Robert Csonga

Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A.

Eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor required for the function of the human immunodeficiency virus type-1 (HIV-1) Rev trans-activator protein. The majority of a set of eIF-5A mutants did not support growth of yeast cells having an inactivated genomic copy of eIF-5A, indicating that the introduced mutation eliminated eIF-5A activity. Two nonfunctional mutants, eIF-5AM13 and eIF-5AM14, retained their binding capacity for the HIV-1 Rev response element: Rev complex. Both mutants were constitutively expressed in human T cells. When these T cells were infected with replication-competent HIV-1, virus replication was inhibited. The eIF-5AM13 and eIF-5AM14 proteins blocked Rev trans-activation and Rev-mediated nuclear export.

Early activation of sphingosine kinase in mast cells and recruitment to FcεRI are mediated by its interaction with Lyn kinase

ABSTRACT Sphingosine kinase has been recognized as an essential signaling molecule that mediates the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase and generation of sphingosine-1-phosphate have been linked to the initial rise in Ca2+, released from internal stores, and to degranulation. These events either precede or are concomitant with the activation of phospholipase C-γ and the generation of inositol trisphosphate. Here we show that sphingosine kinase type 1 (SPHK1) interacts directly with the tyrosine kinase Lyn and that this interaction leads to the recruitment of this lipid kinase to the high-affinity receptor for immunoglobulin E (FcεRI). The interaction of SPHK1 with Lyn caused enhanced lipid and tyrosine kinase activity. After FcεRI triggering, enhanced sphingosine kinase activity was associated with FcεRI in sphingolipid-enriched rafts of mast cells. Bone marrow-derived mast cells from Lyn−/ − mice, compared to syngeneic wild-type cells, were defective in the initial induction of SPHK1 activity, and the defect was overcome by retroviral Lyn expression. These findings position the activation of SPHK1 as an FcεRI proximal event.

Molecular characterization of a cDNA encoding functional human deoxyhypusine synthase and chromosomal mapping of the corresponding gene locus

Deoxyhypusine synthase is essentially required for the post‐translational formation of hypusine, a modification of a specific lysine residue in eukaryotic initiation factor 5A, which appears to be pivotal for cell proliferation. From a human peripheral blood mononuclear cells cDNA library we isolated two independent sequences encoding biologically active deoxyhypusine synthase. DNA sequence analysis revealed a 369 amino acid protein with a molecular mass of 41.055 kDa. This recombinant deoxyhypusine synthase showed significant catalytic activity in synthesis of deoxyhypusine after in vitro transcription and translation as well as upon expression in Escherichia coli. Using a panel of somatic rodent‐human cell hybrids we localized the deoxyhupusine synthase gene to human chromosome 19.

Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay

Hypusine synthesis in the eukaryotic initiation factor 5A is a unique two‐step posttranslational modification. After deoxyhypusine is generated by the deoxyhypusine synthase, the deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the formation of mature hypusine. A rapid assay for monitoring the deoxyhypusine hydroxylase activity was established, employing the oxidative cleavage of the hypusyl residue and subsequent extraction of the generated aldehydes. As metal ion chelators have been reported to inhibit the deoxyhypusine hydroxylase, the mechanism of this inhibition and the effect of transition metal ions on the enzyme activity were investigated. A ferric ion appears to be essential for enzymatic activity, the inhibition of which is entirely attributed to the metal ion bunding capacity of the chelators.

TNF–· and IL–5 Gene Induction in IgE plus Antigen–Stimulated Mast Cells Require Common and Distinct Signaling Pathways

Background: Mast cells produce a variety of cytokines and chemokines in a timely and tightly controlled fashion if stimulated via the FcεRI. Evidence is accumulating that the transcriptional induction of the corresponding genes and the release of these mediators are dependent on common and mediator–specific components of the signal transduction and transcription factor machinery. Methods: We addressed this issue by comparing the effects of mitogen activated protein (MAP) kinase pathway inhibitors and protein kinase C (PKC) inhibitors on the induction of TNF–α and IL–5 after IgE plus antigen (Ag) stimulation in CPII mouse mast cells using Western blot analyses and transient transfections of reporter gene plasmids. Results: TNF–α shows a strict dependence on the MAP kinase pathway, while IL–5 is either activated by PMA–dependent PKCs or along the MAP kinase pathway. In addition, both mediators are sensitive to PKCμ inhibition, suggesting involvement of this atypical, non–PMA dependent PKC in the overall induction process. Conclusion: While the two cytokines were recently shown to be regulated by a member of the nuclear factor of activated T–cells (NF–AT) transcription factor family, activator protein 1 (AP1) was identified as a cofactor at the TNF–α promoter while a GATA family member comprised the cofactor at the IL–5 promoter. This suggests that the differences in requirement for signal transduction cascades are the result of a different usage of NF–AT cofactors for transcription of each cytokine in mast cells.

Glycosphingolipid-Induced Relocation of Lyn and Syk into Detergent-Resistant Membranes Results in Mast Cell Activation

Sphingosine, sphingosine-1-phosphate, and the more complex sphingolipid ceramide exert strong immunomodulatory effects on a variety of leukocytes. However, little is known regarding such a potential of glycosphingolipids, a class of sugar derivatives of sphingosine. Here we demonstrate that galactosylsphingosine, one of the smallest representatives of this group, accumulates in the detergent-resistant membranes resulting in the relocation of the tyrosine kinases Lyn and Syk into this compartment. The result of this is an enhanced tyrosine phosphorylation and kinase activity leading to priming and activation of mast cells by conveying a weak yet significant activation of the mitogen-activated protein kinase pathway(s). In comparison to IgE/Ag triggering, galactosylsphingosine stimulates the mitogen-activated protein kinase pathway more rapidly and favors c-Jun NH2-terminal kinase 1 activation over extracellular signal-regulatory kinase 1 and 2. At the transcription factor level, this “ultratransient signaling event” results in an activation of JunD as the predominant AP-1 component. In this respect, the effects of galactosylsphingosine are clearly distinct from the signaling elicited by other sphingolipids without the sugar moiety, such as sphingosine-1-phosphate.