Rapid, transient induction of ER stress in the liver and kidney after acute exposure to heavy metal: evidence from transgenic sensor mice

Endoplasmic reticulum (ER) stress-responsive alkaline phosphatase (ES-TRAP) serves as a sensitive indicator for ER stress. In response to heavy metals including cadmium, nickel and cobalt, hepatocytes and renal tubular cells expressing ES-TRAP exhibited ER stress and decreased ES-TRAP activity. In ES-TRAP transgenic mice, acute exposure to cadmium showed rapid, transient decreases in the activity of serum ES-TRAP. It was inversely correlated with the induction of endogenous ER stress markers in the liver and kidney. Our result provides first evidence for the acute, reversible induction of ER stress in vivo after exposure to heavy metal.     

Assembly of lysine 63-linked ubiquitin conjugates by phosphorylated alpha-synuclein implies Lewy body biogenesis

alpha-Synuclein (alpha-syn) and ubiquitin (Ub) are major protein components deposited in Lewy bodies (LBs) and Lewy neurites, which are pathologic hallmarks of idiopathic Parkinson disease (PD). Almost 90% of alpha-syn in LBs is phosphorylated at serine 129 (Ser(129)). However, the role of Ser(129)-phosphorylated alpha-syn in the biogenesis of LBs remains unclear. Here, we show that compared with coexpression of wild type (WT)alpha-syn and Ub, coexpression of phospho-mimic mutant alpha-syn (S129D) and Ub in neuro2a cells results in an increase of Ub-conjugates and the formation of ubiquitinated inclusions. Furthermore, S129D alpha-syn fails to increase the Ub-conjugates and form ubiquitinated inclusions in the presence of a K63R mutant Ub. In addition, as compared with WT alpha-syn, S129D alpha-syn increased cytoplasmic and neuritic aggregates of itself in neuro2a cells treated with H(2)O(2) and serum deprivation. These results suggest that the contribution of Ser(129)-phosphorylated alpha-syn to the Lys(63)-linked Ub-conjugates and aggregation of itself may be involved in the biogenesis of LBs in Parkinson disease and other related synucleinopathies.     

Interleukin-1 (IL-1)-induced TAK1-dependent Versus MEKK3-dependent NFkappaB activation pathways bifurcate at IL-1 receptor-associated kinase modification

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is phosphorylated after it is recruited to the receptor, subsequently ubiquitinated, and eventually degraded upon IL-1 stimulation. Although a point mutation changing lysine 134 to arginine (K134R) in IRAK abolished IL-1-induced IRAK ubiquitination and degradation, mutations of serines and threonines adjacent to lysine 134 to alanines ((S/T)A (131-144)) reduced IL-1-induced IRAK phosphorylation and abolished IRAK ubiquitination. Through the study of these IRAK modification mutants, we uncovered two parallel IL-1-mediated signaling pathways for NFkappaB activation, TAK1-dependent and MEKK3-dependent, respectively. These two pathways bifurcate at the level of IRAK modification. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classical NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through IkappaBalpha phosphorylation and subsequent dissociation from NFkappaB but without IkappaBalpha degradation. These results provide significant insight to our further understanding of NFkappaB activation pathways.     

Inhibition of the dopamine D1 receptor signaling by PSD-95

Dopamine D1 receptors play an important role in movement, reward, and learning and are implicated in a number of neurological and psychiatric disorders. These receptors are concentrated in dendritic spines of neurons, including the spine head and the postsynaptic density. D1 within spines is thought to modulate the local channels and receptors to control the excitability and synaptic properties of spines. The molecular mechanisms mediating D1 trafficking, anchorage, and function in spines remain elusive. Here we show that the synaptic scaffolding protein PSD-95 thought to play a role in stabilizing glutamate receptors in the postsynaptic density, interacts with D1 and regulates its trafficking and function. Interestingly, the D1-PSD-95 interaction does not require the well characterized domains of PSD-95 but is mediated by the carboxyl-terminal tail of D1 and the NH(2) terminus of PSD-95, a region that is recognized only recently to participate in protein-protein interaction. Co-expression of PSD-95 with D1 in mammalian cells inhibits the D1-mediated cAMP accumulation without altering the total expression level or the agonist binding properties of the receptor. The diminished D1 signaling is mediated by reduced D1 expression at the cell surface as a consequence of an enhanced constitutive, dynamin-dependent endocytosis. In addition, genetically engineered mice lacking PSD-95 show a heightened behavioral response to either a D1 agonist or the psychostimulant amphetamine. These studies demonstrate a role for a glutamatergic scaffold in dopamine receptor signaling and trafficking and identify a new potential target for the modulation of abnormal dopaminergic function.     

Src docks to A-kinase anchoring protein gravin, regulating beta2-adrenergic receptor resensitization and recycling

Gravin (AKAP12) is a membrane-associated scaffold that provides docking for protein kinases, phosphatases, and adaptor molecules obligate for resensitization and recycling of beta(2)-adrenergic receptors. Gravin binds to the cell membrane in a Ca(2+)-sensitive manner and to receptors through well characterized protein-protein interactions. Although the interaction of serine/threonine, cyclic AMP-dependent protein kinase with protein kinase A-anchoring proteins is well described and involves a kinase regulatory subunit binding domain in the C terminus of these proteins, far less is known about tyrosine kinase docking to members of this family of scaffolds. The non-receptor tyrosine kinase Src regulates resensitization of beta(2)-adrenergic receptors and docks to gravin. Gravin displays nine proline-rich domains distributed throughout the molecule. One class I ligand for Src homology domain 3 docking, found in the N terminus ((10)RXPXXP(15)) of gravin, is shown to bind Src. Binding of Src to gravin activates the intrinsic tyrosine kinase of Src. Mutagenesis/deletion of the class I ligand (P15A,P16A) on the N terminus of gravin abolishes both the docking of Src to gravin as well as the receptor resensitization and recycling catalyzed by gravin. The Src-binding peptide-(1-51) of gravin behaves as a dominant-negative for AKAP gravin regulation of receptor resensitization/recycling. The tyrosine kinase Src plays an essential role in the AKAP gravin-mediated receptor resensitization and recycling, an essential aspect of receptor biology.     

Methods of using click chemistry in the discovery of enzyme inhibitors

This protocol describes the step-by-step procedures for the efficient assembly of bidentate inhibitor libraries of a target enzyme, using the so-called 'click chemistry' between an alkyne-bearing core group and an azide-modified peripheral group, followed by direct biological screening for the identification of potential 'hits'. The reaction is highlighted by its modularity, high efficiency (approximately 100% yield in most cases) and tolerance toward many functional groups present in the fragments, as well as biocompatibility (typically carried out in aqueous conditions with small amounts of biocompatible catalysts). The approach consists of three steps: (i) chemical synthesis of alkyne-bearing protein tyrosine phosphatase or matrix metalloprotease core groups and diverse azide-modified peripheral groups; (ii) click chemistry to assemble the bidentate inhibitor libraries; and (iii) direct screening of the libraries with target enzymes using 384-well microplate assays. Following the chemical synthesis of the core and peripheral groups and optimization of the click chemistry conditions (approximately 1 week), steps (ii) and (iii) take 3 d to complete (approximately 1-2 d for library assembly and 1 d for inhibitor screening).     

True phosphorus digestibility and the endogenous phosphorus outputs associated with brown rice for weanling pigs measured by the simple linear regression analysis technique

The objectives of this study were to determine true phosphorus (P) digestibility, degradability of phytate-P complex and the endogenous P outputs associated with brown rice feeding in weanling pigs by using the simple linear regression analysis technique. Six barrows with an average initial body weight of 12.5 kg were fitted with a T-cannula and fed six diets according to a 6 × 6 Latin-square design. Six maize starch-based diets, containing six levels of P at 0.80, 1.36, 1.93, 2.49, 3.04, and 3.61 g/kg per kg dry-matter (DM) intake (DMI), were formulated with brown rice. Each experimental period lasted 10 days. After a 7-day adaptation, all faecal samples were collected on days 8 and 9. Ileal digesta samples were collected for a total of 24 h on day 10. The apparent ileal and faecal P digestibility values of brown rice were affected ( P < 0.01) by the P contents in the assay diets. The apparent ileal and faecal P digestibility values increased from − 48.0 to 36.7% and from − 35.6 to 40.0%, respectively, as P content increased from 0.80 to 3.61 g/kg DMI. Linear relationships ( P < 0.05), expressed as g/kg DMI, between the apparent ileal and faecal digestible P and dietary levels of P, suggested that true P digestibility and the endogenous P outputs associated with brown rice feeding could be determined by using the simple regression analysis technique. There were no differences ( P>0.05) in true P digestibility values (57.7 ± 5.4 v. 58.2 ± 5.9%), phytate P degradability (76.4 ± 6.7 v. 79.0 ± 4.4%) and the endogenous P outputs (0.812 ± 0..096 v. 0.725 ± 0.083 g/kg DMI) between the ileal and the faecal levels. The endogenous faecal P output represented 14 and 25% of the National Research Council (1998) recommended daily total and available P requirements in the weanling pig, respectively. About 58% of the total P in brown rice could be digested and absorbed by the weanling pig. Our results suggest that the large intestine of the weanling pigs does not play a significant role in the digestion of P in brown rice. Diet formulation on the basis of total or apparent P digestibility with brown rice may lead to P overfeeding and excessive P excretion in pigs.     

Study of gene function based on spatial co-expression in a high-resolution mouse brain atlas

Background  

The Allen Brain Atlas (ABA) project systematically profiles three-dimensional high-resolution gene expression in postnatal mouse brains for thousands of genes. By unveiling gene behaviors at both the cellular and molecular levels, ABA is becoming a unique and comprehensive neuroscience data source for decoding enigmatic biological processes in the brain. Given the unprecedented volume and complexity of the in situ hybridization image data, data mining in this area is extremely challenging. Currently, the ABA database mainly serves as an online reference for visual inspection of individual genes; the underlying rich information of this large data set is yet to be explored by novel computational tools. In this proof-of-concept study, we studied the hypothesis that genes sharing similar three-dimensional expression profiles in the mouse brain are likely to share similar biological functions.