We have studied regulation of the multidrug resistance protein 2 (mrp2) during bile duct ligation (BDL) in the rat. In hepatocytes isolated after 16, 48, and 72 hours of BDL, mrp2-mediated dinitrophenyl-glutathione (DNP-GS) transport was decreased to 65%, 33%, and 33% of control values, respectively. The impaired mrp2-mediated transport coincided with strongly decreased mrp2 protein levels, without any significant changes in mrp2 RNA levels. Restoration of bile flow after a 48-hour BDL period resulted in a slow recovery of mrp2-mediated transport and protein levels. The multidrug resistance protein 2 (mrp2), previously called canalicular multispecific organic anion transporter, is an adenosine triphosphate (ATP)-dependent transporter that mediates the biliary excretion of a wide variety of endogenous and xenobiotic compounds. 1 The mrp2 protein has been extensively characterized in transport-mutant rats (the TR Ϫ rat, derived from the Wistar strain), 1 and the Eisai hyperbilirubinemic rat (EHBR rat, derived from the Sprague-Dawley rat strain), 2 which lack this protein owing to mutations in the mrp2 gene. [3][4][5] In humans, mutations in the orthologous MRP2 gene cause the Dubin-Johnson syndrome, 6-8 an autosomal recessive defect in the hepatobiliary excretion of a broad range of organic anions. 1,9 The human MRP2 and rat mrp2 are members of the ATP-binding cassette transporter family, and are abundantly and specifically expressed in the canalicular membrane of the hepatocyte. 3,4 Substrates transported by this protein include conjugates of glucuronide, sulphate, and glutathione, and complexes (or cotransport) of heavy metals and oxyanions with glutathione. 1 Recently, we have shown an important role for mrp2 in the transport of reduced glutathione (GSH), 10 which probably serves a physiological function in maintenance of the bile acid-independent flow.Extrahepatic or obstructive cholestasis is a pathological condition caused by biliary obstruction leading to impaired bile flow. 11-13 As a result, bile salt and non-bile salt organic anions (including taurocholate and bilirubin-glucuronides) accumulate in hepatocytes and regurgitate into the circulation. Morphological changes associated with obstructive cholestasis include dilation of the canaliculus, loss of microvilli, and rearrangement of tight-junctional structures. [11][12][13] Kawaguchi et al. 14 have recently shown that alterations in tight-junctional structures are heterogenously distributed throughout the liver lobule during bile duct ligation (BDL) in the rat.Several studies have been published that describe an association between activity and expression levels of canalicular transporters and obstructive cholestasis using the BDL model in the rat. P-glycoprotein (P-gp) expression levels and activity are increased during BDL, 15 with a concomitant increase in both mdr1a and mdr1b RNA levels. 15 In addition, P-gp (partly) redistributes to the pericanalicular vesicles of the hepatocyte. 15,16 Trauner et al. 17 have shown that BDL in the rat is associated ...

Polo-like kinase 1 is an important regulator of cell cycle progression whose over-expression is often associated with oncogenesis. Polo-like kinase 1 hence represents an attractive target for cancer intervention. BI 2536 (Boehringer Ingelheim, Ingelheim, Germany), a Polo-like kinase 1 inhibitor currently in clinical trials, exhibits nanomolar potency against Polo-like kinase isoforms and high selectivity against other kinases. We have previously published the crystal structures of the Polo-like kinase 1 domain in complex with AMPPNP and an Aurora A inhibitor. In this work, we present the co-crystal structure of Polo-like kinase 1 with BI 2536. The structure, in combination with selectivity data for BI 2536 and related compounds, illustrates important features for potency and selectivity. In particular, we show that the methoxy group of BI 2536 is an important specificity determinant against non-Polo-like kinases by taking advantage of a small pocket generated by Leu 132 in the hinge region of Polo-like kinase 1. The work presented here provides a framework for structure-based drug design of Polo-like kinase 1-specific inhibitors.

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Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 A resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the kcat of the reaction, with little change in the apparent Km for the protein or nucleotide substrates (kcat = 0.0094, 0.0376, and 0.0049 s-1 and Km(ATP) = 3.2, 4.0, and 3.0 microM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1-specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.

Microglia serve as the innate immune cells of the central nervous system (CNS) by providing continuous surveillance of the CNS microenvironment and initiating defense mechanisms to protect CNS tissue. Upon injury, microglia transition into an activated state altering their transcriptional profile, transforming their morphology, and producing pro-inflammatory cytokines. These activated microglia initially serve a beneficial role, but their continued activation drives neuroinflammation and neurodegeneration. Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the CNS, and activated microglia and macrophages play a significant role in mediating disease pathophysiology and progression. Colony-stimulating factor-1 receptor (CSF1R) and its ligand CSF1 are elevated in CNS tissue derived from MS patients. We performed a large-scale RNA-sequencing experiment and identified CSF1R as a key node of disease progression in a mouse model of progressive MS. We hypothesized that modulating microglia and infiltrating macrophages through the inhibition of CSF1R will attenuate deleterious CNS inflammation and reduce subsequent demyelination and neurodegeneration. To test this hypothesis, we generated a novel potent and selective small-molecule CSF1R inhibitor (sCSF1Rinh) for preclinical testing. sCSF1Rinh blocked receptor phosphorylation and downstream signaling in both microglia and macrophages and altered cellular functions including proliferation, survival, and cytokine production. In vivo, CSF1R inhibition with sCSF1Rinh attenuated neuroinflammation and reduced microglial proliferation in a murine acute LPS model. Furthermore, the sCSF1Rinh attenuated a disease-associated microglial phenotype and blocked both axonal damage and neurological impairments in an experimental autoimmune encephalomyelitis (EAE) model of MS. While previous studies have focused on microglial depletion following CSF1R inhibition, our data clearly show that signaling downstream of this receptor can be beneficially modulated in the context of CNS injury. Together, these data suggest that CSF1R inhibition can reduce deleterious microglial proliferation and modulate microglial phenotypes during neuroinflammatory pathogenesis, particularly in progressive MS.

The enzymatic A1 chain of cholera toxin retrotranslocates across the endoplasmic reticulum membrane into the cytosol, where it induces toxicity. Almost all other retrotranslocation substrates are modified by the attachment of polyubiquitin chains and moved into the cytosol by the ubiquitin-interacting p97 ATPase complex. The cholera toxin A1 chain, however, can induce toxicity in the absence of ubiquitination, and the motive force that drives retrotranslocation is not known. Here, we use adenovirus expressing dominant-negative mutants of p97 to test whether p97 is required for toxin action. We find that cholera toxin still functions with only a small decrease in potency in cells that cannot retrotranslocate other substrates at all. These results suggest that p97 does not provide the primary driving force for extracting the A1 chain from the endoplasmic reticulum, a finding that is consistent with a requirement for polyubiquitination in p97 function. Cholera toxin (CT)1 enters host cells by co-opting two fundamental aspects of cell function that reverse membrane and protein transport in the secretory pathway. First, the toxin binds to a membrane lipid that carries it retrograde from plasma membrane to the endoplasmic reticulum (ER) (1). After arrival in the ER, a fragment of CT, the A1 chain, then enters the cytosol by hijacking the cellular machinery for degradation of terminally misfolded proteins (2, 3). Such proteins are recognized by ER luminal chaperones, transported to the cytosol, and degraded by the proteasome. This pathway is termed retrotranslocation, or ERAD for ER-associated protein degradation. Unlike misfolded proteins, the A1 chain escapes degradation by the proteasome (4) and induces toxicity by activating adenylyl cyclase.CT is a member of the AB5 family of toxins, where the A subunit couples non-covalently with a homopentameric B subunit. The B subunit of CT is responsible for binding to ganglioside GM1 in the cell membrane, and this complex carries the A subunit into the ER (1). The A subunit has two domains, a compact N-terminal A1 chain that contains the enzymatic activity of the toxin and an extended C-terminal A2 chain that tethers the A and B subunits together. The A1 and A2 chains are linked by a flexible peptide loop containing a serine-protease cleavage site subtended by a single disulfide bond. Cleavage at this site occurs after secretion from the Vibrio, and this is required for toxicity.There is a small change in the conformation of the toxin caused by proteolytic cleavage of the peptide loop linking the A1 and A2 chains that renders the A1 chain susceptible to attack by the ER luminal chaperone protein-disulfide isomerase. In its reduced form, protein-disulfide isomerase binds, unfolds, and dissociates the A1 chain from the remainder of the holotoxin (i.e. the A2 chain and B subunit) (3). Upon oxidation, protein-disulfide isomerase releases the A1 chain, presumably still in its unfolded conformation, to the retrotranslocation machinery. The A1 chain may cross the ER membrane by pas...

Phosphodiesterases (PDEs) modulate signaling by cyclic nucleotides in diverse processes such as cardiac contractility, platelet aggregation, lipolysis, glycogenolysis, and smooth muscle contraction. Cyclic guanosine monophosphate (cGMP) stimulated human phosphodiesterase 2 (PDE2) is expressed mainly in brain and heart tissues. PDE2A is involved in the regulation of blood pressure and fluid homeostasis by the atrial natriuretic peptide (ANP), making PDE2-type enzymes important targets for drug discovery. The design of more potent and selective inhibitors of PDE2A for the treatment of heart disease would be greatly aided by the identification of active site residues in PDE2A that determine substrate and inhibitor selectivity. The identification of active site residues through traditional mutational studies involves the time-consuming and tedious purification of a large number of mutant proteins from overexpressing cells. Here we report an alternative approach to rapidly produce active site mutants of human PDE2A and identify their enzymatic properties using a wheat germ in vitro translation (IVT, also known as cell-free translation) system. We also present the crystal structure of the catalytic domain of human PDE2A determined at 1.7 A resolution, which provided a framework for the rational design of active site mutants. Using a rapid IVT approach for expression of human PDE2A mutants, we identified the roles of active site residues Asp811, Gln812, Ile826, and Tyr827 in inhibitor and substrate selectivity for PDE2A.

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