Publications by authors named "Maryna Kapustina"

19 Publications

  • Page 1 of 1

Sirtuin 6 (SIRT6) regulates redox homeostasis and signaling events in human articular chondrocytes.

Free Radic Biol Med 2021 Apr 16;166:90-103. Epub 2021 Feb 16.

Division of Rheumatology, Allergy and Immunology and the Thurston Arthritis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address:

The nuclear localized protein deacetylase, SIRT6, has been identified as a crucial regulator of biological processes that drive aging. Among these processes, SIRT6 can promote resistance to oxidative stress conditions, but the precise mechanisms remain unclear. The objectives of this study were to examine the regulation of SIRT6 activity by age and oxidative stress and define the role of SIRT6 in maintaining redox homeostasis in articular chondrocytes. Although SIRT6 levels did not change with age, SIRT6 activity was significantly reduced in chondrocytes isolated from older adults. Using dimedone-based chemical probes that detect oxidized cysteines, we identified that SIRT6 is oxidized in response to oxidative stress conditions, an effect that was associated with reduced SIRT6 activity. Enhancement of SIRT6 activity through adenoviral SIRT6 overexpression specifically increased the basal levels of two antioxidant proteins, peroxiredoxin 1 (Prx1) and sulfiredoxin (Srx) and decreased the levels of an inhibitor of antioxidant activity, thioredoxin interacting protein (TXNIP). Conversely, in chondrocytes derived from mice with cartilage specific Sirt6 knockout, Sirt6 loss decreased Prx1 levels and increased TXNIP levels. SIRT6 overexpression decreased nuclear-generated HO levels and oxidative stress-induced accumulation of nuclear phosphorylated p65. Our data demonstrate that SIRT6 activity is altered with age and oxidative stress conditions associated with aging. SIRT6 contributes to chondrocyte redox homeostasis by regulating specific members of the Prx catalytic cycle. Targeted therapies aimed at preventing the age-related decline in SIRT6 activity may represent a novel strategy to maintain redox balance in joint tissues and decrease catabolic signaling events implicated in osteoarthritis (OA).
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http://dx.doi.org/10.1016/j.freeradbiomed.2021.01.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009856PMC
April 2021

Stimulator of interferon genes (STING) is an essential proviral host factor for human rhinovirus species A and C.

Proc Natl Acad Sci U S A 2020 11 15;117(44):27598-27607. Epub 2020 Oct 15.

Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;

Human rhinoviruses (RVs) are positive-strand RNA viruses that cause respiratory tract disease in children and adults. Here we show that the innate immune signaling protein STING is required for efficient replication of members of two distinct RV species, RV-A and RV-C. The host factor activity of STING was identified in a genome-wide RNA interference (RNAi) screen and confirmed in primary human small airway epithelial cells. Replication of RV-A serotypes was strictly dependent on STING, whereas RV-B serotypes were notably less dependent. Subgenomic RV-A and RV-C RNA replicons failed to amplify in the absence of STING, revealing it to be required for a step in RNA replication. STING was expressed on phosphatidylinositol 4-phosphate (PI4P)-enriched membranes and was enriched in RV-A16 compared with RV-B14 replication organelles isolated in isopycnic gradients. The host factor activity of STING was species-specific, as murine STING (mSTING) did not rescue RV-A16 replication in STING-deficient cells. This species specificity mapped primarily to the cytoplasmic, ligand-binding domain of STING. Mouse-adaptive mutations in the RV-A16 2C protein allowed for robust replication in cells expressing mSTING, suggesting a role for 2C in recruiting STING to RV-A replication organelles. Palmitoylation of STING was not required for RV-A16 replication, nor was the C-terminal tail of STING that mediates IRF3 signaling. Despite co-opting STING to promote its replication, interferon signaling in response to STING agonists remained intact in RV-A16 infected cells. These data demonstrate a surprising requirement for a key host mediator of innate immunity to DNA viruses in the life cycle of a small pathogenic RNA virus.
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http://dx.doi.org/10.1073/pnas.2014940117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959528PMC
November 2020

A new light chain for myosin-7.

J Biol Chem 2020 07;295(28):9297-9298

Dept. of Cell Biology and Physiology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA

Recent research has revealed that an adhesion complex based on cadherins and the motor protein myosin-7b (MYO7B) links the tips of intestinal microvilli. Choi now report that a largely uncharacterized protein known as calmodulin-like protein 4 (CALML4) is a component of this adhesion complex and functions as a light chain for myosin-7b. Because the intermicrovillar adhesion complex is homologous to the myosin-7a (MYO7A)-based Usher syndrome complex and Choi also report that CALML4 can bind to myosin-7a, this work also has important implications for research on myosin-7a and hereditary deaf-blindness.
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http://dx.doi.org/10.1074/jbc.H120.014595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363122PMC
July 2020

Gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis A virus.

Nat Microbiol 2020 09 25;5(9):1069-1078. Epub 2020 May 25.

Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

The Picornaviridae are a diverse family of positive-strand RNA viruses that includes numerous human and veterinary pathogens. Among these, hepatitis A virus (HAV), a common cause of acute hepatitis in humans, is unique in that it is hepatotropic and is released from hepatocytes without lysis in small vesicles that resemble exosomes. These quasi-enveloped virions are infectious and are the only form of virus that can be detected in the blood during acute infection. By contrast, non-enveloped naked virions are shed in faeces and stripped of membranes by bile salts during passage through the bile ducts to the gut. How these two distinct types of infectious hepatoviruses enter cells to initiate infection is unclear. Here, we describe a genome-wide forward screen that shows that glucosylceramide synthase and other components of the ganglioside synthetic pathway are crucial host factors that are required for cellular entry by hepatoviruses. We show that gangliosides-preferentially disialogangliosides-function as essential endolysosome receptors that are required for infection by both naked and quasi-enveloped virions. In the absence of gangliosides, both virion types are efficiently internalized through endocytosis, but capsids fail to uncoat and accumulate within LAMP1 endolysosomes. Gangliosides relieve this block, binding to the capsid at low pH and facilitating a late step in entry involving uncoating and delivery of the RNA genome to the cytoplasm. These results reveal an atypical cellular entry pathway for hepatoviruses that is unique among picornaviruses.
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http://dx.doi.org/10.1038/s41564-020-0727-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483933PMC
September 2020

Going with the Flow (or Not).

Biophys J 2019 09 5;117(5):791-792. Epub 2019 Aug 5.

Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. Electronic address:

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http://dx.doi.org/10.1016/j.bpj.2019.07.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732662PMC
September 2019

Simultaneous quantification of actin monomer and filament dynamics with modeling-assisted analysis of photoactivation.

J Cell Sci 2016 12 9;129(24):4633-4643. Epub 2016 Nov 9.

Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610, USA

Photoactivation allows one to pulse-label molecules and obtain quantitative data about their behavior. We have devised a new modeling-based analysis for photoactivatable actin experiments that simultaneously measures properties of monomeric and filamentous actin in a three-dimensional cellular environment. We use this method to determine differences in the dynamic behavior of β- and γ-actin isoforms, showing that both inhabit filaments that depolymerize at equal rates but that β-actin exists in a higher monomer-to-filament ratio. We also demonstrate that cofilin (cofilin 1) equally accelerates depolymerization of filaments made from both isoforms, but is only required to maintain the β-actin monomer pool. Finally, we used modeling-based analysis to assess actin dynamics in axon-like projections of differentiating neuroblastoma cells, showing that the actin monomer concentration is significantly depleted as the axon develops. Importantly, these results would not have been obtained using traditional half-time analysis. Given that parameters of the publicly available modeling platform can be adjusted to suit the experimental system of the user, this method can easily be used to quantify actin dynamics in many different cell types and subcellular compartments.
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http://dx.doi.org/10.1242/jcs.194670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201019PMC
December 2016

Modeling the Excess Cell Surface Stored in a Complex Morphology of Bleb-Like Protrusions.

PLoS Comput Biol 2016 Mar 25;12(3):e1004841. Epub 2016 Mar 25.

Department of Mathematics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

Cells transition from spread to rounded morphologies in diverse physiological contexts including mitosis and mesenchymal-to-amoeboid transitions. When these drastic shape changes occur rapidly, cell volume and surface area are approximately conserved. Consequently, the rounded cells are suddenly presented with a several-fold excess of cell surface whose area far exceeds that of a smooth sphere enclosing the cell volume. This excess is stored in a population of bleb-like protrusions (BLiPs), whose size distribution is shown by electron micrographs to be skewed. We introduce three complementary models of rounded cell morphologies with a prescribed excess surface area. A 2D Hamiltonian model provides a mechanistic description of how discrete attachment points between the cell surface and cortex together with surface bending energy can generate a morphology that satisfies a prescribed excess area and BLiP number density. A 3D random seed-and-growth model simulates efficient packing of BLiPs over a primary rounded shape, demonstrating a pathway for skewed BLiP size distributions that recapitulate 3D morphologies. Finally, a phase field model (2D and 3D) posits energy-based constitutive laws for the cell membrane, nematic F-actin cortex, interior cytosol, and external aqueous medium. The cell surface is equipped with a spontaneous curvature function, a proxy for the cell surface-cortex couple, that is a priori unknown, which the model "learns" from the thin section transmission electron micrograph image (2D) or the "seed and growth" model image (3D). Converged phase field simulations predict self-consistent amplitudes and spatial localization of pressure and stress throughout the cell for any posited stationary morphology target and cell compartment constitutive properties. The models form a general framework for future studies of cell morphological dynamics in a variety of biological contexts.
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http://dx.doi.org/10.1371/journal.pcbi.1004841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807848PMC
March 2016

Spatiotemporal relationships between the cell shape and the actomyosin cortex of periodically protruding cells.

Cytoskeleton (Hoboken) 2015 Jun;72(6):268-81

Department of Cell Biology and Physiology, School of Medicine, UNC Chapel Hill, North Carolina.

We investigate the dynamics of cell shape and analyze the actin and myosin distributions of cells exhibiting cortical density traveling waves. These waves propagate by repeated cycles of cortical compression (folding) and dilation (unfolding) that lead to periodic protrusions (oscillations) of the cell boundary. The focus of our detailed analysis is the remarkable periodicity of this phenotype, in which both the overall shape transformation and distribution of actomyosin density are repeated from cycle to cycle even though the characteristics of the shape transformation vary significantly for different regions of the cell. We show, using correlation analysis, that during traveling wave propagation cortical actin and plasma membrane densities are tightly coupled at each point along the cell periphery. We also demonstrate that the major protrusion appears at the wave trailing edge just after the actin cortex density has reached a maximum. Making use of the extraordinary periodicity, we employ latrunculin to demonstrate that sequestering actin monomers can have two distinct effects: low latrunculin concentrations can trigger and enhance traveling waves but higher concentrations of this drug retard the waves. The fundamental mechanism underlying this periodically protruding phenotype, involving folding and unfolding of the cortex-membrane couple, is likely to hold important clues for diverse phenomena including cell division and amoeboid-type migration.
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http://dx.doi.org/10.1002/cm.21229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4529805PMC
June 2015

Two functionally distinct sources of actin monomers supply the leading edge of lamellipodia.

Cell Rep 2015 Apr 10;11(3):433-45. Epub 2015 Apr 10.

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA; Department of Neurology, Emory University School of Medicine, Atlanta, GA 30322, USA; Center for Neurodegenerative Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA. Electronic address:

Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin) regulated by the ordered assembly from and disassembly into actin monomers (G-actin). Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer-binding protein thymosin β4 (Tβ4) for optimal leading-edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it does not interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions.
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http://dx.doi.org/10.1016/j.celrep.2015.03.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418508PMC
April 2015

Gleevec, an Abl family inhibitor, produces a profound change in cell shape and migration.

PLoS One 2013 2;8(1):e52233. Epub 2013 Jan 2.

Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America.

The issue of how contractility and adhesion are related to cell shape and migration pattern remains largely unresolved. In this paper we report that Gleevec (Imatinib), an Abl family kinase inhibitor, produces a profound change in the shape and migration of rat bladder tumor cells (NBTII) plated on collagen-coated substrates. Cells treated with Gleevec adopt a highly spread D-shape and migrate more rapidly with greater persistence. Accompanying this more spread state is an increase in integrin-mediated adhesion coupled with increases in the size and number of discrete adhesions. In addition, both total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) revealed a band of small punctate adhesions with rapid turnover near the cell leading margin. These changes led to an increase in global cell-substrate adhesion strength, as assessed by laminar flow experiments. Gleevec-treated cells have greater RhoA activity which, via myosin activation, led to an increase in the magnitude of total traction force applied to the substrate. These chemical and physical alterations upon Gleevec treatment produce the dramatic change in morphology and migration that is observed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052233PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3534684PMC
August 2013

Compression and dilation of the membrane-cortex layer generates rapid changes in cell shape.

J Cell Biol 2013 Jan;200(1):95-108

Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.

Rapid changes in cellular morphology require a cell body that is highly flexible yet retains sufficient strength to maintain structural integrity. We present a mechanism that meets both of these requirements. We demonstrate that compression (folding) and subsequent dilation (unfolding) of the coupled plasma membrane-cortex layer generates rapid shape transformations in rounded cells. Two- and three-dimensional live-cell images showed that the cyclic process of membrane-cortex compression and dilation resulted in a traveling wave of cortical actin density. We also demonstrate that the membrane-cortex traveling wave led to amoeboid-like cell migration. The compression-dilation hypothesis offers a mechanism for large-scale cell shape transformations that is complementary to blebbing, where the plasma membrane detaches from the actin cortex and is initially unsupported when the bleb extends as a result of cytosolic pressure. Our findings provide insight into the mechanisms that drive the rapid morphological changes that occur in many physiological contexts, such as amoeboid migration and cytokinesis.
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http://dx.doi.org/10.1083/jcb.201204157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542801PMC
January 2013

RhoA regulates calcium-independent periodic contractions of the cell cortex.

Biophys J 2010 Aug;99(4):1053-63

Department of Cell and Developmental Biology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

When microtubules are depolymerized in spreading cells, they experience morphological oscillations characterized by a period of about a minute, indicating that normal interactions between the microfilament and microtubule systems have been significantly altered. This experimental system provides a test bed for the development of both fine- and coarse-grained models of complex motile processes, but such models need to be adequately informed by experiment. Using criteria based on Fourier transform analysis, we detect spontaneous oscillations in spreading cells. However, their amplitude and tendency to operate at a single frequency are greatly enhanced by microtubule depolymerization. Knockdown of RhoA and addition of various inhibitors of the downstream effector of RhoA, Rho kinase, block oscillatory behavior. Inhibiting calcium fluxes from endoplasmic reticulum stores and from the extracellular medium does not significantly affect the ability of cells to oscillate, indicating that calcium plays a subordinate regulatory role compared to Rho. We characterized the dynamic structure of the oscillating cell by light, fluorescence, and electron microscopy, showing how oscillating cells are dynamically polarized in terms of their overall morphology, f-actin and phosphorylated myosin light chain distribution, and nuclear position and shape. Not only will these studies guide future experiments, they will also provide a framework for the development of refined mathematical models of the oscillatory process.
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http://dx.doi.org/10.1016/j.bpj.2010.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920727PMC
August 2010

Modeling capping protein FRAP and CALI experiments reveals in vivo regulation of actin dynamics.

Cytoskeleton (Hoboken) 2010 Aug;67(8):519-34

Department of Cell and Developmental Biology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7090, USA.

To gain insights on cellular mechanisms regulating actin polymerization, we used the Virtual Cell to model fluorescence recovery after photobleaching (FRAP) and chromophore-assisted laser inactivation (CALI) experiments on EGFP-capping protein (EGFP-CP). Modeling the FRAP kinetics demonstrated that the in vivo rate for the dissociation of CP from actin filaments is much faster (approximately 0.1 s(-1)) than that measured in vitro (0.01-0.0004 s(-1)). The CALI simulation revealed that in order to induce sustainable changes in cell morphology after CP inactivation, the cells should exhibit anticapping ability. We included the VASP protein as the anticapping agent in the modeling scheme. The model predicts that VASP affinity for barbed ends has a cooperative dependence on the concentration of VASP-barbed end complexes. This dependence produces a positive feedback that stabilizes the complexes and allows sustained growth at clustered filament tips. We analyzed the range of laser intensities that are sufficient to induce changes in cell morphology. This analysis demonstrates that FRAP experiments with EGFP-CP can be performed safely without changes in cell morphology, because, the intensity of the photobleaching beam is not high enough to produce the critical concentration of free barbed ends that will induce filament growth before diffusional replacement of EGFP-CP occurs.
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http://dx.doi.org/10.1002/cm.20463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929670PMC
August 2010

In silico generation of alternative hypotheses using causal mapping (CMAP).

PLoS One 2009 29;4(4):e5378. Epub 2009 Apr 29.

Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

Previously, we introduced causal mapping (CMAP) as an easy to use systems biology tool for studying the behavior of biological processes that occur at the cellular and molecular level. CMAP is a coarse-grained graphical modeling approach in which the system of interest is modeled as an interaction map between functional elements of the system, in a manner similar to portrayals of signaling pathways commonly used by molecular cell biologists. CMAP describes details of the interactions while maintaining the simplicity of other qualitative methods (e.g., Boolean networks).In this paper, we use the CMAP methodology as a tool for generating hypotheses about the mechanisms that regulate molecular and cellular systems. Furthermore, our approach allows competing hypotheses to be ranked according to a fitness index and suggests experimental tests to distinguish competing high fitness hypotheses. To motivate the CMAP as a hypotheses generating tool and demonstrate the methodology, we first apply this protocol to a simple test-case of a three-element signaling module. Our methods are next applied to the more complex phenomenon of cortical oscillations observed in spreading cells. This analysis produces two high fitness hypotheses for the mechanism that underlies this dynamic behavior and suggests experiments to distinguish the hypotheses. The method can be widely applied to other cellular systems to generate and compare alternative hypotheses based on experimentally observed data and using computer simulations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005378PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2671158PMC
July 2009

Independent saturation of three TrpRS subsites generates a partially assembled state similar to those observed in molecular simulations.

Proc Natl Acad Sci U S A 2009 Feb 27;106(6):1790-5. Epub 2009 Jan 27.

Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA.

Two new crystal structures of Bacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) afford evidence that a closed interdomain hinge angle requires a covalent bond between AMP and an occupant of either pyrophosphate or tryptophan subsite. They also are within experimental error of a cluster of structures observed in a nonequilibrium molecular dynamics simulation showing partial active-site assembly. Further, the highest energy structure in a minimum action pathway computed by using elastic network models for Open and Pretransition state (PreTS) conformations for the fully liganded TrpRS monomer is intermediate between that simulated structure and a partially disassembled structure from a nonequilibrium molecular dynamics trajectory for the unliganded PreTS. These mutual consistencies provide unexpected validation of inferences drawn from molecular simulations.
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http://dx.doi.org/10.1073/pnas.0812752106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2644116PMC
February 2009

Mechanical and biochemical modeling of cortical oscillations in spreading cells.

Biophys J 2008 Jun 7;94(12):4605-20. Epub 2008 Mar 7.

Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Actomyosin-based cortical contractility is a common feature of eukaryotic cells and is involved in cell motility, cell division, and apoptosis. In nonmuscle cells, oscillations in contractility are induced by microtubule depolymerization during cell spreading. We developed an ordinary differential equation model to describe this behavior. The computational model includes 36 parameters. The values for all but two of the model parameters were taken from experimental measurements found in the literature. Using these values, we demonstrate that the model generates oscillatory behavior consistent with current experimental observations. The rhythmic behavior occurs because of the antagonistic effects of calcium-induced contractility and stretch-activated calcium channels. The model makes several experimentally testable predictions: 1), buffering intracellular calcium increases the period and decreases the amplitude of cortical oscillations; 2), increasing the number or activity of stretch activated channels leads to an increase in period and amplitude of cortical oscillations; 3), inhibiting Ca(2+) pump activity increases the period and amplitude of oscillations; and 4), a threshold exists for the calcium concentration below which oscillations cease.
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http://dx.doi.org/10.1529/biophysj.107.121335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2397354PMC
June 2008

A conformational transition state accompanies tryptophan activation by B. stearothermophilus tryptophanyl-tRNA synthetase.

Structure 2007 Oct;15(10):1272-84

Department of Cell Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

B. stearothermophilus tryptophanyl-tRNA synthetase catalysis proceeds via high-energy protein conformations. Unliganded MD trajectories of the pretransition-state complex with Mg(2+)ATP and the (post) transition-state analog complex with adenosine tetraphosphate relax rapidly in opposite directions, the former regressing, the latter progressing along the structural reaction coordinate. The two crystal structures (rmsd 0.7 A) therefore lie on opposite sides of a conformational free-energy maximum as the chemical transition state forms. SNAPP analysis illustrates the complexity of the associated long-range conformational coupling. Switching interactions in four nonpolar core regions are locally isoenergetic throughout the transition. Different configurations, however, propagate their effects to unfavorable, longer-range interactions at the molecular surface. Designed mutation shows that switching interactions enhance the rate, perhaps by destabilizing the ground state immediately before the transition state and limiting nonproductive diffusion before and after the chemical transition state, thereby reducing the activation entropy. This paradigm may apply broadly to energy-transducing enzymes.
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http://dx.doi.org/10.1016/j.str.2007.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2693061PMC
October 2007

Computational studies of tryptophanyl-tRNA synthetase: activation of ATP by induced-fit.

J Mol Biol 2006 Oct 1;362(5):1159-80. Epub 2006 Sep 1.

Department of Biochemistry and Biophysics, CB 7260, University of North Carolina, Chapel Hill, NC 27599-7260, USA.

Catalysis of amino acid activation by Bacillus stearothermophilus tryptophanyl-tRNA synthetase involves three allosteric states: (1) Open; (2) closed pre-transition state (PreTS); and (3) closed products (Product). The interconversions of these states entail significant domain motions driven by ligand binding. We explore the application of molecular dynamics simulations to investigate ligand-linked conformational stability changes associated with this catalytic cycle. Multiple molecular dynamics trajectories (5 ns) for 11 distinct liganded and unliganded monomer configurations show that the PreTS conformation is unstable in the absence of ATP, reverting within approximately 600 ps nearly to the Open conformation. In contrast, Open and Product state trajectories were stable, even without ligands, confirming the previous suggestion that catalysis entails destabilization of the protein conformation, driven by ATP-binding energies developed as the PreTS state assembles during induced-fit. The simulations suggest novel mechanistic details associated with both induced-fit (Open-PreTS) and catalysis (PreTS-Product). Notably, Mg2+ -ATP interactions are coupled to interactions between ATP and active-site lysine side-chains via mechanisms that cannot be captured under the molecular mechanics approximations, and which therefore require restraining potentials for stable simulation. Simulations of Mg2+. ATP-bound PreTS complexes with restraining potentials and with a virtual K111A mutant confirm that these coupling interactions are necessary to sustain the PreTS conformation and, in turn, provide a new model for how the PreTS conformation activates ATP for catalysis. These results emphasize the central role of the PreTS state as a high-energy intermediate structure along the catalytic pathway and suggest that Mg2+ and the KMSKS loop function cooperatively during catalysis.
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http://dx.doi.org/10.1016/j.jmb.2006.06.078DOI Listing
October 2006

Structure alignment via Delaunay tetrahedralization.

Proteins 2005 Jul;60(1):66-81

Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

A novel protein structure alignment technique has been developed reducing much of the secondary and tertiary structure to a sequential representation greatly accelerating many structural computations, including alignment. Constructed from incidence relations in the Delaunay tetrahedralization, alignments of the sequential representation describe structural similarities that cannot be expressed with rigid-body superposition and complement existing techniques minimizing root-mean-squared distance through superposition. Restricting to the largest substructure superimposable by a single rigid-body transformation determines an alignment suitable for root-mean-squared distance comparisons and visualization. Restricted alignments of a test set of histones and histone-like proteins determined superpositions nearly identical to those produced by the established structure alignment routines of DaliLite and ProSup. Alignment of three, increasingly complex proteins: ferredoxin, cytidine deaminase, and carbamoyl phosphate synthetase, to themselves, demonstrated previously identified regions of self-similarity. All-against-all similarity index comparisons performed on a test set of 45 class I and class II aminoacyl-tRNA synthetases closely reproduced the results of established distance matrix methods while requiring 1/16 the time. Principal component analysis of pairwise tetrahedral decomposition similarity of 2300 molecular dynamics snapshots of tryptophanyl-tRNA synthetase revealed discrete microstates within the trajectory consistent with experimental results. The method produces results with sufficient efficiency for large-scale multiple structure alignment and is well suited to genomic and evolutionary investigations where no geometric model of similarity is known a priori.
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http://dx.doi.org/10.1002/prot.20479DOI Listing
July 2005