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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.

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

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.

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.

Gonadotropin releasing hormone (GnRH) from the hypothalamus regulates the synthesis and secretion of gonadotropin hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH regulates steroidogenesis in both sexes and ovulation in females, while FSH stimulates folliculogenesis in females and spermatogenesis in males. LH and FSH are heterodimers of a common alpha subunit and a unique beta subunit, which provides biological specificity and is the rate limiting factor in hormone synthesis. Immediate early genes, early growth response 1 (Egr1) and fos proto-oncogene (cFos) are critical in the induction of LH-beta and FSH-beta subunits by GnRH, respectfully. However, gaps exist in our understanding of developmental initiation and hormonal regulation of gonadotropin gene expression. Specifically, epigenetic mechanisms that may play a role in beta subunit transcriptional regulation are unknown. The aim of this work was to identify transcriptional cofactors that are recruited to gonadotropin beta-subunit promoters with or without GnRH. Transcription factors interact with cofactors that recruit chromatin remodeling enzymes in order to regulate transcription. Identification of cofactors may explain tight regulation of gonadotropin hormone levels in reproductive physiology. Previous studies identified regions on the beta-subunit promoters that are necessary for GnRH responsiveness. These regions were used to pull down interacting proteins that bind to these response elements using nuclear extracts from the immortalized mature gonadotrope cell line, LβT2. Using a discovery proteomics approach, we identified different transcriptional cofactors that are recruited to beta subunit promoters with or without GnRH. Approximately 1500-2000 proteins were identified per pulldown. As expected, proteins known to interact with beta subunit promoters, such as Egr1, cFos and cJun were identified in the DNA pulldown experiments as positive controls. We identified 63 proteins unique for LH-beta promoter under control conditions and 60 unique for FSH-beta promoter, of which 7 proteins for LH-beta and 8 proteins for FSH-beta may play a role as corepressors. We further identified 97 proteins that were pulled down with the LH-beta promoter following GnRH treatment, of which 9 proteins were also pulled down with Egr1 as potential coactivator candidates. We also identified 72 proteins that were pulled down with the FSH-beta promoter following GnRH treatment, of which 6 proteins were pulled down with cFos as potential coactivator candidates. Functional studies to identify roles of these cofactors in gonadotropin hormone expression are in progress. The identification of epigenetic regulators will allow for better understanding of the transcriptional regulation of gonadotropin beta-subunit gene expression, which is critical for reproductive function.

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

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

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

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.

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.

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

Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile that oxidizes ferrous iron or sulfur compounds to obtain energy in the presence of various ions. To investigate the potassium ion response of A. ferrooxidans, we conducted a proteomics analysis. We identified eight proteins that were differentially expressed in the presence of high potassium concentration, including four up-regulated and four down-regulated proteins. Transcription levels of the genes encoding differential expressed proteins were subsequently analyzed by Northern blot in the presence of high potassium concentration. Among the up-regulated proteins, GDP-mannose 4,6-dehydratase, ribose 5-phosphate isomerase A and ribose-phosphate pyrophosphokinase were known to be implicated in the synthesis of glycocalyx, suggesting that the formation of glycocalyx might be involved in the A. ferrooxidans response to high potassium concentration. Thickening of the glycocalyx layer was also observed in cells cultivated under high potassium concentration via transmission electronic microscopy (TEM) analysis. Among the down-regulated proteins, ATP synthase F1 delta subunit and ATP synthase F1 beta subunit were two important components of ATP synthase. ATP synthase (P-ATPase) is directly linked to the transport of potassium into the cell, thus Acidithiobacillus ferrooxidans might just reduce the quantity of ATP synthase to offset the high potassium level in the culture medium. Therefore, the results obtained here provide some new clues to improve our understanding of the response of A. ferrooxidans to high potassium concentration.

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.

FUS (fused in sarcoma) proteinopathy is a group of neurodegenerative diseases characterized by the formation of inclusion bodies containing the FUS protein, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Previous studies show that mitochondrial damage is an important aspect of FUS proteinopathy. However, the molecular mechanisms by which FUS induces mitochondrial damage remain to be elucidated. Our biochemical and genetic experiments demonstrate that FUS interacts with the catalytic subunit of mitochondrial ATP synthase (ATP5B), disrupts the formation of ATP synthase complexes, and inhibits mitochondrial ATP synthesis. FUS expression activates the mitochondrial unfolded protein response (UPR(mt)). Importantly, down-regulating expression of ATP5B or UPR(mt) genes in FUS transgenic flies ameliorates neurodegenerative phenotypes. Our data show that mitochondrial impairment is a critical early event in FUS proteinopathy, and provide insights into the pathogenic mechanism of FUS-induced neurodegeneration.

Rationale: Familial hydatidiform mole is extremely rare while familial gestational trophoblastic neoplasia (GTN) has never been reported. Inspired by 2 biological sisters with postmolar GTN and liver toxicity, we reviewed susceptible maternal-effect genes and explored the role of possible drug transporter genes in the development of GTN. Patient concerns: We reported one Chinese family where the two sisters developed postmolar GTN while experiencing fast remission and significant hepatic toxicity from actinomycin D chemotherapy. Diagnoses: The index pregnancy was diagnosed with curettage. The following GTN was confirmed when there was a rise in beta-hCG for three consecutive weekly measurements over at least a period of 2 weeks. Computed tomography was used to identify lung metastasis. The elder sister was diagnosed with gestational trophoblastic neoplasia (III: 2) while the younger sister was diagnosed as III: 3 according to WHO scoring system. Interventions: Patients were treated with actinomycin D of 10 μg/kg intravenously for 5 days every 2 weeks. When hepatic toxicity was indicated, polyene phosphatidyl choline and magnesium isoglycyrrhizinate were prescribed. Outcomes: Both patients responded extremely well to the 5-day actinomycin D regimen. Beta-hCG remained less than 2 mIU/ml after 5 cycles while computed tomography scan showed downsized pulmonary nodules. Both experienced significant rise in ALT and AST levels that could be ameliorated with corresponding medication. Monthly followed-up showed negative beta-hCG levels and normal liver enzyme levels. Lessons: We speculated that the known or unknown NLRP7 and KHDC3L mutations might be correlated with drug disposition in liver while liver drug transporters such as P-glycoprotein family that are also expressed in trophoblasts might be correlated to GTN susceptibility. Future genomic profiles of large samples alike using next generation sequencing are needed to confirm our hypothesis and discover yet unknown genes.

Visualizing fluorescent proteins is essential for understanding cellular function. While advances in microscopy can now resolve individual molecules, determining whether the labeled molecules report native behaviors and how the measured behaviors can be coupled to cellular outputs remains challenging. Here, we used integrin alpha-beta heterodimers – which connect extracellular matrix (ECM) and the cytoskeleton – to quantify the mobility and conformation of labeled integrins. We found that while unlabeled and labeled integrins all localized to adhesions and support anchorage-dependent cell function, integrin mobility decreased when the beta rather than the alpha subunit was labeled. In contrast to unlabeled and alpha labeled subunits, beta labeled subunits changed cellular behavior; decreasing protrusive activity and increasing adhesion size and the extent of cell spreading. Labeling the beta subunit changed the integrin conformation, extending the molecule and exposing an epitope that is revealed by activation with Mn2+ treatment. Our findings indicate labeling induced changes in dynamic integrin behavior alter molecular conformation as well as cellular adhesion-dependent function to demonstrate a coupling between molecular inputs and distinct cellular outputs. This article has an associated First Person interview with the first author of the paper., Summary: Our findings indicate labeling induced changes in dynamic integrin behaviors alter molecular conformation and cellular adhesion-dependent function to demonstrate a coupling between molecular inputs and distinct cellular outputs.

A 39-year-old woman with non–small cell lung cancer underwent preprocedural pregnancy testing. Results showed increased levels of human chorionic gonadotropin, but she reported not being sexually active. Repeat testing after treatment with antibody-blocking agent was similar. View this table: Table 1. Quantitative hCG assays. 1. What forms of human chorionic gonadotropin (hCG) are measured in most quantitative hCG …

The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme's rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F1-catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ(0) cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.

F-ATP synthases are described to have mechanisms which regulate the unnecessary depletion of ATP pool during an energy limited state of the cell. Mg-ADP inhibition is one of the regulatory features where Mg-ADP gets entrapped in the catalytic site, preventing the binding of ATP and further inhibiting ATP hydrolysis. Knowledge about the existence and regulation of the related archaeal-type A1AO ATP synthases (A3B3CDE2FG2ac) is limited. We demonstrate MgADP inhibition of the enzymatically active A3B3D- and A3B3DF complexes of Methanosarcina mazei Gö1 A-ATP synthase and reveal the importance of the amino acids P235 and S238 inside the P-loop (GPFGSGKTV) of the catalytic A subunit. Substituting these two residues by the respective P-loop residues alanine and cysteine (GAFGCGKTV) of the related eukaryotic V-ATPase increases significantly the ATPase activity of the enzyme variant and abolishes MgADP inhibition. The atomic structure of the P235A, S238C double mutant of subunit A of the Pyrococcus horikoshii OT3 A-ATP synthase provides details of how these critical residues affect nucleotide-binding and ATP hydrolysis in this molecular engine. The qualitative data are confirmed by quantitative results derived from fluorescence correlation spectroscopy experiments. MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Accepted version

F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.

OBJETIVO: Determinar la correlación entre niveles de gonadotropina coriónica beta medidos en el segundo trimestre de la gestación y el riesgo de presentación de preeclampsia. DISEÑO: Estudio prospectivo, descriptivo. MATERIAL: A 85 gestantes en un segundo trimestre se les determinó la sub unidad beta de la gonadotropina coriónica sérica, siendo seguidas hasta el término. RESULTADOS: Edad materna promedio 29,4 añoss, peso pregestacional promedio 55,7 kilos. Edad gestacional media a la toma de la muestra 20,6 semanas, 38 nulíparas y 47 multíparas. La concentración plasmática de la gonadotropina coriónica beta tuvo una media de 32,988 mUI/mL y un rango de 5000 - 140000 mUI/mL. Edad gestacional promedio al parto 38 semanas, 53 partos vaginales y 32 partos por cesárea. La preeclampsia se presentó en 14 de 85 gestantes (16%), 10 con preeclampsia leve y 4 con preeclampsia severa. La gonadotropina coriónica beta en la preeclampsia severa tuvo un rango de 60000 - 140000 mUI/mL y en la leve de 53000 – 71000 mUI/mL, diferencia altamente significativa. CONCLUSIÓN: El valor de la gonadotropina coriónica beta en el segundo trimestre de la gestación, podría ser considerado un factor de riesgo para predecir la posible ocurrencia de la preeclampsia. Habría que realizar estudios más amplios para negar o confirmar esta hipótesis.

Like the evolutionary related F1FO ATP synthases and V1VO ATPases, the A1AO ATP synthases from archaea are multisubunit, membrane-bound transport machines that couple ion flow to the synthesis of ATP. Although the subunit composition is known for at least two species, nothing is known so far with respect to the function of individual subunits or amino acid residues. To pave the road for a functional analysis of A1AO ATP synthases, we have cloned the entire operon from Methanosarcina mazei into an expression vector and produced the enzyme in Escherichia coli. Inverted membrane vesicles of the recombinants catalyzed ATP synthesis driven by NADH oxidation as well as artificial driving forces. Δ μ ˜ H + as well as ΔpH were used as driving forces which is consistent with the inhibition of NADH-driven ATP synthesis by protonophores. Exchange of the conserved glutamate in subunit c led to a complete loss of ATP synthesis, proving that this residue is essential for H+ translocation. Exchange of two conserved arginine residues in subunit a has different effects on ATP synthesis. The role of these residues in ion translocation is discussed.

The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs) in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs.

Living organisms rely on the FoF1 ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the Fo motor uses a transmembrane electrochemical ion gradient to create clockwise torque that overcomes F1 ATPase-driven counterclockwise torque at high ATP is a major unresolved question. Using single FoF1 molecules embedded in lipid bilayer nanodiscs, we now report the observation of Fo-dependent rotation of the c10 ring in the ATP synthase (clockwise) direction against the counterclockwise force of ATPase-driven rotation that occurs upon formation of a leash with Fo stator subunit a. Mutational studies indicate that the leash is important for ATP synthase activity and support a mechanism in which residues aGlu-196 and cArg-50 participate in the cytoplasmic proton half-channel to promote leash formation.

ATP, the universal energy currency of cells, is produced by F-type ATP synthases, which are ancient, membrane-bound nanomachines. F-type ATP synthases use the energy of a transmembrane electrochemical gradient to generate ATP by rotary catalysis. Protons moving across the membrane drive a rotor ring composed of 8-15 c-subunits. A central stalk transmits the rotation of the c-ring to the catalytic F1 head, where a series of conformational changes results in ATP synthesis. A key unresolved question in this fundamental process is how protons pass through the membrane to drive ATP production. Mitochondrial ATP synthases form V-shaped homodimers in cristae membranes. Here we report the structure of a native and active mitochondrial ATP synthase dimer, determined by single-particle electron cryomicroscopy at 6.2 Å resolution. Our structure shows four long, horizontal membrane-intrinsic α-helices in the a-subunit, arranged in two hairpins at an angle of approximately 70° relative to the c-ring helices. It has been proposed that a strictly conserved membrane-embedded arginine in the a-subunit couples proton translocation to c-ring rotation. A fit of the conserved carboxy-terminal a-subunit sequence places the conserved arginine next to a proton-binding c-subunit glutamate. The map shows a slanting solvent-accessible channel that extends from the mitochondrial matrix to the conserved arginine. Another hydrophilic cavity on the lumenal membrane surface defines a direct route for the protons to an essential histidine-glutamate pair. Our results provide unique new insights into the structure and function of rotary ATP synthases and explain how ATP production is coupled to proton translocation.