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The present study was designed to investigate comparative expressions of follicle-stimulating hormone beta subunit (FSHβ) and nuclear receptor coactivator 1 (NCOA1) genes by real-time polymerase chain reaction, using polymerase chain reaction single-strand conformation polymorphism methods to investigate the effects of gene polymorphisms on reproductive traits, including total number of kits born (TNB) and number of born alive (NBA) in pink-eyed white mink. Four single-nucleotide polymorphisms were identified in the FSHβ and NCOA1 genes. The g.1228G>A polymorphism of FSHβ was associated with NBA and TNB (P < 0.01). The g.151536T>C polymorphism of NCOA1 was associated with NBA and TNB (P < 0.01). The NCOA1 mRNA levels in hypothalamus, ovary, and uterus during the first half of gestation were higher than during the middle term and last half of gestation (P < 0.01). The FSHβ mRNA levels in the hypothalamus and uterus were higher during the first half of gestation than during the middle term and last half of gestation (P < 0.05). In conclusion, the g.1866T>C polymorphism of FSHβ and the g.151536T>C polymorphism of NCOA1 could be molecular markers for reproductive traits, and expressions of FSHβ and NCOA1 might be involved in the regulation of embryo attachment mechanisms in pink-eyed white mink breeding.

Mucosal and skin cancers are associated with infections by human papillomaviruses (HPV). The manner how viral oncoproteins hijack the host cell metabolism to meet their own energy demands and how this may contribute to tumorigenesis is poorly understood. We now show that the HPV oncoprotein E7 of HPV8, HPV11 and HPV16 directly interact with the beta subunit of the mitochondrial ATP-synthase (ATP5B), which may therefore represent a conserved feature across different HPV genera. By measuring both glycolytic and mitochondrial activity we observed that the association of E7 with ATP5B was accompanied by reduction of glycolytic activity. Interestingly, there was a drastic increase in spare mitochondrial respiratory capacity in HPV8-E7 and an even more profound increase in HPV16-E7 expressing cells. In addition, we could show that ATP5B levels were unchanged in betaHPV positive skin cancers. However, comparing HPV-positive and HPV-negative oropharyngeal squamous cell carcinomas (OPSCC) we noticed that, while ATP5B expression levels did not correlate with patient overall survival in HPV-negative OPSCC, there was a strong correlation within the HPV16-positive OPSCC patient group. These novel findings provide evidence that HPV targets the host cell energy metabolism important for viral life cycle and HPV-mediated tumorigenesis.

The sodium-potassium ATPase (NKA) establishes ion gradients that facilitate many physiological processes. In the heart, NKA activity is regulated by its interaction with phospholemman (PLM, FXYD1). Here we used a novel fluorescence lifetime-based assay to investigate the structure, stoichiometry, and affinity of the NKA-PLM regulatory complex. We observed concentration dependent association of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit followed by lower affinity alpha-alpha and alpha-PLM interactions. The data provide the first evidence that the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits in intact cells. Docking and molecular dynamics simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with stoichiometry (alpha-beta-PLM)2.

The voltage-gated Na+ channel regulates the initiation and propagation of the action potential in excitable cells. The major cardiac isoform NaV1.5, encoded by SCN5A, comprises a monomer with four homologous repeats (I-IV) that each contain a voltage sensing domain (VSD) and pore domain. In native myocytes, NaV1.5 forms a macromolecular complex with NaVβ subunits and other regulatory proteins within the myocyte membrane to maintain normal cardiac function. Disturbance of the NaV complex may manifest as deadly cardiac arrhythmias. Although SCN5A has long been identified as a gene associated with familial atrial fibrillation (AF) and Brugada Syndrome (BrS), other genetic contributors remain poorly understood. Emerging evidence suggests that mutations in the non-covalently interacting NaVβ1 and NaVβ3 are linked to both AF and BrS. Here, we investigated the molecular pathologies of 8 variants in NaVβ1 and NaVβ3. Our results reveal that NaVβ1 and NaVβ3 variants contribute to AF and BrS disease phenotypes by modulating both NaV1.5 expression and gating properties. Most AF-linked variants in the NaVβ1 subunit do not alter the gating kinetics of the sodium channel, but rather modify the channel expression. In contrast, AF-related NaVβ3 variants directly affect channel gating, altering voltage-dependent activation and the time course of recovery from inactivation via the modulation of VSD activation.

Sea stars can adhere to various underwater substrata using an adhesive secretion of which Sfp1 is a major component. Sfp1 is a multimodular protein composed of four subunits (Sfp1 Alpha, Beta, Delta, and Gamma) displaying different functional domains. We recombinantly produced two fragments of Sfp1 comprising most of its functional domains: the C-terminal part of the Beta subunit (rSfp1 Beta C-term) and the Delta subunit (rSfp1 Delta). Surface plasmon resonance analyses of protein adsorption onto different model surfaces showed that rSfp1 Beta C-term exhibits a significantly higher adsorption than the fibrinogen control on hydrophobic, hydrophilic protein-resistant, and charged self-assembled monolayers, while rSfp1 Delta adsorbed more on negatively charged and on protein-resistant surfaces compared to fibrinogen. Truncated recombinant rSfp1 Beta C-term proteins were produced in order to investigate the role of the different functional domains in the adsorption of this protein. The analysis of their adsorption capacities on glass showed that two mechanisms are involved in rSfp1 Beta C-term adsorption: (1) one mediated by the EGF-like domain and involving Ca2+ and Mg2+ ions, and (2) one mediated by the sequence of Sfp1 Beta with no homology with known functional domain in databases, in the presence of Na+, Ca2+ and Mg2+ ions.

The sodium potassium ATPase (Na+/K+ ATPase) is essential for the maintenance of a low intracellular Na+ and a high intracellular K+ concentration. Loss of function of the Na+/K+ ATPase due to mutations in Na+/K+ ATPase genes, anoxic conditions, depletion of ATP or inhibition of the Na+/K+ ATPase function using cardiac glycosides such as digitalis, causes a depolarization of the resting membrane potential. While in non-excitable cells, the uptake of glucose and amino acids is decreased if the function of the Na+/K+ ATPase is compromised, in excitable cells the symptoms range from local hyper-excitability to inactivating depolarization. Although several studies have demonstrated the differential expression of the various Na+/K+ ATPase alpha and beta isoforms in the brain tissue of rodents, their expression profile during development has yet to be thoroughly investigated. An immunohistochemical analysis of postnatal day 19 mouse brain showed ubiquitous expression of Na+/K+ ATPase isoforms α1, β1 and β2 in both neurons and glial cells, whereas α2 was expressed mostly in glial cells and the α3 and β3 isoforms were expressed in neurons. Furthermore, we examined potential changes in the relative expression of the different Na+/K+ ATPase isoforms in different brain areas of postnatal day 6 and in adult 9 months old animals using immunoblot analysis. Our results show a significant up-regulation of the α1 isoform in cortex, hippocampus and cerebellum, whereas, the α2 isoform was significantly up-regulated in midbrain. The β3 isoform showed a significant up-regulation in all brain areas investigated. The up-regulation of the α3 isoform matched that of the β2 isoform which were both significantly up-regulated in cortex, hippocampus and midbrain, suggesting that the increased maturation of the neuronal network is accompanied by an increase in expression of α3/β2 complexes in these brain structures.

The β subunit of bovine luteinizing hormone (LH) was crystallized and its structure solved to 3.15 ​Å resolution by molecular replacement using human chorionic gonadotropin (hCG) β subunit as search model. The asymmetric unit contains two copies of the β subunit that are related by a non-crystallographic symmetry (NCS) two-fold axis, both copies of which contain proteolytic cleavages after amino acid 100. It is noteworthy that the oligosaccharide moieties covalently attached at asparagine 13 were particularly pronounced in the electron density, allowing seven sugar residues to be defined. The α subunit of LH, which is common to all glycosylated gonadotropin hormones, was placed by superposition of hCG on the LH beta subunits, thereby yielding a model for the intact hormone., Graphical abstract A model of luteinizing hormone based on the known α subunit and the β subunit determined here by X-ray crystallography.Image 1, Highlights • The X-ray crystallographic structure of the beta subunit of bovine luteinizing hormone (LH) has been determined at 3.15 A resolution • A model for the entire LH has been proposed based on the beta subunit structure reported here and the known structure if the common alpha subunit • A large amount (7 sugar residues on one molecule in the asymmetric unit, 5 on the other) of the oligosaccharide attached at asn 13 was visible in electron density maps • Each of the two molecules in the asymmetric unit, which contains an NCS twofold axis, is associated with two molecules of beta octylglucoside • The beta subunit has a proteolytic cleavage between amino acids 100 and 101, which releases the “seatbelt” peptide, though that peptide remains associated through an S–S bridge to the core.

Gestational trophoblastic diseases (GTD) are group of pregnancy-related tumors characterized by abnormal levels of ‘β-hCG’ with higher incidence in South-East Asia, especially India. Our laboratory has reported that wild-type BRCA1 transcriptionally regulates β-hCG in triple negative breast cancers (TNBCs). These factors culminated into analysis of BRCA1 status in GTD, which would emanate into elucidation of BRCA1- β-hCG relationship and unraveling etio-pathology of GTD. BRCA1 level in GTD is down-regulated due to the over-expression of DNMT3b and subsequent promoter hypermethylation, when compared to the normal placentae accompanied with its shift in localization. There is an inverse correlation of serum β-hCG levels with BRCA1 mRNA expression. The effects of methotrexate (MTX), which is the first-line chemotherapeutic used for GTD treatment, when analyzed in comparison with plumbagin (PB) revealed that PB alone is efficient than MTX alone or MTX-PB in combination, in showing selective cytotoxicity against GTD. Interestingly, PB increases BRCA1 levels post-treatment, altering DNMT3b levels and resultant BRCA1 promoter methylation. Also, cohort study analyzed the incidence of GTD at Sree Avittom Thirunal (SAT) Hospital, Thiruvananthapuram, which points out that 11.5% of gestational trophoblastic neoplasia (GTN) cases were referred to Regional Cancer Centre, Thiruvananthapuram, for examination of breast lumps. This has lend clues to supervene the risk of GTD patients towards BRCA1-associated diseases and unveil novel therapeutic for GTD, a plant-derived naphthoquinone, PB, already reported as selectively cytotoxic against BRCA1 defective tumors.

Purpose Primary breast and prostate cancers can be cured, but metastatic disease cannot. Identifying cell factors that predict metastatic potential could guide both prognosis and treatment. Methods We used Cell-SELEX to screen an RNA aptamer library for differential binding to prostate cancer cell lines with high vs. low metastatic potential. Mass spectroscopy, immunoblot, and immunohistochemistry were used to identify and validate aptamer targets. Aptamer properties were tested in vitro, in xenograft models, and in clinical biopsies. Gene expression datasets were queried for target associations in cancer. Results We identified a novel aptamer (Apt63) that binds to the beta subunit of F1Fo ATP synthase (ATP5B), present on the plasma membrane of certain normal and cancer cells. Apt63 bound to plasma membranes of multiple aggressive breast and prostate cell lines, but not to normal breast and prostate epithelial cells, and weakly or not at all to non-metastasizing cancer cells; binding led to rapid cell death. A single intravenous injection of Apt63 induced rapid, tumor cell-selective binding and cytotoxicity in MDA-MB-231 xenograft tumors, associated with endonuclease G nuclear translocation and DNA fragmentation. Apt63 was not toxic to non-transformed epithelial cells in vitro or adjacent normal tissue in vivo. In breast cancer tissue arrays, plasma membrane staining with Apt63 correlated with tumor stage (p

Background The most important factor for a successful pregnancy after in vitro fertilization is embryo quality. The aim of this study was to explore the possibility that using the immunomagnetic reduction (IMR) assay to quantitatively measure β-subunit of human chorionic gonadotropin (β-hCG) in blastocyst culture media to differentiate embryo quality. Methods This was a prospective case-control study including 28 samples of blastocyst culture media. We used single-step blastocyst culture and IMR assay to analyze β-hCG concentrations in culture media. We also explored the relationship between IMR signals of β-hCG and morphological grading of blastocysts. Results β-hCG concentration-dependent IMR signals were highly correlated with blastocyst morphological quality (Spearman correlation coefficient: 0.731). Receiver-operating characteristic curve analysis showed a cut-off IMR value to differentiate embryo quality of 0.873%, with an area under the curve of 0.947, sensitivity of 0.882 and specificity of 0.818. Furthermore, subanalysis also revealed a positive correlation between β-hCG concentration-dependent IMR signals and trophectoderm grading, with a Spearman correlation coefficient of 0.576. Conclusions An IMR assay can quantitatively measure β-hCG in blastocyst culture media, and may be a potential clinical tool to assist in the assessment of good blastocyst quality before embryo transfer.

Pentameric GABAA receptors are composed from 19 possible subunits. The GABAA β subunit is unique because the β1 and β3 subunits can assemble and traffic to the cell surface as homomers, whereas most of the other subunits, including β2, are heteromers. The intracellular domain (ICD) of the GABAA subunits has been implicated in targeting and clustering GABAA receptors at the plasma membrane. Here, we sought to test whether and how the ICD is involved in functional expression of the β3 subunit. Since θ is the most homologous to β but does not form homomers, we created two reciprocal chimeric subunits, swapping the ICD between the β3 and θ subunits, and expressed them in HEK293 cells. Surface expression was detected with immunofluorescence and functional expression was quantified using whole-cell patch-clamp recording with fast perfusion. Results indicate that, unlike β3, neither the β3/θIC nor the θ/β3IC chimera can traffic to the plasma membrane when expressed alone; however, when expressed in combination with either wild-type α3 or β3, the β3/θIC chimera was functionally expressed. This suggests that the ICD of α3 and β3 each contain essential anterograde trafficking signals that are required to overcome ER retention of assembled GABAA homo- or heteropentamers.

Sheath blight caused by Rhizotonia solani is described as the second major disease affecting rice. Genetic resistance to R. solani the ideal control measure is hampered because of the difficulty in identifying adequate resistance sources under typical selection conditions. Proteomic analysis techniques using two-dimensional gels (2D-PAGE) allow the study or monitoring of global changes in protein expression under normal and stress conditions. In this work, we compared rice leaves protein expression patterns of two Venezuelan varieties 12, 24 and 48 h after inoculation with R. solani. Approximately 400 and 300 protein spots stained with Sypro Ruby were reproducibly resolved across gel replicates, for PALMAR and FONAIAP-2000, respectively. Forty proteins out of a total 49 were identified for PALMAR variety, with thirty-two up-regulated protein spots and 8 down-regulated. Twenty-six proteins out of a total 33 were identified for FONAIAP-2000 variety, with seven up-regulated protein spots and 19 down-regulated. RuBisCo was the protein most identified (48% and 82% of the detected proteins for PALMAR and FONAIAP-2000, respectively). Other identified proteins showing variations were ATPase beta subunit, UDP-glucose anthocyanin 5-O-glucosyltransferase, RNA-binding protein, putative transkelotase 1, putative ferredoxin-NAPD(H) oxide-reductase, and putative 33kDa oxygen evolving protein photosystem II. Based on our results, rice response to R. solani could be described where energy is required to induce a defense and it is supplied by proteins involved in energy metabolism. According to this, proteomic could provide information and insights on the response of rice to challenge with R. solani and other pathogens. El tizón de la vaina causado por Rhizotonia solani se describe como la segunda enfermedad importante que afecta al arroz. La resistencia genética a R. solani, la medida de control ideal, se ve obstaculizada debido a la dificultad de identificar fuentes de resistencia adecuadas en condiciones típicas de selección. Las técnicas de análisis proteómico que utilizan geles bidimensionales (2D-PAGE) permiten el estudio o seguimiento de cambios globales en la expresión de proteínas en condiciones normales y de estrés. En este trabajo, comparamos los patrones de expresión de proteínas en hojas de arroz de dos variedades venezolanas 12, 24 y 48 h después de la inoculación con R. solani. Aproximadamente 400 y 300 manchas de proteína teñidas con Sypro Ruby se resolvieron de forma reproducible a través de réplicas de gel, para PALMAR y FONAIAP-2000, respectivamente. Se identificaron cuarenta proteínas de un total de 49 para la variedad PALMAR, con treinta y dos manchas proteicas reguladas al alza y 8 reguladas a la baja. Se identificaron 26 proteínas de un total de 33 para la variedad FONAIAP-2000, con siete manchas proteicas reguladas al alza y 19 reguladas a la baja. RuBisCo fue la proteína más identificada (48% y 82% de las proteínas detectadas para PALMAR y FONAIAP-2000, respectivamente). Otras proteínas identificadas que mostraron variaciones fueron la subunidad beta de la ATPasa, la UDP-glucosa antocianina 5-O-glucosiltransferasa, la proteína de unión al ARN, la supuesta transkelotasa 1, la supuesta ferredoxina-NAPD (H) óxido-reductasa y el supuesto fotosistema II de la proteína que evoluciona el oxígeno de 33 kDa. Con base en nuestros resultados, la respuesta del arroz a R. solani podría describirse donde se requiere energía para inducir una defensa y es suministrada por proteínas involucradas en el metabolismo energético. Según esto, proteomic podría proporcionar información y conocimientos sobre la respuesta del arroz al desafío con R. solani y otros patógenos.

This article aims to provide a comprehensive review of the clinical features and genetics basis of multi-system pseudohypoaldosteronism type 1 caused by SCNN1B gene mutations.

We recently characterized two paralogs of the thyrotropin (TSH) beta subunit in Atlantic salmon,tshβaandtshβb, issued from teleost-specific whole genome duplication. The transcript expression oftshβb, but not oftshβa, peaks at the time of smoltification, which revealed a specific involvement oftshβbparalog in this metamorphic event.Tshβaandtshβbare expressed by distinct pituitary cells in salmon, likely related to TSH cells from thepars distalisandpars tuberalis, respectively, in mammals and birds. The present study aimed at investigating the neuroendocrine and endocrine factors potentially involved in the differential regulation oftshβaandtshβbparalogs, using primary cultures of Atlantic salmon pituitary cells. The effects of various neurohormones and endocrine factors potentially involved in the control of development, growth, and metabolism were tested. Transcript levels oftshβaandtshβbwere measured by qPCR, as well as those of growth hormone (gh), for comparison and validation. Corticotropin-releasing hormone (CRH) stimulatedtshβatranscript levels in agreement with its potential role in the thyrotropic axis in teleosts, but had no effect ontshβbparalog, while it also stimulatedghtranscript levels. Thyrotropin-releasing hormone (TRH) had no effect on neithertshβparalogs norgh. Somatostatin (SRIH) had no effects on bothtshβparalogs, while it exerted a canonical inhibitory effect onghtranscript levels. Thyroid hormones [triiodothyronine (T3) and thyroxine (T4)] inhibited transcript levels of bothtshβparalogs, as well asgh, but with a much stronger effect ontshβathan ontshβbandgh.Conversely, cortisol had a stronger inhibitory effect ontshβbthantshβa, while no effect ongh. Remarkably, insulin-like growth factor 1 (IGF1) dose-dependently stimulatedtshβbtranscript levels, while it had no effect ontshβa, and a classical inhibitory effect ongh. This study provides the first data on the neuroendocrine factors involved in the differential regulation of the expression of the twotshβparalogs. It suggests that IGF1 may be involved in triggering the expression peak of thetshβbparalog at smoltification, thus representing a potential internal signal in the link between body growth and smoltification metamorphosis.

Bone resorption by osteoclasts is an energy consuming activity, which depends on mitochondrial ATP. ATP5B, a mitochondrial ATP synthase beta subunit, is a catalytic core involved in producing ATP. Here, we investigated the contribution of ATP5B in osteoclast differentiation and joint destruction. ATP5B (LV-ATP5B) targeting or non-targeting (LV-NC) siRNA containing lentivirus particles were transduced into bone marrow macrophage derived osteoclasts or locally administered to arthritic mouse joints. Inhibition of ATP5B reduced the expression of osteoclast related genes and proteins, suppressed bone resorption by significantly impairing F-actin formation and decreased the levels of adhesion-associated proteins. In addition, ATP5B deficiency caused osteoclast mitochondrial dysfunction and, impaired the secretion of vacuole protons and MMP9. Importantly, inhibition of ATP5B expression, protected arthritis mice from joint destructions although serum levels of inflammatory mediators (TNF-α, IL-1β) and IgG2α antibodies were unaffected. These results demonstrate an essential function of ATP5B in osteoclast differentiation and bone resorption, and suggest it as a potential therapeutic target for protecting bones in RA.

The current study aims to evaluate the modulatory effect of zinc oxide nanoparticles (ZnO NPs) on the bioenergetic signature biomarkers in the Ehrlich ascitic carcinoma (EAC) model. To achieve this goal, 90 female albino mice were included in this study and were divided into six equal groups (n =15 per group): saline-treated group, ZnO NP-treated, EACs-bearing mice, and three groups of EACs-bearing mice treated with ZnO NPs at a dose of 20 mg/kg every other day, 10 mg/kg every other day, 10 mg/kg every day, respectively, for 14 days. The tissues from treated groups and control groups were homogenized and used for the assay of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and F1 beta subunit of adenosine triphosphate (ATP) synthase levels, as well as the determination of lactate level. The survival time of mice was improved in all ZnO NP-treated groups, especially in EACs-bearing mice treated with ZnO NPs at a dose of 10 mg/kg every other day. This improvement was associated with an increased F1 beta subunit of ATP synthase level and a decreased GAPDH level. Also, the lactate level was significantly decreased in all treated groups when compared with the untreated group. The overall effect was the increased bioenergetic signature as compared with EC.These results implied that ZnO NPs have a significant efficacy against cancer cells and they significantly increased the bioenergetic signature.

Purpose:Human chorionic gonadotropin stimulates fetal testosterone production and contributes to normal development of male genitalia. Using population based data we hypothesized that differences in maternal free beta human chorionic gonadotropin may be associated with hypospadias.Materials and Methods:Data were obtained from the Paris Registry of Congenital Malformations (REMAPAR) (2011 to 2016). The initial study population included 3,172 pregnant women who gave birth to a singleton live born male infant with a congenital malformation. After exclusion of cases with unknown beta human chorionic gonadotropin and those with chromosomal or genetic abnormalities, the study population included 194 boys with isolated hypospadias and 1,075 controls. For cases with operative notes (125) we obtained data on type (proximal/distal) of hypospadias. Using quantile regression we compared median values of multiple of median beta human chorionic gonadotropin measured for first trimester Down syndrome screening (10th to 13th gestational weeks) for overall as well as by type of hypospadias vs controls. We also considered possible effects of placental dysfunction (maternal age, intrauterine growth retardation and preterm births) as potential confounding factors.Results:Overall the median beta human chorionic gonadotropin multiple of median was comparable for women who had an infant with hypospadias vs controls (0.99 vs 0.95, p=0.3). However, proximal hypospadias was associated with a statistically significant higher median multiple of median than distal hypospadias or unspecified (1.49 vs 0.92 vs 1.05, p=0.02). The estimates were comparable after adjustment for placental dysfunction.Conclusions:Our findings support the hypothesis that an alteration in maternal beta human chorionic gonadotropin levels is associated with hypospadias. However, this association appears to be limited to proximal hypospadias.

The Ny-1 gene confers hypersensitive response (HR) to Potato virus Y (PVY) in potato cultivars (Solanum tuberosum L.). Tetraploid potato breeding clone PB07–37 possessing the allele Ny-1 in a duplex state was developed. After PVY infection, the size of necrotic lesions in leaves of PB07–37 was reduced by approximately 68% in relation to cultivar Rywal plants displaying Ny-1 in a simplex dosage. Two-dimensional gel electrophoresis (2-DE) was applied for screening PVY-induced proteins in leaves of PB07–37. Compared with non-infected control plants, 60 reproducible PVY-induced proteins were detected using LC-MS/MS analysis, of which 41 were involved in qualitative changes and 19 were differently expressed in inoculated leaves by at least 1.5-fold. Proteins involved in the category of photosynthesis and primary metabolism were the most abundant. The results from PB07–37 (Ny-1 duplex) were compared with data from Rywal (Ny-1 simplex). The protein profiles in the Ny-1 simplex and Ny-1 duplex plants are genotype specific. Only eight proteins were identified in both genotypes. Five of them: ATP synthase CF1 alpha chain, chloroplastic; ATP synthase CF1 beta subunit, chloroplastic; ATP synthase subunit beta, mitochondrial-like; linoleate 13S-lipoxygenase 2–1, chloroplastic; and mitochondrial monodehydroascorbate reductase 5 are known to be involved in the defense response in plants. In our study, however, protein abundance did not correspond to the Ny-1 resistance gene dosage.

Sit4p is a type 2A-related protein phosphatase in Saccharomyces cerevisiae involved in a wide spectrum of cellular functions, including the glucose repression of mitochondrial transcription. Here we report that Sit4p is also involved in post-translational regulation of mitochondrial proteins and identified 9 potential targets. One of these, the ATP synthase (FoF1 complex) beta subunit Atp2p, was characterized and two phosphorylation sites, T124 and T317, were identified. Expression of Atp2p-T124 or T317 phosphoresistant versions in sit4Δ cells decreased Atp2p phosphorylation confirming these as Sit4p-regulated sites. Moreover, Sit4p and Atp2p interacted both physically and genetically. Mimicking phosphorylation at T124 or T317 increased Atp2p levels, resulting in higher abundance/activity of ATP synthase. Similar changes were observed in sit4Δ cells in which Atp2p is endogenously more phosphorylated. Expression of Atp2-T124 or T317 phosphomimetics also increased mitochondrial respiration and ATP levels and extended yeast lifespan. These results suggest that Sit4p-mediated dephosphorylation of Atp2p-T124/T317 downregulates Atp2p alongside with ATP synthase and mitochondrial function. Combination of transcriptional with post-translational regulation during fermentative growth may allow for a more efficient Sit4p repression of mitochondrial respiration.

The voltage-gated sodium ion channel NaV1.7 is crucial in pain signaling. We examined how auxiliary β2 and β3 subunits and the phosphorylation state of the channel influence its biophysical properties and pharmacology. The human NaV1.7α subunit was co-expressed with either β2 or β3 subunits in HEK-293 cells. The β2 subunits and the NaV1.7α, however, were barely associated as evidenced by immunoprecipitation. Therefore, the β2 subunits did not change the biophysical properties of the channel. In contrast, β3 subunit was clearly associated with NaV1.7α. This subunit had a significant degree of glycosylation, and only the fully glycosylated β3 subunit was associated with the NaV1.7α. Electrophysiological characterisation revealed that the β3 subunit had small but consistent effects: a right-hand shift of the steady-state inactivation and faster recovery from inactivation. Furthermore, the β3 subunit reduced the susceptibility of NaV1.7α to several sodium channel blockers. In addition, we assessed the functional effect of NaV1.7α phosphorylation. Inhibition of kinase activity increased channel inactivation, while the blocking phosphatases produced the opposite effect. In conclusion, co-expression of β subunits with NaV1.7α, to better mimic the native channel properties, may be ineffective in cases when subunits are not associated, as shown in our experiments with β2. The β3 subunit significantly influences the function of NaV1.7α and, together with the phosphorylation of the channel, regulates its biophysical and pharmacological properties. These are important findings to take into account when considering the role of NaV1.7 channel in pain signaling.

Mycoplasma synoviae (MS) is an important pathogen which causes huge economic losses to the poultry industry worldwide, and research on MS can provide the foundation for diagnosis, prevention, and treatment of MS infection. In this study, primers designed based on the sequences of pyruvate dehydrogenase complex (PDC) E1 alpha and beta subunit genes (pdhA and pdhB, respectively) of MS 53 strain(AE017245.1) in GenBank were used to amplify the pdhA and pdhB genes of MS WVU1853 strain through PCR. Subsequently, the prokaryotic expression vectors pET-28a(+)-pdhA and pET-28a(+)-pdhB were constructed and expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins rMSPDHA and rMSPDHB were purified, and anti-rMSPDHA and anti-rMSPDHB sera were prepared by immunizing rabbits, respectively. Subcellular localization of PDHA and PDHB in MS cells, binding activity of rMSPDHA and rMSPDHB to chicken plasminogen (Plg) and human fibronectin (Fn), complement-dependent mycoplasmacidal assays, and adherence and adherence inhibition assays were accomplished. The results showed that PDHA and PDHB were distributed both on the surface membrane and within soluble cytosolic fractions of MS cells. The rMSPDHA and rMSPDHB presented binding activity with chicken Plg and human Fn. The rabbit anti-rMSPDHA and anti-rMSPDHB sera had distinct mycoplasmacidal efficacy in the presence of guinea pig complement, and the adherence of MS to DF-1 cells pretreated with Plg was effectively inhibited by treatment with anti-rMSPDHA or anti-rMSPDHB sera. These findings indicated that surface-associated MSPDHA and MSPDHB were adhesion-related factors of MS and that the binding between MSPDHA/MSPDHB and Plg/Fn contributed to MS adhesion to DF-1 cells.

How protons power rotation Synthesis of adenosine triphosphate (ATP) in mitochondria is accomplished by a large molecular machine, the F 1 F O ATP synthase. Proton translocation across the F O region that spans the mitochondrial inner membrane drives ATP synthesis in the F 1 region through a rotational mechanism. Guo et al. present a high-resolution structure of the dimeric F O complex from Saccharomyces cerevisiae , determined by electron microscopy. The structure gives insights into how proton translocation powers rotation and suggests how F O dimers bend the membrane to give mitochondria their characteristic cristae. Science , this issue p. 936

Fo·F1H+-ATPase/synthase in coupled plasma membrane vesicles of Paracoccus denitrificans catalyzes ATP hydrolysis and/or ATP synthesis with comparable enzyme turnover. Significant difference in pH-profile of these alternative activities is seen: decreasing pH from 8.0 to 7.0 results in reversible inhibition of hydrolytic activity, whereas ATP synthesis activity is not changed. The inhibition of ATPase activity upon acidification results from neither change in ADP(Mg2+)-induced deactivation nor the energy-dependent enzyme activation. Vmax, not apparent KmATP is affected by lowering the pH. Venturicidin noncompetitively inhibits ATP synthesis and coupled ATP hydrolysis, showing significant difference in the affinity to its inhibitory site depending on the direction of the catalysis. This difference cannot be attributed to variations of the substrate-enzyme intermediates for steady-state forward and back reactions or to possible equilibrium between ATP hydrolase and ATP synthase Fo·F1 modes of the opposite directions of catalysis. The data are interpreted as to suggest that distinct non-equilibrated molecular isoforms of Fo·F1 ATP synthase and ATP hydrolase exist in coupled energy-transducing membranes.

THIS STUDY aimed to detect the effect of stress conditions imposed by different treatments of NaCl and jasmonic acid (JA) on the expression pattern and assembly of RuBisCO large subunit (RbcL) and RuBisCO enzyme complex using SDS-PAGE and HDN-PAGE techniques, respectively. Both Total Soluble Proteins (TSPs) and Chloroplast Proteins (ChPs) were variably affected by each treatment. Notably, a unique protein band was detected in the seedlings treated with 150 mM NaCl for 5 days. Surprisingly, this band was not detected under any other salt treatment. Using nano HPLC-ESI-MS/MS MALDI-TOF protein sequencing, this band was identified as downshifted RbcL at approximately 50 kDa in addition to the original one was separated at approximately 55 kDa on SDS-PAGE. The expression of RbcL protein decreased in a dose dependent manner in the plants treated with JA for one and five days. All treatments did not affect the RuBisCO complex assembly at approximately 660 kDa, but triggered stress-induced ChPs at approximately 440 kDa and 1 MDa concomitant with NaCl treatments. RbcL band isolated from intact chloroplast was associated with Coupling Factor (CF1) beta subunit of ATP synthase as revealed by MALDI-TOF protein sequencing analysis. Possible interpretations of these findings have been further discussed in this study.

Background: Sickle cell disease is a genetic disease caused by a point mutation in the beta globin gene on chromosome 11 leading to the substitution of valine in place of glutamic acid in beta subunit of globin molecule. The vaso-occlusive crisis, or sickle cell crisis, is a common painful complication of sickle cell disease in adolescents and adults. Many studies have suggested the alteration in intracellular concentration of various ions associated with sickle cell disease.Aims and Objectives: This study aims to evaluate the activity of red blood cell membrane ATPase activity of sickle cell crisis patients in comparison to sickle cell patients in steady state.Material and Methods: Twenty patients of sickle cell crisis were included in the study. Twenty sickle cell patients in steady state were also registered who served as controls. The RBC Membrane ATPase activity were estimated and expressed as μmol of Pi/μg of protein/hour.Results: The activity of membrane ATPase were found to be significantly high in patients of sickle cell crisis in comparison to steady state ones.Conclusion: Membrane ATPases play a crucial role in the pathophysiology of sickle cell crisis. This study can be a based upon for future studies to understand the pathophysiology of sickle cell crisis and development of diagnostic and treatment modalities for the same.Asian Journal of Medical Sciences Vol.8(5) 2017 27-30

Although cell phones have been used worldwide, some adverse and toxic effects were reported for this communication technology apparatus. To analyze in vivo effects of exposure to radiofrequency-electromagnetic field (RF-EMF) on protein expression in rat testicular proteome, 20 Sprague-Dawley rats were exposed to 900 MHz RF-EMF for 0, 1, 2, or 4 h/day for 30 consecutive days. Protein content of rat testes was separated by high-resolution two-dimensional electrophoresis using immobilized pH gradient (pI 4–7, 7 cm) and 12% acrylamide and identified by MALDI-TOF/TOF-MS. Two protein spots were found differentially overexpressed (P

Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Here we studied the mitochondrial F1FO-ATP synthase (or Complex V) from the photosynthetic eukaryote E. gracilis in detail. The enzyme was purified by a two-step chromatographic procedure and its subunit composition was resolved by a three-dimensional gel electrophoresis (BN/SDS/SDS). Twenty-two different subunits were identified by mass-spectrometry analyses among which the canonical α, β, γ, δ, ε, and OSCP subunits, and at least seven subunits previously found in Trypanosoma. The ADP/ATP carrier was also associated to the ATP synthase into a dimeric ATP synthasome. Single-particle analysis by transmission electron microscopy of the dimeric ATP synthase indicated that the structures of both the catalytic and central rotor parts are conserved while other structural features are original. These new features include a large membrane-spanning region joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring, the latter having not been reported for any mitochondrial F-ATPase.

ATP-binding cassette (ABC) transporters use the energy of ATP binding and hydrolysis to move a large variety of compounds across biological membranes. P-glycoprotein, involved in multidrug resistance, is the most investigated eukaryotic family member. Although a large number of biochemical and structural approaches have provided important information, the conformational dynamics underlying the coupling between ATP binding/hydrolysis and allocrite transport remains elusive. To tackle this issue, we performed molecular dynamic simulations for different nucleotide occupancy states of Sav1866, a prokaryotic P-glycoprotein homologue. The simulations reveal an outward-closed conformation of the transmembrane domain that is stabilized by the binding of two ATP molecules. The hydrolysis of a single ATP leads the X-loop, a key motif of the ATP binding cassette, to interfere with the transmembrane domain and favor its outward-open conformation. Our findings provide a structural basis for the unidirectionality of transport in ABC exporters and suggest a ratio of one ATP hydrolyzed per transport cycle., An outward-closed state of the Sav1866 efflux pump is stabilized by the binding of two ATP molecules. Hydrolysis at one ATP binding site favors the outward-open conformation. An export mechanism is proposed based on these two states.

In archaea the A1AO ATP synthase uses a transmembrane electrochemical potential to generate ATP, while the soluble A1 domain (subunits A3B3DF) alone can hydrolyse ATP. The three nucleotide-binding AB pairs form a barrel-like structure with a central orifice that hosts the rotating central stalk subunits DF. ATP binding, hydrolysis and product release cause a conformational change inside the A:B-interface, which enforces the rotation of subunits DF. Recently, we reported that subunit F is a stimulator of ATPase activity. Here, we investigated the nucleotide-dependent conformational changes of subunit F relative to subunit D during ATP hydrolysis in the A3B3DF complex of the Methanosarcina mazei Gö1 A-ATP synthase using single-molecule Förster resonance energy transfer. We found two conformations for subunit F during ATP hydrolysis. NMRC (Natl Medical Research Council, S’pore) Accepted version

Adenosine triphosphate (ATP) is mainly produced in the mitochondrion and used as a universal energy source for various cellular events. Various fluorescent probes for ATP have been established successfully, but most of them are not appropriate for monitoring the fluctuation of the mitochondrial ATP level. Herein, a fluorescent probe named Mito-Rh is first synthesized and used to recognize ATP in mitochondrion. In the probe, rhodamine, diethylenetriamine, and triphenylphosphonium are selected as fluorophore, reaction site, and mitochondrion-targeting group, respectively. Probe Mito-Rh shows high sensitivity to ATP with 81-fold fluorescence enhancement, and the detection range (0.1-10 mM) can match the concentration level of ATP in the mitochondrion. Moreover, Mito-Rh provides excellent selectivity toward ATP over other biological anions (ADP, AMP, GTP, CTP, UTP) owing to a concurrent effect of dual recognition sites (hydrogen bond and π-π stacking). In particular, the probe can localize in mitochondrion specifically and demonstrates utility in the real-time detection of mitochondrial ATP concentration changes.

To demonstrate the ability of the Nativis signal transduction technology (Butters et al. 2014) to modulate the expression of algae mRNA and protein, we tested if we can alter specific enzyme levels in Chlamydomonas reinhardtii. We inhibited the synthesis of the enzyme tryptophan synthase beta subunit (MAA7) by applying the signal derived from a published siRNA (Zhao et al. 2009). With lower levels of MAA7, Chlamydomonas reinhardtii can grow in the presence of the prodrug 5-Fluoroindole (5-FI), because less 5-Fluoroin-dole can be converted to the toxic 5-Fluoro-L-tryptophan (5-FT). We find a 24% (±5%) increase of growth with the signal versus no signal. To see if that effect was due to the reduction of the amount of mRNA encoding MAA7, we used Real-Time Quantitative PCR (RT-QPCR) to measure the levels of MAA7 mRNA. To normalize the MAA7 mRNA level, we compared them to the levels of a mRNA that is not affected by the signal (G protein beta subunit-like polypeptide, Cblp). Two conditions increase the effectiveness of the signal. One can either treat the cell cultures during the logarithmic growth phase (starting the cultures at density of 0.104 OD at 750 nm). Or one can treat the cultures at a later stage of the logarithmic growth, but treating them for a longer time (8.7% versus 3.5% of the culture time). Under these conditions we found around a 50% decrease in the mRNA levels for MAA7. Treating the cultures at the earlier growth phase or at a later growth phase is less effective, with only a 20% effect.

The Fo-a subunit of the Na+-transporting FoF1 ATP synthase from Propionigenium modestum plays a key role in Na+ transport. It forms half channels that allow Na+ to enter and leave the buried carboxyl group on Fo-c subunits. The essential Arg residue R226, which faces the carboxyl group of Fo-c subunits in the middle of transmembrane helix 5 of the Fo-a subunit, separates the cytoplasmic side and periplasmic half-channels. To elucidate contributions of other amino acid residues of transmembrane helix 5 using hybrid FoF1 (Fo from P. modestum and F1 from thermophilic Bacillus PS3), 25 residues were individually mutated to Cys, and effects of modification with the SH-modifying agent N-ethylmaleimide (NEM) on ATP synthesis and hydrolysis activity were analyzed. NEM significantly inhibited ATP synthesis and hydrolysis as well as proton pumping activities of A214C, G215C, A218C, I223C (cytoplasmic side from R226), and N230C (periplasmic side from R226) mutants and inhibited ATP synthesis activity of the K219C mutant (cytoplasmic side from R226). Thus, these residues contribute to the integrity of the Na+ half channel, and both half channels are present in the Fo-a subunit.

The beta subunit of human chorionic gonadotropin (hCG beta) is encoded by six genes (CGB) classified as type I and type II. CGB mRNA is produced in large amounts by trophoblastic tissues and in small amounts by several cancerous tissues including prostate cancer and by a few benign tissues, including the prostate. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to study the expression levels of all CGB mRNAs together (total CGB mRNA) and the two types of CGB mRNA separately in non-cancerous (n = 74) and cancerous prostatic tissue obtained by radical prostatectomy (n = 193). RNA was isolated from formalin-fixed paraffin-embedded (FFPE) samples and mRNA levels of CGB were correlated with disease-specific survival. Total CGB mRNA concentrations were significantly lower (p

International audience; The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is a heterodimer made of two non-covalently linked subunits. The α-subunit structure, hCGα, has 2 N-glycosylation sites, while the beta subunit, hCGβ, has 2 N- and 4 O-glycosylation sites. This leads to numerous glycoforms. A method based on the analysis of hCG glycoforms at the intact level by nano-reversed phase liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) with an Orbitrap analyzer was previously developed using a recombinant hCG-based drug, Ovitrelle®, as standard. It allowed the detection of about 30 hCGα glycoforms, but didn't allow the detection of hCGβ glycoforms. This method was thus here significantly modified (addition of a pre-concentration step of the sample to increase the sample volume from 70 nl to 1 µl, optimization of the gradient slope and the nature and content of the acidic additive in the mobile phase). It led to an improvement of the separation of hCGα and hCGβ glycoforms, which allowed for the first time the detection of 33 hCGβ glycoforms at intact level. In addition, a higher number of hCGα glycoforms (42 in total, i.e. a 40% increase) was detected. The figures of merit of this new method were next assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.02 and 0.95% (n = 3), with an average value of 0.36% for the alpha glycoforms and between 0.01 and 1.08% (n = 3) with an average value of 0.23% for the beta glycoforms. The RSDs of the relative peak area measured on the extracted ion chromatogram of each glycoform were below 20% (n = 3), with an average value of 9.8%, thus allowing semi-relative quantification. Therefore, this method has a high potential for rapid quality control aiming for the detection and comparison of glycoforms present in glycoprotein-based pharmaceutical preparations.

The amiloride-sensitive sodium channel beta subunit (SCNN1B) gene encodes the beta subunit of the epithelial sodium channel, which is involved in blood pressure homeostasis. The aim of the present study was to investigate the association between SCNN1B gene promoter methylation and essential hypertension (EH), and to explore whether SCNN1B methylation was altered by antihypertensive therapy. The present study recruited 282 individuals: 94 controls, 94 incident cases and 94 prevalent cases. Subsequently, the methylation status of six CpG sites in the SCNN1B promoter region was measured using bisulfite pyrosequencing technology. Among the six CpG sites, a significant difference in CpG1 and CpG2 methylation levels were detected between controls and incident cases (CpG1: β‑standardized=0.17, adjusted P=0.015; CpG2: β‑standardized=‑0.41, adjusted P=0.001). In addition, a significant difference was detected in CpG1 methylation levels between incident cases and prevalent cases (β‑standardized=‑0.252, adjusted P=3.77E‑04). The present study also demonstrated that CpG1 and CpG2 methylation levels were significantly lower in males compared with in females (CpG1: t=‑3.180, P=0.002; CpG2: t=‑2.148, P=0.033). CpG1 methylation was also shown to be positively correlated with age (controls: r=0.285, P=0.008; incident cases: r=0.401, P=0.0001; prevalent cases: r=0.367, P=0.001). These results indicated a significant association between EH and SCNN1B methylation, which was affected by age, gender and antihypertensive therapy.

Designing novel antigens to rise specific antibodies for Thyroid Stimulating Hormone (TSH) detection is of great significance. A novel fusion protein consisting of the C termini sequence of TSH beta subunit and a fusion sequence was designed and produced for rabbit immunization. Thereafter, the produced antibodies were purified and characterized for TSH detection. Our results indicate that the produced antibody is capable of sensitive and specific detection of TSH with low cross reactivity. This study underscores the applicability of designed fusion protein for specific and sensitive polyclonal antibody production and the importance of selecting an amenable region of the TSH for immunization.

The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a "down" state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an "up" state, where the α-helices, devoid of ATP, enter the α3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme's hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual "open" conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis.

Epidemiological studies suggest an increased fracture risk in patients taking proton pump inhibitors (PPIs) for long term. The underlying mechanism, however, has been disputed. By binding to the gastric proton pump, PPIs inhibit gastric acid secretion. We have previously shown that proton pump (H+/K+ATPase beta subunit) KO mice exhibit reduced bone mineral density (BMD) and inferior bone strength compared with WT mice. Patients using PPIs as well as these KO mice exhibit gastric hypoacidity, and subsequently increased serum concentrations of the hormone gastrin. In this study, we wanted to examine whether inhibition of the gastrin/CCK2 receptor influences bone quality in these mice. KO and WT mice were given either the gastrin/CCK2 receptor antagonist netazepide dissolved in polyethylene glycol (PEG) or only PEG for 1year. We found significantly lower bone mineral content and BMD, as well as inferior bone microarchitecture in KO mice compared with WT. Biomechanical properties by three-point bending test also proved inferior in KO mice. KO mice receiving netazepide exhibited significantly higher cortical thickness, cortical area fraction, trabecular thickness and trabecular BMD by micro-CT compared with the control group. Three-point bending test also showed higher Young’s modulus of elasticity in the netazepide KO group compared with control mice. In conclusion, we observed that the gastrin receptor antagonist netazepide slightly improved bone quality in this mouse model, suggesting that hypergastrinemia may contribute to deteriorated bone quality during acid inhibition.

The thermodynamic H(+)/ATP ratio of the H(+)-ATP synthase from chloroplasts was measured in proteoliposomes after energization of the membrane by an acid base transition (Turina et al. 2003 [13], 418-422). The method is discussed, and all published data obtained with this system are combined and analyzed as a single dataset. This meta-analysis led to the following results. 1) At equilibrium, the transmembrane ΔpH is energetically equivalent to the transmembrane electric potential difference. 2) The standard free energy for ATP synthesis (reference reaction) is ΔG°(ref)=33.8±1.3kJ/mol. 3) The thermodynamic H(+)/ATP ratio, as obtained from the shift of the ATP synthesis equilibrium induced by changing the transmembrane ΔpH (varying either pH(in) or pH(out)) is 4.0±0.1. The structural H(+)/ATP ratio, calculated from the ratio of proton binding sites on the c-subunit-ring in F(0) to the catalytic nucleotide binding sites on the β-subunits in F(1), is c/β=14/3=4.7. We infer that the energy of 0.7 protons per ATP that flow through the enzyme, but do not contribute to shifting the ATP/(ADP·Pi) ratio, is used for additional processes within the enzyme, such as activation, and/or energy dissipation, due e.g. to internal uncoupling. The ratio between the thermodynamic and the structural H(+)/ATP values is 0.85, and we conclude that this value represents the efficiency of the chemiosmotic energy conversion within the chloroplast H(+)-ATP synthase.

Key points SLC17A9 proteins function as a lysosomal ATP transporter responsible for lysosomal ATP accumulation. P2X4 receptors act as lysosomal ion channels activated by luminal ATP. SLC17A9-mediated ATP transport across the lysosomal membrane is suppressed by Bafilomycin A1, the V-ATPase inhibitor. SLC17A9 mainly uses voltage gradient but not pH gradient generated by the V-ATPase as the driving force to transport ATP into the lysosome to activate P2X4. Abstract The lysosome contains abundant ATP which plays important roles in lysosome functions and in cell signalling. Recently, solute carrier family 17 member 9 (SLC17A9, also known as VNUT for vesicular nucleotide transporter) proteins were suggested to function as a lysosomal ATP transporter responsible for lysosomal ATP accumulation, and P2X4 receptors were suggested to be lysosomal ion channels that are activated by luminal ATP. However, the molecular mechanism of SLC17A9 transporting ATP and the regulatory mechanism of lysosomal P2X4 are largely unknown. In this study, we report that SLC17A9-mediated ATP transport across lysosomal membranes is suppressed by Bafilomycin A1, the V-ATPase inhibitor. By measuring P2X4 activity, which is indicative of ATP transport across lysosomal membranes, we further demonstrated that SLC17A9 mainly uses voltage gradient but not pH gradient as the driving force to transport ATP into lysosomes. This study provides a molecular mechanism for lysosomal ATP transport mediated by SLC17A9. It also suggests a regulatory mechanism of lysosomal P2X4 by SLC17A9.

Thylakoid membrane-localized chloroplast ATP synthases use the proton motive force generated by photosynthetic electron transport to produce ATP from ADP. Although it is well known that the chloroplast ATP synthase is composed of more than 20 proteins with α3β3γ1ε1δ1I1II1III14IV1 stoichiometry, its biogenesis process is currently unclear. To unravel the molecular mechanisms underlying the biogenesis of chloroplast ATP synthase, we performed extensive screening for isolating ATP synthase mutants in Arabidopsis (Arabidopsis thaliana). In the recently identified bfa3 (biogenesis factors required for ATP synthase 3) mutant, the levels of chloroplast ATP synthase subunits were reduced to approximately 25% of wild-type levels. In vivo labeling analysis showed that assembly of the CF1 component of chloroplast ATP synthase was less efficient in bfa3 than in the wild type, indicating that BFA3 is required for CF1 assembly. BFA3 encodes a chloroplast stromal protein that is conserved in higher plants, green algae, and a few species of other eukaryotic algae, and specifically interacts with the CF1β subunit. The BFA3 binding site was mapped to a region in the catalytic site of CF1β. Several residues highly conserved in eukaryotic CF1β are crucial for the BFA3–CF1β interaction, suggesting a coevolutionary relationship between BFA3 and CF1β. BFA3 appears to function as a molecular chaperone that transiently associates with unassembled CF1β at its catalytic site and facilitates subsequent association with CF1α during assembly of the CF1 subcomplex of chloroplast ATP synthase.

F-type ATP synthases are reversible machinery that not only synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane, but also can hydrolyze ATP to pump ions under certain conditions. To prevent wasteful ATP hydrolysis, subunit ε in bacterial ATP synthases changes its conformation from the non-inhibitory down- to the inhibitory up-state at a low cellular ATP concentration. Recently, a crystal structure of the ε subunit in complex with ATP was solved in a non-biologically relevant dimeric form. Here, to derive the functional ATP binding site motif, we carried out molecular dynamics simulations and free energy calculations. Our results suggest that the ATP binding site markedly differs from the experimental resolved one; we observe a reorientation of several residues, which bind to ATP in the crystal structure. In addition we find that an Mg(2+) ion is coordinated by ATP, replacing interactions of the second chain in the crystal structure. Thus we demonstrate more generally the influence of crystallization effects on ligand binding sites and their respective binding modes. Furthermore, we propose a role for two highly conserved residues to control the ATP binding/unbinding event, which have not been considered before. Additionally our results provide the basis for the rational development of new biosensors based on subunit ε, as shown previously for novel sensors measuring the ATP concentration in cells.

A1AO ATP synthases couple ion-transport of the AO sector and ATP synthesis/hydrolysis of the A3B3-headpiece via their stalk subunits D and F. Here, we produced and purified stable A3B3D- and A3B3DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Gö1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates (Vmax) of A3B3D and A3B3DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a Vmax of 7.9 s− 1 and 30.4 s− 1, respectively, while the KM is the same for both. Deletions of the N- or C-termini of subunit F abolished the effect of ATP hydrolysis activation. We generated subunit F mutant proteins with single amino acid substitutions and demonstrated that the subunit F residues S84 and R88 are important in stimulating ATP hydrolysis. Hybrid formation of the A3B3D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit ε of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis. NMRC (Natl Medical Research Council, S’pore) Accepted version

Gonadotropin-inhibitory hormone (GnIH) acts to inhibit reproduction at all levels of the hypothalamo-pituitary-gonad axis. GnIH expression and/or immunoreactivity in the hypothalamus increase with acute stress in some birds and mammals, and thus may be involved in stress-induced reproductive inhibition. Much is known about GnIH and stress in seasonal and continuous breeders, but far less is known about these interactions in opportunistic breeders. For opportunistically breeding animals, reproductive readiness is closely associated with unpredictable environmental cues, and thus the GnIH system may be more sensitive to stress. To test this, we collected tissues from zebra finches immediately following capture or after 60 min of restraint. Restraint significantly increased plasma corticosterone in males and females but, contrary to studies on other species, restrained birds had significantly fewer GnIH immunoreactive (GnIH-ir) cell bodies than control birds. GnIH-ir cell number did not differ between the sexes. Stressed females had lower mRNA expression of the beta subunit of follicle stimulating hormone (FSHβ) in the pituitary, suggesting that the reduction in observed GnIH immunoreactivity in females may have been due to increased GnIH release in response to acute stress. GnIH expression increased in the testes, but not the ovaries, of restrained animals. Our data suggest that although GnIH responsiveness to stress appears to be conserved across species, specific tissue response and direction of GnIH regulation is not. Variation in the GnIH response to stress between species might be the result of ecological adaptations or other species differences in the response of the GnIH system to stress.

Specific ATP binding to the ε subunit of thermophilic F1-ATPase has been utilized for the biosensors of ATP in vivo. I report here that the ε subunit containing R103A/R115A mutations can bind ATP with a dissociation constant at 52 nM, which is two orders of magnitude higher affinity than the wild type. The mutant retained specificity for ATP; ADP and GTP bound to the mutant with dissociation constants 16 and 53 μM, respectively. Thus, the mutant would be a good platform for various types of nucleotide biosensor with appropriate modifications.

Tryptophan synthase is a model system for understanding allosteric regulation within enzyme complexes. Amino acid interaction networks were previously delineated in the isolated alpha subunit (αTS) in the absence of the beta subunit (βTS). The amino acid interaction networks were different between the ligand-free enzyme and the enzyme actively catalyzing turnover. Previous X-ray crystallography studies indicated only minor localized changes when ligands bind αTS, and so, structural changes alone could not explain the changes to the amino acid interaction networks. We hypothesized that the network changes could instead be related to changes in conformational dynamics. As such, we conducted nuclear magnetic resonance relaxation studies on different substrate- and products-bound complexes of αTS. Specifically, we collected 15N R2 relaxation dispersion data that reports on microsecond-to-millisecond timescale motion of backbone amide groups. These experiments indicated that there are conformational exchange events throughout αTS. Substrate and product binding change specific motional pathways throughout the enzyme, and these pathways connect the previously identified network residues. These pathways reach the αTS/βTS binding interface, suggesting that the identified dynamic networks may also be important for communication with the βTS subunit.