Inhibition of the inositol kinase Itpkb augments calcium signaling in lymphocytes and reveals a novel strategy to treat autoimmune disease

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca2+ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease

Acute exercise leads to regulation of Telomere-Associated genes and MicroRNA expression in immune Cells

Telomeres are specialized nucleoprotein structures that protect chromosomal ends from degradation. These structures progressively shorten during cellular division and can signal replicative senescence below a critical length. Telomere length is predominantly maintained by the enzyme telomerase. Significant decreases in telomere length and telomerase activity are associated with a host of chronic diseases; conversely their maintenance underpins the optimal function of the adaptive immune system. Habitual physical activity is associated with longer leukocyte telomere length; however, the precise mechanisms are unclear. Potential hypotheses include regulation of telomeric gene transcription and/or microRNAs (miRNAs). We investigated the acute exercise-induced response of telomeric genes and miRNAs in twenty-two healthy males (mean age = 24.1±1.55 years). Participants undertook 30 minutes of treadmill running at 80% of peak oxygen uptake. Blood samples were taken before exercise, immediately post-exercise and 60 minutes post-exercise. Total RNA from white blood cells was submitted to miRNA arrays and telomere extension mRNA array. Results were individually validated in white blood cells and sorted T cell lymphocyte subsets using quantitative real-time PCR (qPCR). Telomerase reverse transcriptase (TERT) mRNA (P = 0.001) and sirtuin-6 (SIRT6) (P<0.05) mRNA expression were upregulated in white blood cells after exercise. Fifty-six miRNAs were also differentially regulated post-exercise (FDR <0.05). In silico analysis identified four miRNAs (miR-186, miR-181, miR-15a and miR-96) that potentially targeted telomeric gene mRNA. The four miRNAs exhibited significant upregulation 60 minutes post-exercise (P<0.001). Telomeric repeat binding factor 2, interacting protein (TERF2IP) was identified as a potential binding target for miR-186 and miR-96 and demonstrated concomitant downregulation (P<0.01) at the corresponding time point. Intense cardiorespiratory exercise was sufficient to differentially regulate key telomeric genes and miRNAs in white blood cells. These results may provide a mechanistic insight into telomere homeostasis and improved immune function and physical health. Funding NHMR

tICA-Metadynamics for Identifying Slow Dynamics in Membrane Permeation

Molecular simulations are commonly used to understand the mechanism of membrane permeation of small molecules, particularly for biomedical and pharmaceutical applications. However, despite significant advances in computing power and algorithms, calculating an accurate permeation free energy profile remains elusive for many drug molecules because it can require identifying the rate-limiting degrees of freedom (i.e., appropriate reaction coordinates). To resolve this issue, researchers have developed machine learning approaches to identify slow system dynamics. In this work, we apply time-lagged independent component analysis (tICA), an unsupervised dimensionality reduction algorithm, to molecular dynamics simulations with well-tempered metadynamics to find the slowest collective degrees of freedom of the permeation process of trimethoprim through a multicomponent membrane. We show that tICA-metadynamics yields translational and orientational collective variables (CVs) that increase convergence efficiency ∼1.5 times. However, crossing the periodic boundary is shown to introduce artifacts in the translational CV that can be corrected by taking absolute values of molecular features. Additionally, we find that the convergence of the tICA CVs is reached with approximately five membrane crossings and that data reweighting is required to avoid deviations in the translational CV

One Descriptor to Fold Them All: Harnessing Intuition and Machine Learning to Identify Transferable Lasso Peptide Reaction Coordinates

Identifying optimal reaction coordinates for complex conformational changes and protein folding remains an outstanding challenge. This study combines collective variable (CV) discovery based on chemical intuition and machine learning with enhanced sampling to converge the folding free energy landscape of lasso peptides, a unique class of natural products with knot-like tertiary structures. This knotted scaffold imparts remarkable stability, making lasso peptides resistant to proteolytic degradation, thermal denaturation, and extreme pH conditions. Although their direct synthesis would enable therapeutic design, it has not yet been possible due to the improbable occurrence of spontaneous lasso folding. Thus, simulations characterizing the folding propensity are needed to identify strategies for increasing access to the lasso architecture by stabilizing the pre-lasso ensemble before isopeptide bond formation. Herein, harmonic linear discriminant analysis (HLDA) is combined with metadynamics-enhanced sampling to discover CVs capable of distinguishing the pre-lasso fold and converging the folding propensity. Intuitive CVs are compared to iterative rounds of HLDA to identify CVs that not only accomplish these goals for one lasso peptide but also seem to be transferable to others, establishing a protocol for the identification of folding reaction coordinates for lasso peptides

CalQuo 2 : Automated Fourier-space, population-level quantification of global intracellular calcium responses

Intracellular calcium acts as a secondary messenger in a wide variety of crucial biological signaling processes. Advances in fluorescence microscopy and calcium sensitive dyes has led to the routine quantification of calcium responses in non-excitable cells. However, the automatization of global intracellular calcium analysis at the single-cell level within a large population simultaneously remains challenging. One software, CalQuo (Calcium Quantification), offers some automatic features in calcium analysis. Here, we present an advanced version of the software package: CalQuo2. CalQuo2 analyzes the calcium response in the Fourier-domain, allowing the number of user-defined filtering parameters to be reduced to one and a greater diversity of calcium responses to be recognized, compared to CalQuo that directly interprets the calcium intensity signal. CalQuo2 differentiates cells that release a single calcium response and those that release oscillatory calcium fluxes. We have demonstrated the use of CalQuo2 by measuring the calcium response in genetically modified Jurkat T-cells under varying ligand conditions, in which we show that peptide:MHCs and anti-CD3 antibodies trigger a fraction of T cells to release oscillatory calcium fluxes that increase with increasing koff rates. These results show that CalQuo2 is a robust and user-friendly tool for characterizing global, single cell calcium responses

Antigen receptor signaling: the Tuscan chronicles.

commentary EMBO conference 2005 "Signaling in the immune system

Hyperactivation of nuclear factor of activated T cells 1 (NFAT1) in T cells attenuates severity of murine autoimmune encephalomyelitis

Nuclear factor of activated T cells (NFAT) proteins are a group of Ca2+-regulated transcription factors residing in the cytoplasm of resting cells. Dephosphorylation by calcineurin results in nuclear translocation of NFAT and subsequent expression of target genes; rephosphorylation by kinases, including casein kinase 1 (CK1), restores NFAT to its latent state in the cytoplasm. We engineered a hyperactivable version of NFAT1 with increased affinity for calcineurin and decreased affinity for casein kinase 1. Mice expressing hyperactivable NFAT1 in their T-cell compartment exhibited a dramatically increased frequency of both IL-17– and IL-10–producing cells after differentiation under Th17 conditions—this was associated with direct binding of NFAT1 to distal regulatory regions of Il-17 and Il-10 gene loci in Th17 cells. Despite higher IL-17 production in culture, the mice were significantly less prone to myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis than controls, correlating with increased production of the immunomodulatory cytokine IL-10 and enhanced accumulation of regulatory T cells within the CNS. Thus, NFAT hyperactivation paradoxically leads to decreased susceptibility to experimental autoimmune encephalomyelitis, supporting previous observations linking defects in Ca2+/NFAT signaling to lymphoproliferation and autoimmune disease