Synapsin associates with cyclophilin B in an ATP- and cyclosporin A-dependent manner

Immunophilins are ubiquitous enzymes responsible for proline isomerisation during protein synthesis and for the chaperoning of several membrane proteins. These activities can be blocked by the immunosuppressants cyclosporin A, FK506 and rapamycin. It has been shown that all three immunosuppressants have neurotrophic activity and can modulate neurotransmitter release, but the molecular basis of these effects is currently unknown. Here, we show that synapsin I, a synaptic vesicle-associated protein, can be purified from Torpedo cholinergic synaptosomes through its affinity to cyclophilin B, an immunophilin that is particularly abundant in brain. The interaction is direct and conserved in mammals, and shows a dissociation constant of about 0.5 microM in vitro. The binding between the two proteins can be disrupted by cyclosporin A and inhibited by physiological concentrations of ATP. Furthermore, cyclophilin B co-localizes with synapsin I in rat synaptic vesicle fractions and its levels in synaptic vesicle-containing fractions are decreased in synapsin knockout mice. These results suggest that immunophilins are involved in the complex protein networks operating at the presynaptic level and implicate the interaction between cyclophilin B and synapsins in presynaptic function.     

Cholesterol-dependent separation of the beta2-adrenergic receptor from its partners determines signaling efficacy: insight into nanoscale organization of signal transduction

Determining the role of lipid raft nanodomains in G protein-coupled receptor signaling remains fraught by the lack of assays directly monitoring rafts in native membranes. We thus combined extensive biochemical and pharmacological approaches to a nanoscale strategy based on bioluminescence resonance energy transfer (BRET) to assess the spatial and functional influence of cholesterol-rich liquid-ordered lipid nanodomains on beta(2) adrenergic receptor (beta(2)AR) signaling. The data revealed that whereas beta(2)AR did not partition within liquid-ordered lipid phase, a pool of G protein and adenylyl cyclase (AC) were sequestered in these domains. Destabilization of the liquid-ordered phase by cholesterol depletion led to a lateral redistribution of Galpha(s) and AC that favored interactions between the receptor and its signaling partners as assessed by BRET. This resulted in an increased basal and agonist-promoted beta(2)AR-stimulated cAMP production that was partially dampened as a result of constitutive protein kinase A-dependent phosphorylation and desensitization of the receptor. This restraining influence of nanodomains on beta(2)AR signaling was further substantiated by showing that liquid-ordered lipid phase stabilization using caveolin overexpression or increasing membrane cholesterol amount led to an inhibition of beta(2)AR-associated signaling. Given the emerging concept that clustering of receptors and effectors into signaling platforms contributes to the efficacy and selectivity of signal transduction, our results support a model whereby cholesterol-promoted liquid-ordered lipid phase-embedding G(s) and AC allows their lateral separation from the receptor, thus restraining the basal activity and controlling responsiveness of beta(2)AR signaling machinery within larger signaling platforms.     

Detection of sugar-binding proteins in membrane-depleted nuclei

Nuclear sugar-binding proteins were detected in membrane-depleted nuclei isolated from hamster BHK cells and mouse L 1210 leukemia cells by means of fluorescein-labelled neoglycoproteins. In fluorescence microscopy, the fluorescence was seen throughout the nucleus but was generally brighter over the nucleoli than over the rest of the nucleus. Flow cytofluorometry analysis demonstrated the presence of nuclear sugar-binding proteins for synthetic glycoproteins associated with different sugar residues. Among the nine neoglycoproteins used, four neoglycoproteins (namely alpha-rhamnosylated, alpha-glucosylated, N-acetyl-beta-glucosaminylated and alpha-mannosylated-6P-serum albumin) strongly labelled nuclei. Various controls strongly argue for the specificity of the nuclear labelling. The possibility that some of the sugar-binding proteins might correspond to endogenous nuclear lectins is considered.     

Translation initiator EIF4G1 mutations in familial Parkinson disease

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.     

Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity

The monomeric model of rhodopsin-like G protein-coupled receptors (GPCRs) has progressively yielded the floor to the concept of GPCRs being oligo(di)mers, but the functional correlates of dimerization remain unclear. In this report, dimers of glycoprotein hormone receptors were demonstrated in living cells, with a combination of biophysical (bioluminescence resonance energy transfer and homogenous time resolved fluorescence/fluorescence resonance energy transfer), functional and biochemical approaches. Thyrotropin (TSHr) and lutropin (LH/CGr) receptors form homo- and heterodimers, via interactions involving primarily their heptahelical domains. The large hormone-binding ectodomains were dispensable for dimerization but modulated protomer interaction. Dimerization was not affected by agonist binding. Observed functional complementation indicates that TSHr dimers may function as a single functional unit. Finally, heterologous binding-competition studies, performed with heterodimers between TSHr and LH/CG-TSHr chimeras, demonstrated the unsuspected existence of strong negative cooperativity of hormone binding. Tracer desorption experiments indicated an allosteric behavior in TSHr and, to a lesser extent, in LH/CGr and FSHr homodimers. This study is the first report of homodimerization associated with negative cooperativity in rhodopsin-like GPCRs. As such, it may warrant revisitation of allosterism in the whole GPCR family.     

Structural aspects of intranuclear matrix disintegration upon RNase digestion of HeLa cell nuclei

The organization of the intranuclear elements observed in histone-depleted (2 M NaCl-extracted) HeLa cell nuclei was investigated by means of electron microscopy and two-dimensional gel electrophoresis. This work was mainly aimed at verifying whether or not an intranuclear skeleton or matrix existed, which could explain the stable attachment of RNA to the residual nuclear structure after high-salt extraction, and its three-dimensional organization. We compared the ultrastructure and the polypeptide composition of RNA-containing and RNA-depleted (RNase-treated) nuclear residues, and we visualized intermediate stages of RNase action on the intranuclear material. We showed that this material was made of two types (fibrillar and granular) of salt-resistant RNP components equally sensitive to RNase when the enzyme was used prior to high-salt extraction. At least in our material and under our experimental conditions, no intranuclear matrix could be distinguished from the residual RNP material. Our results further suggest that formation of such a matrix is a path-dependent phenomenon.     

Localization of NADPH-protochlorophyllide reductase in plastids of barley at different greening stages

The localization of protochorophyllide (Pchlide) and of NADPH-protochlorophyllide oxidoreductase (POR, EC 1.6.99.1) within (etio)chloroplasts has been investigated at selected stages of greening of barley seedlings. Pchlide pigment and POR protein contents were evaluated in different plastid membrane fractions by fluorescence spectroscopy and immunoblot analysis using a monospecific polyclonal antibody raised against the purified enzyme. Fluorescence analysis showed the presence of Pchlide in both the envelope and thylakoid membranes. During greening, the Pchlide content, expressed on a total protein basis, decreased in thylakoid membranes, whereas it increased in the envelope membranes. POR proteins were detected mainly in thylakoid membranes at early greening stages. In contrast, the weak amount of POR proteins was associated more specifically with envelope membranes of mature chloroplasts. Whatever the greening stage, thylakoid-bound Pchlide and POR proteins were more abundant in the thylakoid regions which remained unsolubilized after mild Triton treatment used as standard procedure to prepare PS II particles. This suggests the preferential association of Pchlide and POR to the appressed regions of thylakoids. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cutting Edge: A dual role for type I IFNs during polyinosinic-polycytidylic acid-induced NK cell activation

NK cells are cytotoxic lymphocytes that are most efficient at fulfilling their functions after a phase of priming provided by cytokines and/or accessory cells. Although type I IFNs are known to be important in this process, it remains unclear whether they act directly on NK cells or indirectly on accessory cells. We used adoptive transfer experiments and mixed bone marrow chimeras to dissect the requirement for type I IFN signaling in response to the dsRNA analog polyinosinic-polycytidylic acid. We demonstrate that optimal NK cell priming requires type I IFNs to signal on both NK cells and accessory cells. In the absence of IL-15, the residual NK cell activation was strictly dependent on cell-intrinsic IFNAR signaling in NK cells. Our results suggest that type I IFNs produced following viral infection simultaneously target accessory cells for IL-15 transpresentation and NK cells themselves and that these two pathways cooperate for NK cell priming.     

The ubiquitin-proteasome and the mitochondria-associated apoptotic pathways are sequentially downregulated during recovery after immobilization-induced muscle atrophy

Immobilization produces morphological, physiological, and biochemical alterations in skeletal muscle leading to muscle atrophy and long periods of recovery. Muscle atrophy during disuse results from an imbalance between protein synthesis and proteolysis but also between apoptosis and regeneration processes. This work aimed to characterize the mechanisms underlying muscle atrophy and recovery following immobilization by studying the regulation of the mitochondria-associated apoptotic and the ubiquitin-proteasome-dependent proteolytic pathways. Animals were subjected to hindlimb immobilization for 4-8 days (I4 to I8) and allowed to recover after cast removal for 10-40 days (R10 to R40). Soleus and gastrocnemius muscles atrophied from I4 to I8 to a greater extent than extensor digitorum longus and tibialis anterior muscles. Gastrocnemius muscle atrophy was first stabilized at R10 before being progressively reduced until R40. Polyubiquitinated proteins accumulated from I4, whereas the increased ubiquitination rates and chymotrypsin-like activity of the proteasome were detectable from I6 to I8. Apoptosome and caspase-3 or -9 activities increased at I6 and I8, respectively. The ubiquitin-proteasome-dependent pathway was normalized early when muscle stops to atrophy (R10). By contrast, the mitochondria-associated apoptotic pathway was first downregulated below basal levels when muscle started to recover at R15 and completely normalized at R20. Myf 5 protein levels decreased from I4 to I8 and were normalized at R10. Altogether, our results suggest a two-stage process in which the ubiquitin-proteasome pathway is rapidly up- and downregulated when muscle atrophies and recovers, respectively, whereas apoptotic processes may be involved in the late stages of atrophy and recovery.