Direct transport across the plasma membrane of mammalian cells of Leishmania HASPB as revealed by a CHO export mutant

Leishmania HASPB is a lipoprotein that is exported to the extracellular space from both Leishmania parasites and mammalian cells via an unconventional secretory pathway. Exported HASPB remains anchored in the outer leaflet of the plasma membrane mediated by myristate and palmitate residues covalently attached to the N-terminal SH4 domain of HASPB. HASPB targeting to the plasma membrane depends on SH4 acylation that occurs at intracellular membranes. How acylated HASPB is targeted to the plasma membrane and, in particular, the subcellular site of HASPB membrane translocation is unknown. In order to address this issue, we screened for clonal CHO mutants that are incapable of exporting HASPB. A detailed characterization of such a CHO mutant cell line revealed that the expression level of the HASPB reporter molecule is unchanged compared to CHO wild-type cells; that it is both myristoylated and palmitoylated; and that it is mainly localized to the plasma membrane as judged by confocal microscopy and subcellular fractionation. However, based on a quantitative flow cytometry assay and a biochemical biotinylation assay of surface proteins, HASPB transport to the outer leaflet of the plasma membrane is largely reduced in this mutant. From these data, we conclude that the subcellular site of HASPB membrane translocation is the plasma membrane as the reporter molecule accumulates in this location when export is blocked. Thus, these results allow us to define a two-step process of HASPB cell surface biogenesis in which SH4 acylation of HASPB firstly mediates intracellular targeting to the plasma membrane. In a second step, the plasma membrane-resident machinery, which is apparently disrupted in the CHO mutant cell line, mediates membrane translocation of HASPB. Intriguingly, the angiogenic growth factor FGF-2, another protein secreted by unconventional means, is shown to be secreted normally from the HASPB export mutant cell line. These observations demonstrate that the export machinery component defective in the export mutant cell line functions specifically in the HASPB export pathway

New mutations at the imprinted Gnas cluster show gene dosage effects of Gsα in postnatal growth and implicate XLαs in bone and fat metabolism, but not in suckling

The imprinted Gnas cluster is involved in obesity, energy metabolism, feeding behavior, and viability. Relative contribution of paternally expressed proteins XLαs, XLN1, and ALEX or a double dose of maternally expressed Gsα to phenotype has not been established. In this study, we have generated two new mutants (Ex1A-T-CON and Ex1A-T) at the Gnas cluster. Paternal inheritance of Ex1A-T-CON leads to loss of imprinting of Gsα, resulting in preweaning growth retardation followed by catch-up growth. Paternal inheritance of Ex1A-T leads to loss of imprinting of Gsα and loss of expression of XLαs and XLN1. These mice have severe preweaning growth retardation and incomplete catch-up growth. They are fully viable probably because suckling is unimpaired, unlike mutants in which the expression of all the known paternally expressed Gnasxl proteins (XLαs, XLN1 and ALEX) is compromised. We suggest that loss of ALEX is most likely responsible for the suckling defects previously observed. In adults, paternal inheritance of Ex1A-T results in an increased metabolic rate and reductions in fat mass, leptin, and bone mineral density attributable to loss of XLαs. This is, to our knowledge, the first report describing a role for XLαs in bone metabolism. We propose that XLαs is involved in the regulation of bone and adipocyte metabolism

Leukotrienes inhibit early stages of HIV-1 infection in monocyte-derived microglia-like cells

<p>Abstract</p> <p>Background</p> <p>Microglia are one of the main cell types to be productively infected by HIV-1 in the central nervous system (CNS). Leukotriene B₄(LTB₄) and cysteinyl-leukotrienes such as LTC₄are some of the proinflammatory molecules produced in infected individuals that contribute to neuroinflammation. We therefore sought to investigate the role of leukotrienes (LTs) in HIV-1 infection of microglial cells.</p> <p>Methods</p> <p>To evaluate the role of LTs on HIV-1 infection in the CNS, monocyte-derived microglial-like cells (MDMis) were utilized in this study. Leukotriene-treated MDMis were infected with either fully replicative brain-derived HIV-1 isolates (YU2) or R5-tropic luciferase-encoding particles in order to assess viral production and expression. The efficacy of various steps of the replication cycle was evaluated by means of p24 quantification by ELISA, luciferase activity determination and quantitative real-time polymerase chain reaction (RT-PCR).</p> <p>Results</p> <p>We report in this study that virus replication is reduced upon treatment of MDMis with LTB₄and LTC₄. Additional experiments indicate that these proinflammatory molecules alter the pH-independent entry and early post-fusion events of the viral life cycle. Indeed, LT treatment induced a diminution in integrated proviral DNA while reverse-transcribed viral products remained unaffected. Furthermore, decreased C-C chemokine receptor type 5 (CCR5) surface expression was observed in LT-treated MDMis. Finally, the effect of LTs on HIV-1 infection in MDMis appears to be mediated partly via a signal transduction pathway involving protein kinase C.</p> <p>Conclusions</p> <p>These data show for the first time that LTs influence microglial cell infection by HIV-1, and may be a factor in the control of viral load in the CNS.</p

Insights into the Function of the CRM1 Cofactor RanBP3 from the Structure of Its Ran-Binding Domain

Proteins bearing a leucine-rich nuclear export signal (NES) are exported from the nucleus by the transport factor CRM1, which forms a cooperative ternary complex with the NES-bearing cargo and with the small GTPase Ran. CRM1-mediated export is regulated by RanBP3, a Ran-interacting nuclear protein. Unlike the related proteins RanBP1 and RanBP2, which promote disassembly of the export complex in the cytosol, RanBP3 acts as a CRM1 cofactor, enhancing NES export by stabilizing the export complex in the nucleus. RanBP3 also alters the cargo selectivity of CRM1, promoting recognition of the NES of HIV-1 Rev and of other cargos while deterring recognition of the import adaptor protein Snurportin1. Here we report the crystal structure of the Ran-binding domain (RBD) from RanBP3 and compare it to RBD structures from RanBP1 and RanBP2 in complex with Ran and CRM1. Differences among these structures suggest why RanBP3 binds Ran with unusually low affinity, how RanBP3 modulates the cargo selectivity of CRM1, and why RanBP3 promotes assembly rather than disassembly of the export complex. The comparison of RBD structures thus provides an insight into the functional diversity of Ran-binding proteins

Functional Characterization of the HuR:CD83 mRNA Interaction

Maturation of dendritic cells (DC) is characterized by expression of CD83, a surface protein that appears to be necessary for the effective activation of naïve T-cells and T-helper cells by DC. Lately it was shown that CD83 expression is regulated on the posttranscriptional level by interaction of the shuttle protein HuR with a novel posttranscriptional regulatory RNA element (PRE), which is located in the coding region of the CD83 transcript. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export via the CRM1 pathway. To date, however, the structural basis of this interaction, which potentially involves three distinct RNA recognition motifs (RRM1–3) in HuR and a complex three-pronged RNA stem-loop element in CD83 mRNA, has not been investigated in detail. In the present work we analyzed this interaction in vitro and in vivo using various HuR- and CD83 mRNA mutants. We are able to demonstrate that both, RRM1 and RRM2 are crucial for binding, whereas RRM3 as well as the HuR hinge region contributed only marginally to this protein∶RNA interaction. Furthermore, mutation of uridine rich patches within the PRE did not disturb HuR:CD83 mRNA complex formation while, in contrast, the deletion of specific PRE subfragments from the CD83 mRNA prevented HuR binding in vitro and in vivo. Interestingly, the observed inhibition of HuR binding to CD83 mRNA does not lead to a nuclear trapping of the transcript but rather redirected this transcript from the CRM1- towards the NXF1/TAP-specific nuclear export pathway. Thus, the presence of a functional PRE permits nucleocytoplasmic trafficking of the CD83 transcript via the CRM1 pathway

Cdk Phosphorylation of a Nucleoporin Controls Localization of Active Genes through the Cell Cycle

The localization of active genes to the nuclear periphery is regulated through the cell cycle by Cdk1 phosphorylation of a single nuclear pore protein

Postnatal Changes in the Expression Pattern of the Imprinted Signalling Protein XLαs Underlie the Changing Phenotype of Deficient Mice

The alternatively spliced trimeric G-protein subunit XLαs, which is involved in cAMP signalling, is encoded by the Gnasxl transcript of the imprinted Gnas locus. XLαs deficient mice show neonatal feeding problems, leanness, inertia and a high mortality rate. Mutants that survive to weaning age develop into healthy and fertile adults, which remain lean despite elevated food intake. The adult metabolic phenotype can be attributed to increased energy expenditure, which appears to be caused by elevated sympathetic nervous system activity. To better understand the changing phenotype of Gnasxl deficient mice, we compared XLαs expression in neonatal versus adult tissues, analysed its co-localisation with neural markers and characterised changes in the nutrient-sensing mTOR1-S6K pathway in the hypothalamus. Using a newly generated conditional Gnasxl lacZ gene trap line and immunohistochemistry we identified various types of muscle, including smooth muscle cells of blood vessels, as the major peripheral sites of expression in neonates. Expression in all muscle tissues was silenced in adults. While Gnasxl expression in the central nervous system was also developmentally silenced in some midbrain nuclei, it was upregulated in the preoptic area, the medial amygdala, several hypothalamic nuclei (e.g. arcuate, dorsomedial, lateral and paraventricular nuclei) and the nucleus of the solitary tract. Furthermore, expression was detected in the ventral medulla as well as in motoneurons and a subset of sympathetic preganglionic neurons of the spinal cord. In the arcuate nucleus of Gnasxl-deficient mice we found reduced activity of the nutrient sensing mTOR1-S6K signalling pathway, which concurs with their metabolic status. The expression in these brain regions and the hypermetabolic phenotype of adult Gnasxl-deficient mice imply an inhibitory function of XLαs in energy expenditure and sympathetic outflow. By contrast, the neonatal phenotype of mutant mice appears to be due to a transient role of XLαs in muscle tissues

HIV infection of non-dividing cells: a divisive problem

Understanding how lentiviruses can infect terminally differentiated, non-dividing cells has proven a very complex and controversial problem. It is, however, a problem worth investigating, for it is central to HIV-1 transmission and AIDS pathogenesis. Here I shall attempt to summarise what is our current understanding for HIV-1 infection of non-dividing cells. In some cases I shall also attempt to make sense of controversies in the field and advance one or two modest proposals

In vitro nuclear interactome of the HIV-1 Tat protein

<p>Abstract</p> <p>Background</p> <p>One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86–101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry.</p> <p>Results</p> <p>Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied <it>in silico </it>analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture.</p> <p>Conclusion</p> <p>We have completed the <it>in vitro </it>Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.</p

Microstrip X-Band Array Antenna with Wide Sector Characteristics.

A microstrip antenna for a nord non cooperative Near-Range Radar Network (NRN) for the guidance and control of vehicles on airport maneuvring areas has been developed. The design principles, the technical solution and measurement results are presented