DmeRF system is required for nickel and cobalt resistance in Rhizobium leguminosarum bv. viciae.

A member of the Cation Diffusion Facilitator (CDF) family with high sequence similarity to DmeF (Divalent metal efflux) from Cupridavirus metallidurans was identified in Rhizobium leguminosarum bv. viciae UPM1137. The R. leguminosarum dmeF mutant strain was highly sensitive to Co2+ and moderately sensitive to Ni2+, but its tolerance to other metals such as Zn2+, Cu2+ or Mn2+ was unaffected. An open reading frame located upstream of R. leguminosarum dmeF, designated dmeR, encodes a protein homologous to the nickel and cobalt regulator RcnR from E.coli. Expression of the dmeRF operon was induced by nickel and cobalt ions in free-living cells, likely by alleviating DmeR-mediated transcriptional repression of the operon.

Molecular physiology of nickel and cobalt homeostasis in Rhizobium leguminosarum.

Transition metals such as Fe, Cu, Mn, Ni, or Co are essential nutrients, as they are constitutive elements of a significant fraction of cell proteins. Such metals are present in the active site of many enzymes, and also participate as structural elements in different proteins. From a chemical point of view, metals have a defined order of affinity for binding, designated as the Irving-Williams series (Irving and Williams, 1948) Mg2+ menor que Mn2+ menor que Fe2+ menor que Co2+ menor que Ni2+ menor que Cu2+mayor queZn2+ Since cells contain a high number of different proteins harbouring different metal ions, a simplistic model in which proteins are synthesized and metals imported into a ?cytoplasmic soup? cannot explain the final product that we find in the cell. Instead we need to envisage a complex model in which specific ligands are present in definite amounts to leave the right amounts of available metals and protein binding sites, so specific pairs can bind appropriately. A critical control on the amount of ligands and metal present is exerted through specific metal-responsive regulators able to induce the synthesis of the right amount of ligands (essentially metal binding proteins), import and efflux proteins. These systems are adapted to establish the metal-protein equilibria compatible with the formation of the right metalloprotein complexes. Understanding this complex network of interactions is central to the understanding of metal metabolism for the synthesis of metalloenzymes, a key topic in the Rhizobium-legume symbiosis. In the case of the Rhizobium leguminosarum bv viciae (Rlv) UPM791 -Pisum sativum symbiotic system, the concentration of nickel in the plant nutrient solution is a limiting factor for hydrogenase expression, and provision of high amounts of this element to the plant nutrient solution is required to ensure optimal levels of enzyme synthesis (Brito et al., 1994).

Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake

Bacteria require nickel transporters for the synthesis of Ni-containing metalloenzymes in natural, low nickel habitats. In this work we carry out functional and topological characterization of Rhizobium leguminosarum HupE, a nickel permease required for the provision of this element for [NiFe] hydrogenase synthesis. Expression studies in the Escherichia coli nikABCDE mutant strain HYD723 revealed that HupE is a medium-affinity permease (apparent Km 227 ! 21 nM; Vmax 49 ! 21 pmol Ni2+ min"1 mg"1 bacterial dry weight) that functions as an energy-independent diffusion facilitator for the uptake of Ni(II) ions. This Ni2+ transport is not inhibited by similar cations such as Mn2+, Zn2+, or Co2+, but is blocked by Cu2+. Analysis of site-directed HupE mutants allowed the identification of several residues (H36, D42, H43, F69, E90, H130, and E133) that are essential for HupE-mediated Ni uptake in E. coli cells. By using translational fusions to reporter genes we demonstrated the presence of five transmembrane domains with a periplasmic N-terminal domain and a C-terminal domain buried in the lipid bilayer. The periplasmic N-terminal domain contributes to stability and functionality of the protein

Phosphorylation of synaptic vesicle proteins: modulation of the alpha SNAP interaction with the core complex.

We analyzed whether synaptic membrane trafficking proteins are substrates for casein kinase II, calcium/calmodulin-dependent protein kinase II, and cAMP-dependent protein kinase (PKA), three kinases implicated in the modulation of synaptic transmission. Each kinase phosphorylates a specific set of the vesicle proteins syntaxin 1A, N-ethylmaleimide-sensitive factor (NSF), vesicle-associated membrane protein (VAMP), synaptosome-associated 25-kDa protein (SNAP-25), n-sec1, alpha soluble NSF attachment protein (alpha SNAP), and synaptotagmin. VAMP is phosphorylated by calcium/calmodulin-dependent protein kinase II on serine 61. alpha SNAP is phosphorylated by PKA; however, the beta SNAP isoform is phosphorylated only 20% as efficiently. alpha SNAP phosphorylated by PKA binds to the core docking and fusion complex 10 times weaker than the dephosphorylated form. These studies provide a first glimpse at regulatory events that may be important in modulating neurotransmitter release during learning and memory.

The primary structures of the Archaeon Halobacterium salinarium blue light receptor sensory rhodopsin II and its transducer, a methyl-accepting protein.

Recently, a large family of transducer proteins in the Archaeon Halobacterium salinarium was identified. On the basis of the comparison of the predicted structural domains of these transducers, three distinct subfamilies of transducers were proposed. Here we report isolation, complete gene sequences, and analysis of the encoded primary structures of transducer gene htrII, a member of family B, and its blue light receptor gene (sopII) of sensory rhodopsin II (SRII). The start codon ATG of the 714-bp sopII gene is one nucleotide beyond the termination codon TGA of the 2298-bp htrII gene. The deduced protein sequence of HtrII predicts a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm. HtrII has a common feature with HtrI, the sensory rhodopsin I transducer; like HtrI, HtrII possesses a hydrophilic loop structure just after the second transmembrane segment. The C-terminal 299 residues (765 amino acid residues total) of HtrII show strong homology to the signaling and methylation domain of eubacterial transducer Tsr. The hydropathy plot of the primary structure of SRII indicates seven membrane-spanning alpha-helical segments, a characteristic feature of retinylidene proteins ("rhodopsins") from a widespread family of photoactive pigments. SRII shows high identity with SRI (42%), bacteriorhodopsin (BR) (32%), and halorhodopsin (24%). The crucial positions for retinal binding sites in these proteins are nearly identical, with the exception of Met-118 (numbering according to the mature BR sequence), which is replaced by Val in SRII. In BR, residues Asp-85 and Asp-96 are crucial in proton pumping. In SRII, the position corresponding to Asp-85 in BR is conserved, but the corresponding position of Asp-96 is replaced by an aromatic Tyr. Coexpression of the htrII and sopII genes restores SRII phototaxis to a mutant (Pho81) that contains a deletion in the htrI/sopI and insertion in htrII/sopII regions. This paper describes the first example that both HtrI and HtrII exist in the same halobacterial cell, confirming that different sensory rhodopsins SRI and SRII in the same organism have their own distinct transducers.

Transcriptionally active Stat1 is required for the antiproliferative effects of both interferon alpha and interferon gamma.

Type I (alpha, beta) and type II (gamma) interferons (IFNs) can restrict the growth of many cell types. INF-stimulated gene transcription, a key early event in IFN response, acts through the Janus kinase-signal transducers and activators of transcription pathway, in which both IFN-alpha and IFN-gamma activate the transcription factor Stat1. A cell line lacking Stat1 (U3A) was not growth-arrested by IFN-alpha or IFN-gamma, and experiments were carried out with U3A cells permanently expressing normal or various mutant forms of Stat1 protein. Only cells in which complete Stat1 activity was available (Stat1alpha) were growth-inhibited by IFN-gamma. A mutant that supports 20-30% normal transcription did not cause growth restraint. In contrast, IFN-alpha growth restraint was imposed by cells producing Stat1beta, which lacks transcriptional activation potential. This parallels earlier results showing the truncated Stat1 can function in IFN-alpha gene activation. In addition to experiments on long-term cultured cells, we also found that wild-type primary mouse embryonic fibroblasts were inhibited by IFNs, but fibroblasts from Stat1-deficient mouse embryos were not inhibited by IFNs.

An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective.

Overactivation of calcium-activated neutral protease (calpain) has been implicated in the pathophysiology of several degenerative conditions, including stroke, myocardial ischemia, neuromuscular degeneration, and cataract formation. Alpha-mercaptoacrylate derivatives (exemplified by PD150606), with potent and selective inhibitory actions against calpain, have been identified. PD150606 exhibits the following characteristics: (i) Ki values for mu- and m-calpains of 0.21 microM and 0.37 microM, respectively, (ii) high specificity for calpains relative to other proteases, (iii) uncompetitive inhibition with respect to substrate, and (iv) it does not shield calpain against inactivation by the active-site inhibitor trans-(epoxysuccinyl)-L-leucyl-amido-3-methylbutane, suggesting a nonactive site action for PD150606. The recombinant calcium-binding domain from each of the large or small subunits of mu-calpain was found to interact with PD150606. In low micromolar range, PD15O6O6 inhibited calpain activity in two intact cell systems. The neuroprotective effects of this class of compound were also demonstrated by the ability of PD150606 to attenuate hypoxic/hypoglycemic injury to cerebrocortical neurons in culture and excitotoxic injury to Purkinje cells in cerebellar slices.

Peptide analogs to pathogenic epitopes of the human acetylcholine receptor alpha subunit as potential modulators of myasthenia gravis.

Myasthenia gravis is an autoimmune disease in which T cells specific to epitopes of the autoantigen, the human acetylcholine receptor, play a role. We identified two peptides, p195-212 and p259-271, from the alpha subunit of the receptor, which bound to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) from peripheral blood lymphocytes of myasthenia gravis patients and stimulated lymphocytes of >80% of the patients. We have prepared analogs of these myasthenogenic peptides and tested their ability to bind to MHC class II determinants and to interfere specifically with T-cell stimulation. We first determined relative binding efficiency of the myasthenogenic peptides and their analogs to APCs of patients. We found that single substituted analogs of p195-212 (Ala-207) and p259-271 (Lys-262) could bind to human MHC molecules on APCs as efficiently as the original peptides. Moreover, dual analogs containing the two single substituted analogs in one stretch (either sequentially, Ala-207/Lys-262, or reciprocally, Lys-262/Ala-207) could also bind to APCs of patients, including those that failed to bind one of the single substituted analogs. The single substituted analogs significantly inhibited T-cell stimulation induced by their respective myasthenogenic peptides in >95% of the patients. The dual analogs were capable of inhibiting stimulation induced by either of the peptides: They inhibited the response to p195-212 and p259-271 in >95% and >90% of the patients, respectively. Thus, the dual analogs are good candidates for inhibition of T-cell responses of myasthenia gravis patients and might have therapeutic potential.

A functional Rev-erb alpha responsive element located in the human Rev-erb alpha promoter mediates a repressing activity.

Rev-erb alpha belongs to the nuclear receptor superfamily, which contains receptors for steroids, thyroid hormones, retinoic acid, and vitamin D, as well as "orphan" receptors. No ligand has been found for Rev-erb alpha to date, making it one of these orphan receptors. Similar to some other orphan receptors, Rev-erb alpha has been shown to bind DNA as a monomer on a specific sequence called a Rev-erb alpah responsive element (RevRE), but its transcriptional activity remains unclear. In this paper, we characterize a functional RevRE located in the human Rev-erb alpha promoter itself. We also present evidence that (i) Rev-erb alpha mediates transcriptional repression of its own promoter in vitro, (ii) this repressing effect strictly depends on the binding of Rev-erb alpha to its responsive element and is transferable to a heterologous promoter; and (iii) Rev-erb alpha binds to this responsive sequence as a homodimer.