Alzheimer's associated variant ubiquitin causes inhibition of the 26S proteasome and chaperone expression

Intracellular protein inclusions in Alzheimer's disease and progressive supranuclear palsy contain UBB+1, a variant ubiquitin. UBB+1 is able block the 26S proteasome in cell lines. Proteasome inhibition by drug action has previously been shown to induce a heat-shock response and render protection against stress. We investigated UBB+1 by developing a stable, conditional expression model in SH-SY5Y human neuroblastoma cells. Induction of UBB+1 expression caused proteasome inhibition as was confirmed by reduced ability to process misfolded canavanyl proteins, accumulation of GFPu, a proteasome substrate, and reduced cleavage of a fluorogenic substrate. We show that expression of UBB+1 induces expression of heat-shock proteins. This priming of the chaperone system in these cells promotes a subsequent resistance to tert-butyl hydroperoxide-mediated oxidative stress. We conclude that although UBB+1-expressing cells have a compromised ubiquitin-proteasome system, they are protected against oxidative stress conditions.     

Mitochondrial apoptotic cell death and moderate superoxide generation upon selective activation of non-desensitizing AMPA receptors in hippocampal cultures

In the present work we investigated the effect of selective stimulation of non-desensitizing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in the intracellular processes leading to hippocampal neuronal death and production of reactive oxygen species (ROS). Activation of AMPA receptors in the presence of cyclothiazide (CYZ), a blocker of AMPA receptor desensitization, resulted in the death of approximately 25% of neurones, which was prevented by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(f)quinoxaline (NBQX), an AMPA-preferring receptor antagonist. (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) protected the neurones from necrotic death induced by AMPA or NMDA receptor activation. Neurodegeneration caused by selective activation of non-desensitizing AMPA receptors, in the presence of AMPA, CYZ and MK-801, significantly decreased the number of Co2+-positive neurones, used as a cytochemical marker of Ca2+-permeable AMPA receptors, but maintained intracellular ATP/ADP. The AMPA-mediated apoptotic cell death involved mitochondrial cytochrome c release and the activation of caspases-1 and -3, which was prevented by NBQX. Interestingly, although selective activation of AMPA receptors was not associated with production of intracellular peroxides, a moderate increase in superoxide production was observed upon exposure to antimycin A (AA). Furthermore, increased activity of Mn- superoxide dismutase (SOD) was observed on selective activation of non-desensitizing AMPA receptors. Taken together, these data make important contributions to the elucidation of the downstream pathways activated in AMPA receptor-mediated excitotoxicity in cultured rat hippocampal neurones.     

Productive infection maintains a dynamic steady state of residual viremia in human immunodeficiency virus type 1-infected persons treated with suppressive antiretroviral therapy for five years

To provide insight into the dynamics and source of residual viremia in human immunodeficiency virus (HIV) patients successfully treated with antiretroviral therapy, 14 intensely monitored patients treated with indinavir and efavirenz sustaining HIV RNA at <50 copies/ml for >5 years were studied. Abacavir was added to the regimen of eight patients at year 5. After the first 9 months of therapy, HIV RNA levels had reached a plateau ("residual viremia") that persisted for over 5 years. Levels of residual viremia differed among patients and ranged from 3.2 to 23 HIV RNA copies/ml. Baseline HIV DNA was the only significant pretreatment predictor of residual viremia in regression models including baseline HIV RNA, CD4 count, and patient age. In the four of five patients with detectable viremia who added abacavir to their regimen after 5 years, HIV RNA levels declined rapidly. The estimated half-life of infected cells was 6.7 days. Decrease in activated memory cells and a reduction in gamma interferon production to HIV Gag and p24 antigen in ELISpot assays were observed, consistent with a decrease in HIV replication. Thus, in patients treated with efavirenz plus indinavir, levels of residual viremia were established by 9 months, were predicted by baseline proviral DNA, and remained constant for 5 years. Even after years of highly suppressive therapy, HIV RNA levels declined rapidly after the addition of abacavir, suggesting that productive infection contributes to residual ongoing viremia and can be inhibited with therapy intensification.     

High glucose augments the angiotensin II-induced activation of JAK2 in vascular smooth muscle cells via the polyol pathway

Angiotensin II (Ang II), protein kinase C (PKC), reactive oxygen species (ROS) generated by NADPH oxidase, the activation of Janus kinase 2 (JAK2), and the polyol pathway play important parts in the hyperproliferation of vascular smooth muscle cells (VSMC), a characteristic feature of diabetic macroangiopathy. The precise mechanism, however, remains unclear. This study investigated the relation between the polyol pathway, PKC-beta, ROS, JAK2, and Ang II in the development of diabetic macroangiopathy. VSMC cultured in high glucose (HG; 25 mm) showed significant increases in the tyrosine phosphorylation of JAK2, production of ROS, and proliferation activities when compared with VSMC cultured in normal glucose (5.5 mm (NG)). Both the aldose reductase specific inhibitor (zopolrestat) or transfection with aldose reductase antisense oligonucleotide blocked the phosphorylation of JAK2, the production of ROS, and proliferation of VSMC induced by HG, but it had no effect on the Ang II-induced activation of these parameters in both NG and HG. However, transfection with PKC-beta antisense oligonucleotide, preincubation with a PKC-beta-specific inhibitor (LY379196) or apocynin (NADPH oxidase-specific inhibitor), or electroporation of NADPH oxidase antibodies blocked the Ang II-induced JAK2 phosphorylation, production of ROS, and proliferation of VSMC in both NG and HG. These observations suggest that the polyol pathway hyperactivity induced by HG contributes to the development of diabetic macroangiopathy through a PKC-beta-ROS activation of JAK2.     

Insight into the mechanism of dopamine D1-like receptor activation. Evidence for a molecular interplay between the third extracellular loop and the cytoplasmic tail

A chimeric D1A dopaminergic receptor harboring the cytoplasmic tail (CT) of the D1B subtype (D1A-CTB) has been used previously to show that CT imparts high dopamine (DA) affinity and constitutive activity to the D1B receptors. However, the D1A-CTB chimera, unlike the D1B subtype, exhibits a significantly lower DA potency for stimulating adenylyl cyclase and a drastically lower maximal binding capacity (Bmax). Here, using a functional complementation of chimeric D1-like receptors, we have identified the human D1B receptor regions regulating the intramolecular relationships that lead to an increased DA potency and contribute to Bmax. We demonstrate that the addition of variant residues of the third extracellular loop (EL3) of the human D1B receptor into D1A-CTB chimera leads to a constitutively active mutant receptor displaying an increased DA affinity, potency, and Bmax. These results strongly suggest that constitutively active D1-like receptors can adopt multiple active conformations, notably one that confers increased DA affinity with decreased DA potency and Bmax and another that imparts increased DA affinity with a strikingly increased DA potency and Bmax. Overall, we show that a novel molecular interplay between EL3 and CT regulates multiple active conformations of D1-like receptors and may have potential implications for other G protein-coupled receptor classes.     

5'-AMP-activated protein kinase controls insulin-containing secretory vesicle dynamics

Changes in 5'-AMP-activated protein kinase (AMPK) activity have recently been implicated in the control of insulin secretion by glucose (da Silva Xavier, G., Leclerc, I., Varadi, A., Tsuboi, T., Moule, S. K., and Rutter, G. A. (2003) Biochem. J. 371, 761-774). Here, we examine the possibility that activation of AMPK may regulate distal steps in insulin secretion, including vesicle movement and fusion with the plasma membrane. Vesicle dynamics were imaged in single pancreatic MIN6 beta-cells expressing lumen-targeted pH-insensitive yellow fluorescent protein, neuropeptide Y.Venus, or monomeric red fluorescent protein by total internal reflection fluorescence and Nipkow disc confocal microscopy. Overexpression of a truncated, constitutively active form of AMPK (AMPKalpha1, 1-312, T172D; AMPK CA), inhibited glucose-stimulated (30 versus 3.0 mM) vesicle movements, and decreased the number of vesicles docked or fusing at the plasma membrane, while having no effect on the kinetics of individual secretory events. Expression of the activated form of AMPK also prevented dispersal of the cortical actin network at high glucose concentrations. Monitored in permeabilized cells, where the effects of AMPK CA on glucose metabolism and ATP synthesis were bypassed, AMPK CA inhibited Ca2+ and ATP-induced insulin secretion, and decreased ATP-dependent vesicle movements. These findings suggest that components of the vesicle transport network, including vesicle-associated motor proteins, may be targets of AMPK in beta-cells, dephosphorylation of which is required for vesicle mobilization at elevated glucose concentrations.     

20 S proteasome from Saccharomyces cerevisiae is responsive to redox modifications and is S-glutathionylated

The 20 S proteasome core purified from Saccharomyces cerevisiae is inhibited by reduced glutathione (GSH), cysteine (Cys), or the GSH precursor gamma-glutamylcysteine. Chymotrypsin-like activity was more affected by GSH than trypsin-like activity, whereas the peptidylglutamyl-hydrolyzing activity (caspase-like) was not inhibited by GSH. Cys-sulfenic acid formation in the 20 S core was demonstrated by spectral characterization of the Cys-S(O)-4-nitrobenzo-2-oxa-1,3-diazole adduct, indicating that 20 S proteasome Cys residues might react with reduced sulfhydryls (GSH, Cys, and gamma-glutamylcysteine) through the oxidized Cys-sulfenic acid form. S-Glutahionylation of the 20 S core was demonstrated in vitro by GSH-biotin incorporation and by decreased alkylation with monobromobimane. Compounds such as N-ethylmaleimide (-S-sulfhydril H alkylating), dimedone (-SO sulfenic acid H reactant), or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (either -SH or -SOH reactant) highly inhibited proteasomal chymotrypsin-like activity. In vivo experiments revealed that 20 S proteasome extracted from H(2)O(2)-treated cells showed decreased chymotrypsin-like activity accompanied by S-glutathionylation as demonstrated by GSH release from the 20 S core after reduction with NaBH(4). Moreover, cells pretreated with H(2)O(2) showed decreased reductive capacity assessed by determination of the GSH/oxidized glutathione ratio and increased protein carbonyl levels. The present results indicate that at the physiological level the yeast 20 S proteasome is regulated by its sulfhydryl content, thereby coupling intracellular redox signaling to proteasome-mediated proteolysis.