ATPase Function Research Articles

The CATP-8/P5A-type ATPase functions in multiple pathways during neuronal patterning.

The assembly of neuronal circuits involves the migrations of neurons from their place of birth to their final location in the nervous system, as well as the coordinated growth and patterning of axons and dendrites. In screens for genes required for patterning of the nervous system, we identified the catp-8/P5A-ATPase as an important regulator of neural patterning. P5A-ATPases are part of the P-type ATPases, a family of proteins known to serve a conserved function as transporters of ions, lipids and polyamines in unicellular eukaryotes, plants, and humans. While the function of many P-type ATPases is relatively well understood, the function of P5A-ATPases in metazoans remained elusive. We show here, that the Caenorhabditis elegans ortholog catp-8/P5A-ATPase is required for defined aspects of nervous system development. Specifically, the catp-8/P5A-ATPase serves functions in shaping the elaborately sculpted dendritic trees of somatosensory PVD neurons. Moreover, catp-8/P5A-ATPase is required for axonal guidance and repulsion at the midline, as well as embryonic and postembryonic neuronal migrations. Interestingly, not all axons at the midline require catp-8/P5A-ATPase, although the axons run in the same fascicles and navigate the same space. Similarly, not all neuronal migrations require catp-8/P5A-ATPase. A CATP-8/P5A-ATPase reporter is localized to the ER in most, if not all, tissues and catp-8/P5A-ATPase can function both cell-autonomously and non-autonomously to regulate neuronal development. Genetic analyses establish that catp-8/P5A-ATPase can function in multiple pathways, including the Menorin pathway, previously shown to control dendritic patterning in PVD, and Wnt signaling, which functions to control neuronal migrations. Lastly, we show that catp-8/P5A-ATPase is required for localizing select transmembrane proteins necessary for dendrite morphogenesis. Collectively, our studies suggest that catp-8/P5A-ATPase serves diverse, yet specific, roles in different genetic pathways and may be involved in the regulation or localization of transmembrane and secreted proteins to specific subcellular compartments.

Defects in KCNJ16 Cause a Novel Tubulopathy with Hypokalemia, Salt Wasting, Disturbed Acid-Base Homeostasis, and Sensorineural Deafness.

The transepithelial transport of electrolytes, solutes, and water in the kidney is a well-orchestrated process involving numerous membrane transport systems. Basolateral potassium channels in tubular cells not only mediate potassium recycling for proper Na+,K+-ATPase function but are also involved in potassium and pH sensing. Genetic defects in KCNJ10 cause EAST/SeSAME syndrome, characterized by renal salt wasting with hypokalemic alkalosis associated with epilepsy, ataxia, and sensorineural deafness. A candidate gene approach and whole-exome sequencing determined the underlying genetic defect in eight patients with a novel disease phenotype comprising a hypokalemic tubulopathy with renal salt wasting, disturbed acid-base homeostasis, and sensorineural deafness. Electrophysiologic studies and surface expression experiments investigated the functional consequences of newly identified gene variants. We identified mutations in the KCNJ16 gene encoding KCNJ16, which along with KCNJ15 and KCNJ10, constitutes the major basolateral potassium channel of the proximal and distal tubules, respectively. Coexpression of mutant KCNJ16 together with KCNJ15 or KCNJ10 in Xenopus oocytes significantly reduced currents. Biallelic variants in KCNJ16 were identified in patients with a novel disease phenotype comprising a variable proximal and distal tubulopathy associated with deafness. Variants affect the function of heteromeric potassium channels, disturbing proximal tubular bicarbonate handling as well as distal tubular salt reabsorption.

Role of Reduced Sarco-Endoplasmic Reticulum Ca2+-ATPase Function on Sarcoplasmic Reticulum Ca2+ Alternans in the Intact Rabbit Heart.

Sarcoplasmic reticulum (SR) Ca2+ cycling is tightly regulated by ryanodine receptor (RyR) Ca2+ release and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ uptake during each excitation–contraction coupling cycle. We previously showed that RyR refractoriness plays a key role in the onset of SR Ca2+ alternans in the intact rabbit heart, which contributes to arrhythmogenic action potential duration (APD) alternans. Recent studies have also implicated impaired SERCA function, a key feature of heart failure, in cardiac alternans and arrhythmias. However, the relationship between reduced SERCA function and SR Ca2+ alternans is not well understood. Simultaneous optical mapping of transmembrane potential (Vm) and SR Ca2+ was performed in isolated rabbit hearts (n = 10) using the voltage-sensitive dye RH237 and the low-affinity Ca2+ indicator Fluo-5N-AM. Alternans was induced by rapid ventricular pacing. SERCA was inhibited with cyclopiazonic acid (CPA; 1–10 μM). SERCA inhibition (1, 5, and 10 μM of CPA) resulted in dose-dependent slowing of SR Ca2+ reuptake, with the time constant (tau) increasing from 70.8 ± 3.5 ms at baseline to 85.5 ± 6.6, 129.9 ± 20.7, and 271.3 ± 37.6 ms, respectively (p < 0.05 vs. baseline for all doses). At fast pacing frequencies, CPA significantly increased the magnitude of SR Ca2+ and APD alternans, most strongly at 10 μM (pacing cycle length = 220 ms: SR Ca2+ alternans magnitude: 57.1 ± 4.7 vs. 13.4 ± 8.9 AU; APD alternans magnitude 3.8 ± 1.9 vs. 0.2 ± 0.19 AU; p < 0.05 10 μM of CPA vs. baseline for both). SERCA inhibition also promoted the emergence of spatially discordant alternans. Notably, at all CPA doses, alternation of SR Ca2+ release occurred prior to alternation of diastolic SR Ca2+ load as pacing frequency increased. Simultaneous optical mapping of SR Ca2+ and Vm in the intact rabbit heart revealed that SERCA inhibition exacerbates pacing-induced SR Ca2+ and APD alternans magnitude, particularly at fast pacing frequencies. Importantly, SR Ca2+ release alternans always occurred before the onset of SR Ca2+ load alternans. These findings suggest that even in settings of diminished SERCA function, relative refractoriness of RyR Ca2+ release governs the onset of intracellular Ca2+ alternans.

FANCM regulates repair pathway choice at stalled replication forks

Repair pathway "choice" at stalled mammalian replication forks is an important determinant of genome stability; however, the underlying mechanisms are poorly understood. FANCM encodes a multi-domain scaffolding and motor protein that interacts with several distinct repair protein complexes at stalled forks. Here, we use defined mutations engineered within endogenous Fancm in mouse embryonic stem cells to study how Fancm regulates stalled fork repair. We find that distinct FANCM repair functions are enacted by molecularly separable scaffolding domains. These findings define FANCM as a key mediator of repair pathway choice at stalled replication forks and reveal its molecular mechanism. Notably, mutations that inactivate FANCM ATPase function disable all its repair functions and "trap" FANCM at stalled forks. We find that Brca1 hypomorphic mutants are synthetic lethal with Fancm null or Fancm ATPase-defective mutants. The ATPase function of FANCM may therefore represent a promising "druggable" target for therapy of BRCA1-linked cancer.

PprA Protein Inhibits DNA Strand Exchange and ATP Hydrolysis of Deinococcus RecA and Regulates the Recombination in Gamma-Irradiated Cells.

DrRecA and PprA proteins function are crucial for the extraordinary resistance to γ-radiation and DNA strand break repair in Deinococcus radiodurans. DrRecA mediated homologous recombination help in DNA strand break repair and cell survival, while the PprA protein confers radio-resistance via its roles in DNA repair, genome maintenance, and cell division. Genetically recA and pprA genes interact and constitute an epistatic group however, the mechanism underlying their functional interaction is not clear. Here, we showed the physical and functional interaction of DrRecA and PprA protein both in solution and inside the cells. The absence of the pprA gene increases the recombination frequency in gamma-irradiated D. radiodurans cells and genomic instability in cells growing under normal conditions. PprA negatively regulates the DrRecA functions by inhibiting DrRecA mediated DNA strand exchange and ATPase function in vitro. Furthermore, it is shown that the inhibitory effect of PprA on DrRecA catalyzed DNA strand exchange was not due to sequestration of homologous dsDNA and was dependent on PprA oligomerization and DNA binding property. Together, results suggest that PprA is a new member of recombination mediator proteins (RMPs), and able to regulate the DrRecA function in γ-irradiated cells by protecting the D. radiodurans genome from hyper-recombination and associated negative effects.

NCX-4040, a Unique Nitric Oxide Donor, Induces Reversal of Drug-Resistance in Both ABCB1- and ABCG2-Expressing Multidrug Human Cancer Cells.

Simple SummaryDevelopment of resistance to chemotherapeutics during the treatment of human cancers is a serious problem in the clinic, resulting in a poor treatment outcome and survival. It is believed that overexpression of ABC efflux proteins (e.g., P-gp/ABCB1, BCRP/ABCG2 and MRP/ABCC1) on the tumor cell membrane is one of the main mechanisms for this clinical resistance. Our recent studies indicate that nitric oxide (NO), inhibits ATPase functions of ABC transporters, resulting in reversal of resistance to various anticancer drugs. In this study we have found that nitric oxide and/or active metabolite (s) generated from NCX4040, a nitric oxide donor, inhibited ABC transporter activities by inhibiting their ATPase functions, causing reversal of both adriamycin and topotecan resistance in human MDR tumor cells. We also found that nitric oxide and/or metabolites of NCX4040 significantly enhanced drug accumulations in MDR tumor cells. These studies strongly suggest that tumor specific nitric oxide donors that deliver high amounts of nitric oxide and reactive species to clinical resistant tumors may be extremely useful in treating human tumors overexpressing ABC transporters, including cancer stem cells. In addition, such NO-donors can also be utilized for treating other diseases where drug resistance results from the presence of ATP-dependent transporter proteins.The emergence of multidrug resistance (MDR) in the clinic is a significant problem for a successful treatment of human cancers. Overexpression of various ABC transporters (P-gp, BCRP and MRP’s), which remove anticancer drugs in an ATP-dependent manner, is linked to the emergence of MDR. Attempts to modulate MDR have not been very successful in the clinic. Furthermore, no single agent has been found to significantly inhibit their functions to overcome clinical drug resistance. We have previously shown that nitric oxide (●NO) inhibits ATPase functions of ABC transporters, causing reversal of resistance to clinically active anticancer drugs. In this study, we have used cytotoxicity and molecular docking studies to show that NCX4040, a nitric oxide donor related to aspirin, inhibited the functions of ATPase which resulted in significant reversal of resistance to both adriamycin and topotecan in P-gp- and BCRP-expressing human cancer cell lines, respectively. We also used several other cytotoxic nitric oxide donors, e.g., molsidomine and S-nitroso glutathione; however, both P-gp- and BCRP-expressing cells were found to be highly resistant to these NO-donors. Molecular docking studies showed that NCX4040 binds to the nucleotide binding domains of the ATPase and interferes with further binding of ATP, resulting in decreased activities of these transporters. Our results are extremely promising and suggest that nitric oxide and other reactive species delivered to drug resistant tumor cells by well-designed nitric oxide donors could be useful in sensitizing anticancer drugs in multidrug resistant tumors expressing various ABC transporters.

Investigation of anticancer activities of STA-9090 (ganetespib) as a second generation HSP90 inhibitor in Saos-2 osteosarcoma cells

Osteosarcoma is common childhood tumour type of the bone. Chemotherapy is the most important step in treatment of osteosarcoma. Despite advanced diagnosis methods and target specific cancer therapeutics, osteosarcoma has still a high mortality rate and a tendency to metastasize. Therefore, new therapeutic strategies are evaluated in osteosarcoma treatment in pre-clinical and clinical studies. In the last ten years, heat shock protein 90 (HSP90) has been important biological target to design target specific cancer drugs. HSP90 play vital roles in proper folding, stabilization and maintenance of oncogenic client proteins in tumorigenesis. Therefore, inhibition of HSP90 has been significant therapeutic aspects in cancer drug design. STA-9090 (ganetespib) is a second generation small molecule HSP90 inhibitor which blocks tumurogenesis in cancer cells. STA-9090 inhibited ATP hydrolysis and protein folding process of HSP90. In this study, STA-9090 decreased Saos-2 cell proliferation and IC50 dose of STA-9090 was found out as 18.71 µM and 10.25 µM at 24 h and 48 h, respectively. STA-9090 inhibited HSP90 ATPase function and disrupted oncogenic client protein folding activity. Also, STA-9090 decreased protein level of the HSP90 in osteosarcoma cells. Expression analysis of osteosarcoma and bone metabolism related genes was performed by RT2 Profiler PCR Array. This study has found the down-regulation of the expression levels of oncogenic genes: DKK1, TWIST1, WNT10B, WNT3A, RANK, RANKL, PTH, FGFR1, FGFR2, LTBP2, IL6, TGFβ1, MMP2 and SPARC genes, in STA-9090 treated Saso-2 cells. Furthermore, expression levels of osteosarcoma related genes, OPG, ERα, ERβ, IL15, BMP2 and BMP7, were found to have increased significantly. Biological activities of STA-9090 on Saos-2 cell line show its potential as a target specific drug to inhibit osteosarcoma and its metastasis.

Plasma Membrane H+-ATPase SmPHA4 Negatively Regulates the Biosynthesis of Tanshinones in Salvia miltiorrhiza.

Salvia miltiorrhiza Bunge has been widely used in the treatment of cardiovascular and cerebrovascular diseases, due to the pharmacological action of its active components such as the tanshinones. Plasma membrane (PM) H+-ATPase plays key roles in numerous physiological processes in plants. However, little is known about the PM H+-ATPase gene family in S. miltiorrhiza (Sm). Here, nine PM H+-ATPase isoforms were identified and named SmPHA1–SmPHA9. Phylogenetic tree analysis showed that the genetic distance of SmPHAs was relatively far in the S. miltiorrhiza PM H+-ATPase family. Moreover, the transmembrane structures were rich in SmPHA protein. In addition, SmPHA4 was found to be highly expressed in roots and flowers. HPLC revealed that accumulation of dihydrotanshinone (DT), cryptotanshinone (CT), and tanshinone I (TI) was significantly reduced in the SmPHA4-OE lines but was increased in the SmPHA4-RNAi lines, ranging from 2.54 to 3.52, 3.77 to 6.33, and 0.35 to 0.74 mg/g, respectively, suggesting that SmPHA4 is a candidate regulator of tanshinone metabolites. Moreover, qRT-PCR confirmed that the expression of tanshinone biosynthetic-related key enzymes was also upregulated in the SmPHA4-RNAi lines. In summary, this study highlighted PM H+-ATPase function and provided new insights into regulatory candidate genes for modulating secondary metabolism biosynthesis in S. miltiorrhiza.

Transcriptional co-regulation of plasma membrane H+-ATPase and NADPH oxidase during root growth

During root growth, plasma membrane NADPH oxidase (NOX) and PM H+-ATPase function cooperatively to maintain the membrane electrical balance while mediating cell growth through wall relaxation. A threshold [Ca+2]cyt and H2O2 mediate the feed-forward loop between these two enzymes in Vigna radiata seedlings. The apoplastic superoxide (O2ˉ) being produced by NOX is subsequently dismutated to H2O2 either spontaneously or by the activity of apoplastic Cu/Zn superoxide dismutase (SOD) enzyme. Since SOD utilises apoplastic H+ (extruded from cytosol by PM H+-ATPase) and O2ˉ as substrates, its functioning is inevitably depending on PM H+-ATPase and NOX activities. Further conversion of H2O2 by class III peroxidase (Prx) to hydroxyl radical (OH) is instrumental for wall polysaccharide cleavage and cell wall relaxation. Thus, SOD and Prx activities appear to be co-ordinated with the upstream interplay between NOX and PM H+-ATPase in the regulatory network for root growth (Majumdar and Kar, 2019, J Plant Physiol 232: 248–256). In the present investigation, possible co-regulation among NOX and PM H+-ATPase, at gene expression level, has been studied during root growth of V. radiata seedlings using quantitative real-time PCR (qRT-PCR). It is evident that a transcriptional co-regulation exists between the two enzymes and inhibition of any one enzyme or removal of its product represses gene expression of the other. As observed for their activities, H2O2 and Ca+2 apparently regulate expression of both NOX and PM H+-ATPase genes and the probable mechanism is discussed.

Evolution of Plant Na+-P-Type ATPases: From Saline Environments to Land Colonization.

Soil salinity is one of the major factors obstructing the growth and development of agricultural crops. Eukaryotes have two main transport systems involved in active Na+ removal: cation/H+ antiporters and Na+-P-type ATPases. Key transport proteins, Na+/K+-P-ATPases, are widely distributed among the different taxa families of pumps which are responsible for keeping cytosolic Na+ concentrations below toxic levels. Na+/K+-P-ATPases are considered to be absent in flowering plants. The data presented here are a complete inventory of P-type Na+/K+-P-ATPases in the major branches of the plant kingdom. We also attempt to elucidate the evolution of these important membrane pumps in plants in comparison with other organisms. We were able to observe the gradual replacement of the Na+-binding site to the Ca2+-binding site, starting with cyanobacteria and moving to modern land plants. Our results show that the α-subunit likely evolved from one common ancestor to bacteria, fungi, plants, and mammals, whereas the β-subunit did not evolve in green algae. In conclusion, our results strongly suggest the significant differences in the domain architecture and subunit composition of plant Na+/K+-P-ATPases depending on plant taxa and the salinity of the environment. The obtained data clarified and broadened the current views on the evolution of Na+/K+-P-ATPases. The results of this work would be helpful for further research on P-type ATPase functionality and physiological roles.

Identification of Na+/K+-ATPase α/β isoforms in Rhinella marina tissues by RNAseq and a molecular docking approach at the protein level to evaluate α isoform affinities for bufadienolides

Na+/K+-ATPase (NKA) function is inhibited by Bufadienolides (BD), a group of cardiotonic steroids (CTS) primarily produced by anurans of the Bufonidae family, such as Rhinella marina. This study characterized the presence of α and β NKA subunit isoforms in R. marina via RNAseq in four tissues: oocytes, skin, heart, and skeletal muscle. Transcripts encoding three α-like isoforms (α1, α2, α3) and three β-like isoforms (β1, β2, β4) were identified. The amino acid sequence of α1-like isoform shared 99.4% identity with the α1 isoform previously published for R. marina. Sequences for α2, α3, and β4 from R. marina were previously unavailable. The first extracellular loop in the α2-like isoform in R. marina showed similar substitutions to those found in their susceptible homologues in other taxa (L/Q111T and S119T); in contrast, this same loop in α3-like isoform showed similar substitutions (Q111L and G120R) to those reported for toad-eating animals such as snakes, which suggests relatively lower affinity for CTS. Docking results showed that all three α-like isoforms identified in R. marina transcriptomes have low affinity to CTS compared to the susceptible α1 isoform of Sus scrofa (pig), with α1-like isoform being the most resistant. The tissue-specific RNAseq results showed the following expression of NKA α-like and β-like subunit isoforms: Oocytes expressed α1 and β1; skin α1, β1, and low levels of β2; heart α1, α3, and β1; skeletal muscle α1, β4, with low levels of α2, α3, and β1. R. marina could be used as an important model for future structural, functional and pharmacological studies of NKA and its isoforms.

Mutational analysis of the Hsp70 substrate-binding domain: Correlating molecular-level changes with in vivo function.

Hsp70 is an evolutionarily conserved chaperone involved in maintaining protein homeostasis during normal growth and upon exposure to stresses. Mutations in the β6/β7 region of the substrate-binding domain (SBD) disrupt the SBD hydrophobic core resulting in impairment of the heat-shock response and prion propagation in yeast. To elucidate the mechanisms behind Hsp70 loss of function due to disruption of the SBD, we undertook targeted mutational analysis of key residues in the β6/β7 region. We demonstrate the critical functional role of the F475 residue across yeast cytosolic Hsp70-Ssa family. We identify the size of the hydrophobic side chain at 475 as the key factor in maintaining SBD stability and functionality. The introduction of amino acid variants to either residue 475, or close neighbor 483, caused instability and cleavage of the Hsp70 SBD and subsequent degradation. Interestingly, we found that Hsp70-Ssa cleavage may occur through a vacuolar carboxypeptidase (Pep4)-dependent mechanism rather than proteasomal. Mutations at 475 and 483 result in compromised ATPase function, which reduces protein re-folding activity and contributes to depletion of cytosolic Hsp70 in vivo. The combination of reduced functionality and stability of Hsp70-Ssa results in yeast cells that are compromised in their stress response and cannot propagate the [PSI+ ] prion.

Design of novel peptide inhibitors against the conserved bacterial transcription terminator, Rho

The transcription terminator Rho regulates many physiological processes in bacteria, such as antibiotic sensitivity, DNA repair, RNA remodeling, and so forth, and hence, is a potential antimicrobial target, which is unexplored. The bacteriophage P4 capsid protein, Psu, moonlights as a natural Rho antagonist. Here, we report the design of novel peptides based on the C-terminal region of Psu using phenotypic screening methods. The resultant 38-mer peptides, in addition to containing mutagenized Psu sequences, also contained plasmid sequences, fused to their C termini. Expression of these peptides inhibited the growth of Escherichia coli and specifically inhibited Rho-dependent termination in vivo. Peptides 16 and 33 exhibited the best Rho-inhibitory properties in vivo. Direct high-affinity binding of these two peptides to Rho also inhibited the latter's RNA-dependent ATPase and transcription termination functions in vitro. These two peptides remained functional even if eight to ten amino acids were deleted from their C termini. In silico modeling and genetic and biochemical evidence revealed that these two peptides bind to the primary RNA-binding site of the Rho hexamer near its subunit interfaces. In addition, the gene expression profiles of these peptides and Psu overlapped significantly. These peptides also inhibited the growth of Mycobacteria and inhibited the activities of Rho proteins from Mycobacterium tuberculosis, Xanthomonas, Vibrio cholerae, and Salmonella enterica. Our results showed that these novel anti-Rho peptides mimic the Rho-inhibition function of the ∼42-kDa dimeric bacteriophage P4 capsid protein, Psu. We conclude that these peptides and their C-terminal deletion derivatives could provide a basis on which to design novel antimicrobial peptides.

MutL Protein from the Neisseria gonorrhoeae Mismatch Repair System: Interaction with ATP and DNA

The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a non-canonical nucleotide pair by the MutS protein and the formation of a complex between MutS and MutL. Eukaryotic and most bacterial MutL homologs function as endonucleases that introduce a single-strand break in the daughter strand of the DNA, thus activating the repair process. However, many aspects of the functioning of this protein remain unknown. We studied the ATPase and DNA binding functions of the MutL protein from the pathogenic bacterium Neisseria gonorrhoeae (NgoMutL), which exhibits endonuclease activity. For the first time, the kinetic parameters of ATP hydrolysis by the full-length NgoMutL protein were determined. Its interactions with single- and double-stranded DNA fragments of various lengths were studied. NgoMutL was shown to be able to efficiently form complexes with DNA fragments that are longer than 40 nucleotides. Using modified DNA duplexes harboring a 2-pyridyldisulfide group on linkers of various lengths, we obtained NgoMutL conjugates with DNA for the first time. According to these results, the Cys residues of the wild-type protein are located at a distance of approximately 18-50 Å from the duplex. The efficiency of the affinity modification of Cys residues in NgoMutL with reactive DNAs was shown to decrease in the presence of ATP or its non-hydrolyzable analog, as well as ZnCl2, in the reaction mixture. We hypothesize that the conserved Cys residues of the C-terminal domain of NgoMutL, which are responsible for the coordination of metal ions in the active center of the protein, are involved in its interaction with DNA. This information may be useful in reconstruction of the main stages of MMR in prokaryotes that are different from γ-proteobacteria, as well as in the search for new targets for drugs against N. gonorrhoeae.

Modulating the mechanokinetics of spontaneous contractions of the myometrium of rats using calix[4]arene C-90 – plasma membrane calcium ATPase inhibitor

Introduction. Plasma membrane calcium ATPase is a constitutive structure of cells that functions as a high affinity system of releasing Са2+ ions from the cytoplasm and ensures a long-term maintenance of the basal concentration of these cations in the state of dormancy. Currently, there are no satisfactory means for the pharmacological correction of plasma membrane calcium ATPase function. Thus, elaboration, synthesis, and study of substances with the targeted impact on plasma membrane calcium ATPase are topical issues. Previously, we determined the ability of this calix[4]arene in the concentration of 10 µM to inhibit the contractive activity and to slow down the relaxation of smooth muscle of the myometrium in the NO-dependent way, which considerably decreases the normalized maximal velocity of the relaxation phase. Materials and Methods. The tenzometric methods and mechanokinetic analysis were used to investigate the impact of the cumulative increase in the concentration of calix[4]arene С-90 (10 nM – 100 µM) on the spontaneous contractive activity of the myometrium of rats. The complete profile of spontaneous cycles of contractions-relaxa­tions was studied by the empirical multiparameter method of complex mechanokinetic analysis, elaborated by us (with the consideration of the parameters of time (τ0, τC and τR), force (Fmax, FC and FR), velocity (VC and VR), and impulse (Іmax, ІC and ІR). Results. Calix[4]arene C-90 evoked the dose-dependent inhibition of spontaneous contractive activity of the myometrium preparations. Its high concentrations caused a change in the structure of the contraction act, such as an increase in the duration of the contraction phase, while the duration of the relaxation phase did not show any changes. The multiparameter method of the complex mechanokinetic analysis demonstrated that in the whole range of the investigated concentrations, substance С-90 considerably decreases the indices of force parameters (Fmax, FC and FR) and the values of impulses of force Іmax, IC and IR of the spontaneous contractions of the myometrium. On the background of all the applied concentrations (10-7–10-4 М), calix[4]arene С-90 conditioned the slowing down of the relaxation of spontaneous contractions in uterine muscle prepa­rations of rats, which was reflected in a reliable decrease in the parameter for the maxi­mal velocity of the relaxation phase (VR). Conclusions. The results of the study demonstrate that calix[4]arene С-90 inhibits the processes of Са2+ extrusion from smooth muscle cells myoplasm, probably, impacting plasma membrane calcium ATPase molecules directly. It is noteworthy that C-90 is also likely to inhibit the processes of the intake of these cations to cells from the extracellular medium, causing a decrease in the velocity of force intensification during the contraction phase, and reducing the frequency and force of the spontaneous contractions in the myometrium.

Genome-Wide Identification, Characterization, and Expression Analyses of P-Type ATPase Superfamily Genes in Soybean

P-type ATPases are transmembrane pumps of cations and phospholipids. They are energized by hydrolysis of ATP and play important roles in a wide range of fundamental cellular and physiological processes during plant growth and development. However, the P-type ATPase superfamily genes have not been characterized in soybean. Here, we performed genome-wide bioinformatic and expression analyses of the P-type ATPase superfamily genes in order to explore the potential functions of P-type ATPases in soybean. A total of 105 putative P-type ATPase genes were identified in the soybean genome. Phylogenetic relationship analysis of the P-type ATPase genes indicated that they can be divided into five subfamilies including P1B, P2A/B, P3A, P4 and P5. Proteins belonging to the same subfamily shared conserved domains. Forty-seven gene pairs were related to segmental duplication, which contributed to the expansion of the P-type ATPase genes during the evolution of soybean. Most of the P-type ATPase genes contained hormonal- and/or stress-related cis-elements in their promoter regions. Expression analysis by retrieving RNA-sequencing datasets suggested that almost all of the P-type ATPase genes could be detected in soybean tissues, and some genes showed tissue-specific expression patterns. Nearly half of the P-type ATPase genes were found to be significantly induced or repressed under stresses like salt, drought, cold, flooding, and/or phosphate starvation. Four genes were significantly affected by rhizobia inoculation in root hairs. The induction of two P2B-ATPase genes, GmACA1 and GmACA2, by phosphate starvation was confirmed by quantitative RT-PCR. This study provides information for understanding the evolution and biological functions of the P-type ATPase superfamily genes in soybean.

Machine Learning of Allosteric Effects: The Analysis of Ligand-Induced Dynamics to Predict Functional Effects in TRAP1.

Allosteric molecules provide a powerful means to modulate protein function. However, the effect of such ligands on distal orthosteric sites cannot be easily described by classical docking methods. Here, we applied machine learning (ML) approaches to expose the links between local dynamic patterns and different degrees of allosteric inhibition of the ATPase function in the molecular chaperone TRAP1. We focused on 11 novel allosteric modulators with similar affinities to the target but with inhibitory efficacy between the 26.3 and 76%. Using a set of experimentally related local descriptors, ML enabled us to connect the molecular dynamics (MD) accessible to ligand-bound (perturbed) and unbound (unperturbed) systems to the degree of ATPase allosteric inhibition. The ML analysis of the comparative perturbed ensembles revealed a redistribution of dynamic states in the inhibitor-bound versus inhibitor-free systems following allosteric binding. Linear regression models were built to quantify the percentage of experimental variance explained by the predicted inhibitor-bound TRAP1 states. Our strategy provides a comparative MD–ML framework to infer allosteric ligand functionality. Alleviating the time scale issues which prevent the routine use of MD, a combination of MD and ML represents a promising strategy to support in silico mechanistic studies and drug design.

Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28.

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

ATPase inhibition by omeprazole reveals role of heat shock proteins on testicular torsion.

Testicular torsion leads ischaemic injury and generates reactive oxygen species. Reactive oxygen species triggers lipid peroxidation, protein degradation and DNA damage. These biochemical processes trigger tissue damage. Heat shock proteins (HSPs) are important in spermatogenesis, and this work elucidates role of HSPs at the testicular torsion-detorsion process. A proton-pump inhibitor, omeprazole, tested to reveal the drug's curative effect since HSP functions through ATP hydrolysis. Thirty-two male Wistar Albino rats were divided into four groups: sham, control, omeprazole and serum physiologic groups. Right testis was torsed, while left ones remained untorsed. Protein peroxidation, DNA damage and lipid hydroperoxide levels as well as HSP expression were measured. Further, the effects were visualised with histopathologic imaging. HSP expression increases at the torsed right testis compared to the contralateral testis. Although HSP70 and HSP90 help antioxidant enzymes to keep their native structure, their anti-apoptotic properties accelerate the tissue damage. Omeprazole a proton-pump inhibitor employed to impair electron transfer chain and to inhibit HSP ATPase function. Omeprazole effectively inhibits HSPs and alleviates lipid peroxidation and DNA damage levels both at molecular and at tissue level, and the drug has profound curative effect on testicular torsion recovery.

Na+-K+-ATPase functions in the developing hippocampus: regional differences in CA1 and CA3 neuronal excitability and role in epileptiform network bursting.

The Na+-K+-ATPase (Na+-K+ pump) is essential for setting resting membrane potential and restoring transmembrane Na+ and K+ gradients after neuronal firing, yet its roles in developing neurons are not well understood. This study examined the contribution of the Na+-K+ pump to resting membrane potential and membrane excitability of developing CA1 and CA3 neurons and its role in maintaining synchronous network bursting. Experiments were conducted in postnatal day (P)9 to P13 rat hippocampal slices using whole cell patch-clamp and extracellular field-potential recordings. Blockade of the Na+-K+ pump with strophanthidin caused marked depolarization (23.1 mV) in CA3 neurons but only a modest depolarization (3.3 mV) in CA1 neurons. Regarding other membrane properties, strophanthidin differentially altered the voltage-current responses, input resistance, action-potential threshold and amplitude, rheobase, and input-output relationship in CA3 vs. CA1 neurons. At the network level, strophanthidin stopped synchronous epileptiform bursting in CA3 induced by 0 Mg2+ and 4-aminopyridine. Furthermore, dual whole cell recordings revealed that strophanthidin disrupted the synchrony of CA3 neuronal firing. Finally, strophanthidin reduced spontaneous excitatory postsynaptic current (sEPSC) bursts (i.e., synchronous transmitter release) and transformed them into individual sEPSC events (i.e., nonsynchronous transmitter release). These data suggest that the Na+-K+ pump plays a more profound role in membrane excitability in developing CA3 neurons than in CA1 neurons and that the pump is essential for the maintenance of synchronous network bursting in CA3. Compromised Na+-K+ pump function leads to cessation of ongoing synchronous network activity, by desynchronizing neuronal firing and neurotransmitter release in the CA3 synaptic network. These findings have implications for the regulation of network excitability and seizure generation in the developing brain.NEW & NOTEWORTHY Despite the extensive literature showing the importance of the Na+-K+ pump in various neuronal functions, its roles in the developing brain are not well understood. This study reveals that the Na+-K+ pump differentially regulates the excitability of CA3 and CA1 neurons in the developing hippocampus, and the pump activity is crucial for maintaining network activity. Compromised Na+-K+ pump activity desynchronizes neuronal firing and transmitter release, leading to cessation of ongoing epileptiform network bursting.