β Cell-Specific Deletion of the IL-1 Receptor Antagonist Impairs β Cell Proliferation and Insulin Secretion

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Hepatitis C Virus Impairs p53 via Persistent Overexpression of 3��-Hydroxysterol ��24-Reductase

Persistent infection with hepatitis C virus (HCV) induces tumorigenicity in hepatocytes. To gain insight into the mechanisms underlying this process, we generated monoclonal antibodies on a genome-wide scale against an HCV-expressing human hepatoblastoma-derived cell line, RzM6-LC, showing augmented tumorigenicity. We identified 3β-hydroxysterol Δ24-reductase (DHCR24) from this screen and showed that its expression reflected tumorigenicity. HCV induced the DHCR24 overexpression in human hepatocytes. Ectopic or HCV-induced DHCR24 overexpression resulted in resistance to oxidative stress-induced apoptosis and suppressed p53 activity. DHCR24 overexpression in these cells paralleled the increased interaction between p53 and MDM2 (also known as HDM2), a p53-specific E3 ubiquitin ligase, in the cytoplasm. Persistent DHCR24 overexpression did not alter the phosphorylation status of p53 but resulted in decreased acetylation of p53 at lysine residues 373 and 382 in the nucleus after treatment with hydrogen peroxide. Taken together, these results suggest that DHCR24 is elevated in response to HCV infection and inhibits the p53 stress response by stimulating the accumulation of the MDM2-p53 complex in the cytoplasm and by inhibiting the acetylation of p53 in the nucleus.     

Modeling Effect of a ��-Secretase Inhibitor on Amyloid-�� Dynamics Reveals Significant Role of an Amyloid Clearance Mechanism

Aggregation of the small peptide amyloid beta (A??) into oligomers and fibrils in the brain is believed to be a precursor to Alzheimer??s disease. A?? is produced via multiple proteolytic cleavages of amyloid precursor protein (APP), mediated by the enzymes ??- and ??-secretase. In this study, we examine the temporal dynamics of soluble (unaggregated) A?? in the plasma and cerebral-spinal fluid (CSF) of rhesus monkeys treated with different oral doses of a ??-secretase inhibitor. A dose-dependent reduction of A?? concentration was observed within hours of drug ingestion, for all doses tested. A?? concentration in the CSF returned to its predrug level over the monitoring period. In contrast, A?? concentration in the plasma exhibited an unexpected overshoot to as high as 200% of the predrug concentration, and this overshoot persisted as late as 72 hours post-drug ingestion. To account for these observations, we proposed and analyzed a minimal physiological model for A?? dynamics that could fit the data. Our analysis suggests that the overshoot arises from the attenuation of an A?? clearance mechanism, possibly due to the inhibitor. Our model predicts that the efficacy of A?? clearance recovers to its basal (pretreatment) value with a characteristic time of >48 hours, matching the time-scale of the overshoot. These results point to the need for a more detailed investigation of soluble A?? clearance mechanisms and their interaction with A??-reducing drugs.     

Cancer Preventive Isothiocyanates Induce Selective Degradation of Cellular ��- and ��-Tubulins by Proteasomes

Although it is conceivable that cancer preventive isothiocyanates (ITCs), a family of compounds in cruciferous vegetables, induce cell cycle arrest and apoptosis through a mechanism involving oxidative stress, our study shows that binding to cellular proteins correlates with their potencies of apoptosis induction. More recently, we showed that ITCs bind selectively to tubulins. The differential binding affinities toward tubulin among benzyl isothiocyanate, phenethyl isothiocyanate, and sulforaphane correlate well with their potencies of inducing tubulin conformation changes, microtubule depolymerization, and eventual cell cycle arrest and apoptosis in human lung cancer A549 cells. These results support that tubulin binding by ITCs is an early event for cell growth inhibition. Here we demonstrate that ITCs can selectively induce degradation of both α- and β-tubulins in a variety of human cancer cell lines in a dose- and time-dependent manner. The onset of degradation, a rapid and irreversible process, is initiated by tubulin aggregation, and the degradation is proteasome-dependent. Results indicate that the degradation is triggered by ITC binding to tubulin and is irrelevant to oxidative stress. This is the first report that tubulin, a stable and abundant cytoskeleton protein required for cell cycle progression, can be selectively degraded by a small molecule.Microtubules as a major cytoskeleton component in all eukaryotic cells play essential roles such as maintenance of cell polarity, intracellular traffic, organization, and cell motility (1–4). During cell division, the microtubule-formed mitotic spindle ensures the replicated chromosomes separate evenly at the end of the mitotic phase to the two daughter cells (1). It is because of its essential roles in cell growth that microtubules become a valid target for the development of anti-microtubule drugs against the rapidly growing cancer cells (2), as interference of microtubule dynamics arrests cell cycle progression and induces apoptosis (3). Therefore, microtubules have been considered one of the best targets to date for cancer chemotherapy (4).Isothiocyanates (ITCs)³ are among the best studied chemopreventive small molecules (5). The three most studied ITCs, including benzyl-ITC (BITC; abundant in garden cress), phenethyl-ITC (PEITC; in watercress), and sulforaphane (SFN; in broccoli sprouts), have been shown to induce apoptosis and cell cycle arrest (5–8). Although it is believed that oxidative stress plays a role in cell cycle arrest and apoptosis induced by ITCs (6–12), we found that binding to proteins is a predominant intracellular chemical reaction of ITCs, and their protein binding affinities correlate well with inhibition of cell proliferation and induction of apoptosis (13). Recently, we identified tubulin, the microtubule constituent, as an in vivo target of ITCs by two-dimensional gel electrophoresis and mass spectrometry (14). The growth inhibition of human non-small lung cancer A549 cells by ITCs followed the order of BITC > PEITC > SFN. The same order of potency was seen in their binding affinities toward tubulin, induction of its conformational changes, and inhibition of its polymerization. The study provides the first evidence of an in vivo ITC-tubulin binding adduct, indicating that direct modification of cysteines in tubulin by ITCs, rather than oxidative stress, may trigger cell cycle arrest and apoptosis.Here we report an unexpected novel finding that tubulin is selectively degraded in a variety of human cancer cells treated with ITCs. We provide evidence that the degradation is initiated by its binding with ITCs and mediated by the ubiquitin-proteasome pathway. Tubulin has long been viewed as a stable and abundant protein, and its levels in cells are tightly regulated (15). In the literature, the only studies on cellular tubulin level change are related to “the auto-regulation theory,” i.e. when microtubules collapse, the presence of a massive amount of tubulin monomers would selectively destabilize tubulin mRNA and subsequently decrease tubulin protein synthesis (16–18). To our knowledge, there is no report on tubulin degradation as a result of treatment with any agents. Our studies provide strong evidence that supports tubulin as a target of ITCs for cell growth inhibition, pointing to a new mechanism for the anti-microtubule or anti-mitosis effects of ITCs through covalent binding to tubulin and presenting a platform to study protein stability by modification with small molecules.     

Demonstration of a Direct Interaction between β2-Adrenergic Receptor and Insulin Receptor by BRET and Bioinformatics

Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and β₂-adrenergic receptor (β₂AR), which represent the receptor tyrosine kinases (RTKs) and seven transmembrane receptors (7TMRs), respectively. Studies demonstrating cross-talk between these two receptors and their endogenous coexpression have suggested their possible interactions. To evaluate the effect of IR and prospective heteromerization on β₂AR properties, we showed that IR coexpression had no effect on the ligand binding properties of β₂AR; however, IR reduced β₂AR surface expression and accelerated its internalization. Additionally, both receptors displayed a similar distribution pattern with a high degree of colocalization. To test the possible direct interaction between β₂AR and IR, we employed quantitative BRET² saturation and competition assays. Saturation assay data suggested constitutive β₂AR and IR homo- and heteromerization. Calculated acceptor/donor (AD₅₀) values as a measure of the relative affinity for homo- and heteromer formation differed among the heteromers that could not be explained by a simple dimer model. In heterologous competition assays, a transient increase in the BRET² signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation. To complement the BRET² data, we employed the informational spectrum method (ISM), a virtual spectroscopy method to investigate protein-protein interactions. Computational peptide scanning of β₂AR and IR identified intracellular domains encompassing residues at the end of the 7^th TM domain and C-terminal tail of β₂AR and a cytoplasmic part of the IR β chain as prospective interaction domains. ISM further suggested a high probability of heteromer formation and homodimers as basic units engaged in heteromerization. In summary, our data suggest direct interaction and higher-order β₂AR:IR oligomer formation, likely comprising heteromers of homodimers.     

A Residue in Loop 9 of the ��2-Subunit Stabilizes the Closed State of the GABAA Receptor

In γ-aminobutyric acid type A (GABA_A) receptors, the structural elements that couple ligand binding to channel opening remain poorly defined. Here, site-directed mutagenesis was used to determine if Loop 9 on the non-GABA binding site interface of the β2-subunit may be involved in GABA_A receptor activation. Specifically, residues Gly¹⁷⁰-Gln¹⁸⁵ of the β2-subunit were mutated to alanine, co-expressed with wild-type α1- and γ2S-subunits in human embryonic kidney (HEK) 293 cells and assayed for their activation by GABA, the intravenous anesthetic propofol and the endogenous neurosteroid pregnanolone using whole cell macroscopic recordings. Three mutants, G170A, V175A, and G177A, produced 2.5-, 6.7-, and 5.6-fold increases in GABA EC₅₀ whereas one mutant, Q185A, produced a 5.2-fold decrease in GABA EC₅₀. None of the mutations affected the ability of propofol or pregnanolone to potentiate a submaximal GABA response, but the Q185A mutant exhibited 8.3- and 3.5-fold increases in the percent direct activation by propofol and pregnanolone, respectively. Mutant Q185A receptors also had an increased leak current that was sensitive to picrotoxin, indicating an increased gating efficiency. Further Q185E, Q185L, and Q185W substitutions revealed a strong correlation between the hydropathy of the amino acid at this position and the GABA EC₅₀. Taken together, these results indicate that β2 Loop 9 is involved in receptor activation by GABA, propofol, and pregnanolone and that β2(Q185) participates in hydrophilic interactions that are important for stabilizing the closed state of the GABA_A receptor.     

Phosphoinositide 3-Kinase p110�� Regulates Integrin ��IIb��3 Avidity and the Cellular Transmission of Contractile Forces

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin α_IIbβ₃, necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin α_IIbβ₃ activation (affinity modulation) primarily occurs downstream of G_i-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin α_IIbβ₃ bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110β isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin α_IIbβ₃ activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin α_IIbβ₃ association with the contractile cytoskeleton. Analysis of integrin α_IIbβ₃ adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin α_IIbβ₃ bonds. These studies demonstrate an important role for PI3K p110β in regulating the avidity of high affinity integrin α_IIbβ₃ receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110β inhibitors.     

Brain Microvascular Endothelial Cell Derived Exosomes Potently Ameliorate Cognitive Dysfunction by Enhancing the Clearance of Aβ Through Up-Regulation of P-gp in Mouse Model of AD

Neurochemical Research - Accumulation of amyloid-β (Aβ) peptides in the brain is regarded as a major contributor to the pathogenesis and progression of Alzheimer's disease (AD)....     

A Synthetic Combinatorial Strategy for Developing ��-Conotoxin Analogs as Potent ��7 Nicotinic Acetylcholine Receptor Antagonists

α-Conotoxins are peptide neurotoxins isolated from venomous cone snails that display exquisite selectivity for different subtypes of nicotinic acetylcholine receptors (nAChR). They are valuable research tools that have profound implications in the discovery of new drugs for a myriad of neuropharmacological conditions. They are characterized by a conserved two-disulfide bond framework, which gives rise to two intervening loops of extensively mutated amino acids that determine their selectivity for different nAChR subtypes. We have used a multistep synthetic combinatorial approach using α-conotoxin ImI to develop potent and selective α₇ nAChR antagonists. A positional scan synthetic combinatorial library was constructed based on the three residues of the n-loop of α-conotoxin ImI to give a total of 10,648 possible combinations that were screened for functional activity in an α₇ nAChR Fluo-4/Ca²⁺ assay, allowing amino acids that confer antagonistic activity for this receptor to be identified. A second series of individual α-conotoxin analogs based on the combinations of defined active amino acid residues from positional scan synthetic combinatorial library screening data were synthesized. Several analogs exhibited significantly improved antagonist activity for the α₇ nAChR compared with WT-ImI. Binding interactions between the analogs and the α₇ nAChR were explored using a homology model of the amino-terminal domain based on a crystal structure of an acetylcholine-binding protein. Finally, a third series of refined analogs was synthesized based on modeling studies, which led to several analogs with refined pharmacological properties. Of the 96 individual α-conotoxin analogs synthesized, three displayed ≥10-fold increases in antagonist potency compared with WT-ImI.