Kinetically-defined mechanisms and positions of action of two new modulators of glucocorticoid receptor regulated gene induction [Computational Biology]

October 26th, 2015 by

Most of the steps in, and many of the factors contributing to, glucocorticoid receptor (GR) regulated gene induction are currently unknown. A competition assay, based on a validated chemical kinetic model of steroid hormone action, is now used to identify two new factors (BRD4 and NELF-E) and to define their sites and mechanisms of action. BRD4 is a kinase involved in numerous initial steps of gene induction. Consistent with its complicated biochemistry, BRD4 is shown to alter both the maximal activity (Amax) and the steroid concentration required for half-maximal induction (EC50) of GR-mediated gene expression by acting at a minimum of three different kinetically-defined steps. The action at two of these steps is dependent on BRD4 concentration while the third step requires the association of BRD4 with P-TEFb. BRD4 is also found to bind to NELF-E, a component of the NELF-complex. Unexpectedly, NELF-E modifies GR induction in a manner that is independent of the NELF complex. Several of the kinetically-defined steps of BRD4 in this study are proposed to be related to its known biochemical actions. However, novel actions of BRD4 and of NELF-E in GR controlled gene induction have been uncovered. The model-based competition assay is also unique in being able to order, for the first time, the sites of action of the various reaction components, which is GR < Cdk9 < BRD4 < or at induced gene < NELF-E. This ability to order factor actions will assist efforts to reduce the side-effects of steroid treatments.
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Forkhead Box P1 (FOXP1) Transcription Factor Regulates Hepatic Glucose Homeostasis [Molecular Bases of Disease]

October 26th, 2015 by

Dysregulation of hepatic gluconeogenesis contributes to the pathogenesis of diabetes, yet the detailed molecular mechanisms remain to be fully elucidated. Here we show that FOXP1, a transcriptional repressor, plays a key role in the regulation of systemic glucose homeostasis. Hepatic expression levels of FOXP1 are decreased in diabetic mice. Modest hepatic overexpression of FOXP1 in mice inhibited the expression of gluconeogenic genes, such as peroxisome proliferators-activated receptor gamma coactivator-1 alpha (PGC-1a), phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6PC), leading to a decrease in hepatic glucose production and fasting blood glucose levels in normal mice and different mouse models of diabetes, including db/db diabetic and high-fat diet-induced obese mice. FOXP1 physically interacted with FOXO1 in vivo and competed with FOXO1 for binding to the insulin response element (IRE) in the promoter region of gluconeogenic genes, thereby interfering expression of these genes. These results identify a previously unrecognized role for FOXP1 in the transcriptional control of hepatic glucose homeostasis.

Hepatic Xbp1 Deletion Promotes Endoplasmic Reticulum Stress-Induced Liver Injury and Apoptosis [Protein Structure and Folding]

October 26th, 2015 by Olivares, S., Henkel, A. S.

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), a highly conserved signaling cascade that functions to alleviate stress and promote cell survival. If, however, the cell is unable to adapt and restore homeostasis, the UPR activates pathways that promote apoptotic cell death. The molecular mechanisms governing the critical transition from adaptation and survival to initiation of apoptosis remain poorly understood. We aim to determine the role of hepatic Xbp1, a key mediator of the UPR, in controlling the adaptive response to ER stress in the liver. Liver-specific Xbp1 knockout mice (Xbp1LKO) and Xbp1fl/fl control mice were subjected to varying levels and duration of pharmacologic ER stress. Xbp1LKO and Xbp1fl/fl mice showed robust and equal activation of the UPR acutely after induction of ER stress. By 24 hours, Xbp1fl/fl controls showed complete resolution of UPR activation and no liver injury indicating successful adaptation to the stress. Conversely Xbp1LKO mice showed ongoing UPR activation associated with progressive liver injury, apoptosis, and ultimately fibrosis by day 7 after induction of ER stress. These data indicate that hepatic XBP1 controls the adaptive response of the UPR and is critical to restoring homeostasis in the liver in response to ER stress.

Structure-based discovery of novel small molecule Wnt signaling inhibitors by targeting the cysteine rich domain of Frizzled [Molecular Biophysics]

October 26th, 2015 by

Frizzled is the earliest discovered glycosylated Wnt protein receptor and is critical for the initiation of Wnt signaling. Antagonizing Frizzled is effective in inhibiting the growth of multiple tumor types. The extracellular N-terminus of Frizzled contains a conserved cysteine rich domain that directly interacts with Wnt ligands. Structure-based virtual screening and cell-based assays were used to identify five small molecules that can inhibit canonical Wnt signaling and have low IC50 values in the micromolar range. NMR experiments confirmed that these compounds specifically bind to the Wnt binding site on the Frizzled8 cysteine rich domain with sub micromolar dissociation constants. Our study confirms the feasibility of targeting the Frizzled cysteine rich domain as an effective way of regulating canonical Wnt signaling. These small molecules can be further optimized into more potent therapeutic agents for regulating abnormal Wnt signaling by targeting Frizzled.
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Selenoprotein S-dependent selenoprotein K binding to p97(VCP) is essential for endoplasmic reticulum-associated degradation [Protein Synthesis and Degradation]

October 26th, 2015 by

Cytosolic p97(VCP) is translocated to the ER membrane by binding to selenoprotein S (SelS), which is an ER membrane protein, during endoplasmic reticulum-associated degradation (ERAD). Selenoprotein K (SelK) is another known p97(VCP)-binding selenoprotein, and the expression of both SelS and SelK is increased under ER stress. To understand the regulatory mechanisms of SelS, SelK, and p97(VCP) during ERAD, the interaction of the selenoproteins with p97(VCP) was investigated using N2a cells and HEK293 cells. Both SelS and SelK co-precipitated with p97(VCP); however, the association between SelS and SelK did not occur in the absence of p97(VCP). SelS had the ability to recruit p97(VCP) to the ER membrane, but SelK did not. The interaction between SelK and p97(VCP) did not occur in SelS knockdown cells, whereas SelS interacted with p97(VCP) in the presence or absence of SelK. These results suggest that p97(VCP) is first translocated to the ER membrane via its interaction with SelS, and then SelK associates with the complex on the ER membrane. Thus, the interaction between SelK and p97(VCP) is SelS-dependent, and the resulting ERAD complex (SelS-p97(VCP)-SelK) plays an important role in ERAD and ER stress.
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Trpm7 contributes to intercellular junction formation in mouse urothelium [Membrane Biology]

October 26th, 2015 by

Trpm7 is a divalent cation-permeable channel that has been reported to be involved in magnesium homeostasis as well as cellular adhesion and migration. We generated urothelium-specific Trpm7 knockout (KO) mice to reveal the function of Trpm7 in vivo. A Trpm7 KO was induced by tamoxifen and was confirmed by genomic PCR and immunohistochemistry. By using patch-clamp recordings in primary urothelial cells, we observed that Mg 2+-inhibitable cation currents as well as acid-inducible currents were significantly smaller in Trpm7 KO urothelial cells than in the cells from control mice. Assessment of voiding behavior indicated significantly smaller voided volume in Trpm7 KO mice (mean voided volume 0.28 ± 0.08 g in KO mice, 0.36 ± 0.04 g in control mice, p < 0.05, n = 6-8). Histological analysis showed partial but substantial edema in the submucosal layer of Trpm7 KO mice, most likely due to inflammation. The expression of proinflammatory cytokines TNF-α and IL-1β was significantly higher in Trpm7 KO bladders than in controls. In transmission electron microscopic analysis, immature intercellular junctions were observed in Trpm7 KO urothelium but not in control mice. These results suggest that Trpm7 is involved in the formation of intercellular junctions in mouse urothelium. Immature intercellular junctions in Trpm7 knockout mice might lead to a disruption of barrier function resulting in inflammation and hypersensitive bladder afferent nerves that may affect voiding behavior in vivo.

Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Enzyme DPE1 [Protein Structure and Folding]

October 26th, 2015 by

The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whilst export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1) and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The ″gate″ is the most dynamic loop, and appears to play a role in substrate capture, in particular, in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms.

The high efficiency of Photosystem I in the green alga Chlamydomonas reinhardtii is maintained after the antenna size is substantially increased by the association of Light Harvesting Complex II [Bioenergetics]

October 26th, 2015 by

Photosystems (PS) I and II activities depend on their light-harvesting capacity and trapping efficiency, which vary in different environmental conditions. For optimal functioning, these activities need to be balanced. This is achieved by redistribution of excitation energy between the two photosystems via the association and disassociation of Light Harvesting Complexes (LHC)II, in a process known as state transitions. Here we study the effect of LHCII binding to PSI on its absorption properties and trapping efficiency by comparing time-resolved fluorescence kinetics of PSI-LHCI and PSI-LHCI-LHCII complexes of C. reinhardtii. PSI-LHCI-LHCII of C. reinhardtii is the largest PSI supercomplex isolated so far and contains 7 Lhcbs, in addition to the PSI core and the 9 Lhcas that compose PSI-LHCI, together binding ~320 chlorophylls. The average decay time for PSI-LHCI-LHCII is ~65 ps upon 400 nm excitation (15 ps slower than PSI-LHCI) and ~78 ps upon 475 nm excitation (27 ps slower). The transfer of excitation energy from LHCII to PSI-LHCI occurs in ~60 ps. This relatively slow transfer compared to that from LHCI to the PSI core, suggests loose connectivity between LHCII and PSI-LHCI. Despite the relatively slow transfer, the overall decay time of PSI-LHCI-LHCII remains fast enough to assure a 96% trapping efficiency, which is only 1.4% lower than that of PSI-LHCI, concomitant with an increase of the absorption cross section of 47%. This indicates that, at variance with PSII, the design of PSI allows for a large increase of its light-harvesting capacities.
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Dual Action of Zn2+ on the Transport Cycle of the Dopamine Transporter [Enzymology]

October 26th, 2015 by

The dopamine transporter shapes dopaminergic neurotransmission by clearing extracellular dopamine and by replenishing vesicular stores. The dopamine transporter carries an endogenous binding site for Zn2+, but the nature of the Zn2+-dependent modulation has remained elusive: both, inhibition and stimulation of DAT have been reported. Here, we exploited the high time resolution of patch-clamp recordings to examine the effects of Zn2+ on the transport cycle of DAT: we recorded peak currents associated with substrate translocation and steady-state currents reflecting the forward transport mode of DAT. Zn2+ depressed the peak current but enhanced the steady-state current through DAT. The parsimonious explanation is preferential binding of Zn2+ to the outward facing conformation of DAT, which allows for an allosteric activation of DAT in both, the forward transport mode and substrate exchange mode. We directly confirmed that Zn2+ dissociated more rapidly from the inward- than from the outward-facing state of DAT. Finally, we formulated a kinetic model for the action of Zn2+ on DAT that emulated all current experimental observations and accounted for all previous - in part contradictory - findings. Importantly, the model predicts that the intracellular Na+ concentration determines whether substrate uptake by DAT is stimulated or inhibited by Zn2+. This prediction was directly verified. The mechanistic framework provided by the current model is of relevance for the rational design of allosteric activators of DAT. These are of interest for treating de novo loss-of-function mutations of DAT associated with neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD).

SAS-4 in Trypanosoma brucei controls life cycle transitions by modulating the length of the flagellum attachment zone filament [Cell Biology]

October 26th, 2015 by Hu, H., Zhou, Q., Li, Z.

The evolutionarily conserved centriole/basal body protein SAS-4 regulates centriole duplication in metazoa and basal body duplication in flagellated and ciliated organisms. Here, we report that the SAS-4 homolog in the flagellated protozoan Trypanosoma brucei, TbSAS-4, plays an unusual role in controlling life cycle transitions by regulating the length of the flagellum attachment zone (FAZ) filament, a specialized cytoskeletal structure required for flagellum adhesion and cell morphogenesis. TbSAS-4 is concentrated at the distal tip of the FAZ filament, and depletion of TbSAS-4 in the trypomastigote form disrupts the elongation of the new FAZ filament, generating cells with a shorter FAZ associated with a longer unattached flagellum and re-positioned kinetoplast and basal body, reminiscent of epimastigote-like morphology. Further, we show that TbSAS-4 associates with several FAZ tip proteins, and depletion of TbSAS-4 disrupts the enrichment of several of these FAZ tip proteins at the new FAZ tip, suggesting a role of TbSAS-4 in maintaining the integrity of this FAZ tip protein complex. Together, these results uncover a novel function of TbSAS-4 in regulating the length of the FAZ filament to control basal body positioning and life cycle transitions in T. brucei.
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