Cryo-EM analysis of V/A-ATPase intermediates reveals the transition of the ground-state structure to steady-state structures by sequential ATP binding.

Vacuolar/archaeal-type ATPase (V/A-ATPase) is a rotary ATPase that shares a common rotary catalytic mechanism with FoF1 ATP synthase. Structural images of V/A-ATPase obtained by single-particle cryo-electron microscopy during ATP hydrolysis identified several intermediates, revealing the rotary mechanism under steady-state conditions. However, further characterization is needed to understand the transition from the ground state to the steady state. Here, we identified the cryo-electron microscopy structures of V/A-ATPase corresponding to short-lived initial intermediates during the activation of the ground state structure by time-resolving snapshot analysis. These intermediate structures provide insights into how the ground-state structure changes to the active, steady state through the sequential binding of ATP to its three catalytic sites. All the intermediate structures of V/A-ATPase adopt the same asymmetric structure, whereas the three catalytic dimers adopt different conformations. This is significantly different from the initial activation process of FoF1, where the overall structure of the F1 domain changes during the transition from a pseudo-symmetric to a canonical asymmetric structure (PNAS NEXUS, pgac116, 2022). In conclusion, our findings provide dynamical information that will enhance the future prospects for studying the initial activation processes of the enzymes, which have unknown intermediate structures in their functional pathway.

Structures of multisubunit membrane complexes with the CRYO ARM 200.

Progress in structural membrane biology has been significantly accelerated by the ongoing 'Resolution Revolution' in cryo-electron microscopy (cryo-EM). In particular, structure determination by single-particle analysis has evolved into the most powerful method for atomic model building of multisubunit membrane protein complexes. This has created an ever-increasing demand in cryo-EM machine time, which to satisfy is in need of new and affordable cryo-electron microscopes. Here, we review our experience in using the JEOL CRYO ARM 200 prototype for the structure determination by single-particle analysis of three different multisubunit membrane complexes: the Thermus thermophilus V-type ATPase VO complex, the Thermosynechococcus elongatus photosystem I monomer and the flagellar motor lipopolysaccharide peptidoglycan ring (LP ring) from Salmonella enterica.

Structural basis of unisite catalysis of bacterial F0F1-ATPase.

Adenosine triphosphate (ATP) synthases (F0F1-ATPases) are crucial for all aerobic organisms. F1, a water-soluble domain, can catalyze both the synthesis and hydrolysis of ATP with the rotation of the central γε rotor inside a cylinder made of α 3 ß 3 in three different conformations (referred to as ß E, ß TP, and ß DP). In this study, we determined multiple cryo-electron microscopy structures of bacterial F0F1 exposed to different reaction conditions. The structures of nucleotide-depleted F0F1 indicate that the ε subunit directly forces ß TP to adopt a closed form independent of the nucleotide binding to ß TP. The structure of F0F1 under conditions that permit only a single catalytic ß subunit per enzyme to bind ATP is referred to as unisite catalysis and reveals that ATP hydrolysis unexpectedly occurs on ß TP instead of ß DP, where ATP hydrolysis proceeds in the steady-state catalysis of F0F1. This indicates that the unisite catalysis of bacterial F0F1 significantly differs from the kinetics of steady-state turnover with continuous rotation of the shaft.

Mechanical inhibition of isolated Vo from V/A-ATPase for proton conductance.

V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V1 domain, with proton flow through the Vo membrane domain, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V1 domain, the Vo domain of the eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the Vo domain is prevented, however the molecular mechanism of this inhibition is not fully understood. Using cryo-electron microscopy, we determined the structure of both the holo V/A-ATPase and isolated Vo at near-atomic resolution, respectively. These structures clarify how the isolated Vo domain adopts the auto-inhibited form and how the holo complex prevents formation of the inhibited Vo form.

Cryo-EM studies of the rotary H+-ATPase/synthase from Thermus thermophilus.

Proton-translocating rotary ATPases couple proton influx across the membrane domain and ATP hydrolysis/synthesis in the soluble domain through rotation of the central rotor axis against the surrounding peripheral stator apparatus. It is a significant challenge to determine the structure of rotary ATPases due to their intrinsic conformational heterogeneity and instability. Recent progress of single particle analysis of protein complexes using cryogenic electron microscopy (cryo-EM) has enabled the determination of whole rotary ATPase structures and made it possible to classify different rotational states of the enzymes at a near atomic resolution. Three cryo-EM maps corresponding to different rotational states of the V/A type H+-rotary ATPase from a bacterium Thermus thermophilus provide insights into the rotation of the whole complex, which allow us to determine the movement of each subunit during rotation. In addition, this review describes methodological developments to determine higher resolution cryo-EM structures, such as specimen preparation, to improve the image contrast of membrane proteins.

Reactive Oxygen Species, Superoxide Dimutases, and PTEN-p53-AKT-MDM2 Signaling Loop Network in Mesenchymal Stem/Stromal Cells Regulation.

Mesenchymal stromal/stem cells (MSCs) are multipotent cells that can differentiate to various specialized cells, which have the potential capacity to differentiate properly and accelerate recovery in damaged sites of the body. This stem cell technology has become the fundamental element in regenerative medicine. As reactive oxygen species (ROS) have been reported to adversely influence stem cell properties, it is imperative to attenuate the extent of ROS to the promising protective approach with MSCs’ regenerative therapy. Oxidative stress also affects the culture expansion and longevity of MSCs. Therefore, there is great need to identify a method to prevent oxidative stress and replicative senescence in MSCs. Phosphatase and tensin homologue deleted on chromosome 10/Protein kinase B, PKB (PTEN/AKT) and the tumor suppressor p53 pathway have been proven to play a pivotal role in regulating cell apoptosis by regulating the oxidative stress and/or ROS quenching. In this review, we summarize the current research and our view of how PTEN/AKT and p53 with their partners transduce signals downstream, and what the implications are for MSCs’ biology.

Implications of PI3K/AKT/PTEN Signaling on Superoxide Dismutases Expression and in the Pathogenesis of Alzheimer's Disease.

Alzheimer’s disease is a neurodegenerative sickness, where the speed of personal disease progression differs prominently due to genetic and environmental factors such as life style. Alzheimer’s disease is described by the construction of neuronal plaques and neurofibrillary tangles composed of phosphorylated tau protein. Mitochondrial dysfunction may be a noticeable feature of Alzheimer’s disease and increased production of reactive oxygen species has long been described. Superoxide dismutases (SODs) protect from excess reactive oxygen species to form less reactive hydrogen peroxide. It is suggested that SODs can play a protective role in neurodegeneration. In addition, PI3K/AKT pathway has been shown to play a critical role on the neuroprotection and inhibiting apoptosis via the enhancing expression of the SODs. This pathway appears to be crucial in Alzheimer’s disease because it is related to the tau protein hyper-phosphorylation. Dietary supplementation of several ordinary compounds may provide a novel therapeutic approach to brain disorders by modulating the function of the PI3K/AKT pathway. Understanding these systems may offer a better efficacy of new therapeutic approaches. In this review, we summarize recent progresses on the involvement of the SODs and PI3K/AKT pathway in neuroprotective signaling against Alzheimer’s disease.

Cryo EM structure of intact rotary H+-ATPase/synthase from Thermus thermophilus.

Proton translocating rotary ATPases couple ATP hydrolysis/synthesis, which occurs in the soluble domain, with proton flow through the membrane domain via a rotation of the common central rotor complex against the surrounding peripheral stator apparatus. Here, we present a large data set of single particle cryo-electron micrograph images of the V/A type H+-rotary ATPase from the bacterium Thermus thermophilus, enabling the identification of three rotational states based on the orientation of the rotor subunit. Using masked refinement and classification with signal subtractions, we obtain homogeneous reconstructions for the whole complexes and soluble V1 domains. These reconstructions are of higher resolution than any EM map of intact rotary ATPase reported previously, providing a detailed molecular basis for how the rotary ATPase maintains structural integrity of the peripheral stator apparatus, and confirming the existence of a clear proton translocation path from both sides of the membrane.

General anesthetics cause mitochondrial dysfunction and reduction of intracellular ATP levels.

General anesthetics are indispensable for effective clinical care. Although, the mechanism of action of general anesthetics remains controversial, lipid bilayers and proteins have been discussed as their targets. In this study, we focused on the relationship between cellular ATP levels and general anesthetics. The ATP levels of nematodes and cultured mammalian cells were decreased by exposure to three general anesthetics: isoflurane, pentobarbital, and 1-phenoxy-2-propanol. Furthermore, these general anesthetics abolished mitochondrial membrane potential, resulting in the inhibition of mitochondrial ATP synthesis. These results suggest that the observed decrease of cellular ATP level is a common phenomenon of general anesthetics.

Roles of oncogenes and tumor-suppressor genes in osteoclastogenesis (Review).

Osteoporosis is a bone disease that poses a tremendous burden to health care. The receptor activator of nuclear factor-κB (RANK) and its ligand (RANKL) have been a major focus of this research field. RANKL signaling not only activates a variety of downstream signaling pathways required for osteoclast development, but crosstalk with other signaling pathways also adjusts bone homeostasis both in normal physiology and in bone disease. Consequently, novel drugs specifically targeting RANK-RANKL and their signaling pathways in osteoclasts are expected to revolutionize the treatment of various bone diseases such as osteoporosis. Osteoclasts are the exclusive cells involved in bone resorption. Abnormal activation of osteoclasts can lead to reduced bone density, resulting in osteopenia, osteoporosis and other bone disorders. To date, the mechanism of how osteoclast precursors differentiate into mature osteoclasts remains elusive. Cell proliferation and cell death may be key processes in the progression as well as other cell types. Oncogene products and tumor-suppressor molecules play a pivotal role in regulating the processes, which are important in regulating the configuration of bone disorders. Based on the understanding of these processes, promising alternatives to the use of medications against osteoporosis include specific diets with plant-derived supplements to modulate the expression and/or activity of these molecules. In this review, we summarize the progress of research with a focus on the modulatory roles of oncogene products and tumor-suppressor molecules and suggest the scope of further research concerning the prevention of osteoporosis in this field.

Rotation of artificial rotor axles in rotary molecular motors.

F1- and V1-ATPase are rotary molecular motors that convert chemical energy released upon ATP hydrolysis into torque to rotate a central rotor axle against the surrounding catalytic stator cylinder with high efficiency. How conformational change occurring in the stator is coupled to the rotary motion of the axle is the key unknown in the mechanism of rotary motors. Here, we generated chimeric motor proteins by inserting an exogenous rod protein, FliJ, into the stator ring of F1 or of V1 and tested the rotation properties of these chimeric motors. Both motors showed unidirectional and continuous rotation, despite no obvious homology in amino acid sequence between FliJ and the intrinsic rotor subunit of F1 or V1 These results showed that any residue-specific interactions between the stator and rotor are not a prerequisite for unidirectional rotation of both F1 and V1 The torque of chimeric motors estimated from viscous friction of the rotation probe against medium revealed that whereas the F1-FliJ chimera generates only 10% of WT F1, the V1-FliJ chimera generates torque comparable to that of V1 with the native axle protein that is structurally more similar to FliJ than the native rotor of F1 This suggests that the gross structural mismatch hinders smooth rotation of FliJ accompanied with the stator ring of F1.

n-3 polyunsaturated fatty acids stimulate osteoclastogenesis through PPARγ-mediated enhancement of c-Fos expression, and suppress osteoclastogenesis through PPARγ-dependent inhibition of NFkB activation.

n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been reported to suppress osteoclastogenesis in vivo. In this study, the effect of PUFAs on receptor for activation of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis was examined using bone marrow-derived monocytes/macrophage precursor cells (BMMs) or bone marrow cells (BMCs) in vitro. EPA and DHA stimulated the osteoclastic differentiation of BMMs, but n-6 PUFAs, linoleic acid and arachidonic acid had no effect. The stimulation of osteoclastogenesis of BMMs by EPA and DHA was associated with enhancement of the gene expressions of c-Fos, tartrate-resistant acid phosphatase, cathepsin K and peroxisome proliferator-activated receptor-γ (PPARγ) and the protein levels of c-Fos, PPARγ and nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent-1 (NFATc1). The PPARγ agonists, rosiglitazone and GW1929, also stimulated the osteoclastogenesis of BMMs. The PPARγ antagonists, T0070907 and GW9662, inhibited the stimulations of osteoclastogenesis and c-Fos expression by EPA or DHA. However, EPA and DHA inhibited the osteoclastogenesis in BMCs including BMMs and mesenchymal stem cells (MSCs). This inhibition was associated with suppression of the expression of RANKL and nuclear factor-κB (NFκB)-regulating genes, cyclooxygenase 2, TNFα and IL-6 in BMCs and MSCs. The agonists and antagonists of PPARγ showed that the inhibitions of NFκB transcriptional activity and osteoclastogenesis by EPA and DHA were PPARγ-dependent. These results suggest that EPA and DHA directly act on BMMs and stimulate osteoclastogenesis through enhancing c-Fos expression mediated by PPARγ but suppress osteoclastogenesis through the PPARγ-dependent inhibition of NFκB activation of MSCs in BMCs.

Increase in osteoclastogenesis in an obese Otsuka Long-Evans Tokushima fatty rat model.

In the present study, the effects of obesity on bone metabolism were investigated using a hyperphagic and obese rat model, the Otsuka Long­Evans Tokushima fatty (OLETF) rat, which exhibits normal glycemic control at 8 weeks of age. Body weight, food intake, fat mass, markers of bone resorption, the activities of tartrate­resistant acid phosphatase (TRAP) and cathepsin K, the number of osteoclasts in the proximal tibia, and the serum C­terminal crosslinking telopeptide level were higher in OLETF rats than those in control rats (Long­Evans Tokushima Otsuka; LETO). However, no differences in markers of bone formation, alkaline phosphatase activity, the number of osteoblasts in the proximal tibia or the serum osteocalcin level were observed. mRNA and protein levels of c­fms, receptor for activation of nuclear factor­κB (RANK), RANK ligand (RANKL), TRAP and cathepsin K were significantly increased in OLETF rats, although those levels of macrophage colony­stimulating factor (M­CSF) and osteoprotegerin (OPG) were similar to those in LETO rats. The level of serum tumor necrosis factor α (TNFα), and that of TNFα mRNA in bone, increased in association with the activation of NFκB. Furthermore, a frequency analysis and a colony formation assay respectively showed that the number of osteoclast precursors and the number of colony­forming cells induced by M­CSF each increased in OLETF rats compared with the control group. These results suggested that hyperphagia­induced obesity with normal glycemic control induces the upregulation of osteoclastogenesis that is associated with an increase in the expression of c­fms, RANK and RANKL, which is induced by TNFα, via the activation of NFκB.

The ingenious structure of central rotor apparatus in VoV1; key for both complex disassembly and energy coupling between V1 and Vo.

Vacuolar type rotary H+-ATPases (VoV1) couple ATP synthesis/hydrolysis by V1 with proton translocation by Vo via rotation of a central rotor apparatus composed of the V1-DF rotor shaft, a socket-like Vo-C (eukaryotic Vo-d) and the hydrophobic rotor ring. Reconstitution experiments using subcomplexes revealed a weak binding affinity of V1-DF to Vo-C despite the fact that torque needs to be transmitted between V1-DF and Vo-C for the tight energy coupling between V1 and Vo. Mutation of a short helix at the tip of V1-DF caused intramolecular uncoupling of VoV1, suggesting that proper fitting of the short helix of V1-D into the socket of Vo-C is required for tight energy coupling between V1 and Vo. To account for the apparently contradictory properties of the interaction between V1-DF and Vo-C (weak binding affinity but strict requirement for torque transmission), we propose a model in which the relationship between V1-DF and Vo-C corresponds to that between a slotted screwdriver and a head of slotted screw. This model is consistent with our previous result in which the central rotor apparatus is not the major factor for the association of V1 with Vo (Kishikawa and Yokoyama, J Biol Chem. 2012 24597-24603).

Neuron membrane trafficking and protein kinases involved in autism and ADHD.

A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

Functions and characteristics of PINK1 and Parkin in cancer.

Most of the Parkinson disease (PD) linked genes are also associated with cancers. In particular, phosphatase and tensin homologue-induced kinase 1 (PINK1) and Parkin, both of which are involved in recessively inherited familial forms of PD linked to mitochondrial dysfunction, appear to be abnormally expressed in cancers. Functional studies have revealed that PINK1 recruits Parkin to mitochondria to initiate mitophagy, an important autophagic quality control mechanism that rids the cell of damaged mitochondria. Although PD and cancer are obviously disparate human disorders, there is an evidence for low cancer rates in patients with PD. The relationship between cancer rates and PD might be related to the involvement of common pathways in both diseases. This paper provides a concise overview on the cellular functions of the PINK1 and Parkin.

BRCA1 and p53 tumor suppressor molecules in Alzheimer's disease.

Tumor suppressor molecules play a pivotal role in regulating DNA repair, cell proliferation, and cell death, which are also important processes in the pathogenesis of Alzheimer's disease. Alzheimer's disease is the most common neurodegenerative disorder, however, the precise molecular events that control the death of neuronal cells are unclear. Recently, a fundamental role for tumor suppressor molecules in regulating neurons in Alzheimer's disease was highlighted. Generally, onset of neurodegenerative diseases including Alzheimer's disease may be delayed with use of dietary neuro-protective agents against oxidative stresses. Studies suggest that dietary antioxidants are also beneficial for brain health in reducing disease-risk and in slowing down disease-progression. We summarize research advances in dietary regulation for the treatment of Alzheimer's disease with a focus on its modulatory roles in BRCA1 and p53 tumor suppressor expression, in support of further therapeutic research in this field.

Function of α-synuclein and PINK1 in Lewy body dementia (Review).

α-Synuclein (α-syn) is the major protein component of Lewy bodies, a key pathological characteristic of the degenerating brain. The misfolding and aggregation of α-syn is associated with both the idiopathic and familial forms of Parkinson's disease (PD) and Lewy body dementia (LBD). However, the function of α-syn is poorly understood, as it shows both neurotoxic and neuroprotective activities. Mutations in phosphatase and tensin homologue-induced putative kinase 1 (PINK1) also cause recessively inherited PD. Studies support the notion of neuroprotective roles for PINK1, as it protects cells from damage-induced mitochondrial dysfunction, oxidative stress and cell apoptosis. PINK1 plays an essential role in mitochondrial quality control and its homeostasis is maintained through mitochondrial stabilization. The α-syn aggregation is linked to various aspects of mitochondrial dysfunction and PINK1-related mitophagy. Determination of the molecular pathways that lead to α-syn oligomerization and further aggregation may be the basis for the successful design and development of treatments for these neurodegenerative diseases. The present review summarizes the function of PINK1 underlying α-syn aggregation and the mechanisms through which mitochondrial dysfunction plays a role in this process.

PI3K/AKT/PTEN pathway as a target for Crohn's disease therapy (Review).

The pathogenesis of inflammatory bowel disease (IBD), including Crohn's disease, is a subject of increasing interest. Loss-of-function mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) are strong genetic factors linked to Crohn's disease, which eventually leads to an excessive mucosal inflammatory response directed against components of normal gut microbiota. Reactive oxygen species (ROS) play an important role in inflammation processes, as well as in transduction of signals from receptors for several cytokines, such as tumor necrosis factor α (TNFα). ROS activate nuclear factor-κB (NF-κB) via IκB kinase (IKK) through the PI3K/AKT/PTEN pathway. Therefore, this pathway is recognized to play a key role in Crohn's disease. Loss of function has been demonstrated to occur as an early event in a wide variety of diseases. Given this prevalent involvement in a number of diseases, the molecular development that modulates this pathway has been the subject of several studies. In addition, it has been the focus of extensive research and drug discovery activities. A better understanding of the molecular assemblies may reveal novel targets for the therapeutic development against Crohn's disease.

Certain Diet and Lifestyle May Contribute to Islet ß-cells Protection in Type-2 Diabetes via the Modulation of Cellular PI3K/AKT Pathway.

PI3K/AKT pathway has been shown to play a pivotal role on islet ß-cell protection, enhancing ß-cell survival by stimulating cell proliferation and inhibiting cell apoptosis. Accordingly, this pathway appears to be crucial in type-2 diabetes. Understanding the regulations of this pathway may provide a better efficacy of new therapeutic approaches. In this review, we summarize advances on the involvement of the PI3K/AKT pathway in hypothetical intra-cellular signaling of islet ß-cells. As recent findings may show the nutritional regulation of the survival pathway in the islet ß-cells through activation of the PI3K/AKT pathway, we also review studies on the features of several diets, correlated lifestyle, and its signaling pathway involved in type-2 diabetes. The molecular mechanisms contributing to the disease are the subject of considerable investigation, as a better understanding of the pathogenesis will lead to novel therapies against a condition of the disease.