F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements

Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F₁-ATPase (F₁) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-α₃β₃, while simultaneously monitoring rotations of the central shaft-γ. In the ATP waiting dwell, two of three β-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two β-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F₁ structure and the rotation angle of the motor. Remarkably, a structure of F₁ in an ε-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the αβ-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of β and the loosening/tightening motions at the αβ-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F₁-ATPase.ATP synthase (F₁F_o-ATPase) catalyzes ATP synthesis from ADP and P_i in cells. The isolated F₁ portion is called F₁-ATPase, because it also catalyzes the reverse reaction, ATP hydrolysis (1–3). The α₃β₃γ-catalytic core complex of F₁-ATPase (denoted F₁) is a rotary molecular motor in which three αβ-dimers are arranged around the central γ-shaft (4). Unidirectional rotation of γ is driven by the free energy derived from sequential ATP hydrolysis at catalytic sites in the three αβ-dimers (5–7). Under an external torque, F₁ synthesizes ATP coupled to the rotation of γ in the opposite direction (8). This reversible operation of F₁ is achieved by the tripartite mechanochemical coupling between chemical reactions at the catalytic sites of αβ, conformational changes in the stator ring-α₃β₃, and orientation of γ.A combination of the rotation assay (5, 6) and single-molecule fluorescence imaging techniques (9) has led to a detailed picture of the coupling between chemical reactions in α₃β₃ and the rotary angles (10, 11). One ATP hydrolysis reaction in α₃β₃ drives discrete 80° + 40° substeps of γ in bacterial F₁ (7). The 80° substep is mainly driven by the binding energy of ATP (7, 9). The dwell before the 80° substep is, therefore, named the ATP waiting dwell. Release of the product, ADP, occurs before completion of the 80° substep (9, 10). The angle-dependent affinity of ADP suggests that the ADP release event also contributes part of the energy for the 80° substep (10). The dwell before the 40° substep is called the catalytic dwell; it consists of two rate-limiting events: ATP cleavage and release of the product, P_i (10, 12). The 40° substep is accompanied by a decrease of P_i affinity, with release that, in turn, generates torque (10). The coupling scheme between chemical reactions in α₃β₃ and the rotary angles has, therefore, been almost completely established (10).In the coupling of chemical reactions and α₃β₃-conformations, the key concept is thought to be the binding change mechanism, in which three catalytic sites in F₁ undergo sequential transitions between conformational states with different affinities for nucleotides corresponding to different rotary angles (1). The binding change mechanism is supported by the first crystal structure of F₁, in which two βs adopt the closed form with nucleotides and the other β adopts the open form without a nucleotide (4). The 120° step of γ observed in the rotation assay further supports this mechanism (6).However, we still face significant gaps in the structural ATPase cycle. Previous studies have suggested that F₁ should adopt at least two distinct conformational states for the ATP waiting dwell (ATP waiting form) and the catalytic dwell (catalytic form) based on rotation and tilting angles of γ (13). Furthermore, based on indirect evidence, it has been pointed out that the first crystal structure should represent the catalytic form or forms similar to the catalytic intermediate states (14–18). Although the crystal structures of the α₃β₃γ-complex differ from each other in terms of their nucleotide binding states and detailed configurations of the residues, their global structures are similar to the first crystal structure (19), which leaves the structure of the ATP waiting form unresolved. Closing this gap in the conformational cycle will deepen our understanding of the coupling between chemical reactions, α₃β₃-conformations, conformations, and rotary angles, not least by providing critical input into the theoretical modeling of F₁ (20–26).Here, we use the FRET technique to elucidate the conformational transitions of α₃β₃-conformations in F₁. FRET involves excited-state energy transfer from one fluorescent dye (donor) to another (acceptor) through dipole–dipole interactions (27). Single-pair FRET measurements combined with single-molecule techniques have been used to investigate the dynamics of intramolecular conformational changes or intermolecular interactions at the single-molecule level (28–34), including for F_oF₁-ATP synthase (35–37). We perform single-molecule FRET measurement to monitor distance changes between two fluorescently labeled βs and simultaneously monitor the rotational steps of γ. The FRET data allow us to distinguish the ATP waiting form from the catalytic form and thus, relate these dwelling states to the respective crystal structures. A systematic comparison of the crystal structures reveals the structural basis of the ATPase cycle. This study provides a structural basis for tripartite coupling among chemical reactions, conformations in the stator, and rotary angles in bacterial F₁.     

Anatomy of F1-ATPase powered rotation

F₁-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F₁ structures 2HLD1 and 1H8E (Protein Data Bank).The purified F₁-ATPase is a molecular motor that can hydrolyze ATP to drive counterclockwise (CCW) rotation of the γ-subunit within the (αβ)₃-ring (Fig. 1A). In most living organisms, the F_o component of the F_oF₁ complex uses energy derived from a proton-motive force across a membrane to power F₁-dependent synthesis of ATP from ADP and P_i. Consumption of an ATP at each F₁ catalytic site, primarily composed of a β-subunit, correlates with a 120° rotational power stroke of the γ-subunit separated by a catalytic dwell with an 8-ms duration in Escherichia coli enzyme (1). A second “ATP-binding” dwell can occur after the γ-subunit has rotated ∼30° to 40° from the catalytic at limiting substrate concentrations (2, 3). Thus, three successive catalytic events that include power strokes and dwells are required to complete one revolution of the γ-subunit. Once bound to F₁, ATP is retained for 240° (3) such that the ADP and P_i generated are released two catalytic events later.Open in a separate windowFig. 1.Structural components of the F₁-ATPase molecular motor. (A) Top (from membrane) and side views of F₁ composed of the ring of α (orange) and β (purple) subunits surrounding the γ-subunit rotor (blue and green). (B) Open (β_E) and closed (β_D) conformations of the catalytic site composed of the catalytic domain (tan ribbon) and the lever domain (purple) relative to the γ-subunit coiled-coil (green) and foot (blue) domains. In the Gibbons et al. (32) F₁ structure (PDB ID code 1E79) used here, the γ-subunit foot domain is rotated 7° CCW from the structure determined by Menz et al. (4).The γ-subunit is composed of coiled-coil and globular “foot” domains where the former extends through the core of the (αβ)₃-ring (Fig. 1B). The β-subunits contain a catalytic domain and a C-terminal “lever” domain that is extended or open when the catalytic site is devoid of nucleotide and contracted (closed) when nucleotide is bound. In most F₁ crystal structures (4, 5), the coiled-coil faces the β-subunit with the open lever (β_E) whereas the foot domain extends over the lever domains of catalytic sites that usually contain bound ADP (β_D) and ATP (β_T). Although crystal structures provide excellent pictures of the subunit conformations at one rotational position, the rotational movement of the γ-subunit between these static structures and the mechanism in which ATP fuels this movement occurs remain major unresolved questions.Consensus is currently lacking regarding the relationship of nucleotide occupancy at the three catalytic sites to the catalytic dwell and ATP-binding dwell despite the intense scrutiny this question has received since Boyer and coworkers (6, 7) showed that F₁ operates via an alternating site mechanism. The catalytic dwell includes ATP hydrolysis and is believed to be terminated by the release of phosphate (3). Some single-molecule experiments support a mechanism whereby ATP binding and ADP release are concurrent during the ATP-binding dwell (3). As a result, only two catalytic sites are occupied the majority of the time such that three-site occupancy occurs transiently during the ATP-binding dwell. However, these results are inconsistent with the F₁ structure that contains transition state analogs and has three-site nucleotide occupancy (4). Nucleotide binding studies also strongly support a mechanism in which all three sites must be occupied (8, 9) and are consistent with other single-molecule studies that support alternative three-site mechanisms (10–12). At this time there is no consistent evidence that correlates any of the crystal structures to the prevailing rotational mechanism.The β-subunit lever domain is positioned to push against the γ-foot and the γ–coiled-coil as it opens and closes, respectively (Fig. 1B). The asymmetry of the γ-subunit at these interfaces resembles a camshaft that would be consistent with CCW directionality in response to lever movement. The energy for a 120° power stroke has been proposed to derive from the binding affinity of ATP that is used as ATP binding-induced closing of the β-lever (13) and is supported by experiments in which the lever was truncated (14).Based on single-molecule measurements, it was concluded that F₁ is nearly 100% efficient (15). A necessary outcome of this conclusion is that the 120° power strokes must occur at a constant angular velocity (13). Although a number of simulation studies have modeled rotation of the F₁-ATPase γ-subunit (16–19), only one of these (19) has provided a result showing that the angular velocity should vary during a power stroke. The claim of 100% efficiency (15) that serves as the energetic basis of this power stroke mechanism is unwarranted because the magnitude of the viscous flow coupling to the surface was unknown owing to technical limitations, and the authors erroneously used the value of ΔG° in lieu of ΔG in their calculation. The technical handicap was subsequently overcome by Junge and coworkers (20, 21), who relied on elastic probe curvature instead of rotation speed to calculate the average torque of the power stroke. Similar average torque values were subsequently obtained using rotation under limiting drag conditions, when the drag on the probe was measured directly (22).Based on 100% enzyme efficiency, it was difficult to explain how the energy from ATP binding was able to power 120° of rotation when the catalytic dwell interrupts rotation 80° after ATP binds. It was hypothesized that the remaining binding energy needed to power the final 40° of rotation until the next ATP binds is stored as elastic energy in the closed β-lever, which upon product release pushes on the γ-foot as it opens (13). However, to date, experimental evidence that tests these hypotheses is lacking owing to the inability to measure the rotary motion under conditions where the angular velocity is limited by the internal mechanism of the motor.Here we have resolved the angular velocity as a function of the rotational position using an assay that provides 10-μs time resolution. The results clearly show that the angular velocity is not constant during a power stroke, but undergoes a series of accelerations and decelerations as a function of rotational position. The slower angular velocity observed with the lower-affinity substrate ITP provides direct evidence that substrate binding affinity provides energy to power rotation. The correlation of the angular velocity profile of the final 85° of rotation presented here to the profile resulting from the simulations of Pu and Karplus (19) strongly supports a model in which ATP binding-dependent closure of the lever applies force to the γ-subunit. This also provides evidence that associates the F₁ structures of Kabaleeswaran et al. (23) and Menz et al. (4) with the protein conformations at 35° and 120° because they were used as the reference structures for the simulations of Pu and Karplus (19). The data also show that elevated ADP concentrations increase dwells at ∼35° and decrease the angular velocity between 85° and 120°. This indicates that ADP can remain bound subsequent to the ATP-binding dwell consistent with a three-site mechanism.     

Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin results in reduced sarcolemmal integrity and increased susceptibility to muscle damage. The α₇β₁-integrin is a laminin-binding protein up-regulated in the skeletal muscle of DMD patients and in the mdx mouse model. Transgenic overexpression of the α₇-integrin alleviates muscle disease in dystrophic mice, making this gene a target for pharmacological intervention. Studies suggest laminin may regulate α₇-integrin expression. To test this hypothesis, mouse and human myoblasts were treated with laminin and assayed for α₇-integrin expression. We show that laminin-111 (α₁, β₁, γ₁), which is expressed during embryonic development but absent in normal or dystrophic skeletal muscle, increased α₇-integrin expression in mouse and DMD patient myoblasts. Injection of laminin-111 protein into the mdx mouse model of DMD increased expression of α₇-integrin, stabilized the sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscle from exercised-induced damage. These findings demonstrate that laminin-111 is a highly potent therapeutic agent for the mdx mouse model of DMD and represents a paradigm for the systemic delivery of extracellular matrix proteins as therapies for genetic diseases.     

Correlation between the conformational states of F1-ATPase as determined from its crystal structure and single-molecule rotation

F₁-ATPase is a rotary molecular motor driven by ATP hydrolysis that rotates the γ-subunit against the α₃β₃ ring. The crystal structures of F₁, which provide the structural basis for the catalysis mechanism, have shown essentially 1 stable conformational state. In contrast, single-molecule studies have revealed that F₁ has 2 stable conformational states: ATP-binding dwell state and catalytic dwell state. Although structural and single-molecule studies are crucial for the understanding of the molecular mechanism of F₁, it remains unclear as to which catalytic state the crystal structure represents. To address this issue, we introduced cysteine residues at βE391 and γR84 of F₁ from thermophilic Bacillus PS3. In the crystal structures of the mitochondrial F₁, the corresponding residues in the ADP-bound β (β_DP) and γ were in direct contact. The βE190D mutation was additionally introduced into the β to slow ATP hydrolysis. By incorporating a single copy of the mutant β-subunit, the chimera F₁, α₃β₂β(E190D/E391C)γ(R84C), was prepared. In single-molecule rotation assay, chimera F₁ showed a catalytic dwell pause in every turn because of the slowed ATP hydrolysis of β(E190D/E391C). When the mutant β and γ were cross-linked through a disulfide bond between βE391C and γR84C, F₁ paused the rotation at the catalytic dwell angle of β(E190D/E391C), indicating that the crystal structure represents the catalytic dwell state and that β_DP is the catalytically active form. The former point was again confirmed in experiments where F₁ rotation was inhibited by adenosine-5′-(β,γ-imino)-triphosphate and/or azide, the most commonly used inhibitors for the crystallization of F₁.     

Subunit rotation in Escherichia coli FoF1–ATP synthase during oxidative phosphorylation

We report evidence for proton-driven subunit rotation in membrane-bound F_oF₁–ATP synthase during oxidative phosphorylation. A βD380C/γC87 crosslinked hybrid F₁ having epitope-tagged βD380C subunits (β_flag) exclusively in the two noncrosslinked positions was bound to F_o in F₁-depleted membranes. After reduction of the β–γ crosslink, a brief exposure to conditions for ATP synthesis followed by reoxidation resulted in a significant amount of β_flag appearing in the β–γ crosslinked product. Such a reorientation of γC87 relative to the three β subunits can only occur through subunit rotation. Rotation was inhibited when proton transport through F_o was blocked or when ADP and P_i were omitted. These results establish F_oF₁ as the second example in nature where proton transport is coupled to subunit rotation.     

Enhancing Corrosion and Wear Resistance of AA6061 by Friction Stir Processing with Fe78Si9B13 Glass Particles

The AA6061-T6 aluminum alloy samples including annealed Fe₇₈Si₉B₁₃ particles were prepared by friction stir processing (FSP) and investigated by various techniques. The Fe₇₈Si₉B₁₃-reinforced particles are uniformly dispersed in the aluminum alloy matrix. The XRD results indicated that the lattice parameter of α-Al increases and the preferred orientation factors F of (200) plane of α-Al reduces after friction stir processing. The coefficient of thermal expansion (CTE) for FSP samples increases at first with the temperature but then decreases as the temperature further increased, which can be explained by the dissolving of Mg and Si from β phase and Fe₇₈Si₉B₁₃ particles. The corrosion and wear resistance of FSP samples have been improved compared with that of base metal, which can be attributed to the reduction of grain size and the CTE mismatch between the base metal and reinforced particles by FSP, and the lubrication effect of Fe₇₈Si₉B₁₃ particles also plays a role in improving wear resistance. In particular, the FSP sample with reinforced particles in amorphous state exhibited superior corrosion and wear resistance due to the unique metastable structure.     

A Simple and Efficient Method for Preparing High-Purity α-CaSO4·0.5H2O Whiskers with Phosphogypsum

A simple and efficient approach for the high-purity CaSO₄·2H₂O (DH) whiskers and α-CaSO₄·0.5H₂O (α-HH) whiskers derived from such phosphogypsum (PG) was proposed. The impact of different experimental parameters on supersaturated dissolution–recrystallization and preparation processes of α-CaSO₄·0.5H₂O was elaborated. At 3.5 mol/L HCl concentration, the dissolution temperature and time were 90 °C and 20 min, respectively. After eight cycles and 5–8 times cycles, total crystallization amount of CaSO₄·2H₂O was 21.75 and 9.97 g/100 mL, respectively, from supersaturated HCl solution. The number of cycles affected the shape and amount of the crystal. Higher HCl concentration facilitated CaSO₄·2H₂O dissolution and created a much higher supersaturation, which acted as a larger driving force for phase transformation of CaSO₄·2H₂O to α-CaSO₄·0.5H₂O. The HCl solution system’s optimum experimental conditions for HH whiskers preparation involved acid leaching of CaSO₄·2H₂O sample, with HCl concentration 6.0 mol/L, reaction temperature 80 °C, and reaction time 30 min–60 min. Under the third cycle conditions, α-CaSO₄·0.5H₂O whiskers were uniform in size, clear, and distinct in edges and angles. The length range of α-CaSO₄·0.5H₂O whiskers was from 106 μm to 231 μm and diameter range from 0.43 μm to 1.35 μm, while the longest diameter ratio was 231. Purity of α-CaSO₄·0.5H₂O whiskers was approximately 100%, where whiteness reached 98.6%. The reuse of the solution enables the process to discharge no waste liquid. It provides a new reference direction for green production technology of phosphogypsum.     

Single-molecule analysis of fluorescently labeled G-protein–coupled receptors reveals complexes with distinct dynamics and organization

G-protein–coupled receptors (GPCRs) constitute the largest family of receptors and major pharmacological targets. Whereas many GPCRs have been shown to form di-/oligomers, the size and stability of such complexes under physiological conditions are largely unknown. Here, we used direct receptor labeling with SNAP-tags and total internal reflection fluorescence microscopy to dynamically monitor single receptors on intact cells and thus compare the spatial arrangement, mobility, and supramolecular organization of three prototypical GPCRs: the β₁-adrenergic receptor (β₁AR), the β₂-adrenergic receptor (β₂AR), and the γ-aminobutyric acid (GABA_B) receptor. These GPCRs showed very different degrees of di-/oligomerization, lowest for β₁ARs (monomers/dimers) and highest for GABA_B receptors (prevalently dimers/tetramers of heterodimers). The size of receptor complexes increased with receptor density as a result of transient receptor–receptor interactions. Whereas β₁-/β₂ARs were apparently freely diffusing on the cell surface, GABA_B receptors were prevalently organized into ordered arrays, via interaction with the actin cytoskeleton. Agonist stimulation did not alter receptor di-/oligomerization, but increased the mobility of GABA_B receptor complexes. These data provide a spatiotemporal characterization of β₁-/β₂ARs and GABA_B receptors at single-molecule resolution. The results suggest that GPCRs are present on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be targeted, in a ligand-regulated manner, to different cell-surface microdomains.     

Circulating Lp-PLA2 activity correlates with oxidative stress and cytokines in overweight/obese postmenopausal women not using hormone replacement therapy

Controversy remains regarding whether there is an association between circulating lipoprotein-associated phospholipase A₂ (Lp-PLA₂), cytokines, and oxidative stress in healthy postmenopausal women. We investigated the influence of age on Lp-PLA₂ activity in postmenopausal women not using hormone therapy and the relationship of Lp-PLA₂ enzyme activity to serum cytokine levels and oxidative stress indices. Normal weight (n = 1284) and overweight/obese (n = 707) postmenopausal women not using hormone therapy were categorized into five age groups: 50–54, 55–59, 60–64, 65–69, and 70–89 years. Overweight-obese women showed higher plasma Lp-PLA₂ activity, urinary 8-epi-prostaglandin F_2α (8-epi-PGF_2α), serum interleukin (IL)-6, and smaller LDL particles than normal-weight women after adjusting for age, years postmenopause, smoking, drinking, blood pressure, glucose, insulin, lipid profiles, BMI, and waist circumference. Overweight/obese women 70–89 years old showed higher Lp-PLA₂ activity than those aged 50–54 years, whereas no significant difference in Lp-PLA₂ activity existed across normal-weight female age groups. Overweight/obese women aged ≥65 years showed higher Lp-PLA₂, oxidized LDL (ox-LDL), IL-6, and 8-epi-PGF_2α than age-matched normal-weight controls. Overweight/obese women aged ≥70 years had higher ox-LDL levels than those aged 50–59, and overweight/obese women aged 65–89 showed higher IL-6 and 8-epi-PGF_2α. There were strong positive correlations between Lp-PLA₂ and ox-LDL (r = 0.385, P < 0.001), Lp-PLA₂ and IL-6 (r = 0.293, P < 0.001), and ox-LDL and IL-6 (r = 0.303, P < 0.001) in overweight/obese women; however, these relationships were weak in normal-weight women. These results suggest that aging and obesity-related oxidative and inflammatory mediators are associated with Lp-PLA₂ activity in overweight/obese postmenopausal women not using hormone therapy.     

Trapping the ATP binding state leads to a detailed understanding of the F1-ATPase mechanism

The rotary motor enzyme F_oF₁-ATP synthase uses the proton-motive force across a membrane to synthesize ATP from ADP and P_i (H₂PO₄⁻) under cellular conditions that favor the hydrolysis reaction by a factor of 2 × 10⁵. This remarkable ability to drive a reaction away from equilibrium by harnessing an external force differentiates it from an ordinary enzyme, which increases the rate of reaction without shifting the equilibrium. Hydrolysis takes place in the neighborhood of one conformation of the catalytic moiety F₁-ATPase, whose structure is known from crystallography. By use of molecular dynamics simulations we trap a second structure, which is rotated by 40° from the catalytic dwell conformation and represents the state associated with ATP binding, in accord with single-molecule experiments. Using the two structures, we show why P_i is not released immediately after ATP hydrolysis, but only after a subsequent 120° rotation, in agreement with experiment. A concerted conformational change of the α₃β₃ crown is shown to induce the 40° rotation of the γ-subunit only when the β_E subunit is empty, whereas with P_i bound, β_E serves as a latch to prevent the rotation of γ. The present results provide a rationalization of how F₁-ATPase achieves the coupling between the small changes in the active site of β_DP and the 40° rotation of γ.The molecular motor F_oF₁-ATP synthase is composed of two domains: a transmembrane portion (F_o), the rotation of which is induced by a proton gradient, and a globular catalytic moiety (F₁) that synthesizes and hydrolyzes ATP. The primary function of the proton-motive force acting on F_oF₁-ATP synthase is to provide the torque required to rotate the γ-subunit in the direction for ATP synthesis (1, 2). The catalytic moiety, F₁-ATPase, has an α₃β₃ “crown” composed of three α- and three β-subunits arranged in alternation around the γ-subunit, which has a globular base and an extended coiled-coil portion (3) (Fig. 1A). F₁-ATPase by itself binds ATP and hydrolyzes it to induce rotation of the γ-subunit (in the opposite direction from that for synthesis) on the millisecond time scale under optimum conditions (4, 5). All of the α- and β-subunits bind nucleotides, but only the three β-subunits are catalytically active. The original crystal structure (3) of F₁-ATPase from bovine heart mitochondria (MF₁) led to the identification of three conformations of the β-subunit: β_E (empty), β_TP (ATP analog bound), and β_DP (ADP bound); Fig. 1A. In the known structures of F₁-ATPase, which apparently are near the “catalytic dwell” state, the state in which catalysis occurs (6, 7), the β_E subunit conformation is partly to fully open and is very different from those of the β_TP and β_DP subunits, which are closed and very similar to each other (SI Appendix, SI1).Open in a separate windowFig. 1.(A) F₁-ATPase. The three β-subunits and the γ-subunit are shown (α-subunits are not shown for clarity): β_E (yellow), β_DP (orange), β_TP (gold), and γ (purple). To define the β_DP subunit conformation we use the angle between helix B (βT163-A176) and helix C (βT190-G204). The two helices are highlighted: helix B (blue) and helix C (gray); the B^C angle is depicted as a red angle. The β_DPH6 helix, whose orientation was reported to undergo a 20° change during the 40° substep γ-rotation, is highlighted as red. During the forced rotation simulations with an external torque, the force acts on the C_α atom of MF₁:γM25 (shown as a red sphere). The direction of the force is determined as the cross-product of the radial vector of γM25:C_α and the rotational axis (green). (B) Proposed 360° rotation cycle of F₁-ATPase showing the subunit conformations, as well as the binding–release of ligands and the hydrolysis of ATP. Starting from the binding of an ATP* to the β_E subunit in the ATP waiting state (0°), rotation of the γ-stalk by 200° (80°, 40°, 80°) leads to the transition of β_E (γ = 0°) via β_TP (γ = 80°) to β_DP (γ = 200°), the catalytic dwell state where hydrolysis of ATP* takes place. The hydrolysis product P_i* in the β_DP subunit is not released at this catalytic dwell (200°). Instead, the other hydrolysis product ADP* is released first after a 40° rotation [β_DP (200°) → β_HO (240°)]. Then, β_HO is transformed to β_E and P_i* is released after an additional 80° rotation to another catalytic dwell state (320°); the latter is shown in brackets outside the main cycle (see below). Finally, the release of P_i* from β_E leads to a 40° rotation that completes the 360° cycle (21, 41). The other subunits are going through corresponding cycles offset by 120° (β_DP) and 240° (β_TP), respectively. Here, the prime symbol when it appears on the β_DP and β_E conformations indicates that the conformation of corresponding subunits change slightly in or near the specified reaction steps. The γ-subunit is shown as a yellow oval, and its rotation during the hydrolysis cycle is indicated by a red arrow. The reaction steps occurring in or near the catalytic dwell and corresponding changes of ligands in each β-subunit are also shown in the 320° catalytic dwell: The first state (Left in the 320° catalytic dwell) has a bound ATP in β_DP′, and is thus referred to as a prehydrolysis state (the state before the hydrolysis of ATP during the catalytic dwell). The second state (Middle) represents the state after ATP hydrolysis (posthydrolysis state), and the third state (Right) presents the state after the release of P_i bound in β_E′ (postrelease state).     

Urinary excretion of 9α,11β-prostaglandin F2 and leukotriene E4 in patients with exercise-induced bronchoconstriction

Background

Increased levels of mast cell-derived eicosanoids, such as prostaglandin (PG) D₂ and cysteinyl leukotrienes (CysLTs), have been reported in patients with exercise-induced bronchoconstriction (EIB), suggesting that mast cell activation is involved in the mechanism of EIB. However, it is still controversial since these results have not been reproduced in other studies. The aim of this study was to evaluate the role of PGD₂ and LTE₄ in adult asthma with EIB, as measuring urinary levels of their metabolites—9α,11β-PGF₂ and LTE₄ before and after an exercise challenge test.

Methods

Eight patients with asthma and EIB and five normal controls without EIB were enrolled. Exercise challenge tests comprised of 6 min of treadmill exercise or free running were performed in all study subjects, and urine samples before and 1 h after the challenge were collected. Urinary levels of 9α,11β-PGF₂ and LTE₄ were measured by enzyme immunoassay (EIA).

Results

No significant differences were observed in 9α,11β-PGF₂ and LTE₄ levels before/after the exercise challenge between patients with EIB and normal controls. No significant increases in urinary levels of 9α,11β-PGF₂ or LTE₄ were detected during the exercise challenge in patients with EIB and normal controls. No significant correlations were observed between the percent decrease in forced expiratory volume in 1 s (FEV₁) or percent changes in 9α,11β-PGF₂ and LTE₄ levels after the exercise challenge.

Conclusions

Urinary 9α,11β-PGF₂ and LTE₄ levels did not increase after an exercise challenge in patients with EIB, suggesting that urinary excretion of 9α,11β-PGF₂ and LTE₄ may not be a good marker of mast cell activation in patients with EIB.     

Molybdenum Oxide Nanoparticle Aggregates Grown by Chemical Vapor Transport

In this study, the advanced chemical vapor transport (CVT) method in combination with the quenching effect is introduced for creating molybdenum oxide nanoparticle arrays, composed of the hierarchical structure of fine nanoparticles (NPs), which are vertically grown with a homogeneous coverage on the individual carbon fibers of carbon fiber paper (CFP) substrates. The obtained molybdenum oxide NPs hold a metastable high-temperature γ-Mo₄O₁₁ phase along with a stable α-MoO₃ phase by the quenching effect. Furthermore, such a quenching effect forms thinner and smaller nanoparticle aggregates by suppressing the growth and coalescence of primary particles. The molybdenum oxide nanoparticle aggregates are prepared using two different types of precursors: MoO₃ and a 1:1 (mol/mol) mixture of MoO₃ and activated carbon. The results characterized using X-ray diffraction, Raman spectroscopy, X-ray photoelectron spectroscopy, and Fourier-transform infrared spectroscopy show that the relative amount of α-MoO₃ to γ-Mo₄O₁₁ within the prepared NPs is dependent on the precursor type; a lower amount of α-MoO₃ to γ-Mo₄O₁₁ is obtained in the NPs prepared using the mixed precursor of MoO₃ and carbon. This processing–structure landscape study can serve as the groundwork for the development of high-performance nanomaterials in various electronic and catalytic applications.     

Functional and idling rotatory motion within F1-ATPase

ATP synthase mediates proton flow through its membrane portion, F₀, which drives the synthesis of ATP in its headpiece, F₁. The F₁-portion contains a hexagonal array of three subunits α and three β encircling a central subunit γ, that in turn interacts with a smaller and with F₀. Recently we reported that the application of polarized absorption recovery after photobleaching showed the ATP-driven rotation of γ over at least two, if not three, β. Here we extend probes of such rotation aided by a new theory for assessing continuous versus stepped, Brownian versus unidirectional molecular motion. The observed relaxation of the absorption anisotropy is fully compatible with a unidirectional and stepping rotation of γ over three equidistantly spaced angular positions in the hexagon formed by the alternating subunits α and β. The results strongly support a rotational catalysis with equal participation of all three catalytic sites. In addition we report a limited rotation of γ without added nucleotides, perhaps idling and of Brownian nature, that covers only a narrow angular domain.     

Detailed Inspection of γ-ray,Fast and Thermal Neutrons Shielding Competence of Calcium Oxide or Strontium Oxide Comprising Bismuth Borate Glasses

For both the B₂O₃-Bi₂O₃-CaO and B₂O₃-Bi₂O₃-SrO glass systems, γ-ray and neutron attenuation qualities were evaluated. Utilizing the Phy-X/PSD program, within the 0.015–15 MeV energy range, linear attenuation coefficients (µ) and mass attenuation coefficients (μ/ρ) were calculated, and the attained μ/ρ quantities match well with respective simulation results computed by MCNPX, Geant4, and Penelope codes. Instead of B₂O₃/CaO or B₂O₃/SrO, the Bi₂O₃ addition causes improved γ-ray shielding competence, i.e., rise in effective atomic number (Z_eff) and a fall in half-value layer (HVL), tenth-value layer (TVL), and mean free path (MFP). Exposure buildup factors (EBFs) and energy absorption buildup factors (EABFs) were derived using a geometric progression (G–P) fitting approach at 1–40 mfp penetration depths (PDs), within the 0.015–15 MeV range. Computed radiation protection efficiency (RPE) values confirm their excellent capacity for lower energy photons shielding. Comparably greater density (7.59 g/cm³), larger μ, μ/ρ, Z_eff, equivalent atomic number (Z_eq), and RPE, with the lowest HVL, TVL, MFP, EBFs, and EABFs derived for 30B₂O₃-60Bi₂O₃-10SrO (mol%) glass suggest it as an excellent γ-ray attenuator. Additionally, 30B₂O₃-60Bi₂O₃-10SrO (mol%) glass holds a commensurably bigger macroscopic removal cross-section for fast neutrons (Σ_R) (=0.1199 cm⁻¹), obtained by applying Phy-X/PSD for fast neutrons shielding, owing to the presence of larger wt% of ‘Bi’ (80.6813 wt%) and moderate ‘B’ (2.0869 wt%) elements in it. 70B₂O₃-5Bi₂O₃-25CaO (mol%) sample (B: 17.5887 wt%, Bi: 24.2855 wt%, Ca: 11.6436 wt%, and O: 46.4821 wt%) shows high potentiality for thermal or slow neutrons and intermediate energy neutrons capture or absorption due to comprised high wt% of ‘B’ element in it.     

Anticancer efficacy of monotherapy with antibodies to SIRPα/SIRPβ1 mediated by induction of antitumorigenic macrophages

The interaction of signal regulatory protein α (SIRPα) on macrophages with CD47 on cancer cells is thought to prevent antibody (Ab)-dependent cellular phagocytosis (ADCP) of the latter cells by the former. Blockade of the CD47-SIRPα interaction by Abs to CD47 or to SIRPα, in combination with tumor-targeting Abs such as rituximab, thus inhibits tumor formation by promoting macrophage-mediated ADCP of cancer cells. Here we show that monotherapy with a monoclonal Ab (mAb) to SIRPα that also recognizes SIRPβ1 inhibited tumor formation by bladder and mammary cancer cells in mice, with this inhibitory effect being largely dependent on macrophages. The mAb to SIRPα promoted polarization of tumor-infiltrating macrophages toward an antitumorigenic phenotype, resulting in the killing and phagocytosis of cancer cells by the macrophages. Ablation of SIRPα in mice did not prevent the inhibitory effect of the anti-SIRPα mAb on tumor formation or its promotion of the cancer cell–killing activity of macrophages, however. Moreover, knockdown of SIRPβ1 in macrophages attenuated the stimulatory effect of the anti-SIRPα mAb on the killing of cancer cells, whereas an mAb specific for SIRPβ1 mimicked the effect of the anti-SIRPα mAb. Our results thus suggest that monotherapy with Abs to SIRPα/SIRPβ1 induces antitumorigenic macrophages and thereby inhibits tumor growth and that SIRPβ1 is a potential target for cancer immunotherapy.

Macrophages are innate immune cells that show phenotypic heterogeneity and functional diversity; and they play key roles in development, tissue homeostasis and repair, and in cancer, as well as in defense against pathogens (1–3). In the tumor microenvironment (TME), macrophages are exposed to a variety of stimuli, including cell–cell contact, hypoxia, as well as soluble and insoluble factors such as cytokines, chemokines, metabolites, and extracellular matrix components (2, 4). These environmental cues promote the acquisition by macrophages of protumorigenic phenotypes that facilitate tumor development, progression, and metastasis as well as suppress antitumor immune responses (2, 4). A high density of macrophages within tumor tissue is associated with poor prognosis in patients with various types of cancer, including that of the bladder or breast (5–7). Depletion of macrophages in the TME or the reprogramming of these cells to acquire antitumorigenic phenotypes has been shown to ameliorate the immunosuppressive condition and result in a reduction in tumor burden in both preclinical and clinical studies (2, 4, 8, 9). Macrophages within the TME have therefore attracted much attention as a potential therapeutic target for cancer immunotherapy.Signal regulatory protein α (SIRPα) is a transmembrane protein that possesses one NH₂-terminal immunoglobulin (Ig)-V–like and two Ig-C domains in its extracellular region, as well as immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic region (10, 11). The extracellular region of SIRPα interacts with that of CD47, another member of the Ig superfamily of proteins, with this interaction constituting a means of cell–cell communication. The expression of SIRPα in hematopoietic cells is restricted to the myeloid compartment—including macrophages, neutrophils, and dendritic cells (DCs)—whereas CD47 is expressed in most normal cell types as well as cancer cells (12, 13). The interaction of SIRPα on macrophages with CD47 on antibody (Ab)-opsonized viable cells such as blood cells or cancer cells prevents phagocytosis of the latter cells by the former (13–15), with this negative regulation of macrophages being thought to be mediated by SHP1, a protein tyrosine phosphatase that binds to the cytoplasmic region of SIRPα (14). Indeed, blockade of the CD47–SIRPα interaction by Abs to either SIRPα or CD47, in combination with a tumor-targeting Ab such as rituximab (anti-CD20), was found to enhance the Ab-dependent cellular phagocytosis (ADCP) activity of macrophages for cancer cells that do not express SIRPα, resulting in marked suppression of tumor formation in mice (15–19). Targeting of SIRPα in combination with a tumor-targeting Ab therefore provides a potential approach to cancer immunotherapy dependent on enhancement of the ADCP activity of macrophages for cancer cells. In contrast, the effect of Abs to SIRPα in the absence of a tumor-targeting Ab on the phagocytosis by macrophages of, as well as on tumor formation by, cancer cells that do not express SIRPα was minimal or limited.We have now further examined the antitumor efficacy of a monoclonal Ab (mAb) to mouse SIRPα (MY-1) (20) in immunocompetent mice transplanted subcutaneously with several types of murine cancer cells that do not express SIRPα. This Ab prevents the binding of mouse CD47 to SIRPα and cross-reacts with mouse SIRPβ1 (15). We found that monotherapy with MY-1 efficiently attenuated the growth of tumors formed by bladder or mammary cancer cells. In addition, MY-1 markedly promoted the induction of antitumorigenic macrophages able to target these cancer cells. Furthermore, our results suggest that SIRPβ1 on macrophages likely participated in the antitumorigenic effect of MY-1.     

Vacancy Defects in Ga2O3: First-Principles Calculations of Electronic Structure

First-principles density functional theory (DFT) is employed to study the electronic structure of oxygen and gallium vacancies in monoclinic bulk β-Ga₂O₃ crystals. Hybrid exchange–correlation functional B3LYP within the density functional theory and supercell approach were successfully used to simulate isolated point defects in β-Ga₂O₃. Based on the results of our calculations, we predict that an oxygen vacancy in β-Ga₂O₃ is a deep donor defect which cannot be an effective source of electrons and, thus, is not responsible for n-type conductivity in β-Ga₂O₃. On the other hand, all types of charge states of gallium vacancies are sufficiently deep acceptors with transition levels more than 1.5 eV above the valence band of the crystal. Due to high formation energy of above 10 eV, they cannot be considered as a source of p-type conductivity in β-Ga₂O₃.     

SARS-CoV-2 Infects Human ACE2-Negative Endothelial Cells through an αvβ3 Integrin-Mediated Endocytosis Even in the Presence of Vaccine-Elicited Neutralizing Antibodies

Integrins represent a gateway of entry for many viruses and the Arg-Gly-Asp (RGD) motif is the smallest sequence necessary for proteins to bind integrins. All Severe Acute Respiratory Syndrome Virus type 2 (SARS-CoV-2) lineages own an RGD motif (aa 403–405) in their receptor binding domain (RBD). We recently showed that SARS-CoV-2 gains access into primary human lung microvascular endothelial cells (HL-mECs) lacking Angiotensin-converting enzyme 2 (ACE2) expression through this conserved RGD motif. Following its entry, SARS-CoV-2 remodels cell phenotype and promotes angiogenesis in the absence of productive viral replication. Here, we highlight the α_vβ₃ integrin as the main molecule responsible for SARS-CoV-2 infection of HL-mECs via a clathrin-dependent endocytosis. Indeed, pretreatment of virus with α_vβ₃ integrin or pretreatment of cells with a monoclonal antibody against α_vβ₃ integrin was found to inhibit SARS-CoV-2 entry into HL-mECs. Surprisingly, the anti-Spike antibodies evoked by vaccination were neither able to impair Spike/integrin interaction nor to prevent SARS-CoV-2 entry into HL-mECs. Our data highlight the RGD motif in the Spike protein as a functional constraint aimed to maintain the interaction of the viral envelope with integrins. At the same time, our evidences call for the need of intervention strategies aimed to neutralize the SARS-CoV-2 integrin-mediated infection of ACE2-negative cells in the vaccine era.