John J. Siekierka

A mechanism for the control of protein synthesis by adenovirus VA RNAI

In the absence of VA RNAI, protein synthesis in adenovirus-infected HeLa cells fails because of defective initiation. Earlier work showed that the defect results from phosphorylation of the initiation factor elF-2 on its alpha subunit. We have identified the protein kinase responsible as the dsRNA-activated inhibitor of protein synthesis (DAI). DAI is present in uninfected state. It is activated in cells infected with the adenovirus mutant Ad5 dl331, which produces no VA RNAI, but not in cells infected with wild-type virus. Activation occurs during the late phase of infection with the mutant virus, and the activator appears to be dsRNA produced by symmetrical transcription of the viral genome. VA RNAI antagonizes the activation of DAI by dsRNA, but it cannot inhibit the activity of DAI once activated. We propose a mechanism for VA RNAI action based on its partially double-stranded nature.

Imidazopyrimidines, potent inhibitors of p38 MAP kinase.

The MAP kinase p38 is implicated in the release of the pro-inflammatory cytokines TNF-alpha and IL-1 beta. Inhibition of cytokine release may be a useful treatment for inflammatory conditions such as rheumatoid arthritis and Crohns disease. A novel series of imidazopyrimidines have been discovered that potently inhibit p38 and suppress the production of TNF-alpha in vivo.

Detergent-salt resistance of LAP2α in interphase nuclei and phosphorylation-dependent association with chromosomes early in nuclear assembly implies functions in nuclear structure dynamics

Lamina‐associated polypeptide (LAP) 2 of the inner nuclear membrane (now LAP2β) and LAP2α are related proteins produced by alternative splicing, and contain a common 187 amino acid N‐terminal domain. We show here that, unlike LAP2β, LAP2α behaved like a nuclear non‐membrane protein in subcellular fractionation studies and was localized throughout the nuclear interior in interphase cells. It co‐fractionated with LAP2β in nuclear lamina/matrix‐enriched fractions upon extraction of nuclei with detergent, salt and nucleases. During metaphase LAP2α dissociated from chromosomes and became concentrated around the spindle poles. Furthermore, LAP2α was mitotically phosphorylated, and phosphorylation correlated with increased LAP2α solubility upon extraction of cells in physiological buffers. LAP2α relocated to distinct sites around chromosomes at early stages of nuclear reassembly and intermediarily co‐localized with peripheral lamin B and intranuclear lamin A structures at telophase. During in vitro nuclear assembly LAP2α was dephosphorylated and assembled into insoluble chromatin‐associated structures, and recombinant LAP2α was found to interact with chromosomes in vitro. Some LAP2α may also associate with membranes prior to chromatin attachment. Altogether the data suggest a role of LAP2α in post‐mitotic nuclear assembly and in the dynamic structural organization of the nucleus.

FK-506 and cyclosporin A inhibit highly similar signal transduction pathways in human T lymphocytes

This report compares the ability of cyclosporin A and FK-506 to inhibit human T cell activation triggered via cell surface molecules that utilize different intracellular processes. We stimulated highly purified peripheral blood T lymphocytes with mitogens (Con A and PHA), ionomycin + PMA, or monoclonal antibodies specific for cell surface antigens involved in activation (CD2, CD3, CD28) either in combination with each other or in conjunction with PMA. Using measurements of the proliferative response, IL-2 production, and changes in intracellular Ca2+ ([Ca2+]i), we demonstrate that FK-506 exerts its inhibitory effect on early events of T-cell activation in a manner indistinguishable from that of CsA. An important finding in this study is the strict correlation between those activation pathways that are inhibited by FK-506 and CsA and the requirement that the sensitive pathways induce a measurable rise in [Ca2+]i. This correlation held even for the CD28/CD2 pathway which was previously shown to be calcium-independent; however by employing FACS analysis of [Ca2+]i within individual cells, a subset of cells activated via CD28/CD2 was found to respond with a measurable rise in [Ca2+]i. We also noted that the proliferative response induced by certain stimuli, such as ionomycin + PMA and PHA + PMA, was partially resistant to FK-506 and CsA, while IL-2 production was completely suppressed. The partial FK-506/CsA-resistance of these responses was shown to be determined by the amount of PMA added to the cultures. We conclude from our investigations that FK-506 and CsA inhibit highly similar signal transduction pathways in human T lymphocytes.

FK-506 and cyclosporin A : immunosuppressive mechanism of action and beyond

Cyclosporin A and FK-506 are important therapeutic agents that have found widespread use in preventing graft rejection during tissue transplantation. Research efforts aimed at elucidating the molecular mechanism of action of these drugs have, in addition to defining their immunosuppressive functions, led to the identification of two new gene families whose products may function as components of several diverse signal transduction pathways. In the presence of the immunosuppressive drugs, some members of the receptor families interact with the Ca2+/calmodulin-dependent protein phosphatase 2B, also known as calcineurin. Inhibition of phosphatase activity may effect several downstream biochemical processes. In this way, cyclosporin A and FK-506 have proved to be useful probes of signaling events in both lymphocytic and other cell types.

Potent inhibitors of the MAP kinase p38.

The MAP kinase p38 plays a key role in the biosynthesis of the inflammatory cytokines TNF-alpha and IL-1. We have developed a novel series of potent p38 inhibitors that could lead to new methods of treatment for inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease.

Suppression of HIV-1 viral replication and cellular pathogenesis by a novel p38/JNK kinase inhibitor

Objective: To analyze a novel compound, which inhibits serine-threonine protein kinase p38, for its possible bioactivity against HIV-1 infection. Methods: Proteins involved in cellular signal transduction pathways represent a novel class of host therapeutic targets for infectious diseases. In this regard the serine/threonine kinase p38 MAPK, a member of the mitogen-activated protein (MAP) kinase superfamily of signal transduction molecules may play an important role in HIV-1 infection. We analyzed the ability of this compound (RWJ67657) to inhibit HIV replication in primary T cells and monocytes. Cellular expression of phospho-p38MAPK was studied by Western blot analysis. Blockade of HIV infection induced apoptosis was measured by Annexin V staining. Results: p38 inhibitor RWJ67657 was effective in inhibiting HIV-1 replication in both T-cell and monocyte cell lines, irrespective of the coreceptor used by the virus for entry into the cell. Importantly, both reverse transcriptase and protease resistant escape mutant viruses were effectively suppressed by RWJ67657. In addition, the tested compounds block HIV-induced T-cell apoptosis, a critical means of T-cell depletion linked to AIDS progression. Conclusion: Several steps in the HIV-1 virus life cycle appear to depend on cellular activation, including activation of the p38 pathway. Without activation virus replication is thought to be blocked due to incomplete reverse transcription and a lack of proviral DNA integration. The data collectively illustrate that inhibition of the p38 pathway can affect HIV-1 replication. Interruption of HIV infection by p38 inhibitors underscores the value of exploring antiviral drugs that target host cellular proteins.

A Novel Role for the Immunophilin FKBP52 in Copper Transport

FK506-binding protein 52 (FKBP52) is an immunophilin that possesses peptidylprolyl cis/trans-isomerase (PPIase) activity and is a component of a subclass of steroid hormone receptor complexes. Several recent studies indicate that immunophilins can regulate neuronal survival and nerve regeneration although the molecular mechanisms are poorly understood. To investigate the function of FKBP52 in the nervous system, we employed a yeast two-hybrid strategy using the PPIase domain (domain I) as bait to screen a neonatal rat dorsal root ganglia cDNA expression library. We identified an interaction between FKBP52 domain I and Atox1, a copper-binding metallochaperone. Atox1 interacts with Menkes disease protein and Wilson disease protein (WD) and functions in copper efflux. The interaction between FKBP52 and Atox1 was observed in both glutathione S-transferase pull-down experiments and when proteins were ectopically expressed in human embryonic kidney (HEK) 293T cells and was sensitive to FK506. Interestingly, the FKBP52/Atox1 interaction was enhanced when HEK 293T cells were cultured in copper-supplemented medium and decreased in the presence of the copper chelator, bathocuproine disulfate, suggesting that the interaction is regulated in part by intracellular copper. Overexpression of FKBP52 increased rapid copper efflux in 64Cu-loaded cells, as did the overexpression of WD transporter. Taken together, our present findings suggest that FKBP52 is a component of the copper efflux machinery, and in so, may also promote neuroprotection from copper toxicity.

Opposing roles of serine/threonine kinases MEKK1 and LOK in regulating the CD28 responsive element in T-cells

T-cell activation requires signals from both the T-cell receptor (TcR) and other co-stimulatory molecules such as CD28. TcR- and CD28-mediated signals are integrated during T-cell activation resulting in the expression of cytokine genes such as interleukin-2 (IL-2). An enhancer element (CD28RE) of the IL-2 gene specifically responsive to CD28 signals has been previously identified and characterized. This response element and an adjacent Activated Protein-1 (nuclear factor-interleukin-2B) site together (RE/AP1) were shown to complex with c-rel, AP-1 and other factors. However, details of the signal transduction pathways leading from CD28 to the composite response element remain poorly understood. We present data showing that overexpression of the serine threonine kinase, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase kinase-1 (MEKK1), but not nuclear factor-kappa B inducing kinase, or MAP kinase/ERK kinase-1 (MEK1), can significantly increase the level of CD28RE/AP1-driven luciferase (Luc) reporter gene expression in Jurkat E6-1 cells. A MEKK1 dominant negative mutant blocked such activation induced by stimulation with Raji B cells and the superantigen staphylococcus enterotoxin E (SEE), as well as via CD3/CD28. Mutations in either site of the RE/AP1 element abolished MEKK1-induced Luc expression. Calcineurin inhibitors, CsA and FK520, or inhibitors of p38 kinase (SB 203580), or MEK1 (PD 098059), did not affect MEKK1-induced reporter activation. These results directly implicate MEKK1 in the CD28 signalling pathway that activates the CD28 response element. Co-expression of the lymphocyte-oriented kinase (LOK) kinase attenuated Raji/SEE-induced IL-2 production in Jurkat cells, as well as MEKK1 and Raji/SEE-induced reporter gene activation. These data suggest that MEKK1 and LOK may have opposing roles in regulating the CD28RE/AP1 element.

Drugs Designed To Inhibit Human p38 Mitogen-Activated Protein Kinase Activation Treat Toxoplasma gondii and Encephalitozoon cuniculi Infection

ABSTRACT We recently showed that the pyridinylimidazoles SB203580 and SB202190, drugs designed to block human p38 mitogen-activated protein kinase (MAPK) activation, also inhibited replication of the medically important intracellular parasite Toxoplasma gondii in cultured human fibroblasts through a direct effect on the parasite. We now show that additional pyridinylimidazole and imidazopyrimidine p38 MAPK inhibitors inhibit intracellular T. gondii replication in vitro and protect mice against fatal T. gondii infection. Mice surviving infection following treatment with p38 MAPK inhibitors were resistant to subsequent T. gondii challenge, demonstrating induction of protective immunity. Thus, drugs originally developed to block human p38 MAPK activation are useful for treating T. gondii infection without inducing significant immunosuppression. MAPK inhibitors combined with either of the approved anti-Toxoplasma drugs sulfadiazine and pyrimethamine resulted in improved survival among mice challenged with a fatal T. gondii inoculum. A MAPK inhibitor also treated mice infected with the Microsporidium parasite Encephalitozoon cuniculi, suggesting that MAPK inhibitors represent a novel class of agents that may have a broad spectrum of antiparasitic activity. Preliminary studies implicate a T. gondii MAPK homologue as the target of drug action, suggesting possibilities for more-selective agents.