The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases

<p>Abstract</p> <p>Background</p> <p>The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent <it>K</it>_mof the kinases ranges from less than 0.4 μM to in excess of 1000 μM for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude.</p> <p>Results</p> <p>We have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP) as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE) but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold.</p> <p>Conclusions</p> <p>Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and synthetase enzymes as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the broad range of <it>K</it>_massociated with kinase enzymes.</p

A role for human brain pericytes in neuroinflammation

BACKGROUND: Brain inflammation plays a key role in neurological disease. Although much research has been conducted investigating inflammatory events in animal models, potential differences in human brain versus rodent models makes it imperative that we also study these phenomena in human cells and tissue. METHODS: Primary human brain cell cultures were generated from biopsy tissue of patients undergoing surgery for drug-resistant epilepsy. Cells were treated with pro-inflammatory compounds IFNγ, TNFα, IL-1β, and LPS, and chemokines IP-10 and MCP-1 were measured by immunocytochemistry, western blot, and qRT-PCR. Microarray analysis was also performed on late passage cultures treated with vehicle or IFNγ and IL-1β. RESULTS: Early passage human brain cell cultures were a mixture of microglia, astrocytes, fibroblasts and pericytes. Later passage cultures contained proliferating fibroblasts and pericytes only. Under basal culture conditions all cell types showed cytoplasmic NFκB indicating that they were in a non-activated state. Expression of IP-10 and MCP-1 were significantly increased in response to pro-inflammatory stimuli. The two chemokines were expressed in mixed cultures as well as cultures of fibroblasts and pericytes only. The expression of IP-10 and MCP-1 were regulated at the mRNA and protein level, and both were secreted into cell culture media. NFκB nuclear translocation was also detected in response to pro-inflammatory cues (except IFNγ) in all cell types. Microarray analysis of brain pericytes also revealed widespread changes in gene expression in response to the combination of IFNγ and IL-1β treatment including interleukins, chemokines, cellular adhesion molecules and much more. CONCLUSIONS: Adult human brain cells are sensitive to cytokine challenge. As expected 'classical' brain immune cells, such as microglia and astrocytes, responded to cytokine challenge but of even more interest, brain pericytes also responded to such challenge with a rich repertoire of gene expression. Immune activation of brain pericytes may play an important role in communicating inflammatory signals to and within the brain interior and may also be involved in blood brain barrier (BBB) disruption . Targeting brain pericytes, as well as microglia and astrocytes, may provide novel opportunities for reducing brain inflammation and maintaining BBB function and brain homeostasis in human brain disease

Heterologous expression of plasmodial proteins for structural studies and functional annotation

Malaria remains the world's most devastating tropical infectious disease with as many as 40% of the world population living in risk areas. The widespread resistance of Plasmodium parasites to the cost-effective chloroquine and antifolates has forced the introduction of more costly drug combinations, such as Coartem®. In the absence of a vaccine in the foreseeable future, one strategy to address the growing malaria problem is to identify and characterize new and durable antimalarial drug targets, the majority of which are parasite proteins. Biochemical and structure-activity analysis of these proteins is ultimately essential in the characterization of such targets but requires large amounts of functional protein. Even though heterologous protein production has now become a relatively routine endeavour for most proteins of diverse origins, the functional expression of soluble plasmodial proteins is highly problematic and slows the progress of antimalarial drug target discovery. Here the status quo of heterologous production of plasmodial proteins is presented, constraints are highlighted and alternative strategies and hosts for functional expression and annotation of plasmodial proteins are reviewed

Adult human glia, pericytes and meningeal fibroblasts respond similarly to IFNy but not to TGFβ1 or M-CSF.

The chemokine Interferon gamma-induced protein 10 (IP-10) and human leukocyte antigen (HLA) are widely used indicators of glial activation and neuroinflammation and are up-regulated in many brain disorders. These inflammatory mediators have been widely studied in rodent models of brain disorders, but less work has been undertaken using human brain cells. In this study we investigate the regulation of HLA and IP-10, as well as other cytokines and chemokines, in microglia, astrocytes, pericytes, and meningeal fibroblasts derived from biopsy and autopsy adult human brain, using immunocytochemistry and a Cytometric Bead Array. Interferonγ (IFNγ) increased microglial HLA expression, but contrary to data in rodents, the anti-inflammatory cytokine transforming growth factor β1 (TGFβ1) did not inhibit this increase in HLA, nor did TGFβ1 affect basal microglial HLA expression or IFNγ-induced astrocytic HLA expression. In contrast, IFNγ-induced and basal microglial HLA expression, but not IFNγ-induced astrocytic HLA expression, were strongly inhibited by macrophage colony stimulating factor (M-CSF). IFNγ also strongly induced HLA expression in pericytes and meningeal fibroblasts, which do not basally express HLA, and this induction was completely blocked by TGFβ1, but not affected by M-CSF. In contrast, TGFβ1 did not block the IFNγ-induced increase in IP-10 in pericytes and meningeal fibroblasts. These results show that IFNγ, TGFβ1 and M-CSF have species- and cell type-specific effects on human brain cells that may have implications for their roles in adult human brain inflammation

Differential regulation of HLA and IP-10 in adult human microglia, astrocytes, brain pericytes and meningeal fibroblasts by IFNy, TGFβ₁ and M-CSF.

<p>The T cell pro-inflammatory cytokine IFNy upregulates HLA and IP-10 protein expression in adult human brain glial cells, pericytes and meningeal fibroblasts. Microglial HLA was increased by IFNγ (1 ng/ml for 96 h). M-CSF (25 ng/ml), but not TGFβ₁ (10 ng/ml), was found to decrease microglial HLA expression. Astrocytic expression of HLA was also increased by IFNγ, and not modulated by TGFβ₁ or M-CSF. Brain pericytes and meningeal fibroblasts do not basally express HLA but have a marked induction on exposure to IFNγ, which was blocked by TGFβ₁. IFNγ increased adult human microglia, astrocyte and pericyte expression and release of pro-inflammatory cytokines and chemokines, particularly IP-10. IP-10 may be involved in leukocyte trafficking into the CNS.</p

IFNy-induced expression of HLA-DP, DQ, DR in brain-derived pericytes is inhibited by TGFβ₁ but not by M-CSF.

<p>A) Vehicle-treated pericytes (Hoechst-labelled nuclei) do not express HLA-DP, DQ, DR protein. B) IFNy induces a major up-regulation of HLA-DP, DQ, DR protein (green) in brain pericytes. C) TGFβ₁ treatment alone does not induce expression of HLA-DP, DQ, DR in these cells. However, TGFβ₁ completely inhibits the IFNy-stimulated increase in HLA-DP, DQ, DR (D). M-CSF affects neither basal (E) nor IFNy-induced (F) HLA-DP, DQ, DR expression in brain pericytes. Scale bar = 100 µm. G) HLA-DP, DQ, DR is induced by treatment with IFNy, and inhibited by simultaneous exposure to TGFβ₁ but not M-CSF (N = 12). H) Pericyte cell number (per well) is not influenced by IFNy or M-CSF but is significantly decreased by TGFβ₁ (N = 12).</p

Astrocytic expression of HLA-DP, DQ, DR is increased by IFNy, and not changed by TGFβ₁ or M-CSF.

<p>A) Adult human GFAP +ve astrocytes (red) express variable levels of HLA-DP, DQ, DR (green) in basal conditions without any treatment. B) IFNy (1 ng/ml, 96 h) increased astroglial expression of HLA-DP, DQ, DR. C) TGFβ₁ (10 ng/ml) did not affect astrocyte HLA-DP, DQ, DR expression alone, or when enhanced by IFNy treatment (D). E) M-CSF (25 ng/ml) also did not affect basal HLA-DP, DQ, DR expression in astrocytes or IFNy-enhanced HLA-DP, DQ, DR expression in astrocytes (F). Insets show close-up examples of astrocytes indicated by arrows. Scale bar = 100 µm. G) A significant increase in percentage of HLA-DP, DQ, DR-immunopositive astrocytes is found with IFNy treatment. Neither TGFβ₁ nor M-CSF significantly affect astrocyte HLA-DP, DQ, DR protein expression (N = 12). H) Quantification of GFAP-immunopositive astrocyte cell number (per well) following treatment with IFNy, TGFβ₁ or M-CSF does not result in any significant differences compared to vehicle-treated cells (N = 12).</p