Conformational preferences of modified nucleoside N(2)-methylguanosine (m(2)G) and its derivative N(2), N(2)-dimethylguanosine (m(2)(2)G) occur at 26th position (hinge region) in tRNA

Conformational preferences of the modified nucleosides N²-methylguanosine (m²G) and N², N²-dimethylguanosine (m₂²G) have been studied theoretically by using quantum chemical perturbative configuration interaction with localized orbitals (PCILO) method. Automated complete geometry optimization using semiempirical quantum chemical RM1, along with ab initio molecular orbital Hartree–Fock (HF-SCF), and density functional theory (DFT) calculations has also been made to compare the salient features. Single-point energy calculation studies have been made on various models of m²G26:C/A/U44 and m₂²G26:C/A/U44. The glycosyl torsion angle prefers “syn” (χ = 286°) conformation for m²G and m₂²G molecules. These conformations are stabilized by N(3)–HC2′ and N(3)–HC3′ by replacing weak interaction between O5′–HC(8). The N²-methyl substituent of (m²G26) prefers “proximal” or s-trans conformation. It may also prefer “distal” or s-cis conformation that allows base pairing with A/U44 instead of C at the hinge region. Thus, N²-methyl group of m²G may have energetically two stable s-trans m²G:C/A/U or s-cis m²G:A/U rotamers. This could be because of free rotations around C–N bond. Similarly, N², N²-dimethyl substituent of (m₂²G) prefers “distal” conformation that may allow base pairing with A/U instead of C at 44th position. Such orientations of m²G and m₂²G could play an important role in base-stacking interactions at the hinge region of tRNA during protein biosynthesis process.     

Distribution of G1m(1), G1m(2) and G3m(5) allotypes in Aragon.

The distribution of G1m(1), G1m(2) and G3m(5) allotypes was studied in 700 unrelated individuals from Aragon (North-East Spain). The Gm haplotype frequencies were similar to those reported in French areas next to Aragon.     

G1m(1) and G1m(2) allotypes in Albanian towns of Calabria

The G1m(1) and G1m(2) allotype distribution was analyzed in a population sample from 11 Albanian towns of Calabria. The unusually high frequency of the G1m(1) marker already observed in Calabria as well as the presence of the Gm(2) phenotype were shown. The Calabrian and Albanian populations were similar, but significantly different from other Italian populations.     

Bidirectional Control of Postsynaptic Density-95 (PSD-95) Clustering by Huntingtin

Huntington disease is associated with early alterations in corticostriatal synaptic function that precede cell death, and it is postulated that ameliorating such changes may delay clinical onset and/or prevent neurodegeneration. Although many of these synaptic alterations are thought to be attributable to a toxic gain of function of the mutant huntingtin protein, the role that nonpathogenic huntingtin (HTT) plays in synaptic function is relatively unexplored. Here, we compare the immunocytochemical localization of a major postsynaptic scaffolding protein, PSD-95, in striatal neurons from WT mice and mice overexpressing HTT with 18 glutamine repeats (YAC18, nonpathogenic). We found that HTT overexpression resulted in a palmitoylation- and BDNF-dependent increase in PSD-95 clustering at synaptic sites in striatal spiny projection neurons (SPNs) co-cultured with cortical neurons. Surprisingly, the latter effect was mediated presynaptically, as HTT overexpression in cortical neurons alone was sufficient to increase PSD-95 clustering in the postsynaptic SPNs. In contrast, antisense oligonucleotide knockdown of HTT in WT co-cultures resulted in a significant reduction of PSD-95 clustering in SPNs. Notably, despite these bidirectional changes in PSD-95 clustering, we did not observe an alteration in basal electrophysiological measures of AMPA and NMDA receptors. Thus, unlike in previous studies in the hippocampus, enhanced or decreased PSD-95 clustering alone was insufficient to drive AMPA or NMDA receptors into or out of SPN synapses. In all, our results demonstrate that nonpathogenic HTT can indeed influence synaptic protein localization and uncover a novel role of HTT in PSD-95 distribution.     

Increased levels of Wee-1 kinase in G(2) are necessary for Vpr- and gamma irradiation-induced G(2) arrest

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle arrest at the G(2)/M transition and subsequently apoptosis. Here we examined the potential involvement of Wee-1 in Vpr-induced G(2) arrest. Wee-1 is a cellular protein kinase that inhibits Cdc2 activity, thereby preventing cells from proceeding through mitosis. We previously showed that the levels of Wee-1 correlate with Vpr-mediated apoptosis. Here, we demonstrate that Vpr-induced G(2) arrest correlated with delayed degradation of Wee-1 at G(2)/M. Experimental depletion of Wee-1 by a small interfering RNA directed to wee-1 mRNA alleviated Vpr-induced G(2) arrest and allowed apparently normal progression through M into G(1). Similar results were observed when cells were arrested at G(2) following gamma irradiation. Thus, Wee-1 is integrally involved as a key cellular regulatory protein in the signal transduction pathway for HIV-1 Vpr-induced cell cycle arrest.     

G Protein binding sites on Calnuc (nucleobindin 1) and NUCB2 (nucleobindin 2) define a new class of G(alpha)i-regulatory motifs

Heterotrimeric G proteins are molecular switches modulated by families of structurally and functionally related regulators. GIV (Gα-interacting vesicle-associated protein) is the first non-receptor guanine nucleotide exchange factor (GEF) that activates Gα(i) subunits via a defined, evolutionarily conserved motif. Here we found that Calnuc and NUCB2, two highly homologous calcium-binding proteins, share a common motif with GIV for Gα(i) binding and activation. Bioinformatics searches and structural analysis revealed that Calnuc and NUCB2 possess an evolutionarily conserved motif with sequence and structural similarity to the GEF sequence of GIV. Using in vitro pulldown and competition assays, we demonstrate that this motif binds preferentially to the inactive conformation of Gα(i1) and Gα(i3) over other Gα subunits and, like GIV, docks onto the α3/switch II cleft. Calnuc binding was impaired when Lys-248 in the α3 helix of Gα(i3) was replaced with M, the corresponding residue in Gα(o), which does not bind to Calnuc. Moreover, mutation of hydrophobic residues in the conserved motif predicted to dock on the α3/switch II cleft of Gα(i3) impaired the ability of Calnuc and NUCB2 to bind and activate Gα(i3) in vitro. We also provide evidence that calcium binding to Calnuc and NUCB2 abolishes their interaction with Gα(i3) in vitro and in cells, probably by inducing a conformational change that renders the Gα(i)-binding residues inaccessible. Taken together, our results identify a new type of Gα(i)-regulatory motif named the GBA motif (for Gα-binding and -activating motif), which is conserved across different proteins throughout evolution. These findings provide the structural basis for the properties of Calnuc and NUCB2 binding to Gα subunits and its regulation by calcium ions.     

RDJ2 (DNAJA2) chaperones neural G protein signaling pathways

A number of structurally divergent proteins with J domains, called J proteins, interact with and activate the ATPase of Hsp70s, thereby harnessing the ATPase activity for conformational work on target proteins. The precise role of most mammalian J proteins remains undefined. In this paper, we demonstrate that transient expression of the J protein, Rdj2, in HEK 293 cells increased cellular cyclic adenosine monophosphate (cAMP) levels in the presence of the β-adrenergic agonist isoproterenol. In CNS-derived catecholaminergic neuronal cell line (CAD) neuroblastoma cells, expression of Rdj2 increased isoproterenol-stimulated phosphorylation of cAMP response element binding protein (CREB). Moreover, we have characterized the binding properties of Rdj2 and observed a direct interaction between Rdj2 and receptor-coupled trimeric GTP-binding proteins (G proteins). We further show that the composition of the Rdj2-chaperone complex and the cysteine string protein (CSPα)-chaperone complex, another J protein, is distinct. Our data demonstrate that Rdj2 modulates G protein signaling and further suggest that chaperoning G proteins is an emerging theme of the J protein network.     

Activation of Brain L-glutamate Decarboxylase 65 Isoform (GAD65) by Phosphorylation at Threonine 95 (T95)

Protein phosphorylation plays an important role in regulating soluble L-glutamic acid decarboxylase (GAD) and membrane-associated GAD activity. Previously, we reported the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 (hGAD65) and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrated that protein kinase A (PKA) and protein kinase C isoform ε were the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. In the current study, using MALDI-TOF, a total of four potential phosphorylation sites were identified in GAD65, two of which (threonine-95 (T-95) and Ser-417) were not reported previously. We have identified one specific phosphorylation site, (T95), in hGAD65 that can be phosphorylated by kinase C ε (PKCε) using MALDITOF. When T95 is mutated to alanine, hGAD65 could no longer be phosphorylated by PKCε, and the effect of PKC-mediated activation on hGAD65 is abolished. However, when T95 is mutated to glutamic acid, which mimics the phosphorylation status of hGAD65, the activity was greatly increased. An increase of GAD65 activity by 55 % compared to the wild type hGAD65 was observed indicating that mutation of T95 to glutamic acid mimics the effect of phosphorylation. A model depicting the role of phosphorylation of GAD65 in regulation of GABA neurotransmission is presented.     

The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems

Heat shock protein 70 (hsp70) is a stress-inducible protein that prevents apoptosis induced by a wide range of cytotoxic agents by an as yet undefined mechanism. The caspase family of cysteine proteases have been attributed a central role in the execution of apoptosis. However, several cases of caspase-independent apoptosis have been recently reported, suggesting that caspases may not be necessary for apoptosis in all cells. This study examines the protective role of hsp70 in both caspase-dependent and -independent apoptosis. Hydrogen peroxide (H2O2) used at low and high concentrations in Jurkat T cells induces caspase-dependent and -independent apoptosis, respectively. A hsp70-transfected Jurkat clone was used to observe the protection mediated by hsp70 during these two forms of apoptosis. Results reveal that hsp70 inhibits both caspase-dependent and -independent apoptosis. Furthermore, measurement of caspase-3 activity during caspase-dependent apoptosis revealed that caspase activation was inhibited in hsp70 transfectants. Early apoptotic events, such as mitochondrial depolarization, cytochrome c release, and increased intracellular calcium, were demonstrated to be common to both caspase-dependent and -independent H2O2-induced apoptosis. The inhibition of these events by hsp70 suggests that hsp70 may be an important anti-apoptotic regulator, functioning at a very early stage in the apoptotic pathway.     

Heterologous desensitization of response mediated by selective PKC-dependent phosphorylation of G(i-1) and G(i-2)

This study examined the ability of protein kinase C (PKC) toinduce heterologous desensitization by targeting specific G proteinsand limiting their ability to transduce signals in smooth muscle.Activation of PKC by pretreatment of intestinal smooth muscle cellswith phorbol 12-myristate 13-acetate, cholecystokinin octapeptide, orthe phosphatase 1 and phosphatase 2A inhibitor, calyculin A,selectively phosphorylated G_i-1 and G_i-2,but not G_i-3 or G_o, and blockedinhibition of adenylyl cyclase mediated by somatostatin receptorscoupled to G_i-1 and opioid receptors coupled toG_i-2, but not by muscarinic M₂ and adenosineA₁ receptors coupled to G_i-3. Phosphorylationof G_i-1 and G_i-2 and blockade of cyclaseinhibition were reversed by calphostin C and bisindolylmaleimide, andadditively by selective inhibitors of PKC and PKC. Blockade ofinhibition was prevented by downregulation of PKC. Phosphorylation ofG-subunits by PKC also affected responses mediated by-subunits. Pretreatment of muscle cells withcANP-(4-23), a selective agonist of the natriureticpeptide clearance receptor, NPR-C, which activates phospholipase C(PLC)-3 via the -subunits of G_i-1 andG_i-2, inhibited the PLC- response to somatostatin and[D-Pen^2,5]enkephalin. The inhibition waspartly reversed by calphostin C. Short-term activation of PKC had noeffect on receptor binding or effector enzyme (adenylyl cyclase orPLC-) activity. We conclude that selective phosphorylation ofG_i-1 and G_i-2 by PKC partly accounts forheterologous desensitization of responses mediated by the - and-subunits of both G proteins. The desensitization reflects adecrease in reassociation and thus availability of heterotrimeric G proteins.

     

Transcriptional Regulation of Urokinase Receptor in High-（95D） and Low-metastatic （95C） Human Lung Cancer Cells

The malignant neoplasm is characterized by progres-sive growth and the ability to disseminate tumor cellsbeyond the boundaries of the parent tumor. Tumor in-vasion and metastasis are important aspects of tumorprogression and the formation of tumor and metastasis is aprincipal contributing factor to cancer morbidity andmortality. During this process urokinase (urokinase-typeplasminogen activator, uPA) and its receptor (uPAR) playan important role. uPAR is a 55–60 kD glycoprotein which is a…     

G protein-coupled receptor kinase 2 (GRK2) in migration and inflammation

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors and other plasma membrane receptors stimulated by chemotactic messengers. On top of that, GRK2 has been reported to interact with a variety of signal transduction proteins related to cell migration such as MEK, Akt, PI3Kgamma or GIT. Interestingly, the levels of expression and activity of this kinase are altered in a number of inflammatory disorders (as rheumatoid arthritis or multiple sclerosis), thus suggesting that it may play an important role in the onset or development of these pathologies. This review summarizes the mechanisms involved in the control of GRK2 expression and function and highlights novel functional interactions of this protein that might help to explain how altered GRK2 levels affects cell migration in different cell types and pathological settings.     

G(i-1)/G(i-2)-dependent signaling by single-transmembrane natriuretic peptide clearance receptor

Single-transmembrane natriuretic peptide clearance receptor (NPR-C), which is devoid of a cytoplasmic guanylyl cyclase domain, interacts with pertussis toxin (PTx)-sensitive G proteins to activate endothelial nitric oxide synthase (eNOS) expressed in gastrointestinal smooth muscle cells. We examined the ability of NPR-C to activate other effector enzymes in eNOS-deficient tenia coli smooth muscle cells; these cells expressed NPR-C and NPR-B but not NPR-A. Atrial natriuretic peptide (ANP), the selective NPR-C ligand cANP-(4-23), and vasoactive intestinal peptide (VIP) inhibited (125)I-ANP and (125)I-VIP binding to muscle membranes in a pattern indicating high-affinity binding to NPR-C. Interaction of VIP with NPR-C was confirmed by its ability to inhibit (125)I-ANP binding to membranes of NPR-C-transfected COS-1 cells. In tenia muscle cells, all ligands selectively activated G(i-1) and G(i-2); VIP also activated G(s) via VIP(2) receptors. All ligands stimulated phosphoinositide hydrolysis, which was inhibited by ANP-(1-11), PTx, and antibodies to phospholipase C-beta3 (PLC-beta3) and Gbeta. cANP-(4-23) contracted tenia muscle cells; contraction was blocked by U-73122 and PTx and by antibodies to PLC-beta3 and Gbeta in intact and permeabilized muscle cells, respectively. VIP and ANP contracted muscle cells only after inhibition of cAMP- and cGMP-dependent protein kinases. ANP and cANP-(4-23) inhibited forskolin-stimulated cAMP in a PTx-sensitive fashion. We conclude that NPR-C is coupled to activation of PLC-beta3 via betagamma-subunits of G(i-1) and G(i-2) and to inhibition of adenylyl cyclase via alpha-subunits.     

MAPKAP kinase-2: three's company at the G(2) checkpoint

Elegant studies in fission yeast by and in mammalian cells by offer new insights into the mechanism through which stress-induced p38 activation inhibits mitotic entry in eukaryotic cells.