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Abstract: Retroviral infection triggers the cytoplasmic translocation of two Crm1-dependent shuttle factors, namely the Ini1 (integrase interactor 1, hSNF5) and the promyelocytic leukemia (PML) protein. Blocking nuclear export of shuttle factors by leptomycin B increases the efficiency of retroviral integration, suggesting that some may mediate antiviral activity. While PML was shown to counteract proviral establishment, it remained unclear whether Ini1, a protein implicated in various processes during human immunodeficiency virus replication, has the same potential. Employing RNA interference-mediated knock-down of Ini1, we show here that the simultaneous accumulation of both proteins in the cytoplasm likely reflects two non-interdependent phenomena. Furthermore, Ini1 does not interfere with retroviral integration, as cells lacking Ini1 show no increased infection susceptibility. [Copyright &y& Elsevier]

Estrogen receptor-alpha (ER-α) is a nuclear transcriptional factor that is part of the nuclear receptor superfamily. In this study, we isolated and identified a new LXXLL-containing protein that interacts with the ER-α via a yeast two-hybrid assay. We have termed this protein estrogen receptor repressor-10 (ERR-10). The ERR-10 cDNA is predicted to encode a polypeptide of 94 amino acids, with a molecular mass of about 10 kDa. Although the ERR-10 mRNA transcript is expressed in a wide range of normal human tissues, higher expression levels are found in endocrinal tissues relative to other tissues. We have demonstrated, through immunoprecipitation, Western blot and GST pull-down assays, that ERR-10 associates with ER-α. Moreover, ERR-10 decreased 17β-estrodial-induced activation of ER-α transcriptional activity in transient transfection assays of mammalian cells. The ERR-10 N-terminus, which resembles two LXXLL motifs, is essential for ER-α binding and repression activity. Estrogen modulation of estrogen-responsive gene expression was markedly blocked by ERR-10. These results suggest that ERR-10 is a novel mediator in ER transcriptional activation. [Copyright &y& Elsevier]

Knockout of the translation inhibitor 4E-BP1 induces an overexpression of uncoupling protein-1 (UCP1) [Nature Medicine 7 (2001) 1128]. A possible inverse control of UCP1 and 4E-BP1 expressions in mouse brown adipose tissue was investigated. Cold-exposure, which increases the expression of UCP1, decreased that of 4E-BP1 mRNA in wild type but not in β1/β2/β3-adrenoceptor knockout mice. Administration of the β3-adrenoceptor agonist CL 316 246 decreased 4E-BP1 mRNA by 75% and protein by 41% after 6 and 48 h, respectively. Our data are the first report of a regulation by the β3-adrenoceptor of 4E-BP1 expression. They support a role of the latter in adaptive thermogenesis. [Copyright &y& Elsevier]

Ycf53 is a hypothetical chloroplast open reading frame with similarity to the Arabidopsis nuclear gene GUN4. In plants, GUN4 is involved in tetrapyrrole biosynthesis. We demonstrate that one of the two Synechocystis sp. PCC 6803 ycf53 genes with similarity to GUN4 functions in chlorophyll (Chl) biosynthesis as well: cyanobacterial gun4 mutant cells exhibit lower Chl contents, accumulate protoporphyrin IX and show less activity not only of Mg chelatase but also of Fe chelatase. The possible role of Gun4 for the Mg as well as Fe porphyrin biosynthesis branches in Synechocystis sp. PCC 6803 is discussed. [Copyright &y& Elsevier]

Here, we identify ADP-ribosylation factor (ARF)-like 7 (ARL7) as the only ARF- and ARL-family member whose mRNA-expression is induced by liver X-receptor/retinoid X-receptor agonists or cholesterol loading in human macrophages. Moreover, subcellular distribution of mutant and wild type ARL7-enhanced green fluorescent protein (EGFP) supports that ARL7 may be involved in a vesicular transport step between a perinuclear compartment and the plasma membrane. Therefore, we investigated the effect of ARL7 over-expression on the cholesterol secretory pathway. We found that expression of wild type and dominant active ARL7-EGFP stimulated the rate of apolipoprotein AI-specific cholesterol efflux 1.7- and 2.8-fold. In contrast, expression of the dominant negative form of ARL7-EGFP led to ∼50% inhibition of cholesterol efflux. This data is consistent with a model in which ARL7 is involved in transport between a perinuclear compartment and the plasma membrane apparently linked to the ABCA1-mediated cholesterol secretion pathway. [Copyright &y& Elsevier]

Presenilins (PS1 and PS2) are supposed to be unusual aspartic proteases and components of the γ-secretase complex regulating cleavage of type I proteins. Multiple mutations in PS1 are a major cause of familial early-onset Alzheimer’s disease (AD). We and others recently identified PS-related families of proteins (IMPAS/PSH/signal peptide peptidases (SPP)). The functions of these proteins are yet to be determined. We found that intramembrane protease-associated or intramembrane protease aspartic protein Impas 1 (IMP1)/SPP induces intramembranous cleavage of PS1 holoprotein in cultured cells coexpressing these proteins. Mutations in evolutionary invariant sites in hIMP1 or specific γ-secretase inhibitors abolish the hIMP1-mediated endoproteolysis of PS1. In contrast, neither AD-like mutations in hIMP1 nor in PS1 substrate abridge the PS1 cleavage. The data suggest that IMP1 is a bi-aspartic polytopic protease capable of cleaving transmembrane precursor proteins. These data, to our knowledge, are a first observation that a multipass transmembrane protein or the integral protease per se may be a primary substrate for an intramembranous proteolysis. [Copyright &y& Elsevier]

A psaFJ-null mutant of Synechocystis sp. strain PCC 6803 was characterised. As opposed to similar mutants in chloroplasts of green algae, electron transfer from plastocyanin to photosystem 1 was not affected. Instead, a restraint in full chain photosynthetic electron transfer was correlated to malfunction of photosystem 1 at its stromal side. Our hypothesis is that absence of PsaF causes oxidative stress, which triggers the induction of the ‘iron stress inducible’ operon isiAB. Products are the IsiA chlorophyll-binding protein (CP43′) and the isiB gene product flavodoxin. Supporting evidence was obtained by similar isiAB induction in wild type cells artificially exposed to oxidative stress. [Copyright &y& Elsevier]

The molecular basis of potassium uptake in cyanobacteria has not been elucidated. However, genes known from other bacteria to encode potassium transporters can be identified in the genome of Synechocystis sp. strain PCC 6803. Mutants defective in kdpA and ntpJ were generated and characterized to address the role of the Kdp and KtrAB systems in this strain. KtrAB is crucial for K+ uptake, as the ΔntpJ mutant shows slowed growth, slowed potassium uptake kinetics, and increased salt sensitivity. The ΔkdpA mutant has the same phenotype as the wild type even at limiting potassium, but a ΔkdpAΔntpJ double mutant is not viable, indicating a role of Kdp for potassium uptake when the Ktr system is not functioning. [Copyright &y& Elsevier]

Our understanding of estrogen signaling has undergone a true paradigm shift over recent years, following the discovery in 1995 of a second estrogen receptor, estrogen receptor β (ERβ). In many contexts ERβ appears to antagonize the actions of ERα (yin/yang relationship) although there also exist genes that are specifically regulated by one of the two receptors. Studies of ERβ knockout mice have shown that ERβ exerts important functions in the ovary, central nervous system, mammary gland, prostate gland, hematopoiesis, immune system, vessels and bone. The use of ERβ-specific ligands against certain forms of cancer represents one of the many pharmaceutical possibilities that have been created thanks to the discovery of ERβ. [Copyright &y& Elsevier]

Brazzein is a small, heat-stable, intensely sweet protein consisting of 54 amino acid residues. Based on the wild-type brazzein, 25 brazzein mutants have been produced to identify critical regions important for sweetness. To assess their sweetness, psychophysical experiments were carried out with 14 human subjects. First, the results suggest that residues 29–33 and 39–43, plus residue 36 between these stretches, as well as the C-terminus are involved in the sweetness of brazzein. Second, charge plays an important role in the interaction between brazzein and the sweet taste receptor. [Copyright &y& Elsevier]

A novel gap junction forming protein, mouse connexin-30.2 (Cx30.2) contains 278 amino acid residues, and is 79% identical to human Cx31.9 (GJA11). Northern analysis showed that Cx30.2 is ubiquitously expressed, most prominently in testis. Polyclonal antibodies against Cx30.2 detected a 30 kDa protein in cells overexpressing Cx30.2, and in mouse testis. Immunofluorescence showed that Cx30.2 was expressed in vascular smooth muscle, but also in cell types where Cx31.9 was not detected. These data demonstrate that Cx30.2 is a bona fide gene, and suggest that it is the orthologue of Cx31.9, but that it may have additional roles compared with Cx31.9. [Copyright &y& Elsevier]

Vascular endothelial growth factor (VEGF) is a potent angiogenic mediator in tissue repair. In non-healing human wounds plasmin cleaves and inactivates VEGF165. In the present study, we generated recombinant VEGF165 mutants resistant to plasmin proteolysis. Substitution of Arg110 with Ala110 or Gln110, and Ala111 with Pro111 yielded plasmin-resistant and biologically active VEGF165 mutants. In addition, substitution of Ala111 with Pro111 resulted in a substantial degree of stabilization when incubated in wound fluid obtained from non-healing wounds. These results suggest that the plasmin cleavage site Arg110/Ala111 and the carboxyl-terminal domain play an important role in the mitogenic activity of VEGF165. [Copyright &y& Elsevier]

Thrombopoietin (Tpo) and its receptor, c-mpl, are expressed in murine embryonic stem (ES) cells. ES cells are maintained in a pluripotent state by leukemia inhibitory factor (LIF) via activation of the Janus kinase (Jak)–STAT3 signaling pathway. Tpo, like LIF, activates STAT3. We report that Tpo increases the number of undifferentiated colonies derived from wild type or Shp-2 mutant (Shp-2Δ46–110) ES cells. Tpo plus LIF acted synergistically on the Shp-2Δ46–110 ES cells to maintain undifferentiated colonies but no evidence of synergism via Jak–STAT3 activation was detected. Collectively, these data suggest that Tpo can play a role in preventing ES cell differentiation via Jak–STAT3 activation and perhaps via novel pathways that are enhanced in the absence of functional Shp-2. [Copyright &y& Elsevier]

Bisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme’s Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme. [Copyright &y& Elsevier]

Human immunodeficiency virus type-1 envelope glycoprotein (gp120env) binding to cell surface CD4 receptor triggers a broad range of intracellular effects leading to T cell activation and cell cycle entry. Among these effects we and others previously reported on the nuclear translocation of the nuclear factor-κB (NF-κB) transcription factor. The present work further investigates the signal transduction pathways involved in gp120env-induced NF-κB activation. We demonstrate that gp120env–CD4 interaction stimulates the hyperphosphorylation of IκB-α inhibitory protein. Conversely, overexpression of a dominant-negative IκB-α transgene mutated at S32 and S36 residues, abolishes gp120env-induced NF-κB activation. IκB kinases (IKKs) activity was found to be selectively enhanced following CD4 engagement with gp120env and to mediate the phosphorylation of IκB-α while co-transfection experiments using dominant-negative forms of IKKs inhibited gp120env-induced NF-κB activation. Taken together, these results confirm that IKKs complex play a key role in gp120env-induced NF-κB activation. [Copyright &y& Elsevier]

Cardiac troponin I, the inhibitory subunit of the heterotrimeric cardiac troponin (cTn) complex is phosphorylated by protein kinase A at two serine residues located in its heart-specific N-terminal extension. This flexible arm interacts at different sites within cTn dependent on its phosphorylation degree. Bisphosphorylation is known to induce conformational changes within cTnI which finally lead to a reduction of the calcium affinity of cTnC. However, as we show here, the bisphosphorylated cTnI arm does not interact with cTnC, but with cTnT and/or cTnI. [Copyright &y& Elsevier]

We report that the Vps10p domain receptor sorLA binds the adaptor proteins GGA1 and -2, which take part in Golgi–endosome sorting. The GGAs bind with differential requirements via three critical residues in the C-terminal segment of the sorLA cytoplasmic tail. Unlike in sortilin and the mannose 6-phosphate receptors, the GGA-binding segment in sorLA contains neither an acidic cluster nor a dileucine. Our results support the concept of sorLA as a potential sorting receptor and suggest that key residues in sorLA and sortilin conform to a new type of motif (Ψ–Ψ–X–X–∅) defining minimum requirements for GGA binding to cytoplasmic receptor domains. [Copyright &y& Elsevier]

A novel polypeptide with an apparent molecular mass of 9 kDa was detected after sodium dodecyl sulphate–polyacrylamide gel electrophoresis of Arabidopsis photosystem I (PSI) and was N-terminally sequenced. Corresponding cDNA clones encode a precursor protein of 140 amino acid residues which was imported into isolated intact chloroplasts and processed to the mature protein, designated PSI-O. The mature protein has two transmembrane helices and a calculated mass of 10 104 Da. The PSI-O protein was also shown to be present in PSI isolated from barley and spinach, and was essentially absent in chloroplast grana. Expressed sequences encoding similar proteins are available from many species of plants and green algae. [Copyright &y& Elsevier]