Correction: Tumor Immunotherapy-Related Information on Internet-Based Videos Commonly Used by the Chinese Population: Content Quality Analysis.

[This corrects the article DOI: 10.2196/50561.].

New insights into black carbon light absorption enhancement: A comprehensive analysis of two differential behaviors.

High uncertainty in optical properties of black carbon (BC) involving heterogeneous chemistry has recently attracted increasing attention in the field of atmospheric climatology. To fill the gap in BC optical knowledge so as to estimate more accurate climate effects and serve the response to global warming, it is beneficial to conduct site-level studies on BC light absorption enhancement (Eabs) characteristics. Real-time surface gas and particulate pollutant observations during the summer and winter over Wuhan were utilized for the analysis of Eabs simulated by minimum R squared (MRS), considering two distinct atmospheric conditions (2015 and 2017). In general, differences in aerosol emissions led to Eabs differential behaviors. The summer average of Eabs (1.92 ± 0.55) in 2015 was higher than the winter average (1.27 ± 0.42), while the average (1.11 ± 0.20) in 2017 summer was lower than that (1.67 ± 0.69) in winter. Eabs and RBC (representing the mass ratio of non-refractory constituents to elemental carbon) constraints suggest that Eabs increased with the increase in RBC under the ambient condition enriched by secondary inorganic aerosol (SIA), with a maximum growth rate of 70.6% in 2015 summer. However, Eabs demonstrated a negative trend against RBC in 2017 winter due to the more complicated mixing state. The result arose from the opposite impact of hygroscopic SIA and absorbing OC/irregular distributed coatings on amplifying the light absorbency of BC. Furthermore, sensitivity analysis revealed a robust positive correlation (R > 0.9) between aerosol chemical compositions (including sulfate, nitrate, ammonium and secondary organic carbon), which could be significantly perturbed by only a small fraction of absorbing materials or restructuring BC through gaps filling. The above findings not only deepen the understanding of BC, but also provide useful information for the scientific decision-making in government to mitigate particulate pollution and obtain more precise BC radiative forcing.

Tumor Immunotherapy-Related Information on Internet-Based Videos Commonly Used by the Chinese Population: Content Quality Analysis.

BACKGROUND: Tumor immunotherapy is an innovative treatment today, but there are limited data on the quality of immunotherapy information on social networks. Dissemination of misinformation through the internet is a major social issue. OBJECTIVE: Our objective was to characterize the quality of information and presence of misinformation about tumor immunotherapy on internet-based videos commonly used by the Chinese population. METHODS: Using the keyword "tumor immunotherapy" in Chinese, we searched TikTok, Tencent, iQIYI, and BiliBili on March 5, 2022. We reviewed the 118 screened videos using the Patient Education Materials Assessment Tool-a validated instrument to collect consumer health information. DISCERN quality criteria and the JAMA (Journal of the American Medical Association) Benchmark Criteria were used for assessing the quality and reliability of the health information. The videos' content was also evaluated. RESULTS: The 118 videos about tumor immunotherapy were mostly uploaded by channels dedicated to lectures, health-related animations, and interviews; their median length was 5 minutes, and 79% of them were published in and after 2018. The median understandability and actionability of the videos were 71% and 71%, respectively. However, the quality of information was moderate to poor on the validated DISCERN and JAMA assessments. Only 12 videos contained misinformation (score of >1 out of 5). Videos with a doctor (lectures and interviews) not only were significantly less likely to contain misinformation but also had better quality and a greater forwarding number. Moreover, the results showed that more than half of the videos contain little or no content on the risk factors and management of tumor immunotherapy. Overall, over half of the videos had some or more information on the definition, symptoms, evaluation, and outcomes of tumor immunotherapy. CONCLUSIONS: Although the quality of immunotherapy information on internet-based videos commonly used by Chinese people is moderate, these videos have less misinformation and better content. Caution must be exercised when using these videos as a source of tumor immunotherapy-related information.

Cofilin-mediated actin filament network flexibility facilitates 2D to 3D actomyosin shape change.

The organization of actin filaments (F-actin) into crosslinked networks determines the transmission of mechanical stresses within the cytoskeleton and subsequent changes in cell and tissue shape. Principally mediated by proteins such as α-actinin, F-actin crosslinking increases both network connectivity and rigidity, thereby facilitating stress transmission at low crosslinking yet attenuating transmission at high crosslinker concentration. Here, we engineer a two-dimensional model of the actomyosin cytoskeleton, in which myosin-induced mechanical stresses are controlled by light. We alter the extent of F-actin crosslinking by the introduction of oligomerized cofilin. At pH 6.5, F-actin severing by cofilin is weak, but cofilin bundles and crosslinks filaments. Given its effect of lowering the F-actin bending stiffness, cofilin- crosslinked networks are significantly more flexible and softer in bending than networks crosslinked by α-actinin. Thus, upon local activation of myosin-induced contractile stress, the network bends out-of-plane in contrast to the in-plane compression as observed with networks crosslinked by α-actinin. Here, we demonstrate that local effects on filament mechanics by cofilin introduces novel large-scale network material properties that enable the sculpting of complex shapes in the cell cytoskeleton.

Twist response of actin filaments.

Actin cytoskeleton force generation, sensing, and adaptation are dictated by the bending and twisting mechanics of filaments. Here, we use magnetic tweezers and microfluidics to twist and pull individual actin filaments and evaluate their response to applied loads. Twisted filaments bend and dissipate torsional strain by adopting a supercoiled plectoneme. Pulling prevents plectoneme formation, which causes twisted filaments to sever. Analysis over a range of twisting and pulling forces and direct visualization of filament and single subunit twisting fluctuations yield an actin filament torsional persistence length of ~10 µm, similar to the bending persistence length. Filament severing by cofilin is driven by local twist strain at boundaries between bare and decorated segments and is accelerated by low pN pulling forces. This work explains how contractile forces generated by myosin motors accelerate filament severing by cofilin and establishes a role for filament twisting in the regulation of actin filament stability and assembly dynamics.

The nucleoporin Gle1 activates DEAD-box protein 5 (Dbp5) by promoting ATP binding and accelerating rate limiting phosphate release.

The DEAD-box protein Dbp5 is essential for RNA export, which involves regulation by the nucleoporins Gle1 and Nup159 at the cytoplasmic face of the nuclear pore complex (NPC). Mechanistic understanding of how these nucleoporins regulate RNA export requires analyses of the intrinsic and activated Dbp5 ATPase cycle. Here, kinetic and equilibrium analyses of the Saccharomyces cerevisiae Gle1-activated Dbp5 ATPase cycle are presented, indicating that Gle1 and ATP, but not ADP-Pi or ADP, binding to Dbp5 are thermodynamically coupled. As a result, Gle1 binds Dbp5-ATP > 100-fold more tightly than Dbp5 in other nucleotide states and Gle1 equilibrium binding of ATP to Dbp5 increases >150-fold via slowed ATP dissociation. Second, Gle1 accelerated Dbp5 ATPase activity by increasing the rate-limiting Pi release rate constant ∼20-fold, which remains rate limiting. These data show that Gle1 activates Dbp5 by modulating ATP binding and Pi release. These Gle1 activities are expected to facilitate ATPase cycling, ensuring a pool of ATP bound Dbp5 at NPCs to engage RNA during export. This work provides a mechanism of Gle1-activation of Dbp5 and a framework to understand the joint roles of Gle1, Nup159, and other nucleoporins in regulating Dbp5 to mediate RNA export and other Dbp5 functions in gene expression.

Excessive ROS production and enhanced autophagy contribute to myocardial injury induced by branched-chain amino acids: Roles for the AMPK-ULK1 signaling pathway and α7nAChR.

BACKGROUNDS AND AIMS: Leucine, isoleucine, and valine are diet derived and essential amino acids that are termed branched-chain amino acids (BCAA). BCAA are widely used as dietary supplements to boost muscle growth and enhance exercise performance. However, the effects of BCAA on myocardial function are largely unknown. This study was designed to investigate whether BCAA affect heart function and, if so, to further explore the underlying molecular basis for the observed effects. METHODS AND RESULTS: C57BL/6J mice were randomly divided into two groups, the control group received solvent (water) and the BCAA group received 2% BCAA dissolved in water, for a successive period of 12 weeks. Compared with control, BCAA treatment significantly increased water consumption without changing body weight or diet consumption; heart tissue BCAA levels were increased, markers representative of myocardial injury in heart tissue including c-reactive protein and cardiac muscle troponin were increased ; and creatine kinase, creatine kinase-MB, and lactate dehydrogenase were increased in serum; severe myocardial fibrosis was observed by Masson staining, which was accompanied by increased reactive oxygen species (ROS) production and decreased superoxide dismutase activity in heart tissue; both p-AMPK and p-ULK1 were significantly increased as was autophagy, judged by the presence of LC3 by western blotting and immunofluorescence, increased numbers of autophagosomes were found by transmission electron microscopy in the BCAA group. In vitro, 20 mmol/L BCAA significantly decreased cell viability and increased the production of ROS, as well as the expression of p-AMPK/AMPK and p-ULK1/ULK1 in cultured H9C2 cells. Treatment with the ROS scavenger N-acetyl-L-cysteine (NAC) improved cell viability and reversed ROS changes. Decreased H9C2 cell viability induced with 20 mmol/L BCAA was reversed by either blocking AMPK or inhibition of ULK1. Furthermore, blocking AMPK significantly decreased p-ULK1/ULK1, while inhibition of ULK1 reversed the enhanced expression of LC3-II/LC3-I induced by BCAA. Excessive ROS production and decreased cell viability induced by BCAA were further confirmed in primary cultured murine cardiomyocytes. Pharmacological activation of α7nAChR with PNU-282987 attenuated BCAA-induced injury in primary murine cardiomyocytes. However, this compound failed to suppress BCAA activation of AMPK and autophagy (LC3-II/I ratio). CONCLUSION: These results provide the first evidence that treatment of mice with BCAA induced myocardial injury by triggering excessive ROS production and by enhancing AMPK-ULK1 pathway-dependent autophagy. These findings suggested that inhibition of either ROS production or autophagy may alleviate myocardial injury induced by BCAA.

Force and phosphate release from Arp2/3 complex promote dissociation of actin filament branches.

Networks of branched actin filaments formed by Arp2/3 complex generate and experience mechanical forces during essential cellular functions, including cell motility and endocytosis. External forces regulate the assembly and architecture of branched actin networks both in vitro and in cells. Considerably less is known about how mechanical forces influence the disassembly of actin filament networks, specifically, the dissociation of branches. We used microfluidics to apply force to branches formed from purified muscle actin and fission yeast Arp2/3 complex and observed debranching events in real time with total internal reflection fluorescence microscopy. Low forces in the range of 0 pN to 2 pN on branches accelerated their dissociation from mother filaments more than two orders of magnitude, from hours to <1 min. Neither force on the mother filament nor thermal fluctuations in mother filament shape influenced debranching. Arp2/3 complex at branch junctions adopts two distinct mechanical states with different sensitivities to force, which we name "young/strong" and "old/weak." The "young/strong" state 1 has adenosine 5'-diphosphate (ADP)-P i bound to Arp2/3 complex. Phosphate release converts Arp2/3 complex into the "old/weak" state 2 with bound ADP, which is 20 times more sensitive to force than state 1. Branches with ADP-Arp2/3 complex are more sensitive to debranching by fission yeast GMF (glia maturation factor) than branches with ADP-P i -Arp2/3 complex. These findings suggest that aging of branch junctions by phosphate release from Arp2/3 complex and mechanical forces contribute to disassembling "old" actin filament branches in cells.

Active cargo positioning in antiparallel transport networks.

Cytoskeletal filaments assemble into dense parallel, antiparallel, or disordered networks, providing a complex environment for active cargo transport and positioning by molecular motors. The interplay between the network architecture and intrinsic motor properties clearly affects transport properties but remains poorly understood. Here, by using surface micropatterns of actin polymerization, we investigate stochastic transport properties of colloidal beads in antiparallel networks of overlapping actin filaments. We found that 200-nm beads coated with myosin Va motors displayed directed movements toward positions where the net polarity of the actin network vanished, accumulating there. The bead distribution was dictated by the spatial profiles of local bead velocity and diffusion coefficient, indicating that a diffusion-drift process was at work. Remarkably, beads coated with heavy-mero-myosin II motors showed a similar behavior. However, although velocity gradients were steeper with myosin II, the much larger bead diffusion observed with this motor resulted in less precise positioning. Our observations are well described by a 3-state model, in which active beads locally sense the net polarity of the network by frequently detaching from and reattaching to the filaments. A stochastic sequence of processive runs and diffusive searches results in a biased random walk. The precision of bead positioning is set by the gradient of net actin polarity in the network and by the run length of the cargo in an attached state. Our results unveiled physical rules for cargo transport and positioning in networks of mixed polarity.

Nup159 Weakens Gle1 Binding to Dbp5 But Does Not Accelerate ADP Release.

Dbp5, DDX19 in humans, is an essential DEAD-box protein involved in mRNA export, which has also been linked to other cellular processes, including rRNA export and translation. Dbp5 ATPase activity is regulated by several factors, including RNA, the nucleoporin proteins Nup159 and Gle1, and the endogenous small-molecule inositol hexakisphosphate (InsP6). To better understand how these factors modulate Dbp5 activity and how this modulation relates to in vivo RNA metabolism, a detailed characterization of the Dbp5 mechanochemical cycle in the presence of those regulators individually or together is necessary. In this study, we test the hypothesis that Nup159 controls the ADP-bound state of Dbp5. In addition, the contributions of Mg2+ to the kinetics and thermodynamics of ADP binding to Dbp5 were assessed. Using a solution based in vitro approach, Mg2+ was found to slow ADP and ATP release from Dbp5 and increased the overall ADP and ATP affinities, as observed with other NTPases. Furthermore, Nup159 did not accelerate ADP release, while Gle1 actually slowed ADP release independent of Mg2+. These findings are not consistent with Nup159 acting as a nucleotide exchange factor to promote ADP release and Dbp5 ATPase cycling. Instead, in the presence of Nup159, the interaction between Gle1 and ADP-bound Dbp5 was found to be reduced by ~18-fold, suggesting that Nup159 alters the Dbp5-Gle1 interaction to aid Gle1 release from Dbp5.

Identification of a novel autophagic inhibitor cepharanthine to enhance the anti-cancer property of dacomitinib in non-small cell lung cancer.

Inhibition of autophagy is a promising strategy for non-small cell lung cancer (NSCLC) treatment, which is in the clinical trials. However, only chloroquine is used in clinic as an autophagic inhibitor and the inhibitory effect of chloroquine on autophagy is finite. Therefore, the development of an alternative autophagic inhibitor for NSCLC therapy becomes necessary. In the present study, cepharanthine (CEP), an alkaloid extracted from Stephania cepharantha Hayata, was identified as a novel autophagic inhibitor in NSCLC cells. The potential mechanism of the CEP-inhibited autophagy was by blockage of autophagosome-lysosome fusion and inhibition of lysosomal cathepsin B and cathepsin D maturation. Furthermore, we found for the first time that dacomitinib (DAC), a second-generation epidermal growth factor receptor inhibitor that in the phase III clinical trials for NSCLC treatment, induced a protective autophagy to decrease its anti-cancer effect. Combined treatment with CEP increased the anti-proliferative and apoptotic effects of DAC in vitro and enhanced the anti-cancer effect of DAC in NCI-H1975 xenograft mice. Collectively, CEP might be further developed as an autophagic inhibitor, and combined treatment of CEP and DAC could offer an effective strategy for NSCLC treatment.

Induction of reactive oxygen species-stimulated distinctive autophagy by chelerythrine in non-small cell lung cancer cells.

Chelerythrine (CHE), a natural benzo[c]phenanthridine alkaloid, shows anti-cancer effect through a number of mechanisms. Herein, the effect and mechanism of the CHE-induced autophagy, a type II programmed cell death, in non-small cell lung cancer (NSCLC) cells were studied for the first time. CHE induced cell viability decrease, colony formation inhibition, and apoptosis in a concentration-dependent manner in NSCLC A549 and NCI-H1299 cells. In addition, CHE triggered the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II). The CHE-induced expression of LC3-II was further increased in the combination treatment with chloroquine (CQ), an autophagy inhibitor, and large amounts of red-puncta were observed in the CHE-treated A549 cells with stable expression of mRFP-EGFP-LC3, indicating that CHE induces autophagy flux. Silence of beclin 1 reversed the CHE-induced expression of LC3-II. Inhibition of autophagy remarkably reversed the CHE-induced cell viability decrease and apoptosis in NCI-H1299 cells but not in A549 cells. Furthermore, CHE triggered reactive oxygen species (ROS) generation in both cell lines. A decreased level of ROS through pretreatment with N-acetyl-L-cysteine reversed the CHE-induced cell viability decrease, apoptosis, and autophagy. Taken together, CHE induced distinctive autophagy in A549 (accompanied autophagy) and NCI-H1299 (pro-death autophagy) cells and a decreased level of ROS reversed the effect of CHE in NSCLC cells in terms of cell viability, apoptosis, and autophagy.

Osimertinib induces autophagy and apoptosis via reactive oxygen species generation in non-small cell lung cancer cells.

Osimertinib (OSI), also known as AZD9291, is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has been approved for the treatment of non-small cell lung cancer (NSCLC) patients harboring EGFR T790M mutation. Herein, we indicated for the first time that OSI increased the accumulations of cytoplasmic vacuoles, the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II), and the formation of GFP-LC3 puncta in various cancer cells. The OSI-induced expression of LC3-II was further increased when combined treatment with chloroquine (CQ), an autophagy inhibitor, and the mRFP-EGFP-LC3 plasmid-transfected cells exposed to OSI led to the production of more red-fluorescent puncta than green-fluorescent puncta, indicating OSI induced autophagic flux in the NSCLC cells. Knockdown of EGFR showed no effect on the OSI-induced expression of LC3-II in NCI-H1975 cells. In addition, OSI increased reactive oxygen species (ROS) generation and scavenge of ROS via pretreatment with N-acetyl-l-cysteine (NAC), catalase (CAT), or vitamin E (Vita E) significantly inhibited OSI-induced the accumulations of cytoplasmic vacuoles, the expression of LC3-II, as well as the formation of GFP-LC3 puncta. Combinative treatment with CQ could not remarkably change the OSI-induced cell viability decrease, whereas the OSI-induced cell viability decrease and apoptosis could be reversed through pretreatment with NAC, CAT, and Vita E, respectively. Taken together, this is the first report that OSI induces an accompanied autophagy and the generation of ROS is critical for the OSI-induced autophagy, cell viability decrease, and apoptosis in NSCLC cells.

Architecture and Connectivity Govern Actin Network Contractility.

Actomyosin contractility plays a central role in a wide range of cellular processes, including the establishment of cell polarity, cell migration, tissue integrity, and morphogenesis during development. The contractile response is variable and depends on actomyosin network architecture and biochemical composition. To determine how this coupling regulates actomyosin-driven contraction, we used a micropatterning method that enables the spatial control of actin assembly. We generated a variety of actin templates and measured how defined actin structures respond to myosin-induced forces. We found that the same actin filament crosslinkers either enhance or inhibit the contractility of a network, depending on the organization of actin within the network. Numerical simulations unified the roles of actin filament branching and crosslinking during actomyosin contraction. Specifically, we introduce the concept of "network connectivity" and show that the contractions of distinct actin architectures are described by the same master curve when considering their degree of connectivity. This makes it possible to predict the dynamic response of defined actin structures to transient changes in connectivity. We propose that, depending on the connectivity and the architecture, network contraction is dominated by either sarcomeric-like or buckling mechanisms. More generally, this study reveals how actin network contractility depends on its architecture under a defined set of biochemical conditions.

P(I) Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5).

mRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. Although Dbp5 has measurable ATPase activity alone, several regulatory factors (e.g., RNA, nucleoporin proteins, and the endogenous small molecule InsP6) modulate catalytic activity in vitro and in vivo to facilitate mRNA export. An analysis of the intrinsic and regulator-activated Dbp5 ATPase cycle is necessary to define how these factors control Dbp5 and mRNA export. Here, we report a kinetic and equilibrium analysis of the Saccharomyces cerevisiae Dbp5 ATPase cycle, including the influence of RNA on Dbp5 activity. These data show that ATP binds Dbp5 weakly in rapid equilibrium with a binding affinity (KT~4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (KD~0.4 mM). The overall intrinsic steady-state cycling rate constant (kcat) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RNA increases kcat and rate-limiting Pi release 20-fold, although Pi release continues to limit steady-state cycling in the presence of RNA, in conjunction with RNA binding. Together, this work identifies RNA binding and Pi release as important biochemical transitions within the Dbp5 ATPase cycle and provides a framework for investigating the means by which Dbp5 and mRNA export is modulated by regulatory factors.

Molecular basis of purinergic signal metabolism by ectonucleotide pyrophosphatase/phosphodiesterases 4 and 1 and implications in stroke.

NPP4 is a type I extracellular membrane protein on brain vascular endothelium inducing platelet aggregation via the hydrolysis of Ap3A, whereas NPP1 is a type II extracellular membrane protein principally present on the surface of chondrocytes that regulates tissue mineralization. To understand the metabolism of purinergic signals resulting in the physiologic activities of the two enzymes, we report the high resolution crystal structure of human NPP4 and explore the molecular basis of its substrate specificity with NPP1. Both enzymes cleave Ap3A, but only NPP1 can hydrolyze ATP. Comparative structural analysis reveals a tripartite lysine claw in NPP1 that stabilizes the terminal phosphate of ATP, whereas the corresponding region of NPP4 contains features that hinder this binding orientation, thereby inhibiting ATP hydrolysis. Furthermore, we show that NPP1 is unable to induce platelet aggregation at physiologic concentrations reported in human blood, but it could stimulate platelet aggregation if localized at low nanomolar concentrations on vascular endothelium. The combined studies expand our understanding of NPP1 and NPP4 substrate specificity and range and provide a rational mechanism by which polymorphisms in NPP1 confer stroke resistance.

NPP4 is a procoagulant enzyme on the surface of vascular endothelium.

Ap3A is a platelet-dense granule component released into the extracellular space during the second wave of platelet aggregation on activation. Here, we identify an uncharacterized enzyme, nucleotide pyrophosphatase/phosphodiesterase-4 (NPP4), as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion. We demonstrate that NPP4 is present on the surface of vascular endothelium, where it hydrolyzes Ap3A into AMP and ADP, and Ap4A into AMP and ATP. Platelet aggregation assays with citrated platelet-rich plasma reveal that the primary and secondary waves of aggregation and dense granule release are strongly induced by nanomolar NPP4 in a concentration-dependent manner in the presence of Ap3A, while Ap3A alone initiates a primary wave of aggregation followed by rapid disaggregation. NPP2 and an active site NPP4 mutant, neither of which appreciably hydrolyzes Ap3A, have no effect on platelet aggregation and secretion. Finally, by using ADP receptor blockade we confirm that NPP4 mediates platelet aggregation via release of ADP from Ap3A and activation of ADP receptors. Collectively, these studies define the biologic and enzymatic basis for NPP4 and Ap3A activity in platelet aggregation in vitro and suggest that NPP4 promotes hemostasis in vivo by augmenting ADP-mediated platelet aggregation at the site of vascular injury.

Kinetic analysis of autotaxin reveals substrate-specific catalytic pathways and a mechanism for lysophosphatidic acid distribution.

Autotaxin (ATX) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), initiating signaling cascades leading to cancer metastasis, wound healing, and angiogenesis. Knowledge of the pathway and kinetics of LPA synthesis by ATX is critical for developing quantitative physiological models of LPA signaling. We measured the individual rate constants and pathway of the LPA synthase cycle of ATX using the fluorescent lipid substrates FS-3 and 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-LPC. FS-3 binds rapidly (k(1) ≥500 µm(-1) s(-1)) and is hydrolyzed slowly (k(2) = 0.024 s(-1)). Release of the first hydrolysis product is random and rapid (≥1 s(-1)), whereas release of the second is slow and rate-limiting (0.005-0.007 s(-1)). Substrate binding and hydrolysis are slow and rate-limiting with LPC. Product release is sequential with choline preceding LPA. The catalytic pathway and kinetics depend strongly on the substrate, suggesting that ATX kinetics could vary for the various in vivo substrates. Slow catalysis with LPC reveals the potential for LPA signaling to spread to cells distal to the site of LPC substrate binding by ATX. An ATX mutant in which catalytic threonine at position 210 is replaced with alanine binds substrate weakly, favoring a role for Thr-210 in binding as well as catalysis. FTY720P, the bioactive form of a drug currently used to treat multiple sclerosis, inhibits ATX in an uncompetitive manner and slows the hydrolysis reaction, suggesting that ATX inhibition plays a significant role in lymphocyte immobilization in FTY720P-based therapeutics.

Mechanism of Mss116 ATPase reveals functional diversity of DEAD-Box proteins.

Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein that is essential for efficient in vivo splicing of all group I and group II introns and for activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function. We therefore measured the rate and equilibrium constants underlying Mss116 ATP utilization and nucleotide-linked RNA binding. RNA accelerates the Mss116 steady-state ATPase ∼7-fold by promoting rate-limiting ATP hydrolysis such that inorganic phosphate (P(i)) release becomes (partially) rate-limiting. RNA binding displays strong thermodynamic coupling to the chemical states of the Mss116-bound nucleotide such that Mss116 with bound ADP-P(i) binds RNA more strongly than Mss116 with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steady-state cycling is the strong RNA-binding Mss116-ADP-P(i) state. Strong RNA binding allows Mss116 to fulfill its biological role in the stabilization of group II intron folding intermediates. ATPase cycling allows for transient population of the weak RNA-binding ADP state of Mss116 and linked dissociation from RNA, which is required for the final stages of intron folding. In cases where Mss116 functions as a helicase, the data collectively favor a model in which ATP hydrolysis promotes a weak-to-strong RNA binding transition that disrupts stable RNA duplexes. The subsequent strong-to-weak RNA binding transition associated with P(i) release dissociates Mss116-RNA complexes, regenerating free Mss116.

Pathway of ATP utilization and duplex rRNA unwinding by the DEAD-box helicase, DbpA.

DEAD-box RNA helicase proteins use the energy of ATP hydrolysis to drive the unwinding of duplex RNA. However, the mechanism that couples ATP utilization to duplex RNA unwinding is unknown. We measured ATP utilization and duplex RNA unwinding by DbpA, a non-processive bacterial DEAD-box RNA helicase specifically activated by the peptidyl transferase center (PTC) of 23S rRNA. Consumption of a single ATP molecule is sufficient to unwind and displace an 8 base pair rRNA strand annealed to a 32 base pair PTC-RNA "mother strand" fragment. Strand displacement occurs after ATP binding and hydrolysis but before P(i) product release. P(i) release weakens binding to rRNA, thereby facilitating the release of the unwound rRNA mother strand and the recycling of DbpA for additional rounds of unwinding. This work explains how ATPase activity of DEAD-box helicases is linked to RNA unwinding.