Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia

Extracellular nucleotides may be important regulators of bile ductular secretion, because cholangiocytes express P2Y ATP receptors and nucleotides are found in bile. However, the expression, distribution, and function of specific P2Y receptor subtypes in cholangiocytes are unknown. Thus our aim was to determine the subtypes, distribution, and role in secretion of P2Y receptors expressed by cholangiocytes. The molecular subtypes of P2Y receptors were determined by RT-PCR. Functional studies measuring cytosolic Ca2+ (Ca) signals and bile ductular pH were performed in isolated, microperfused intrahepatic bile duct units (IBDUs). PCR products corresponding to P2Y1, P2Y2, P2Y4, P2Y6, and P2X4 receptor subtypes were identified. Luminal perfusion of ATP into IBDUs induced increases in Ca that were inhibited by apyrase and suramin. Luminal ATP, ADP, 2-methylthioadenosine 5'-triphosphate, UTP, and UDP each increased Ca. Basolateral addition of adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), but not ATP, to the perifusing bath increased Ca. IBDU perfusion with ATP-gamma-S induced net bile ductular alkalization. Cholangiocytes express multiple P2Y receptor subtypes that are expressed at the apical plasma membrane domain. P2Y receptors are also expressed on the basolateral domain, but their activation is attenuated by nucleotide hydrolysis. Activation of ductular P2Y receptors induces net ductular alkalization, suggesting that nucleotide signaling may be an important regulator of bile secretion by the liver.     

DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.     

Isolation and characterization of cholangiocyte primary cilia

Primary cilia are distinct organelles expressed by many vertebrate cells, including cholangiocytes; however, their functions remain obscure. To begin to explore the physiological role of these organelles in the liver, we described the morphology and structure of cholangiocyte cilia and developed new approaches for their isolation. Primary cilia were present only in bile ducts and were not observed in hepatocytes or in hepatic arterial or portal venous endothelial cells. Each cholangiocyte possesses a single cilium that extends from the apical membrane into the bile duct lumen. In addition, the length of the cilia was proportional to the bile duct diameter. We reproducibly isolated enriched fractions of cilia from normal rat and mouse cholangiocytes by two different approaches as assessed by scanning electron, transmission electron, and confocal microscopy. The purity of isolated ciliary fractions was further analyzed by Western blot analysis using acetylated tubulin as a ciliary marker and P2Y(2) as a nonciliary cell membrane marker. These novel techniques produced enriched ciliary fractions of sufficient purity and quantity for light and electron microscopy and for biochemical analyses. They will permit further assessment of the role of primary cilia in normal and pathological conditions.     

Structural and evolutionary classification of G/U wobble basepairs in the ribosome

We present a comprehensive structural, evolutionary and molecular dynamics (MD) study of the G/U wobble basepairs in the ribosome based on high-resolution crystal structures, including the recent Escherichia coli structure. These basepairs are classified according to their tertiary interactions, and sequence conservation at their positions is determined. G/U basepairs participating in tertiary interactions are more conserved than those lacking any interactions. Specific interactions occurring in the G/U shallow groove pocket--like packing interactions (P-interactions) and some phosphate backbone interactions (phosphate-in-pocket interactions)--lead to higher G/U conservation than others. Two salient cases of unique phylogenetic compensation are discovered. First, a P-interaction is conserved through a series of compensatory mutations involving all four participating nucleotides to preserve or restore the G/U in the optimal orientation. Second, a G/U basepair forming a P-interaction and another one forming a phosphate-in-pocket interaction are replaced by GNRA loops that maintain similar tertiary contacts. MD simulations were carried out on eight P-interactions. The specific GU/CG signature of this interaction observed in structure and sequence analysis was rationalized, and can now be used for improving sequence alignments.     

Cations and hydration in catalytic RNA: molecular dynamics of the hepatitis delta virus ribozyme

The hepatitis delta virus (HDV) ribozyme is an RNA enzyme from the human pathogenic HDV. Cations play a crucial role in self-cleavage of the HDV ribozyme, by promoting both folding and chemistry. Experimental studies have revealed limited but intriguing details on the location and structural and catalytic functions of metal ions. Here, we analyze a total of approximately 200 ns of explicit-solvent molecular dynamics simulations to provide a complementary atomistic view of the binding of monovalent and divalent cations as well as water molecules to reaction precursor and product forms of the HDV ribozyme. Our simulations find that an Mg2+ cation binds stably, by both inner- and outer-sphere contacts, to the electronegative catalytic pocket of the reaction precursor, in a position to potentially support chemistry. In contrast, protonation of the catalytically involved C75 in the precursor or artificial placement of this Mg2+ into the product structure result in its swift expulsion from the active site. These findings are consistent with a concerted reaction mechanism in which C75 and hydrated Mg2+ act as general base and acid, respectively. Monovalent cations bind to the active site and elsewhere assisted by structurally bridging long-residency water molecules, but are generally delocalized.     

Mechanical and biochemical modeling of cortical oscillations in spreading cells

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 Ca²⁺ pump activity increases the period and amplitude of oscillations; and 4), a threshold exists for the calcium concentration below which oscillations cease.     

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

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.