Gossypol is a di-sesquiterpene natural-product in the form of a functionalised binaphthyl and is isolated from cotton plants. The compound has long been known to exhibit anti-malarial and other ...biological activities. Previous studies have indicated that compounds of this type target
Plasmodium falciparum lactate dehydrogenase (
pfLDH), an essential enzyme for energy generation within the parasite. In this study, we report that simple naphthalene-based compounds, the core of the gossypol structure, exhibit weak inhibition of the parasite lactate dehydrogenase. Crystal structures of the complexes formed by binding of these naphthalene-based compounds to their target enzyme have been used to delineate the molecular features likely to form the gossypol binding site. Two modes of binding are observed: one overlapping the pyruvate but not the co-factor site, the other bridging the binding sites for the co-factor nicontinamide group and pyruvate substrate. This latter site encompasses molecular features unique to
Plasmodium forms of LDH and is likely to represent the mode of binding for gossypol derivatives that show selectivity for the parasite enzymes. We also report a substrate analogue that unexpectedly binds within the adenine pocket of the co-factor groove. Although these core pharmacophore-like molecules only exhibit low levels of inhibitory activity, these molecular snapshots provide a rational basis for renewed structure-based development of naphthalene-based compounds as anti-malarial agents.
Micro-RNA-155 inhibits IFN-γ signaling in CD4⁺ T cells Banerjee, Arnob; Schambach, Felix; DeJong, Caitlin S ...
European journal of immunology,
2010, January 2010, 2010-Jan, 2010-01-00, 20100101, Letnik:
40, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Micro-RNA (miR) are increasingly recognized as critical regulators of tissue-specific patterns of gene expression. CD4⁺ T cells lacking miR-155, for example, exhibit bias towards Th2 differentiation, ...indicating that the absence of individual miR could alter CD4⁺ T-cell differentiation. We now show that miR-155 is induced upon T-cell activation and that it promotes Th1 differentiation when over-expressed in activated CD4⁺ T cells. Antagonism of miR-155 leads to induction of IFN-γ receptor α-chain (IFN-γRα, and a functional miR-155 target site is identified within the 3′ untranslated region of IFN-γRα. These results identify IFN-γRα as a second miR-155 target in T cells and suggest that miR-155 contributes to Th1 differentiation in CD4⁺ T cells by inhibiting IFN-γ signaling.
A hallmark of mammalian immunity is the heterogeneity of cell fate that exists among pathogen-experienced lymphocytes. We show that a dividing T lymphocyte initially responding to a microbe exhibits ...unequal partitioning of proteins that mediate signaling, cell fate specification, and asymmetric cell division. Asymmetric segregation of determinants appears to be coordinated by prolonged interaction between the T cell and its antigen-presenting cell before division. Additionally, the first two daughter T cells displayed phenotypic and functional indicators of being differentially fated toward effector and memory lineages. These results suggest a mechanism by which a single lymphocyte can apportion diverse cell fates necessary for adaptive immunity.
Immunity to intracellular pathogens requires dynamic balance between terminal differentiation of short-lived, cytotoxic effector CD8+ T cells and self-renewal of central-memory CD8+ T cells. We now ...show that T-bet represses transcription of IL-7Ralpha and drives differentiation of effector and effector-memory CD8+ T cells at the expense of central-memory cells. We also found T-bet to be overexpressed in CD8+ T cells that differentiated in the absence of CD4+ T cell help, a condition that is associated with defective central-memory formation. Finally, deletion of T-bet corrected the abnormal phenotypic and functional properties of "unhelped" memory CD8+ T cells. T-bet, thus, appears to function as a molecular switch between central- and effector-memory cell differentiation. Antagonism of T-bet may, therefore, represent a novel strategy to offset dysfunctional programming of memory CD8+ T cells.
Autoimmunity is thought to reflect an imbalance between regulatory T helper lymphocytes (Treg) and pathogenic, IL-17-secreting T helper (Th17) cells. Induction of both adaptive Treg and Th17 cells ...requires signalling from TGF-β. We now show that, in the context of TGF-β signalling, all-trans retinoic acid (ATRA) leads to increased induction of CD4⁺ T cells expressing the Treg specification factor forkhead box protein P3 (FoxP3) and decreased frequency of cells expressing IL-17, even in the presence of IL-6. Using a specific agonist and antagonist, as well as retroviral over-expression, we also provide evidence that the effects of ATRA are likely to be at least partially mediated by the nuclear retinoic acid receptor-α (RARα). These findings indicate that signalling through a specific nuclear retinoic acid receptor can favour the decision to adopt the Treg fate at the expense of Th17 fate. Specific agonists of RARα could, therefore, be considered candidates for the treatment of autoimmunity.
Micro-RNAs comprise a class of small noncoding RNAs which have been found to be important regulators of cellular differentiation in multiple species. Previous analysis of micro-RNA expression in the ...murine hematopoietic system has suggested a role in cell differentiation and the maintenance of cell identity. Naïve progenitor CD4+ T cells respond to a combination of appropriate antigen and other specific signals by undergoing proliferation and further differentiation into one of at least two subsets. T helper 1 (TH1) cells produce high levels of the cytokine IFN-γ and T helper 2 (TH2) cells produce high levels of IL-4, optimizing them for control of intracellular and extracellular pathogens, respectively. It is currently not known whether micro-RNA molecules influence CD4+ T cell differentiation. We have used oligonucleotide arrays to analyze micro-RNA expression profiles of freshly isolated murine CD4+ T cells compared to cells differentiating into TH1 and TH2 subsets. Expression profiles were found to differ significantly between naïve and stimulated CD4+ cells, with fewer differences between TH1 and TH2 subsets. Promising candidate micro-RNAs are being further evaluated by northern blot and genetic studies. Micro-RNA-155 is upregulated on stimulation of CD4+ T cells in multiple oligonucleotide array assays. Micro-RNA-155 is encoded by the BIC oncogene and has been implicated in lymphomagenesis as well as in other malignancies. We have verified the induction of micro-RNA-155 in stimulated helper T cells by northern blot and are studying the effects of this micro-RNA on CD4+ T cell differentiation. Our observations support a role for micro-RNAs in helper T cell differentiation during the immune response.
Immunity to intracellular pathogens requires dynamic balance between terminal differentiation of short-lived, cytotoxic effector CD8 super(+) T cells and self-renewal of central-memory CD8 super(+) T ...cells. We now show that T-bet represses transcription of IL-7R alpha and drives differentiation of effector and effector-memory CD8 super(+) T cells at the expense of central-memory cells. We also found T-bet to be overexpressed in CD8 super(+) T cells that differentiated in the absence of CD4 super(+) T cell help, a condition that is associated with defective central-memory formation. Finally, deletion of T-bet corrected the abnormal phenotypic and functional properties of "unhelped" memory CD8 super(+) T cells. T-bet, thus, appears to function as a molecular switch between central- and effector-memory cell differentiation. Antagonism of T-bet may, therefore, represent a novel strategy to offset dysfunctional programming of memory CD8 super(+) T cells.
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Abstract
Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, ...the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms' efficacy on small and large external datasets, and provide a versatile combined on- and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs.
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