ATPase Family AAA Domain Research Articles

Bioinformatics analysis identifies hub genes and pathways in nasopharyngeal carcinoma.

The aim of the present study was to identify genes associated with and the underlying mechanisms in nasopharyngeal carcinoma (NPC) using microarray data. GSE12452 and GSE34573 gene expression profiles were obtained from the Gene Expression Omnibus (GEO) database. GEO2R was utilized to obtain differentially expressed genes (DEGs). In addition, the Database for Annotation, Visualization and Integrated Discovery was used to perform pathway enrichment analyses for DEGs using the Gene Ontology (GO) annotation along with the Kyoto Encyclopedia of Genes and Genomes (KEGG). Furthermore, Cytoscape was used to perform module analysis of the protein-protein interaction (PPI) network and pathways of the hub genes were studied. A total of 298 genes were ascertained as DEGs in the two datasets. To functionally categorize these DEGs, we obtained 82 supplemented GO terms along with 7 KEGG pathways. Subsequently, a PPI network consisting of 10 hub genes with high degrees of interaction was constructed. These hub genes included cyclin-dependent kinase (CDK) 1, structural maintenance of chromosomes (SMC) 4, kinetochore-associated (KNTC) 1, kinesin family member (KIF) 23, aurora kinase A (AURKA), ATAD (ATPase family AAA domain containing) 2, NDC80 kinetochore complex component, enhancer of zeste 2 polycomb repressive complex 2 subunit, BUB1 mitotic checkpoint serine/threonine kinase and protein regulator of cytokinesis 1. CDK1, SMC4, KNTC1, KIF23, AURKA and ATAD2 presented with high areas under the curve in receiver operator curves, suggesting that these genes may be diagnostic markers for nasopharyngeal carcinoma. In conclusion, it was proposed that CDK1, SMC4, KNTC1, KIF23, AURKA and ATAD2 may be involved in the tumorigenesis of NPC. Furthermore, they may be utilized as molecular biomarkers in early diagnosis of NPC.

Differentially Expressed Mitochondrial Proteins in Human MCF7 Breast Cancer Cells Resistant to Paclitaxel.

Identification of novel proteins with changed expression in resistant cancer cells could be helpful in elucidation mechanisms involved in the development of acquired resistance to paclitaxel. In this study, we carried out a 2D-PAGE using the mitochondrial-enriched fraction from paclitaxel-resistant MCF7/PacR cells compared to original paclitaxel-sensitive MCF7 breast cancer cells. Differentially expressed proteins were identified employing mass spectrometry. We found that lysosomal cathepsin D and mitochondrial abhydrolase-domain containing protein 11 (ABHD11) had decreased expression in MCF7/PacR cells. On the other hand, mitochondrial carbamoyl-phosphate synthetase 1 (CPS1) and ATPase family AAA-domain containing protein 3A and 3B (ATAD3A, ATAD3B) were overexpressed in MCF7/PacR cells. Further, we showed that there was no difference in localization of CPS1 in MCF7 and MCF7/PacR cells. We demonstrated a significant increase in the number of CPS1 positive MCF7/PacR cells, using FACS analysis, compared to the number of CPS1 positive MCF7 cells. Silencing of CPS1 expression by specific siRNA had no significant effect on the resistance of MCF7/PacR cells to paclitaxel. To summarize, we identified several novel proteins of a mitochondrial fraction whose role in acquired resistance to paclitaxel in breast cancer cells should be further assessed.

ATAD2 is associated with malignant characteristics of pancreatic cancer cells.

Pancreatic cancer is one of the most aggressive human cancers and is associated with a poor prognosis. To develop a novel strategy for pancreatic cancer treatment, it is essential to elucidate the molecular mechanisms underlying the invasion and proliferation of cancer cells. ATPase family AAA domain containing protein 2 (ATAD2) is a highly conserved protein with an AAA+ domain and a bromodomain. Accumulating studies have demonstrated that ATAD2 is associated with the progression of multiple cancers. The present study demonstrated that ATAD2 depletion suppressed cell invasion and migration. In addition, ATAD2 knockdown suppressed anchorage-independent growth of pancreatic cancer cells. Finally, ATAD2 depletion was demonstrated to sensitize pancreatic cancer cells to gemcitabine. The results of the present study indicate that ATAD2 is involved in the malignant characteristics of pancreatic cancer.

ATAD3A on the Path to Cancer.

The ATPase family AAA-domain containing protein 3A (ATAD3A), a nuclear-encoded mitochondrial enzyme, is involved in diverse cellular processes, including mitochondrial dynamics, cell death and cholesterol metabolism. Overexpression and/or mutation of the ATAD3A gene have been observed in different types of cancer, associated with cancer development and progression. The dysregulated ATAD3A acts as a broker of a mitochondria-endoplasmic reticulum connection in cancer cells, and inhibition of this enzyme leads to tumor repression and enhanced sensitivity to chemotherapy and radiation. As such, ATAD3A is a promising drug target in cancer treatment.

Mitochondrial ATAD3A regulates milk biosynthesis and proliferation of mammary epithelial cells from dairy cow via the mTOR pathway

ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane protein, which is essential for cell growth and metabolism. The mechanism by which ATAD3A acts is still not fully understood. In this study, we explored the regulatory role of ATAD3A on milk biosynthesis and proliferation of bovine mammary epithelial cell. We showed that ATAD3A is localized in mitochondria and the expression of ATAD3A was up-regulated in response to extracellular stimuli such as amino acids and hormones. We observed that ATAD3A positively regulated milk protein, fat, and lactose biosynthesis, and cell proliferation. We further revealed that ATAD3A promoted the expressions of mTOR, SREBP-1c, and Cyclin D1, and triggers mTOR phosphorylation. In summary, our data reveal that ATAD3A regulates the mTOR, SREBP-1c, and Cyclin D1 signaling pathways for milk biosynthesis and cell proliferation.

Abstract 5084: Potent and isoform-selective ATAD2 bromodomain inhibitor with unprecedented chemical structure and mode of action

Abstract ATAD2 (ATPase family AAA-domain containing protein 2, also called ANCCA) is an epigenetic regulator that binds to chromatin through its bromodomain (BD), a motif specialized for acetyl-lysine recognition. ATAD2 directly associates with multiple transcription factors such as ERα, AR, E2F, and Myc; hence, ATAD2 has been proposed to act as a co-factor for oncogenic transcription factors. Furthermore, we have recently reported a novel role for ATAD2 during DNA replication, uncovering interactions between ATAD2 and histone acetylation marks on newly synthesized histone H4. High expression of ATAD2 strongly correlates with poor patient prognosis in multiple tumor types, including gastric, endometrial, hepatocellular, ovarian, breast and lung cancers. However, the exact function of ATAD2 in these tumor types remains unclear. A more thorough validation of ATAD2 as a therapeutic target is hampered by the lack of isoform-selective, potent and cellularly active ATAD2 inhibitors. A systematic assessment of crystal structures of BD-containing protein family predicted that development of selective inhibitors of ATAD2 would be challenging. In line with this prediction, only limited progress in developing lead compounds targeting ATAD2 has been reported so far. A few notable exceptions relied on fragments as starting points, however, their weak potency, insufficient selectivity against other BDs, permeability limitations or modest cellular activity have curbed their further development towards drug candidates. Here we embarked on a novel strategy to identify ATAD2 inhibitors: 11 different DNA-encoded libraries adding up to 67 billion unique encoded compounds were combined and incubated with ATAD2 BD followed by two rounds of affinity-mediated selection. This approach provided with several series of binders, for which specific target engagement of their SMOL moiety upon off-DNA synthesis was confirmed in biochemical and biophysical assays. Several rounds of potency optimization led to the identification of BAY-850, a highly potent and ATAD2 (isoform A) mono-selective inhibitor, which holds an amine substituted 3-(2-furyl)benzamide core. This compound shows - as revealed by size exclusion chromatography and native mass spectrometry - a novel mode of action for a BD inhibitor based on specific target dimerization. In a cellular fluorescence recovery after photobleaching (FRAP) assay BAY-850 displaced wild-type ATAD2 from the chromatin to the same extent as the genetic mutagenesis of ATAD2 BD. In contrast, chemically very similar inactive control compounds showed no major effects on ATAD2 association with the chromatin. These results qualify BAY-850 as the first biologically active ATAD2 isoform A-specific chemical probe, which will enable further elucidation of the cancer biology of this intriguing protein. Citation Format: Amaury E. Fernández-Montalván, Markus Berger, Benno Kuropka, Seong Joo Koo, Volker Badock, Joerg Weiske, Simon J. Holton, Apirat Chaikuad, Laura Díaz-Sáez, James Bennett, Oleg Federov, Kilian Huber, Paolo Centrella, Matthew A. Clark, Christoph E. Dumelin, Eric A. Sigel, Holly S. Soutter, Dawn M. Troast, Ying Zhang, John W. Cuozzo, Anthony D. Keefe, Didier Roche, Vincent Rodeschini, Jan Hübner, Hilmar Weinmann, Ingo V. Hartung, Matyas Gorjanacz. Potent and isoform-selective ATAD2 bromodomain inhibitor with unprecedented chemical structure and mode of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5084. doi:10.1158/1538-7445.AM2017-5084

Abstract 5239: Probing the cancer epigenome: empowering target validation by open innovation

Abstract Low reproducibility of published target validation studies as well as the frequent failure of genetic knock-down effects to phenocopy those of small molecule inhibitors have been recognized as road blocks for cancer drug discovery. Academic and industrial institutions have started to address these issues by providing access to high quality small molecular probes for novel targets of interest. Here we discuss probe discovery challenges and quality criteria based on the generation of three novel inhibitors for epigenetic targets. ATAD2 (ATPase family AAA-domain containing protein 2) is an epigenetic regulator that binds to chromatin through its bromodomain (BD). ATAD2 has been proposed to act as a co-factor for oncogenic transcription factors such as ERα and Myc. A more thorough validation of ATAD2 as a therapeutic target has been hampered by the lack of appropriate ATAD2 inhibitors. Here we disclose a structurally unprecedented series of ATAD2 BD inhibitors identified from a DNA-encoded library screen. Optimization delivered BAY-850, a highly potent and exceptionally selective ATAD2 BD inhibitor, which fully recapitulates effects seen by genetic mutagenesis studies in a cellular assay. The three BD and PHD-finger (BRPF) family members are found in histone acetyltransferase complexes. Whereas bromodomain inhibitors with dual activity against BRPF1 and 2 have been described before, we now disclose BAY-299, the first nanomolar inhibitor of the BRPF2 BD with high selectivity against its paralogs. Isoform selectivity was confirmed in cellular protein-protein interaction assays and rationalized based on X-Ray structures. BAY-598, a highly selective, cellularly active and orally bioavailable inhibitor of the protein lysine methyl transferase SMYD2, had been disclosed previously (Stresemann et al., AACR 2015). Development of BAY-598 allowed the identification of new methylation targets of SMYD2 as well as a proposed role of SMYD2 in pancreatic cancer. These results support further development of small molecule inhibitors as research tools to probe the functional role of novel epigenetic targets and underscore the power of open innovation for advancing our understanding of cancer target biology. Citation Format: Ingo V. Hartung, Cheryl Arrowsmith, Volker Badock, Naomi Barak, Markus Berger, Peter J. Brown, Clara D. Christ, Erik Eggert, Ursula Egner, Oleg Fedorov, Amaury E. Fernandez-Montalvan, Matyas Gorjanacz, Andrea Haegebarth, Bernard Haendler, Roman C. Hillig, Simon H. Holton, Kilian V. Huber, Seong J. Koo, Antonius ter Laak, Susanne Mueller, Anke Mueller-Fahrnow, Cora Scholten, Stephan Siegel, Timo Stellfeld, Detlef Stoeckigt, Carlo Stresemann, Masoud Vedadi, Joerg Weiske, Hilmar Weinmann. Probing the cancer epigenome: empowering target validation by open innovation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5239. doi:10.1158/1538-7445.AM2017-5239

ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia.

De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.

Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes

ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane protein implicated in mitochondrial dynamics, nucleoid organization, protein translation, cell growth, and cholesterol metabolism. We identified a recurrent de novo ATAD3A c.1582C>T (p.Arg528Trp) variant by whole-exome sequencing (WES) in five unrelated individuals with a core phenotype of global developmental delay, hypotonia, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. We also describe two families with biallelic variants in ATAD3A, including a homozygous variant in two siblings, and biallelic ATAD3A deletions mediated by nonallelic homologous recombination (NAHR) between ATAD3A and gene family members ATAD3B and ATAD3C. Tissue-specific overexpression of borR534W, the Drosophila mutation homologous to the human c.1582C>T (p.Arg528Trp) variant, resulted in a dramatic decrease in mitochondrial content, aberrant mitochondrial morphology, and increased autophagy. Homozygous null bor larvae showed a significant decrease of mitochondria, while overexpression of borWT resulted in larger, elongated mitochondria. Finally, fibroblasts of an affected individual exhibited increased mitophagy. We conclude that the p.Arg528Trp variant functions through a dominant-negative mechanism that results in small mitochondria that trigger mitophagy, resulting in a reduction in mitochondrial content. ATAD3A variation represents an additional link between mitochondrial dynamics and recognizable neurological syndromes, as seen with MFN2, OPA1, DNM1L, and STAT2 mutations.

Structural and Functional Analysis of the interaction between PCNA and ATAD5

Proliferating cell nuclear antigen (PCNA) is a DNA sliding clamp that coordinates DNA replication and repair. PCNA is loaded on and unloaded off of DNA during replication. It is loaded onto DNA by the replication factor C (RFC) complex. The mechanism of PCNA unloading is not fully understood however. ATPase family AAA domain containing protein 5 (ATAD5) interacts with subunits of RFC to form a RFC-like complex that functions in unloading PCNA. Reduced expression of ATAD5 extends the life of replication factories by retaining PCNA and other repliosome proteins on chromatin. ATAD5 also plays a role in maintaining genomic stability, as mutations to ATAD5 cause genomic instability in mice and human cells. The region of ATAD5 that interacts with PCNA had yet to be determined. Proteins interacting with PCNA usually have a PCNA-interaction protein (PIP) motif termed the PIP-box. Through sequence analysis, we identified a putative PIP-box in ATAD5. Using GST pull-down experiments, we found that the putative PIP-box bound to PCNA. Mutation of the key residues in the PIP-box abolished the binding. The interaction was also confirmed by isothermal titration calorimetry (ITC) analysis of PCNA and a synthetic peptide containing the PIP-box. The binding constant (Kd) of the PCNA-peptide interaction is 6.2μM, determined by the ITC assays. To further characterize the interaction, we co-crystallized PCNA and the ATAD5 peptide. The complex crystals diffracted to a resolution of 3.5Å. The crystals belong to the space group H3, with unit-cell parameters a = 83.6Å and c = 211.4Å. Our molecular replacement solution indicates that the peptide binds on PCNA at the conserved PIP-box binding pocket. Taken together, our work demonstrates that ATAD5 does contain a PIP motif that directly interacts with PCNA. The structure-function studies on the interaction will provide insights into the molecular mechanism of PCNA unloading and the role of ATAD5 in DNA replication and repair.

Expression analysis of ATAD3 isoforms in rodent and human cell lines and tissues

ATAD3 (ATPase family AAA-Domain containing protein 3) is a mitochondrial inner membrane ATPase with unknown but vital functions. Initial researches have focused essentially on the major p66-ATAD3 isoform, but other proteins and mRNAs are described in the data banks. Using a set of anti-peptide antibodies and by the use of rodent and human cell lines and organs, we tried to detail ATAD3 gene expression profiles and to verify the existence of the various ATAD3 isoforms. In rodent, the single ATAD3 gene is expressed as a major isoform of 67kDa, (ATAD3l; long), in all cells and organs studied. A second isoform, p57-ATAD3s (small), is expressed specifically throughout brain development and in adult, and overexpressed around the peri-natal period. p57-ATAD3s is also expressed in neuronal and glial rodent cell lines, and during in vitro differentiation of primary cultured rat oligodendrocytes. Other smaller isoforms were also detected in a tissue-specific manner. In human and primates, ATAD3 paralogues are encoded by three genes (ATAD3A, 3B and 3C), each of them presenting several putative variants. Analyzing the expression of ATAD3A and ATAD3B with four specific anti-peptide antibodies, and comparing their expressions with in vitro expressed ATAD3 cDNAs, we were able to observe and define five isoforms. In particular, the previously described p72-ATAD3B is confirmed to be in certain cases a phosphorylated form of ATAD3As. Moreover, we observed that the ATAD3As phosphorylation level is regulated by insulin and serum. Finally, exploring ATAD3 mRNA expression, we confirmed the existence of an alternative splicing in rodent and of several mRNA isoforms in human.Considering these observations, we propose the development of a uniform denomination for ATAD3 isoforms in rodent and human.

An alternative import pathway of AIF to the mitochondria

In eukaryotic cells, transport of the newly synthesized proteins and phospholipids to the appropriate subcellular target compartments is essential for maintaining organelle morphology and cell survival. In animal cells, mitochondria are major organelles containing DNA genome that encodes only for a small fraction of their proteins, which are required for the organelle function. Most mitochondrial proteins are encoded by the nuclear genes and imported to the mitochondria following protein synthesis. Apoptosis-inducing factor (AIF), an essential FAD-dependent NADH oxidase for the oxidative phosphorylation, is located in the intermembranous space and contains mitochondrial localization signals. However, the import mechanism of AIF to the mitochondria is not yet studied. Using sucrose gradient ultracentrifugation and immunoblotting, AIF was detected in fractions of the endoplasmic reticulum, mitochondria-associated membranes (MAM) and mitochondria, and AIF from these fractions was resistant to trypsin in the absence of digitonin, suggesting that AIF could be protected by phospholipids. Knockdown of dynamin-related protein 1 (DRP1kd) expression reduced AIF levels in the mitochondria, but increased AIF concentrations in the MAM. Knockdown of mitofusin-2 (Mfn-2kd) or ATPase family AAA domain containing 3A (ATAD3Akd) expression, however, reduced AIF levels in the mitochondria and increased the number of transport vesicles that contained AIF in the cytosol, indicating that ATAD3A and Mfn-2 were respectively essential for the import and fusion of transport vesicles into the mitochondria. Here we show that AIF is imported from the endoplasmic reticulum to the mitochondria via mitochondria-associated membranes and transport vesicles.

Human papillomavirus infection and expression of ATPase family AAA domain containing 3A, a novel anti-autophagy factor, in uterine cervical cancer

Our aim was to determine the association of human papillomavirus (HPV) infection with the expression of ATPase family AAA domain containing 3A (ATAD3A), an anti-autophagy factor, in uterine cervical cancer (UCC). The HPV genotype was determined by an Easychip HPV blot assay. ATAD3A expression was determined by immunohistochemical staining. High-risk HPV (hrHPV) was detected in 184 (88.9%) of 207 UCC cases. ATAD3A expression was detected in 164 (79.2%) UCC cases. A significant correlation was found between ATAD3A expression and the presence of hrHPV (p<0.001), FIGO stage (p=0.014), lymph node involvement (p=0.001), c-MET expression (p<0.001), interleukin-8 (p=0.03) and patient survival (p=0.0016). Interestingly, silencing of E6/E7 expression decreased ATAD3A expression and cell survival. Moreover, knockdown of ATAD3A (ATAD3Akd) expression or addition of resveratrol, increased cellular autophagy and apoptosis and reduced drug resistance. Resveratrol reduced ATAD3A expression, and increased abrasion of the mitochondrial outer membrane as well as numbers of autophagosomes, the phenomena that were frequently found in ATAD3Akd cells. In conclusion, our results show that HPV infection correlates with increased ATAD3A expression and drug resistance in UCC. Persistent HPV infection may stabilize ATAD3A expression to inhibit cell autophagy and apoptosis as well as to increase drug resistance.

ATPase family AAA domain containing 3A is an anti-apoptotic factor and a secretion regulator of PSA in prostate cancer

In order to investigate the clinical value of ATPase family AAA domain containing 3A (ATAD3A), a potential anti-apoptotic factor in prostate cancer (PCa), immunohistochemistry was used to measure ATAD3A expression in pathological specimens from 86 Chinese patients and in 183 tissue-array samples from American patients. The effect of ATAD3A on the expression of prostate specific antigen (PSA) and drug resistance in PCa cell lines was determined by in vitro experiments. ATAD3A was detected in 74 of 86 (86.0%) Chinese specimens and in 145 of 183 (79.2%) American patient samples. No difference was found in ATAD3A expression between these two patient groups. In vitro, silencing of ATAD3A expression reduced PSA secretion and cisplatin resistance, suggesting that ATAD3A was associated with PSA secretion and drug resistance in PCa.

Neodymium-YAG laser iridotomy in angle closure glaucoma: preliminary study.

A prospective short-term preliminary clinical study to evaluate the efficacy and immediate complications of Q-switched Nd-YAG laser iridotomy in the treatment of acute and chronic angle closure glaucoma is described. The follow-up period ranged from four to 10 months. Of 40 eyes treated 36 (90%) required a single lasing session for patency (19, one application; 17, two applications), and four eyes (10%) required two sessions. Closure of the iridotomy site following Nd-YAG lasing due to pigment epithelium proliferation occurred in 10% of eyes, an incidence remarkably lower than that of argon laser iridotomy. Transitory closure or diminution of a prior patent iridotomy during the first hour after lasing was observed in 6.7% of eyes. Patency was again noted up to three weeks later and remained unchanged. Immediate postoperative complications included a marked increase in ocular pressure (42% of eyes), minimal transitory bleeding, and transitory localised corneal oedema at the lasing site. Persistent iridocorneal adhesion at the lasing site was noted in three eyes, and localised lenticular opacities, of non-progressive type, were observed in one eye.