ATPase Regulation Research Articles

Mechanisms of Human Erythrocytes Volume Stabilization

Functional completeness of erythrocytes depends on high deformability of these cells, that allows them to pass through narrow tissue capillaries. The erythrocytes high deformability is provided due to maintenance of discoid shape with an optimal cell surface area to volume ratio. In its turn this ratio is maintained due to cell volume stabilization at a given cell surface area. In this work, using mathematical simulation, we studied role of Na/K-ATPase, calcium activated potassium channels and adenylate metabolism in human erythrocyte volume stabilization at increase in cell membrane permeability to cations. The simulation took into account a contribution of glycolytic metabolites and adenylates to cytoplasm osmotic pressure. It was shown that the presence in the cell of Na/K-ATPase and two opposite transmembrane gradients of Na+ and K+ ions provide a significantly improved cell volume stabilization at the increase in cell membrane permeability, compared with hypothetical cells, in which the osmotic balance between cell and extracellular compartment is provided due to a gradient of only one ion (Na+). In this case the erythrocyte volume deviates from the optimal value by less than 10% at change in cell membrane permeability from 50 to 200% of its normal value. In this case, however, the intracellular ion concentrations may change significantly (by several times). The adenylate metabolism system can provide an additional regulation of transport ATPases due to regulation of intracellular ATP levels. Under these conditions stabilization of steady-state values of intracellular ion concentrations (ion homeostasis) and of cell volume in the range of cell membrane permeability changes from 50 to 1500% of the normal value. In this case, however, the cell volume and intracellular ion concentrations may significantly deviate from the stabilized values during transitional processes. Simultaneous function of both, ion transport systems and adenylate metabolism allows to provide ion homeostasis and efficient erythrocyte volume stabilization (within 5% deviation from the optimal value) both in steady-state conditions and during transitional processes at increase in cell membrane permeability up to 10-15 times compared with the normal value.

Evolutionary Dynamics and Molecular Mechanisms of HORMA Domain Protein Signaling

Controlled assembly and disassembly of multi-protein complexes is central to cellular signaling. Proteins of the widespread and functionally diverse HORMA family nucleate assembly of signaling complexes by binding short peptide motifs through a distinctive safety-belt mechanism. HORMA proteins are now understood as key signaling proteins across kingdoms, serving as infection sensors in a bacterial immune system and playing central roles in eukaryotic cell cycle, genome stability, sexual reproduction, and cellular homeostasis pathways. Here, we describe how HORMA proteins' unique ability to adopt multiple conformational states underlies their functions in these diverse contexts. We also outline how a dedicated AAA+ ATPase regulator, Pch2/TRIP13, manipulates HORMA proteins' conformational states to activate or inactivate signaling in different cellular contexts. The emergence of Pch2/TRIP13 as a lynchpin for HORMA protein action in multiple genome-maintenance pathways accounts for its frequent misregulation in human cancers and highlights TRIP13 as a novel therapeutic target.

Interactions of ubiquitin and CHMP5 with the V domain of HD-PTP reveals role for regulation of Vps4 ATPase.

The family of Bro1 proteins coordinates the activity of the Endosomal Sorting Complexes Required for Transport (ESCRTs) to mediate a number of membrane remodeling events. These events culminate in membrane scission catalyzed by ESCRT-III, whose polymerization and disassembly is controlled by the AAA-ATPase, Vps4. Bro1-family members Alix and HD-PTP as well as yeast Bro1 have central “V” domains that noncovalently bind Ub and connect ubiquitinated proteins to ESCRT-driven functions such as the incorporation of ubiquitinated membrane proteins into intralumenal vesicles of multivesicular bodies. Recently, it was discovered that the V domain of yeast Bro1 binds the MIT domain of Vps4 to stimulate its ATPase activity. Here we determine the structural basis for how the V domain of human HD-PTP binds ubiquitin. The HD-PTP V domain also binds the MIT domain of Vps4, and ubiquitin binding to the HD-PTP V domain enhances its ability to stimulate Vps4 ATPase activity. Additionally, we found that V domains of both HD-PTP and Bro1 bind CHMP5 and Vps60, respectively, providing another potential molecular mechanism to alter Vps4 activity. These data support a model whereby contacts between ubiquitin, ESCRT-III, and Vps4 by V domains of the Bro1 family may coordinate late events in ESCRT-driven membrane remodeling events.

Research advances on the role of Na+-K+-ATPase regulation in pulmonary edema clearance of acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a critical disease with high mortality, and currently there is no specific treatment. ARDS is characterized by refractory hypoxemia secondary to pulmonary edema, but the underlying mechanism is not yet fully understood. Alveolar edema fluid is mainly actively transported and reabsorbed by sodium-water transport system. The sodium pump (Na+-K+-ATPase-mediated Na+ transport) on the basal side of type II alveolar epithelial cells (AT II) is the main driving force for pulmonary edema clearance. Na+-K+-ATPase regulation is affected by many regulatory factors through a variety of ways, among which "long-term regulation" mechanism plays an important role, including positively regulating the gene transcription and protein expression of Na+-K+-ATPase. Na+-K+-ATPase can also be degraded by ubiquitin-proteasome pathway (UPP) and autophagy lysosome pathway to affect its abundance and enzyme activity, meanwhile, Na+-K+-ATPase α1 plays a key role in sodium water transport. We review the "long-term regulation" mechanism of Na+-K+-ATPase related pathways in pulmonary edema clearance and explore the possibility of new therapies for ARDS based on this mechanism, so as to provide new targets for the treatment of ARDS.

Structural and Functional Analysis of Disease-Linked p97 ATPase Mutant Complexes

IBMPFD/ALS is a genetic disorder caused by a single amino acid mutation on the p97 ATPase, promoting ATPase activity and cofactor dysregulation. The disease mechanism underlying p97 ATPase malfunction remains unclear. To understand how the mutation alters the ATPase regulation, we assembled a full-length p97R155H with its p47 cofactor and first visualized their structures using single-particle cryo-EM. More than one-third of the population was the dodecameric form. Nucleotide presence dissociates the dodecamer into two hexamers for its highly elevated function. The N-domains of the p97R155H mutant all show up configurations in ADP- or ATPγS-bound states. Our functional and structural analyses showed that the p47 binding is likely to impact the p97R155H ATPase activities via changing the conformations of arginine fingers. These functional and structural analyses underline the ATPase dysregulation with the miscommunication between the functional modules of the p97R155H.

MPK6 Kinase Regulates Plasma Membrane H+-ATPase Activity in Cold Acclimation.

Cold and freezing stresses severely affect plant growth, development, and survival rate. Some plant species have evolved a process known as cold acclimation, in which plants exposed to temperatures above 0 °C trigger biochemical and physiological changes to survive freezing. During this response, several signaling events are mediated by transducers, such as mitogen activated protein kinase (MAPK) cascades. Plasma membrane H+-ATPase is a key enzyme for the plant cell life under regular and stress conditions. Using wild type and mpk3 and mpk6 knock out mutants in Arabidopsis thaliana, we explored the transcriptional, translational, and 14-3-3 protein regulation of the plasma membrane H+-ATPase activity under the acclimation process. The kinetic analysis revealed a differential profiling of the H+-ATPase activity depending on the presence or absence of MPK3 or MPK6 under non-acclimated or acclimated conditions. Negative regulation of the plasma membrane H+-ATPase activity was found to be exerted by MPK3 in non-acclimated conditions and by MPK6 in acclimated conditions, describing a novel form of regulation of this master ATPase. The MPK6 regulation involved changes in plasma membrane fluidity. Moreover, our results indicated that MPK6 is a critical regulator in the process of cold acclimation that leads to freezing tolerance and further survival.

Targeting allostery in the Dynein motor domain with small molecule inhibitors

Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dynein's unusually large size, four distinct nucleotide-binding sites, and conformational dynamics pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterized S.cerevisiae dynein construct by pyrazolo-pyrimidinone-based compounds. These data, along with functional assays of dynein motility and mutagenesis studies, suggest that the compounds inhibit dynein by engaging the regulatory ATPase sites in the AAA3 and AAA4 domains, and not by interacting with dynein's main catalytic site in the AAA1 domain. A double Walker B mutation of the AAA3 and AAA4 sites substantially reduces enzyme activity, suggesting that targeting these regulatory domains is sufficient to inhibit dynein. Our findings reveal how chemical inhibitors can be designed to disrupt allosteric communication across dynein's AAA domains.

The Human Rogdi Protein Is the Functional Homologue of Yeast Rav2 and Can Promote V‐ATPase Assembly

Mutations in the human Rogdi protein cause Kohlschutter-Tonz syndrome, which is characterized by early-onset seizures, developmental defects, and defective deposition of tooth enamel (amelogenesis imperfecta). The cellular function of Rogdi is not known. The yeast RAVE (regulator of ATPase of vacuoles and endosomes) complex and mammalian Rabconnectin-3 complexes catalyze assembly of certain subpopulations of the V-ATPase proton pump and thus help to regulate pH homeostasis. The yeast RAVE complex is composed of three subunits: Rav1, Rav2, and Skp1. Skp1 is a multifunctional scaffold protein, but both Rav1 and Rav2 act specifically in V-ATPase assembly. Rabconnectin-3 complexes have been reported to contain two larger subunits, Rbcn3α and 3β; both subunits are predicted to have structural similarity to Rav1. No mammalian homologue of yeast Rav2 had been identified. However, despite limited sequence homology, we find that the yeast Rav2 sequence can be modeled with very high confidence on a recent structure of human Rogdi (H. Lee et al. (2017) Sci. Rep. 7:3972). Expression of human Rogdi in yeast complements the V-ATPase-associated growth defects of a yeast rav2∆ mutant. Complementation requires the presence of Rav1, suggesting that Rogdi acts as part of the RAVE complex rather than bypassing RAVE function. Consistent with this, Rogdi copurifies with a FLAG-tagged Rav1 protein. Yeast Rav2 binds to the N-terminal β-propeller region of Rav1. Two-hybrid assays indicate that Rogdi can interact with the same region of Rav1, as well as binding to the N-terminal β-propeller regions of Rbcn3α and 3β isoforms. Based on these data, we hypothesize that Rogdi is a subunit of mammalian Rabconnectin-3 complexes. WDR72 is a possible Rbcn3β isoform in humans. Mutations in the N-terminal β-propeller domain of WDR72 also cause amelogenesis imperfecta, as well as distal renal tubule acidosis, a disease associated with defects in V-ATPase activity. We are testing whether these mutations affect the interaction between Rogdi and WDR72, potentially linking Rogdi with both Rabconnectin-3 and V-ATPase function.

Dissecting Regulatory Interactions with Cytosolic N‐terminal Domain of V‐ATPase a‐subunit Isoforms

V-ATPases acidify lysosomes, endosomes, Golgi apparatus, and regulated secretory granules. Each of these organelles has a distinct luminal pH, but it is not fully understood how this pH is established or how V-ATPase subpopulations respond specifically to extracellular stimuli. The V-ATPase a-subunit of the integral membrane Vo subcomplex occupies a critical position in the enzyme and is a major focus of regulatory interactions. The Vo a-subunit is present as organelle- and tissue-specific isoforms in most organisms. All isoforms consist of a cytosolic N-terminal domain (aNT) and an integral membrane C-terminal domain. The aNT domains have a “dumbbell” shape with proximal and distal globular subdomains connected by a coiled-coil. We are dissecting the role of aNT in isoform-specific V-ATPase regulation. The two organelle-specific a-subunit isoforms in yeast, Vph1 and Stv1, reside in the lysosome-like vacuole and Golgi apparatus, respectively. These isoforms differ in their dependence on the RAVE (regulator of ATPase of vacuoles and endosomes) complex. Vph1-containing V-ATPases require the RAVE complex for their assembly, but Stv1-containing V-ATPases assemble stably in the absence of RAVE. We have generated chimeras of Vph1NT and Stv1NT, using information from recent structural studies. Stv1NT does not bind to RAVE subunits in vitro, but introduction of the proximal domain of Vph1NT fully restores the RAVE interaction, implicating this region of Vph1 in RAVE-dependent assembly. The two isoforms also interact with distinct phosphoinositide phospholipids enriched in their organelle of residence; Stv1NT binds tightly to Golgi PI4P and Vph1NT binds to vacuolar PI(3,5)P2. Mutational data identified a PI4P binding site in the proximal domain of Stv1NT, and chimeras of Stv1NT and Vph1NT support this is as the major binding site. Mutations in the Vph1NT distal domain disrupt PI(3,5)P2-dependent V-ATPase regulation, but results with chimeras indicate that PI(3,5)P2 binding to the Vph1NT distal domain is much weaker than PI4P binding to Stv1NT. The human genome encodes four a-subunit genes that also exhibit isoform-specific regulation. The a2 isoform is enriched in the Golgi/endosomes and the a1 isoform resides in endosomes and lysosomes. The a2NT domain binds specifically to PI4P-containing liposomes in vitro and the a1NT domain interacts with PI3P and PI(3,5)P2, two lipids enriched in endosomes and lysosomes. These data indicate that Vo a-subunit isoforms contain an “isoform code” that imparts V-ATPase subpopulations with distinct properties.

Abstract PS16-32: Obg-like ATPase 1 enhances chemoresistance of breast cancer via activation of TGF-β/Smad axis cascades

Abstract Background: Understanding the molecular mechanism of drug resistance helps identify aneffective target for therapy of breast cancer. We investigated in this study the regulatory role ofObg-like ATPase 1 playing in multiple drug resistance of breast cancer.Methods: Paclitaxel resistant cell line (MCF-7-PTR) was developed by a continuous increasingpaclitaxel concentration. MTT assay was used to validate either acquired resistant or OLA1modified cell lines. qRT-PCR, immunoblot, apoptosis and cell cycle assays were performed toassess expression of genes and proteins in cell lines. A series of in vitro assays was performed inthe cells with RNAi-mediated knockdown to elucidate the regulatory role of OLA1 in breast cancer.Findings: We demonstrated that OLA1 was highly correlated with either acquired or intrinsicresistance of breast cancer. Further study showed that escalated expression of OLA1 promotedEMT process in tumor cells through TGF-β/Smad signaling cascades, resulting in the enhancedexpression of anti-apoptosis-related proteins (cleaved caspase3, Bax, Bcl-2) and the strengthendepolymerization of microtubules in tumor cells. Our findings revealed that OLA1 enhanced theanti-apoptotic ability and elucidated a regulatory role of OLA1 in promoting drug resistance ofbreast cancer.Interpretation: OLA1 is highly correlated with drug resistance of breast cancer. Chemo-sensitivity of the disease can be thus enhanced significantly by knocked down OLA1,which ledto inactivation of the TGF-β/Smad signaling cascades, polymerized microtubules, and promotedcell apoptosis. Our data suggest that OLA1 may be developed as a potential target to improvechemotherapy of patients with breast cancer.Keywords: breast cancer; Obg-like ATPase 1; multidrug resistance; γ-tubulin; paclitaxel Citation Format: Gary Xiao, Jianzhou Liu, Xiaoyu Miao. Obg-like ATPase 1 enhances chemoresistance of breast cancer via activation of TGF-β/Smad axis cascades [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-32.

Tissue-specific protective properties of lithium: comparison of rat kidney, erythrocytes and brain.

Lithium (Li) represents a first choice mood stabilizer for bipolar disorder (BD). Despite extensive clinical use, questions regarding its mechanism of action and pathological mechanism of renal function impairment by Li remain open. The present study aimed to improve our knowledge in this area paying special attention to the relationship between the length of Li action, lipid peroxidation (LP), and Na+/K+-ATPase properties. The effects of therapeutic Li doses, administered daily to male Wistar rats for 1 (acute), 7 (short term) and 28 days (chronic), were studied. For this purpose, Na+/K+-ATPase activity measurements, [3H]ouabain binding and immunoblot analysis of α-Na+/K+-ATPase were performed. Li-induced LP was evaluated by determining the malondialdehyde concentration by HPLC. Sleep deprivation (SD) was used as an experimental approach to model the manic phase of BD. Results obtained from the kidney were compared to those obtained from erythrocytes and different brain regions in the same tested animals. Whereas treatment with therapeutic Li concentration did not bring any LP damage nor significant changes of Na+/K+-ATPase expression and [3H]ouabain binding in the kidney, it conferred strong protection against this type of damage in the forebrain cortex. Importantly, the observed changes in erythrocytes indicated changes in forebrain cortices. Thus, different resistance to SD-induced changes of LP and Na+/K+-ATPase was detected in the kidney, erythrocytes and the brain of Li-treated rats. Our study revealed the tissue-specific protective properties of Li against LP and Na+/K+-ATPase regulation.

α2-3 Sialic acid binding and uptake by human monocyte-derived dendritic cells alters metabolism and cytokine release and initiates tolerizing T cell programming.

SummaryDendritic cells (DCs) are key in the initiation of the adaptive T cell responses to tailor adequate immunity that corresponds to the type of pathogen encountered. Oppositely, DCs control the resolution phase of inflammation and are able to induce tolerance after receiving anti-inflammatory cytokines or upon encounter of self-associated molecular patterns, such as α2-3 linked sialic acid (α2-3sia).Objective: We here investigated whether α2-3sia, that bind immune inhibitory Siglec receptors, would alter signaling and reprogramming of LPS-stimulated human monocyte-derived DCs (moDCs).Methods and Results: Transcriptomic analysis of moDCs stimulated with α2-3sia-conjugated dendrimers revealed differentially expressed genes related to metabolic pathways, cytokines, and T cell differentiation. An increase in genes involved in ATPase regulator activity, oxidoreductase activity, and glycogen metabolic processes was detected. Metabolic extracellular flux analysis confirmed a more energetic moDC phenotype upon α2-3sia binding as evidenced by an increase in both glycolysis and mitochondrial oxidative phosphorylation. TH1 differentiation promoting genes IFNL and IL27, were significantly downregulated in the presence of α2-3sia. Functional assays confirmed that α2-3sia binding to moDCs induced phosphorylation of Siglec-9, reduced production of inflammatory cytokines IL-12 and IL-6, and increased IL-10. Surprisingly, α2-3sia-differentiated moDCs promoted FoxP3+CD25+/-CD127- regulatory T cell differentiation and decreased FoxP3-CD25-CD127- effector T cell proliferation.Conclusions: In conclusion, we demonstrate that α2-3sia binding to moDCs, phosphorylates Siglec-9, alters metabolic pathways, cytokine signaling, and T cell differentiation processes in moDCs and promotes regulatory T cells. The sialic acid-Siglec axis on DCs is therefore, a novel target to induce tolerance and to explore for immunotherapeutic interventions aimed to restore inflammatory processes.

How to limit the speed of a motor: the intricate regulation of the XPB ATPase and translocase in TFIIH.

The superfamily 2 helicase XPB is an integral part of the general transcription factor TFIIH and assumes essential catalytic functions in transcription initiation and nucleotide excision repair. The ATPase activity of XPB is required in both processes. We investigated the interaction network that regulates XPB via the p52 and p8 subunits with functional mutagenesis based on our crystal structure of the p52/p8 complex and current cryo-EM structures. Importantly, we show that XPB’s ATPase can be activated either by DNA or by the interaction with the p52/p8 proteins. Intriguingly, we observe that the ATPase activation by p52/p8 is significantly weaker than the activation by DNA and when both p52/p8 and DNA are present, p52/p8 dominates the maximum activation. We therefore define p52/p8 as the master regulator of XPB acting as an activator and speed limiter at the same time. A correlative analysis of the ATPase and translocase activities of XPB shows that XPB only acts as a translocase within the context of complete core TFIIH and that XPA increases the processivity of the translocase complex without altering XPB’s ATPase activity. Our data define an intricate network that tightly controls the activity of XPB during transcription and nucleotide excision repair.

Transcriptomic investigation reveals toxic damage due to tilmicosin and potential resistance against tilmicosin in primary chicken myocardial cells

Tilmicosin is widely used to treat respiratory infections in animals and has been reported to induce cardiac damage and even sudden death. However, its exact mechanisms, especially in chickens, remain unclear. This study confirmed the dose-dependent damaging effect of tilmicosin on primary chicken myocardial cells. Primary chicken myocardial cells treated with tilmicosin (0.5 μg/mL) for 0 h, 12 h, and 48 h were subjected to RNA sequencing and bioinformatics analysis. Transcriptomic analysis revealed that cytokine-cytokine receptor interactions, calcium signaling pathway, peroxisomes, phagosomes, mitogen-activated protein kinase (MAPK) signaling pathway, and oxidative phosphorylation were significantly and differentially affected after 12 h or 48 h of tilmicosin treatment. Further evidence demonstrated consistently increased proinflammatory factors, peroxidation, and ferroptosis, and intracellular ion imbalance was caused by tilmicosin for 12 h, but this imbalance had recovered at 48 h. Meanwhile, intracellular resistance to tilmicosin-induced toxicity involved the active regulation of cyclooxygenase-1 and ATPase H+/K+–transporting beta subunit at 48 h, sustained activation of MAPK12, and downregulation of dual specificity phosphatase 10 at 12 h. In summary, this study suggests that tilmicosin exerts its cardiotoxicity in primary chicken myocardial cells through multiple mechanisms and finds several intracellular molecular targets to resist the toxicity.

Allosteric coupling between \u03b1-rings of the 20S proteasome

Proteasomal machinery performs essential regulated protein degradation in eukaryotes. Classic proteasomes are symmetric, with a regulatory ATPase docked at each end of the cylindrical 20S. Asymmetric complexes are also present in cells, either with a single ATPase or with an ATPase and non-ATPase at two opposite ends. The mechanism that populates these different proteasomal complexes is unknown. Using archaea homologs, we construct asymmetric forms of proteasomes. We demonstrate that the gate conformation of the two opposite ends of 20S are coupled: binding one ATPase opens a gate locally, and also opens the opposite gate allosterically. Such allosteric coupling leads to cooperative binding of proteasomal ATPases to 20S and promotes formation of proteasomes symmetrically configured with two identical ATPases. It may also promote formation of asymmetric complexes with an ATPase and a non-ATPase at opposite ends. We propose that in eukaryotes a similar mechanism regulates the composition of the proteasomal population.

The Adaptive Potential of the Middle Domain of Yeast Hsp90

The distribution of fitness effects (DFEs) of new mutations across different environments quantifies the potential for adaptation in a given environment and its cost in others. So far, results regarding the cost of adaptation across environments have been mixed, and most studies have sampled random mutations across different genes. Here, we quantify systematically how costs of adaptation vary along a large stretch of protein sequence by studying the distribution of fitness effects of the same ≈2,300 amino-acid changing mutations obtained from deep mutational scanning of 119 amino acids in the middle domain of the heat shock protein Hsp90 in five environments. This region is known to be important for client binding, stabilization of the Hsp90 dimer, stabilization of the N-terminal-Middle and Middle-C-terminal interdomains, and regulation of ATPase–chaperone activity. Interestingly, we find that fitness correlates well across diverse stressful environments, with the exception of one environment, diamide. Consistent with this result, we find little cost of adaptation; on average only one in seven beneficial mutations is deleterious in another environment. We identify a hotspot of beneficial mutations in a region of the protein that is located within an allosteric center. The identified protein regions that are enriched in beneficial, deleterious, and costly mutations coincide with residues that are involved in the stabilization of Hsp90 interdomains and stabilization of client-binding interfaces, or residues that are involved in ATPase–chaperone activity of Hsp90. Thus, our study yields information regarding the role and adaptive potential of a protein sequence that complements and extends known structural information.

Abstract 3900: The BRG1 ATPase helps to maintain the de-differentiation phenotype in alveolar rhabdomyosarcoma

Abstract The alveolar subtype of rhabdomyosarcoma (aRMS) is a highly malignant soft tissue tumor, which commonly occurs in childhood and is associated with the skeletal muscle lineage. aRMS is characterized by the expression of the fusion transcription factor PAX3-FOXO1, acting as the main oncogenic driver. Despite the expression of master regulatory factors (MRFs) of skeletal muscle differentiation, aRMS cells are blocked from executing the terminal differentiation program. Since PAX3-FOXO1 plays a role in repressing differentiation, in part by induction of epigenetic changes, we were interested to gain additional knowledge about epigenetic cofactors that mediate this activity of the fusion protein. Here, we identified BRG1, the major catalytic subunit of SWI/SNF complexes, to be essential for tumor cell proliferation in aRMS and the most important regulatory ATPase. We further report that the physiological effects of interference with SWI/SNF complex function involve transcriptional activation of the myogenic differentiation program, accompanied by altered morphology and cell cycle profiles. Proteomic studies suggest that PAX3-FOXO1 is not an integral part of SWI/SNF complexes, but still has close proximity indicating potential functional interaction through chromatin interfaces. Genome-wide ChIP binding profiles in aRMS cells revealed associations of BRG1 with motifs of myogenic factors including MYOG, MYF5, and MYOD, suggesting functional interactions with MRFs. Our data is consistent with a model where BRG1 containing complexes have a repressive function at regulatory elements of downstream differentiation target genes. Ongoing work aims to further characterize BRG1-mediated changes on genome architecture to understand its contribution to differentiation blockade in the background of fusion positive aRMS. We conclude that BRG1 is an essential target to overcome differentiation blockade of aRMS cells. While other SWI/SNF complex members have been proposed previously to be potentially important for RMS oncogenesis, these proteins generally lack druggability. In contrast, efforts to develop BRG1 specific compounds are already underway, including PROTAC compounds. Preclinical studies using these tools will provide novel insights into therapeutic applicability to target SWI/SNF complexes in RMS. Citation Format: Dominik Laubscher, Benjamin Z. Stanton, Berkley Gryder, Sudipto Das, Thorkell Andresson, Bernd Roschitzki, Witold Wolski, QuyAi Ngo, Joana G. Marques, Marco Wachtel, Christopher Vakoc, Javed Khan, Beat Schäfer. The BRG1 ATPase helps to maintain the de-differentiation phenotype in alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3900.

A genome-wide identification, characterization and functional analysis of salt-related long non-coding RNAs in non-model plant Pistacia vera L. using transcriptome high throughput sequencing

Long non-coding RNAs (lncRNAs) play crucial roles in regulating gene expression in response to plant stresses. Given the importance regulatory roles of lncRNAs, providing methods for predicting the function of these molecules, especially in non-model plants, is strongly demanded by researchers. Here, we constructed a reference sequence for lncRNAs in P. vera (Pistacia vera L.) with 53220 transcripts. In total, we identified 1909 and 2802 salt responsive lncRNAs in Ghazvini, a salt tolerant cultivar, after 6 and 24 h salt treatment, respectively and 1820 lncRNAs in Sarakhs, a salt sensitive cultivar, after 6 h salt treatment. Functional analysis of these lncRNAs by several hybrid methods, revealed that salt responsive NAT-related lncRNAs associated with transcription factors, CERK1, LEA, Laccase genes and several genes involved in the hormone signaling pathways. Moreover, gene ontology (GO) enrichment analysis of salt responsive target genes related to top five selected lncRNAs showed their involvement in the regulation of ATPase, cation transporter, kinase and UDP-glycosyltransferases genes. Quantitative real-time PCR (qRT-PCR) experiment results of lncRNAs, pre-miRNAs and mature miRNAs were in accordance with our RNA-seq analysis. In the present study, a comparative analysis of differentially expressed lncRNAs and microRNA precursors between salt tolerant and sensitive pistachio cultivars provides valuable knowledge on gene expression regulation under salt stress condition.

Chemo-Mechanical Coupling in the Transport Cycle of a Heme ABC Transporter

The heme importer from pathogenic bacteria is a member of the ATP-binding cassette (ABC) transporter family, which uses the energy of ATP-binding and hydrolysis for extensive conformational changes. Previous studies have indicated that conformational changes after heme translocation are triggered by ATP-binding to nucleotide binding domains (NBDs) and then, in turn, induce conformational transitions of the transmembrane domains (TMDs). In this study, we applied a template-based iterative all-atom molecular dynamics (MD) simulation to predict the ATP-bound outward-facing conformation of the Burkholderia cenocepacia heme importer BhuUV-T. The resulting model showed a stable conformation of the TMD with the cytoplasmic gate in the closed state and the periplasmic gate in the open state. Furthermore, targeted MD simulation predicted the intermediate structure of an occluded form (Occ) with bound ATP, in which both ends of the heme translocation channel are closed. The MD simulation of the predicted Occ revealed that Ser147 on the ABC signature motifs (LSGG[Q/E]) of NBDs occasionally flips and loses the active conformation required for ATP-hydrolysis. The flipping motion was found to be coupled to the inter-NBD distance. Our results highlight the functional significance of the signature motif of ABC transporters in regulation of ATPase and chemo-mechanical coupling mechanism.

Biochemical regulation and structural analysis of copper-transporting ATPase in a human hepatoma cell line for Wilson disease.

Hepatic copper levels differ among patients with Wilson disease (WD) and normal individuals depending on the dietary intake, copper bioavailability, and genetic factors. Copper chloride (CuCl2 ) caused dose-dependent reduction in cell viability of human teratocarcinoma (HepG2) cell line, measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cells were exposed to different concentrations of CuCl2 in log doses and maximum cell viability reduction was recorded at 15 µg/mL. Toxic dose of CuCl2 is potent inducer of reactive oxygen species (ROS). Apoptosis as a pattern of cell death was confirmed through sub-G1 fraction and morphological changes such as mitochondrial depolarization, endoplasmic reticulum and lysosomal destabilization, phosphatidylserine translocation, and DNA damage. Our transcriptional and translational results strongly support apoptotic cell death. Using the available data present in dbSNP and bioinformatics tools, three nonsynonymous single nucleotide polymorphisms (nsSNPs) were identified as deleterious, reducing the stability of protein ATP7B. Structural analysis of native and mutant ATP7B proteins was investigated using molecular dynamics simulation (MDS) approach. Mutation in ATP7B gene might disturb the structural conformation and catalytic function of the ATP7B protein may be inducing WD. Hence, excess dietary intake of copper chloride must be avoided for safety of health to prevent from WD.