Browsing by Subject "Adenosine Triphosphate"
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Item Open AccessA kinesin motor in a force-producing conformation.(BMC Struct Biol, 2010-07-05) Heuston, Elisabeth; Bronner, C Eric; Kull, F Jon; Endow, Sharyn ABACKGROUND: Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states. RESULTS: Here we show that mutation of a single residue in the kinesin-14 Ncd causes the motor to release ADP and hydrolyze ATP faster than wild type, but move more slowly along microtubules in gliding assays, uncoupling nucleotide hydrolysis from force generation. A crystal structure of the motor shows a large rotation of the stalk, a conformation representing a force-producing stroke of Ncd. Three C-terminal residues of Ncd, visible for the first time, interact with the central beta-sheet and dock onto the motor core, forming a structure resembling the kinesin-1 neck linker, which has been proposed to be the primary force-generating mechanical element of kinesin-1. CONCLUSIONS: Force generation by minus-end Ncd involves docking of the C-terminus, which forms a structure resembling the kinesin-1 neck linker. The mechanism by which the plus- and minus-end motors produce force to move to opposite ends of the microtubule appears to involve the same conformational changes, but distinct structural linkers. Unstable ADP binding may destabilize the motor-ADP state, triggering Ncd stalk rotation and C-terminus docking, producing a working stroke of the motor. Item Open AccessA mathematical model for persistent post-CSD vasoconstriction.(PLoS computational biology, 2020-07-15) Xu, Shixin; Chang, Joshua C; Chang, Joshua C; Chow, Carson C; Brennan, KC; Huang, HuaxiongCortical spreading depression (CSD) is the propagation of a relatively slow wave in cortical brain tissue that is linked to a number of pathological conditions such as stroke and migraine. Most of the existing literature investigates the dynamics of short term phenomena such as the depolarization and repolarization of membrane potentials or large ion shifts. Here, we focus on the clinically-relevant hour-long state of neurovascular malfunction in the wake of CSDs. This dysfunctional state involves widespread vasoconstriction and a general disruption of neurovascular coupling. We demonstrate, using a mathematical model, that dissolution of calcium that has aggregated within the mitochondria of vascular smooth muscle cells can drive an hour-long disruption. We model the rate of calcium clearance as well as the dynamical implications on overall blood flow. Based on reaction stoichiometry, we quantify a possible impact of calcium phosphate dissolution on the maintenance of F0F1-ATP synthase activity. Item Open AccessDelivery of an ectonucleotidase inhibitor with ROS-responsive nanoparticles overcomes adenosine-mediated cancer immunosuppression.(Science translational medicine, 2022-06) Mao, Chengqiong; Yeh, Stacy; Fu, Juan; Porosnicu, Mercedes; Thomas, Alexandra; Kucera, Gregory L; Votanopoulos, Konstantinos I; Tian, Shaomin; Ming, XinTumor evasion of immune destruction is associated with the production of immunosuppressive adenosine in the tumor microenvironment (TME). Anticancer therapies can trigger adenosine triphosphate (ATP) release from tumor cells, causing rapid formation of adenosine by the ectonucleotidases CD39 and CD73, thereafter exacerbating immunosuppression in the TME. The goal of this study was to develop an approach to facilitate cancer therapy-induced immunogenic cell death including ATP release and to limit ATP degradation into adenosine, in order to achieve durable antitumor immune response. Our approach was to construct reactive oxygen species (ROS)-producing nanoparticles that carry an ectonucleotidase inhibitor ARL67156 by electronic interaction and phenylboronic ester. Upon near-infrared irradiation, nanoparticle-produced ROS induced ATP release from MOC1 cancer cells in vitro and triggered the cleavage of phenylboronic ester, facilitating the release of ARL67156 from the nanoparticles. ARL67156 prevented conversion of ATP to adenosine and enhanced anticancer immunity in an MOC1-based coculture model. We tested this approach in mouse tumor models. Nanoparticle-based ROS-responsive drug delivery reprogramed the immunogenic landscape in tumors, eliciting tumor-specific T cell responses and tumor regression, conferring long-term survival in mouse models. We demonstrated that TME reprograming sets the stage for response to anti-programmed cell death protein 1 (PD1) immunotherapy, and the combination resulted in tumor regression in a 4T1 breast cancer mouse model that was resistant to PD1 blockade. Furthermore, our approach also induced immunological effects in patient-derived organotypic tumor spheroid model, suggesting potential translation of our nanoparticle approach for treating human cancers. Item Open AccessEvidence for an electrostatic mechanism of force generation by the bacteriophage T4 DNA packaging motor.(Nat Commun, 2014-06-17) Migliori, Amy D; Keller, Nicholas; Alam, Tanfis I; Mahalingam, Marthandan; Rao, Venigalla B; Arya, Gaurav; Smith, Douglas EHow viral packaging motors generate enormous forces to translocate DNA into viral capsids remains unknown. Recent structural studies of the bacteriophage T4 packaging motor have led to a proposed mechanism wherein the gp17 motor protein translocates DNA by transitioning between extended and compact states, orchestrated by electrostatic interactions between complimentarily charged residues across the interface between the N- and C-terminal subdomains. Here we show that site-directed alterations in these residues cause force dependent impairments of motor function including lower translocation velocity, lower stall force and higher frequency of pauses and slips. We further show that the measured impairments correlate with computed changes in free-energy differences between the two states. These findings support the proposed structural mechanism and further suggest an energy landscape model of motor activity that couples the free-energy profile of motor conformational states with that of the ATP hydrolysis cycle. Item Open AccessBcl2l1 Deficiency in Osteoblasts Reduces the Trabecular Bone Due to Enhanced Osteoclastogenesis Likely through Osteoblast Apoptosis.(International journal of molecular sciences, 2023-12) Moriishi, Takeshi; Kawai, Yosuke; Fukuyama, Ryo; Matsuo, Yuki; He, You-Wen; Akiyama, Haruhiko; Asahina, Izumi; Komori, ToshihisaBcl2l1 (Bcl-XL) belongs to the Bcl-2 family, Bcl2 and Bcl2-XL are major anti-apoptotic proteins, and the apoptosis of osteoblasts is a key event for bone homeostasis. As the functions of Bcl2l1 in osteoblasts and bone homeostasis remain unclear, we generated osteoblast-specific Bcl2l1-deficient (Bcl2l1fl/flCre) mice using 2.3-kb Col1a1 Cre. Trabecular bone volume and the trabecular number were lower in Bcl2l1fl/flCre mice of both sexes than in Bcl2l1fl/fl mice. In bone histomorphometric analysis, osteoclast parameters were increased in Bcl2l1fl/flCre mice, whereas osteoblast parameters and the bone formation rate were similar to those in Bcl2l1fl/fl mice. TUNEL-positive osteoblastic cells and serum TRAP5b levels were increased in Bcl2l1fl/flCre mice. The deletion of Bcl2l1 in osteoblasts induced Tnfsf11 expression, whereas the overexpression of Bcl-XL had no effect. In a co-culture of Bcl2l1-deficient primary osteoblasts and wild-type bone-marrow-derived monocyte/macrophage lineage cells, the numbers of multinucleated TRAP-positive cells and resorption pits increased. Furthermore, serum deprivation or the deletion of Bcl2l1 in primary osteoblasts increased apoptosis and ATP levels in the medium. Therefore, the reduction in trabecular bone in Bcl2l1fl/flCre mice may be due to enhanced bone resorption through osteoblast apoptosis and the release of ATP from apoptotic osteoblasts, and Bcl2l1 may inhibit bone resorption by preventing osteoblast apoptosis. Item Open AccessOverexpression of the cardiac beta(2)-adrenergic receptor and expression of a beta-adrenergic receptor kinase-1 (betaARK1) inhibitor both increase myocardial contractility but have differential effects on susceptibility to ischemic injury.(Circ Res, 1999-11-26) Cross, HR; Steenbergen, C; Lefkowitz, RJ; Koch, WJ; Murphy, ECardiac beta(2)-adrenergic receptor (beta(2)AR) overexpression is a potential contractile therapy for heart failure. Cardiac contractility was elevated in mice overexpressing beta(2)ARs (TG4s) with no adverse effects under normal conditions. To assess the consequences of beta(2)AR overexpression during ischemia, perfused hearts from TG4 and wild-type mice were subjected to 20-minute ischemia and 40-minute reperfusion. During ischemia, ATP and pH fell lower in TG4 hearts than wild type. Ischemic injury was greater in TG4 hearts, as indicated by lower postischemic recoveries of contractile function, ATP, and phosphocreatine. Because beta(2)ARs, unlike beta(1)ARs, couple to G(i) as well as G(s), we pretreated mice with the G(i) inhibitor pertussis toxin (PTX). PTX treatment increased basal contractility in TG4 hearts and abolished the contractile resistance to isoproterenol. During ischemia, ATP fell lower in TG4+PTX than in TG4 hearts. Recoveries of contractile function and ATP were lower in TG4+PTX than in TG4 hearts. We also studied mice that overexpressed either betaARK1 (TGbetaARK1) or a betaARK1 inhibitor (TGbetaARKct). Recoveries of function, ATP, and phosphocreatine were higher in TGbetaARK1 hearts than in wild-type hearts. Despite basal contractility being elevated in TGbetaARKct hearts to the same level as that of TG4s, ischemic injury was not increased. In summary, beta(2)AR overexpression increased ischemic injury, whereas betaARK1 overexpression was protective. Ischemic injury in the beta(2)AR overexpressors was exacerbated by PTX treatment, implying that it was G(s) not G(i) activity that enhanced injury. Unlike beta(2)AR overexpression, basal contractility was increased by betaARK1 inhibitor expression without increasing ischemic injury, thus implicating a safer potential therapy for heart failure. Item Open AccessThe potential repertoire of the innate immune system in the bladder: expression of pattern recognition receptors in the rat bladder and a rat urothelial cell line (MYP3 cells).(International urology and nephrology, 2015-12) Hughes, Francis M; Turner, David P; Todd Purves, JThe urothelium is a frontline sensor of the lower urinary tract, sampling the bladder lumen and stimulating an immune response to infectious and noxious agents. Pattern recognition receptors (PRRs) recognize such agents and coordinate the innate response, often by forming inflammasomes that activate caspase-1 and the release of interleukin-1. We have shown the presence of one PRR (NLRP3) in the urothelia and its central role in the inflammatory response to cyclophosphamide. The purpose of this study was to (1) assess the likely range of the PPR response by assessing the repertoire present in the rat bladder and (2) determine the utility of the MYP3 rat urothelia cell line for in vitro studies by assessing its PPR repertoire and functional responsiveness.Immunohistochemistry was performed for seven PPRs (NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4 and AIM2) on bladder sections and MYP3 cells. For functionality, MYP3 cells were challenged with the quintessential NLRP3 activator ATP and assessed for caspase-1 activation.All PPRs examined were expressed in the bladder and localized to the urothelial layer with several also in the detrusor (none in the interstitia). MYP3 cells also expressed all PRRs with a variable intracellular location. ATP-stimulated caspase-1 activity in MYP3 cells in a dose-dependent manner was reduced by knockdown of NLRP3 expression.The results suggest that the bladder possesses the capacity to initiate an innate immune response to a wide array of uropathological agents and the MYP3 cells will provide an excellent investigational tool for this field. Item Open AccessVDAC2 as a novel target for heart failure: Ca2+ at the sarcomere, mitochondria and SR.(Cell calcium, 2022-06) Rosenberg, PaulDespite a growing number of successful therapies, heart failure remains the most common cause of death and disability worldwide. Thus, new and novel therapeutic strategies are urgently needed. Mitochondria of cardiomyocytes generate ATP that is needed to power cardiac contraction. Mitochondrial-derived ATP activate myosin ATPase at the sarcomere and the sarcoplasmic reticular (SR) ATPase Ca2+ pump, both which intersect with Ca2+ during contraction. Failure to maintain the relationship between mitochondria and SR can lead to cardiomyocyte dysfunction and heart failure. Here, we discuss recent discoveries that reveal Ca2+ transport via the voltage dependent anion channel (VDAC) into the mitochondria can favorably impact cardiac contraction and prevent cardiac arrhythmias. In a broader view, discussion of the opening of a new era for HF therapeutics that will address the sarcomere, SR and mitochondria as a functional unit.