assay, and the interaction between circ-ATAD1 and premature miR-618 was analyzed by RNA pull-down assay. The roles of circ-ATAD1, premature miR-618, and mature miR-618 in regulating CRC cell proliferation were explored by CCK-8 assay. RESULTS: Circ-ATAD1 was upregulated in CRC and predicted poor survival. In addition, circ-ATAD1 was inversely correlated with mature miR-618 but not premature miR-618. In CRC cells, circ-ATAD1 overexpression decreased the level of mature miR-618 but not premature miR-618. Circ-ATAD1 was detected in both the nucleus and cytoplasm. A direct interaction between circ-ATAD1 and miR-618 was observed. Moreover, circ-ATAD1 overexpression reduced the inhibitory effects of miR-618 overexpression on cell proliferation. CONCLUSION: Circ-ATAD1 is overexpressed in CRC and may suppress miR-618 maturation to participate in CRC.
Subject(s)
ATPases Associated with Diverse Cellular Activities/genetics , Colorectal Neoplasms , MicroRNAs , Cell Line, Tumor , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular/geneticsABSTRACT
BACKGROUND: Circ-ATAD1 plays an oncogenic role in gastric cancer. However, its roles in other cancers are unclear. We aimed to analyze the role of circ-ATAD1 in osteosarcoma (OS). METHODS: The expression levels of circ-ATAD1, mature miR-154-5p, and premature miR-154-5p in paired OS and non-tumor tissues from 56 OS patients were determined using RT-qPCR. Nuclear fractionation assay was performed to analyze the subcellular location of circ-ATAD1. The interaction between circ-ATAD1 and premature miR-154-5p was analyzed using RNA pull-down assay. The role of circ-ATAD1 in regulating miR-154-5p maturation was analyzed using RT-qPCR in cells with overexpression. Transwell assays were performed to analyze the roles of circ-ATAD1 and miR-154-5p in regulating OS cell invasion and migration. RESULTS: Circ-ATAD1 was overexpressed in OS compared to non-tumor tissues and was detected in the nuclei of OS cells. Mature miR-154-5p, but not premature miR-154-5p, was downregulated in OS tissues compared to non-tumor tissues and was inversely correlated with circ-ATAD1. In OS cells, circ-ATAD1 overexpression decreased the expression of mature miR-154-5p, but not premature miR-154-5p. Transwell assay analysis showed that circ-ATAD1 overexpression increased cell invasion and migration, and mature miR-154-5p overexpression suppressed these cell behaviors. In addition, circ-ATAD1 overexpression reduced the effects of mature miR-154-5p overexpression on cell behaviors. CONCLUSIONS: Circ-ATAD1 is overexpressed in OS and suppresses miR-154-5p maturation to increase cell invasion and migration.
Subject(s)
ATPases Associated with Diverse Cellular Activities/metabolism , Bone Neoplasms/metabolism , MicroRNAs/metabolism , Osteosarcoma/metabolism , Polymerase Chain Reaction/methods , RNA, Circular/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Adult , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Female , Humans , Male , MicroRNAs/genetics , Osteosarcoma/genetics , Osteosarcoma/pathology , RNA, Circular/genetics , Young AdultABSTRACT
A previous study has reported the oncogenic role of circular RNA (circ)-ATAD1 in gastric cancer. The aim of the present study was to investigate the role of circ-ATAD1 in acute myeloid leukemia (AML). Bone marrow mononuclear cells were collected from 60 patients with AML and 60 healthy controls, followed by RNA isolation and reverse transcription-quantitative PCR to assess the expression of circ-ATAD1 and microRNA (miR)-34b. A subcellular fractionation assay was used to determine the subcellular location of circ-ATAD1 in AML cells. Furthermore, circ-ATAD1 and miR-34b were overexpressed in AML cells to study crosstalk between the two molecules. The effect of circ-ATAD1 overexpression on miR-34b gene methylation was also analyzed by methylation-specific PCR, and the roles of circ-ATAD1 and miR-34b in the regulation of AML cell proliferation were analyzed by BrdU assay. circ-ATAD1 expression was found to be elevated, and inversely correlated with that of miR-34b, in patients with AML. Subcellular fractionation assays showed that circ-ATAD1 was specifically expressed in the nucleus. In addition, circ-ATAD1 overexpression in AML cells decreased miR-34b expression and increased miR-34b gene methylation. Moreover, AML cell proliferation was increased by circ-ATAD1 overexpression, but decreased by miR-34b overexpression, and the effect of circ-ATAD1 overexpression on AML cell proliferation was reduced by miR-34b overexpression. Together, these results indicate circ-ATAD1 as a nucleus-specific circRNA in AML, which promotes AML cell proliferation by downregulating miR-34b via methylation.
ABSTRACT
The oncogenic function of circ-ATAD1 has been characterized in gastric cancer, while its role in cervical squamous cell carcinoma (CSCC) is unclear. This study explored the role of circ-ATAD1 in CSCC. To evaluate the differential expression of circ-ATAD1, mature miR-218, and premature miR-218 in CSCC, a total of 62 CSCC patients were subjected to biopsies to collect CSCC and paired normal tissues. Gene expression levels were quantified by RT-qPCRs. Nuclear fractionation assay was performed to analyze the subcellular location of circ-ATAD1. CSCC cells were used to perform cell transfections to explore the crosstalk between circ-ATAD1 and miR-218. The roles of circ-ATAD1 and miR-218 in CSCC cell behaviors were explored by BrdU assay, Transwell assay, cell apoptosis assay, and cell stemness assay. CSCC tissues exhibited upregulated expression of circ-ATAD1, which was localized to both nucleus and cytoplasm. Mature miR-218 was downregulated in CSCC tissues and was inversely correlated with circ-ATAD1, while premature miR-218 was not differentially expressed in CSCC. Upregulation of circ-ATAD1 in CSCC cells decreased the expression levels of mature miR-218, but not that of premature miR-218. In addition, overexpression of circ-ATAD1 increased cell proliferation and decreased cell apoptosis, while overexpression of miR-218 decreased cell proliferation and increased cell apoptosis, and it also attenuated the effects of overexpression of circ-ATAD1 on cell proliferation. However, CSCC cell invasion, migration, and stemness were not affected by circ-ATAD1 and miR-218. Circ-ATAD1 is upregulated in CSCC and may regulate cell proliferation and apoptosis by suppressing the maturation of miR-218.
Subject(s)
ATPases Associated with Diverse Cellular Activities/biosynthesis , Apoptosis/physiology , Carcinoma, Squamous Cell/metabolism , Cell Proliferation/physiology , MicroRNAs/biosynthesis , Uterine Cervical Neoplasms/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Female , Humans , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , RNA, Circular/biosynthesis , RNA, Circular/genetics , Up-Regulation/physiology , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathologyABSTRACT
Precise distribution of proteins is essential to sustain the viability of cells. A complex network of protein synthesis and targeting factors cooperate with protein quality control systems to ensure protein homeostasis. Defective proteins are inevitably degraded by the ubiquitin-proteasome system and lysosomes. However, due to overlapping targeting information and limited targeting fidelity, certain proteins become mislocalized. In this review, we present the idea that transmembrane dislocases recognize and remove mislocalized membrane proteins from cellular organelles. This enables other targeting attempts and prevents degradation of mislocalized but otherwise functional proteins. These transmembrane dislocases can be found in the outer mitochondrial membrane (OMM) and endoplasmic reticulum (ER). We highlight common principles regarding client recognition and outline open questions in our understanding of transmembrane dislocases.
Subject(s)
Endoplasmic Reticulum , Mitochondrial Membranes , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Mitochondrial Membranes/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein TransportABSTRACT
Tail-anchored (TA) proteins have an N-terminal domain in the cytosol and a C-terminal transmembrane domain anchored to a variety of organelle membranes. TA proteins are recognized by targeting factors at the transmembrane domain and C-terminal sequence and are guided to distinct membranes. The promiscuity of targeting sequences and the dysfunction of targeting pathways cause mistargeting of TA proteins. TA proteins are under surveillance by quality control pathways. For resident TA proteins at mitochondrial and ER membranes, intrinsic instability or stimuli induced degrons of the cytosolic and transmembrane domains are sensed by quality control factors to initiate degradation of TA proteins. These pathways are summarized as TA protein degradation-Cytosol (TAD-C) and TAD-Membrane (TAD-M) pathways. For mistargeted and a subset of solitary TA proteins at mitochondrial and peroxisomal membranes, a unique pathway has been revealed in recent years. Msp1/ATAD1 is an AAA-ATPase dually-localized to mitochondrial and peroxisomal membranes. It directly recognizes mistargeted and solitary TA proteins and dislocates them out of membrane. Dislocated substrates are subsequently ubiquitinated by the ER-resident Doa10 ubiquitin E3 ligase complex for degradation. We summarize and discuss the substrate recognition, dislocation and degradation mechanisms of the Msp1 pathway.
Subject(s)
Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Peroxisomes/metabolism , ATPases Associated with Diverse Cellular Activities/metabolism , Adenosine Triphosphatases/metabolism , Humans , Protein Domains , Protein Transport , Proteolysis , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking affects multiple brain functions, such as learning and memory. We have previously shown that Thorase plays an important role in the internalization of AMPARs from the synaptic membrane. Here, we show that N-methyl-d-aspartate receptor (NMDAR) activation leads to increased S-nitrosylation of Thorase and N-ethylmaleimide-sensitive factor (NSF). S-nitrosylation of Thorase stabilizes Thorase-AMPAR complexes and enhances the internalization of AMPAR and interaction with protein-interacting C kinase 1 (PICK1). S-nitrosylated NSF is dependent on the S-nitrosylation of Thorase via trans-nitrosylation, which modulates the surface insertion of AMPARs. In the presence of the S-nitrosylation-deficient C137L Thorase mutant, AMPAR trafficking, long-term potentiation, and long-term depression are impaired. Overall, our data suggest that both S-nitrosylation and interactions of Thorase and NSF/PICK1 are required to modulate AMPAR-mediated synaptic plasticity. This study provides critical information that elucidates the mechanism underlying Thorase and NSF-mediated trafficking of AMPAR complexes.
Subject(s)
ATPases Associated with Diverse Cellular Activities/metabolism , Cell Membrane/metabolism , N-Ethylmaleimide-Sensitive Proteins/metabolism , Receptors, AMPA/metabolism , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Animals , Cell Cycle Proteins/metabolism , Cysteine/metabolism , Endocytosis/drug effects , Glutathione/metabolism , HEK293 Cells , Humans , Mice, Inbred C57BL , Mice, Knockout , N-Methylaspartate/pharmacology , Neuronal Plasticity , Nitric Oxide/metabolism , Nitrosation , Protein Binding , Protein Multimerization , Protein Transport , S-Nitrosoglutathione/metabolismABSTRACT
Circular RNAs (circRNAs) has been shown to be involved in the progression of various malignancies. Nevertheless, the mechanism of dysregulated circRNAs in gastric cancer (GC) remains to be understood. CircRNA microarray was utilized for identifying circRNA expression profiles in GC tissues. Circ-ATAD1 expression was measured by qRT-PCR. The clinical significance of circ-ATAD1 was analyzed by Fisher's exact test, Kaplan-Meier plots, and Cox regression model. The function of circ-ATAD1 was explored by using CCK-8, clone formation, flow cytometric and transwell experiments. RNA sequencing, bioinformatics, RNA pulldown, chromatin immunoprecipitation followed by sequencing, and dual-luciferase reporter assays were applied to determine the regulatory networks of circ-ATAD1 in GC cells. Circ-ATAD1 expression was increased in cancerous tissues. The prognostic value of circ-ATAD1 was identified in GC patients. For GC cells, circ-ATAD1 increased cell progression by sponging miR-140-3p to upregulate YY1. Additionally, YY1 directly bound to the promoter of PCIF1, thereby activating its transcription. Collectively, circ-ATAD1 plays an important role in GC tumorigenesis and progression and might be an important biomarker/therapeutic target for GC.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , MicroRNAs/genetics , Nuclear Proteins/genetics , RNA, Circular/genetics , Stomach Neoplasms/pathology , YY1 Transcription Factor/genetics , Adaptor Proteins, Signal Transducing/metabolism , Aged , Apoptosis/genetics , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , Humans , Kaplan-Meier Estimate , Male , MicroRNAs/metabolism , Middle Aged , Nuclear Proteins/metabolism , Prognosis , Promoter Regions, Genetic , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Stomach Neoplasms/mortality , YY1 Transcription Factor/metabolismABSTRACT
The conserved AAA-ATPase Msp1 is embedded in the outer mitochondrial membrane and removes mislocalized tail-anchored (TA) proteins upon dysfunction of the guided entry of tail-anchored (GET) pathway. It remains unclear how Msp1 recognizes its substrates. Here, we extensively characterize Msp1 and its substrates, including the mitochondrially targeted Pex15Δ30, and full-length Pex15, which mislocalizes to mitochondria upon dysfunction of Pex19 but not the GET pathway. Moreover, we identify two new substrates, Frt1 and Ysy6. Our results suggest that mislocalized TA proteins expose hydrophobic surfaces in the cytoplasm and are recognized by Msp1 through conserved hydrophobic residues. Introducing a hydrophobic patch into mitochondrial TA proteins transforms them into Msp1 substrates. In addition, Pex15Δ30 and Frt1 contain basic inter-membrane space (IMS) residues critical for their mitochondrial mistargeting. Remarkably, Msp1 recognizes this feature through the acidic D12 residue in its IMS domain. This dual-recognition mechanism involving interactions at the cytoplasmic and IMS domains of Msp1 and substrates greatly facilitates substrate recognition and is required by Msp1 to safeguard mitochondrial functions.
Subject(s)
Adenosine Triphosphatases/metabolism , Mitochondria/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Amino Acid Sequence , Fungal Proteins , Gene Expression , Genes, Reporter , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Protein Transport , Proteolysis , Recombinant Fusion Proteins , Sequence DeletionABSTRACT
Neuronal preconditioning in vitro or in vivo with a stressful but non-lethal stimulus leads to new protein expression that mediates a profound neuroprotection against glutamate excitotoxicity and experimental stroke. The proteins that mediate neuroprotection are relatively unknown and under discovery. Here we find that the expression of the AAA + ATPase Thorase is induced by preconditioning stimulation both in vitro and in vivo. Thorase provides neuroprotection in an ATP-dependent manner against oxygen-glucose deprivation (OGD) neurotoxicity or glutamate N-Methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity in vitro. Knock-down of Thorase prevents the establishment of preconditioning induced neuroprotection against OGD or NMDA neurotoxicity. Transgenic overexpression of Thorase provides neuroprotection in vivo against middle cerebral artery occlusion (MCAO)-induced stroke in mice, while genetic deletion of Thorase results in increased injury in vivo following stroke. These results define Thorase as a neuroprotective protein and understanding Thorase signaling could offer a new therapeutic strategy for the treatment of neurologic disorders.
Subject(s)
ATPases Associated with Diverse Cellular Activities/metabolism , Infarction, Middle Cerebral Artery/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Adenosine Triphosphatases , Animals , Brain Ischemia/genetics , Brain Ischemia/metabolism , Brain Ischemia/pathology , Cells, Cultured , Female , Gene Deletion , Glucose/metabolism , Infarction, Middle Cerebral Artery/genetics , Infarction, Middle Cerebral Artery/pathology , Ischemic Preconditioning , Male , Mice , Neurons/metabolism , Neuroprotection , Oxygen/metabolism , Stroke/genetics , Stroke/metabolism , Stroke/pathology , Up-RegulationABSTRACT
Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane.
Subject(s)
Adenosine Triphosphatases/metabolism , Membrane Proteins/metabolism , Mitochondrial Membranes/enzymology , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Conserved Sequence , Hydrolysis , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Models, Molecular , Mutation , Protein Domains , Protein Structure, Quaternary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Structure-Activity RelationshipABSTRACT
Mutations in the PEX1 gene, which encodes a protein required for peroxisome biogenesis, are the most common cause of the Zellweger spectrum diseases. The recognition that Pex1p shares a conserved ATP-binding domain with p97 and NSF led to the discovery of the extended family of AAA+-type ATPases. So far, four AAA+-type ATPases are related to peroxisome function. Pex6p functions together with Pex1p in peroxisome biogenesis, ATAD1/Msp1p plays a role in membrane protein targeting and a member of the Lon-family of proteases is associated with peroxisomal quality control. This review summarizes the current knowledge on the AAA+-proteins involved in peroxisome biogenesis and function.