The 7q11.23 Protein DNAJC30 Interacts with ATP Synthase and Links Mitochondria to Brain Development.

Despite the known causality of copy-number variations (CNVs) to human neurodevelopmental disorders, the mechanisms behind each gene's contribution to the constellation of neural phenotypes remain elusive. Here, we investigated the 7q11.23 CNV, whose hemideletion causes Williams syndrome (WS), and uncovered that mitochondrial dysfunction participates in WS pathogenesis. Dysfunction is facilitated in part by the 7q11.23 protein DNAJC30, which interacts with mitochondrial ATP-synthase machinery. Removal of Dnajc30 in mice resulted in hypofunctional mitochondria, diminished morphological features of neocortical pyramidal neurons, and altered behaviors reminiscent of WS. The mitochondrial features are consistent with our observations of decreased integrity of oxidative phosphorylation supercomplexes and ATP-synthase dimers in WS. Thus, we identify DNAJC30 as an auxiliary component of ATP-synthase machinery and reveal mitochondrial maladies as underlying certain defects in brain development and function associated with WS.

Structure of mycobacterial ATP synthase bound to the tuberculosis drug bedaquiline.

Tuberculosis-the world's leading cause of death by infectious disease-is increasingly resistant to current first-line antibiotics1. The bacterium Mycobacterium tuberculosis (which causes tuberculosis) can survive low-energy conditions, allowing infections to remain dormant and decreasing their susceptibility to many antibiotics2. Bedaquiline was developed in 2005 from a lead compound identified in a phenotypic screen against Mycobacterium smegmatis3. This drug can sterilize even latent M. tuberculosis infections4 and has become a cornerstone of treatment for multidrug-resistant and extensively drug-resistant tuberculosis1,5,6. Bedaquiline targets the mycobacterial ATP synthase3, which is an essential enzyme in the obligate aerobic Mycobacterium genus3,7, but how it binds the intact enzyme is unknown. Here we determined cryo-electron microscopy structures of M. smegmatis ATP synthase alone and in complex with bedaquiline. The drug-free structure suggests that hook-like extensions from the α-subunits prevent the enzyme from running in reverse, inhibiting ATP hydrolysis and preserving energy in hypoxic conditions. Bedaquiline binding induces large conformational changes in the ATP synthase, creating tight binding pockets at the interface of subunits a and c that explain the potency of this drug as an antibiotic for tuberculosis.

Chromatophores efficiently promote light-driven ATP synthesis and DNA transcription inside hybrid multicompartment artificial cells.

The construction of energetically autonomous artificial protocells is one of the most ambitious goals in bottom-up synthetic biology. Here, we show an efficient manner to build adenosine 5'-triphosphate (ATP) synthesizing hybrid multicompartment protocells. Bacterial chromatophores from Rhodobacter sphaeroides accomplish the photophosphorylation of adenosine 5'-diphosphate (ADP) to ATP, functioning as nanosized photosynthetic organellae when encapsulated inside artificial giant phospholipid vesicles (ATP production rate up to ∼100 ATP∙s-1 per ATP synthase). The chromatophore morphology and the orientation of the photophosphorylation proteins were characterized by cryo-electron microscopy (cryo-EM) and time-resolved spectroscopy. The freshly synthesized ATP has been employed for sustaining the transcription of a DNA gene, following the RNA biosynthesis inside individual vesicles by confocal microscopy. The hybrid multicompartment approach here proposed is very promising for the construction of full-fledged artificial protocells because it relies on easy-to-obtain and ready-to-use chromatophores, paving the way for artificial simplified-autotroph protocells (ASAPs).

The pathogenic m.8993 T > G mutation in mitochondrial ATP6 gene prevents proton release from the subunit c-ring rotor of ATP synthase.

The human ATP synthase is an assembly of 29 subunits of 18 different types, of which only two (a and 8) are encoded in the mitochondrial genome. Subunit a, together with an oligomeric ring of c-subunit (c-ring), forms the proton pathway responsible for the transport of protons through the mitochondrial inner membrane, coupled to rotation of the c-ring and ATP synthesis. Neuromuscular diseases have been associated to a number of mutations in the gene encoding subunit a, ATP6. The most common, m.8993 T > G, leads to replacement of a strictly conserved leucine residue with arginine (aL156R). We previously showed that the equivalent mutation (aL173R) dramatically compromises respiratory growth of Saccharomyces cerevisiae and causes a 90% drop in the rate of mitochondrial ATP synthesis. Here, we isolated revertants from the aL173R strain that show improved respiratory growth. Four first-site reversions at codon 173 (aL173M, aL173S, aL173K and aL173W) and five second-site reversions at another codon (aR169M, aR169S, aA170P, aA170G and aI216S) were identified. Based on the atomic structures of yeast ATP synthase and the biochemical properties of the revertant strains, we propose that the aL173R mutation is responsible for unfavorable electrostatic interactions that prevent the release of protons from the c-ring into a channel from which protons move from the c-ring to the mitochondrial matrix. The results provide further evidence that yeast aL173 (and thus human aL156) optimizes the exit of protons from ATP synthase, but is not essential despite its strict evolutionary conservation.

Energy conservation involving 2 respiratory circuits.

Chemiosmosis and substrate-level phosphorylation are the 2 mechanisms employed to form the biological energy currency adenosine triphosphate (ATP). During chemiosmosis, a transmembrane electrochemical ion gradient is harnessed by a rotary ATP synthase to phosphorylate adenosine diphosphate to ATP. In microorganisms, this ion gradient is usually composed of [Formula: see text], but it can also be composed of Na+ Here, we show that the strictly anaerobic rumen bacterium Pseudobutyrivibrio ruminis possesses 2 ATP synthases and 2 distinct respiratory enzymes, the ferredoxin:[Formula: see text] oxidoreductase (Rnf complex) and the energy-converting hydrogenase (Ech complex). In silico analyses revealed that 1 ATP synthase is [Formula: see text]-dependent and the other Na+-dependent, which was validated by biochemical analyses. Rnf and Ech activity was also biochemically identified and investigated in membranes of P. ruminis Furthermore, the physiology of the rumen bacterium and the role of the energy-conserving systems was investigated in dependence of 2 different catabolic pathways (the Embden-Meyerhof-Parnas or the pentose-phosphate pathway) and in dependence of Na+ availability. Growth of P. ruminis was greatly stimulated by Na+, and a combination of physiological, biochemical, and transcriptional analyses revealed the role of the energy conserving systems in P. ruminis under different metabolic scenarios. These data demonstrate the use of a 2-component ion circuit for [Formula: see text] bioenergetics and a 2nd 2-component ion circuit for Na+ bioenergetics in a strictly anaerobic rumen bacterium. In silico analyses infer that these 2 circuits are prevalent in a number of other strictly anaerobic microorganisms.

Conserved role of ATP synthase in mammalian cilia.

We previously found that ATP synthases localize to male-specific sensory cilia and control the ciliary response by regulating polycystin signalling in Caenorhabditis elegans. Herein, we discovered that the ciliary localization of ATP synthase is evolutionarily conserved in mammals. We showed that the ATP synthase subunit F1ß is colocalized with the cilia marker acetylated α-tubulin in both mammalian renal epithelial cells (MDCK) and normal mouse cholangiocytes (NMCs). Treatment with ATP synthase inhibitor oligomycin impaired ciliogenesis in MDCK cells, and F1ß was co-immunoprecipitated with PKD2 in mammalian cells. Our study provides evidence for the evolutionarily conserved localization of ATP synthase in cilia from worm to mammals. Defects in ATP synthase can lead to ciliary dysfunction, which may be a potential mechanism of polycystic kidney disease.

Multiomics Reveals Ectopic ATP Synthase Blockade Induces Cancer Cell Death via a lncRNA-mediated Phospho-signaling Network.

The EGFR tyrosine kinase inhibitor gefitinib is commonly used for lung cancer patients. However, some patients eventually become resistant to gefitinib and develop progressive disease. Here, we indicate that ecto-ATP synthase, which ectopically translocated from mitochondrial inner membrane to plasma membrane, is considered as a potential therapeutic target for drug-resistant cells. Quantitative multi-omics profiling reveals that ecto-ATP synthase inhibitor mediates CK2-dependent phosphorylation of DNA topoisomerase IIα (topo IIα) at serine 1106 and subsequently increases the expression of long noncoding RNA, GAS5. Additionally, we also determine that downstream of GAS5, p53 pathway, is activated by ecto-ATP synthase inhibitor for regulation of programed cell death. Interestingly, GAS5-proteins interactomic profiling elucidates that GAS5 associates with topo IIα and subsequently enhancing the phosphorylation level of topo IIα. Taken together, our findings suggest that ecto-ATP synthase blockade is an effective therapeutic strategy via regulation of CK2/phospho-topo IIα/GAS5 network in gefitinib-resistant lung cancer cells.

The structure of the catalytic domain of the ATP synthase from Mycobacterium smegmatis is a target for developing antitubercular drugs.

The crystal structure of the F1-catalytic domain of the adenosine triphosphate (ATP) synthase has been determined from Mycobacterium smegmatis which hydrolyzes ATP very poorly. The structure of the α3ß3-component of the catalytic domain is similar to those in active F1-ATPases in Escherichia coli and Geobacillus stearothermophilus However, its ε-subunit differs from those in these two active bacterial F1-ATPases as an ATP molecule is not bound to the two α-helices forming its C-terminal domain, probably because they are shorter than those in active enzymes and they lack an amino acid that contributes to the ATP binding site in active enzymes. In E. coli and G. stearothermophilus, the α-helices adopt an "up" state where the α-helices enter the α3ß3-domain and prevent the rotor from turning. The mycobacterial F1-ATPase is most similar to the F1-ATPase from Caldalkalibacillus thermarum, which also hydrolyzes ATP poorly. The ßE-subunits in both enzymes are in the usual "open" conformation but appear to be occupied uniquely by the combination of an adenosine 5'-diphosphate molecule with no magnesium ion plus phosphate. This occupation is consistent with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic products are probably the basis of the inhibition of ATP hydrolysis. It can be envisaged that specific as yet unidentified small molecules might bind to the F1 domain in Mycobacterium tuberculosis, prevent ATP synthesis, and inhibit the growth of the pathogen.

Recent progress in the use of mitochondrial membrane permeability transition pore in mitochondrial dysfunction-related disease therapies.

Mitochondria have various cellular functions, including ATP synthesis, calcium homeostasis, cell senescence, and death. Mitochondrial dysfunction has been identified in a variety of disorders correlated with human health. Among the many underlying mechanisms of mitochondrial dysfunction, the opening up of the mitochondrial permeability transition pore (mPTP) is one that has drawn increasing interest in recent years. It plays an important role in apoptosis and necrosis; however, the molecular structure and function of the mPTP have still not been fully elucidated. In recent years, the abnormal opening up of the mPTP has been implicated in the development and pathogenesis of diverse diseases including ischemia/reperfusion injury (IRI), neurodegenerative disorders, tumors, and chronic obstructive pulmonary disease (COPD). This review provides a systematic introduction to the possible molecular makeup of the mPTP and summarizes the mitochondrial dysfunction-correlated diseases and highlights possible underlying mechanisms. Since the mPTP is an important target in mitochondrial dysfunction, this review also summarizes potential treatments, which may be used to inhibit pore opening up via the molecules composing mPTP complexes, thus suppressing the progression of mitochondrial dysfunction-related diseases.

Comparative genomics reveals the molecular determinants of rapid growth of the cyanobacterium Synechococcus elongatus UTEX 2973.

Cyanobacteria are emerging as attractive organisms for sustainable bioproduction. We previously described Synechococcus elongatus UTEX 2973 as the fastest growing cyanobacterium known. Synechococcus 2973 exhibits high light tolerance and an increased photosynthetic rate and produces biomass at three times the rate of its close relative, the model strain Synechococcus elongatus 7942. The two strains differ at 55 genetic loci, and some of these loci must contain the genetic determinants of rapid photoautotrophic growth and improved photosynthetic rate. Using CRISPR/Cpf1, we performed a comprehensive mutational analysis of Synechococcus 2973 and identified three specific genes, atpA, ppnK, and rpaA, with SNPs that confer rapid growth. The fast-growth-associated allele of each gene was then used to replace the wild-type alleles in Synechococcus 7942. Upon incorporation, each allele successively increased the growth rate of Synechococcus 7942; remarkably, inclusion of all three alleles drastically reduced the doubling time from 6.8 to 2.3 hours. Further analysis revealed that our engineering effort doubled the photosynthetic productivity of Synechococcus 7942. We also determined that the fast-growth-associated allele of atpA yielded an ATP synthase with higher specific activity, while that of ppnK encoded a NAD+ kinase with significantly improved kinetics. The rpaA SNPs cause broad changes in the transcriptional profile, as this gene is the master output regulator of the circadian clock. This pioneering study has revealed the molecular basis for rapid growth, demonstrating that limited genetic changes can dramatically improve the growth rate of a microbe by as much as threefold.

Identification of two segments of the γ subunit of ATP synthase responsible for the different affinities of the catalytic nucleotide-binding sites.

ATP synthase uses a rotary mechanism to couple transmembrane proton translocation to ATP synthesis and hydrolysis, which occur at the catalytic sites in the ß subunits. In the presence of Mg2+, the three catalytic sites of ATP synthase have vastly different affinities for nucleotides, and the position of the central γ subunit determines which site has high, medium, or low affinity. Affinity differences and their changes as rotation progresses underpin the ATP synthase catalytic mechanism. Here, we used a series of variants with up to 45- and 60-residue-long truncations of the N- and C-terminal helices of the γ subunit, respectively, to identify the segment(s) responsible for the affinity differences of the catalytic sites. We found that each helix carries an affinity-determining segment of ∼10 residues. Our findings suggest that the affinity regulation by these segments is transmitted to the catalytic sites by the DELSEED loop in the C-terminal domain of the ß subunits. For the N-terminal truncation variants, presence of the affinity-determining segment and therefore emergence of a high-affinity binding site resulted in WT-like catalytic activity. At the C terminus, additional residues outside of the affinity-determining segment were required for optimal enzymatic activity. Alanine substitutions revealed that the affinity changes of the catalytic sites required no specific interactions between amino acid side chains in the γ and α3ß3 subunits but were caused by the presence of the helices themselves. Our findings help unravel the molecular basis for the affinity changes of the catalytic sites during ATP synthase rotation.

Beneficial factors for biomineralization by ureolytic bacterium Sporosarcina pasteurii.

BACKGROUND: The ureolytic bacterium Sporosarcina pasteurii is well-known for its capability of microbially induced calcite precipitation (MICP), representing a great potential in constructional engineering and material applications. However, the molecular mechanism for its biomineralization remains unresolved, as few studies were carried out. RESULTS: The addition of urea into the culture medium provided an alkaline environment that is suitable for S. pasteurii. As compared to S. pasteurii cultivated without urea, S. pasteurii grown with urea showed faster growth and urease production, better shape, more negative surface charge and higher biomineralization ability. To survive the unfavorable growth environment due to the absence of urea, S. pasteurii up-regulated the expression of genes involved in urease production, ATPase synthesis and flagella, possibly occupying resources that can be deployed for MICP. As compared to non-mineralizing bacteria, S. pasteurii exhibited more negative cell surface charge for binding calcium ions and more robust cell structure as nucleation sites. During MICP process, the genes for ATPase synthesis in S. pasteurii was up-regulated while genes for urease production were unchanged. Interestingly, genes involved in flagella were down-regulated during MICP, which might lead to poor mobility of S. pasteurii. Meanwhile, genes in fatty acid degradation pathway were inhibited to maintain the intact cell structure found in calcite precipitation. Both weak mobility and intact cell structure are advantageous for S. pasteurii to serve as nucleation sites during MICP. CONCLUSIONS: Four factors are demonstrated to benefit the super performance of S. pasteurii in MICP. First, the good correlation of biomass growth and urease production of S. pasteurii provides sufficient biomass and urease simultaneously for improved biomineralization. Second, the highly negative cell surface charge of S. pasteurii is good for binding calcium ions. Third, the robust cell structure and fourth, the weak mobility, are key for S. pasteurii to be nucleation sites during MICP.

The influence of l-carnitine on the expression of miRNAs in asthenospermia spermatozoa and the network regulation of the associated molecules.

l-carnitine is a natural compound that is indispensable for energy metabolism in mammals. The efficiency and safety of l-carnitine in improving sperm activity, enhancing epididymal function and treating male infertility has been widely acknowledged by clinicians. CircRNAs can regulate gene expression at the transcriptional or post-transcriptional level by serving as a molecular sponge of miRNAs with miRNA response elements. However, the detailed mechanism linking miRNA, circRNA and asthenospermia remains unclear. The present study demonstrated that hsa-miR-27b-3p, hsa-miR-151a-5p and hsa-miR-206 play an important role in the effects of l-carnitine treatment of the spermatozoa in asthenospermia patients. Furthermore, the target mRNAs of hsa-miR-206 were analysed by GO and KEGG. The results show that the target mRNAs of hsa-miR-206 may change the activity of ATP synthase and participate in the cAMP signalling pathway and the calcium signalling pathway, which may play an important role in sperm motility.

The Warburg hypothesis and weak ELF biointeractions.

The Warburg observation concerning ATP generation in cancer cells is analyzed with regard to the likely involvement of H+ resonance effects on the angular velocity of the ATP synthase rotor. It is reasonable to expect that the variety of diseases associated with mitochondrial dysfunction may in part be related to the ATP synthase rate of rotation. Experimental measurements of ATP synthase rotational rates, as found in the literature, are consistent with what might be expected from the ion cyclotron resonance (ICR) frequencies of protons moving under a Lorentz force determined by the approximate surface intensity of the geomagnetic field (~26-65 ?T). One research approach proposes that applying the electronic sum of two critical multiple resonance frequencies simultaneously may serve to more closely resemble real world biochemical changes as compared to applying these frequencies sequentially. Accordingly, it is suggested that applying the sum of the two individual resonance frequencies corresponding to H+ and H3O+ has the capacity to both increase proton density as well as couple to ATP synthase rotation. Not only will this tend to stabilize the F0 rotational rate but also perhaps make it feasible to control this rate, thereby providing a new and potentially efficacious means of treating diseases connected to mitochondrial dysfunction electromagnetically.

Purification of a Crenarchaeal ATP Synthase in the Light of the Unique Bioenergetics of Ignicoccus Species.

In this study, the ATP synthase of Ignicoccus hospitalis was purified, characterized, and structurally compared to the respective enzymes of the other Ignicoccus species, to shed light on energy conservation in this unique group of archaea. The crenarchaeal genus Ignicoccus comprises three described species, i.e., I. hospitalis and Ignicoccus islandicus from hot marine sediments near Iceland and Ignicoccus pacificus from a hydrothermal vent system in the Pacific Ocean. This genus is unique among all archaea due to the unusual cell envelope, consisting of two membranes that enclose a large intermembrane compartment (IMC). I. hospitalis is the best studied member of this genus, mainly because it is the only known host for the potentially parasitic archaeon Nanoarchaeum equitansI. hospitalis grows chemolithoautotrophically, and its sole energy-yielding reaction is the reduction of elemental sulfur with molecular hydrogen, forming large amounts of hydrogen sulfide. This reaction generates an electrochemical gradient, which is used by the ATP synthase, located in the outer cellular membrane, to generate ATP inside the IMC. The genome of I. hospitalis encodes nine subunits of an A-type ATP synthase, which we could identify in the purified complex. Although the maximal in vitro activity of the I. hospitalis enzyme was measured around pH 6, the optimal stability of the A1AO complex seemed to be at pH 9. Interestingly, the soluble A1 subcomplexes of the different Ignicoccus species exhibited significant differences in their apparent molecular masses in native electrophoresis, although their behaviors in gel filtration and chromatography-mass spectrometry were very similar.IMPORTANCE The Crenarchaeota represent one of the major phyla within the Archaea domain. This study describes the successful purification of a crenarchaeal ATP synthase. To date, all information about A-type ATP synthases is from euryarchaeal enzymes. The fact that it has not been possible to purify this enzyme complex from a member of the Crenarchaeota until now points to significant differences in stability, possibly caused by structural alterations. Furthermore, the study subject I. hospitalis has a particular importance among crenarchaeotes, since it is the only known host of N. equitans The energy metabolism in this system is still poorly understood, and our results can help elucidate the unique relationship between these two microbes.

The Impacts of Phosphorus Deficiency on the Photosynthetic Electron Transport Chain.

Phosphorus (P) is an essential macronutrient, and P deficiency limits plant productivity. Recent work showed that P deficiency affects electron transport to photosystem I (PSI), but the underlying mechanisms are unknown. Here, we present a comprehensive biological model describing how P deficiency disrupts the photosynthetic machinery and the electron transport chain through a series of sequential events in barley (Hordeum vulgare). P deficiency reduces the orthophosphate concentration in the chloroplast stroma to levels that inhibit ATP synthase activity. Consequently, protons accumulate in the thylakoids and cause lumen acidification, which inhibits linear electron flow. Limited plastoquinol oxidation retards electron transport to the cytochrome b6f complex, yet the electron transfer rate of PSI is increased under steady-state growth light and is limited under high-light conditions. Under P deficiency, the enhanced electron flow through PSI increases the levels of NADPH, whereas ATP production remains restricted and, hence, reduces CO2 fixation. In parallel, lumen acidification activates the energy-dependent quenching component of the nonphotochemical quenching mechanism and prevents the overexcitation of photosystem II and damage to the leaf tissue. Consequently, plants can be severely affected by P deficiency for weeks without displaying any visual leaf symptoms. All of the processes in the photosynthetic machinery influenced by P deficiency appear to be fully reversible and can be restored in less than 60 min after resupply of orthophosphate to the leaf tissue.

Diffusiophoresis in Cells: A General Nonequilibrium, Nonmotor Mechanism for the Metabolism-Dependent Transport of Particles in Cells.

The more we learn about the cytoplasm of cells, the more we realize that the cytoplasm is not uniform but instead is highly inhomogeneous. In any inhomogeneous solution, there are concentration gradients, and particles move either up or down these gradients due to a mechanism called diffusiophoresis. I estimate that inside metabolically active cells, the dynamics of particles can be strongly accelerated by diffusiophoresis, provided that they are at least tens of nanometers across. The dynamics of smaller objects, such as single proteins, are largely unaffected.

Biophysical Mechanism of Protein Export by Bacterial Type III Secretion System.

Type III secretion system (T3SS) is a protein injection nano-machine consisting of syringe and needle-like structure spanning bacterial inner and outer membranes. Bacteria insert the tip of T3SS needle to host cell membranes, and deliver effector proteins directly into host cells via T3SS to prime the host cell environment for infection. Thus inhibition of T3SS would be a potent strategy for suppressing bacterial infection. We previously demonstrated that T3SS needle rotates by proton-motive force (PMF) with the same mechanism as two evolutionally related rotary protein motors, flagellum and ATP synthase (FASEB J., 27, 2013, Ohgita et al.). Inhibition of needle rotation resulted in suppression of effector secretion, indicating the requirement of needle rotation for effector export. Simulation analysis of protein export by the T3SS needle suggests the importance of a hydrophobic helical groove formed by the conserved aromatic residue in the needle components. Based on these results, we have proposed a novel model of protein export by the T3SS needle, in which effector proteins are exported by PMF-dependent needle rotation oppositely to the hydrophobic helical groove in the needle. Quantitative examinations of the correlation between the speeds of T3SS rotation and the amount of effector export support this model. In this review, we summarize our current understanding of T3SS, and discuss our novel model of the protein export mechanism of T3SS based on the needle rotation.