No abstract

Abolhassani et al. show that CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of CD27 deficiency. CD70–CD27 interactions play a nonredundant role regulating humoral- and cell-mediated immunity in humans, especially for control of EBV.

Autophagy is an evolutionarily conserved mechanism for the gross disposal of intracellular proteins in mammalian cells and dysfunction in this pathway has been associated with human disease. Although the serine threonine kinase Akt is suggested to play a role in this process, little is known about the molecular mechanisms by which Akt induces autophagy. Using a yeast two-hybrid screen, Phafin2 (EAPF or PLEKHF2), a lysosomal protein with a unique structure of N-terminal PH (pleckstrin homology) domain and C-terminal FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain was found to interact with Akt. A sucrose gradient fractionation experiment revealed that both Akt and Phafin2 co-existed in the same lysosome enriched fraction after autophagy induction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosome after induction of autophagy. BiFC analysis using PtdIns (3)P interaction defective mutant of Phafin2 demonstrated that lysosomal accumulation of the Akt-Phafin2 complex and subsequent induction of autophagy were lysosomal PtdIns (3)P dependent events. Furthermore, in murine macrophages, both Akt and Phafin2 were required for digestion of fluorescent bacteria and/or LPS-induced autophagy. Taken together, these findings establish that lysosomal accumulation of Akt and Phafin2 is a critical step in the induction of autophagy via an interaction with PtdIns (3)P.

Magnesium transporter 1 (MAGT1) critically mediates magnesium homeostasis in eukaryotes and is highly-conserved across different evolutionary branches. In humans, loss-offunction mutations in the MAGT1 gene cause X-linked magnesium deficiency with Epstein-Barr virus (EBV) infection and neoplasia (XMEN), a disease that has a broad range of clinical and immunological consequences. We have previously shown that EBV susceptibility in XMEN is associated with defective expression of the antiviral natural-killer group 2 member D (NKG2D) protein and abnormal Mg 2؉ transport. New evidence suggests that MAGT1 is the human homolog of the yeast OST3/ OST6 proteins that form an integral part of the N-linked glycosylation complex, although the exact contributions of these perturbations in the glycosylation pathway to disease pathogenesis are still unknown. Using MS-based glycoproteomics, along with CRISPR/Cas9-KO cell lines, natural killer cell-killing assays, and RNA-Seq experiments, we now demonstrate that humans lacking functional MAGT1 have a selective deficiency in both immune and nonimmune glycoproteins, and we identified several critical glycosylation defects in important immune-response proteins and in the expression of genes involved in immunity, particularly CD28. We show that MAGT1 function is partly interchangeable with that of the paralog protein tumorsuppressor candidate 3 (TUSC3) but that each protein has a different tissue distribution in humans. We observed that MAGT1dependent glycosylation is sensitive to Mg 2؉ levels and that reduced Mg 2؉ impairs immune-cell function via the loss of specific glycoproteins. Our findings reveal that defects in protein glycosylation and gene expression underlie immune defects in an inherited disease due to MAGT1 deficiency.MAGT1 is an evolutionally conserved Mg 2ϩ -specific ion transport facilitator found in all animals and has been shown to participate in the multienzyme complex responsible for enzymatic coupling of N-glycans onto peptide substrates (1, 2). Null mutations in the MAGT1 gene lead to the rare primary immunodeficiency "X-linked immunodeficiency with Mg 2ϩ defect, Epstein-Barr virus (EBV) 7 infection and neoplasia" (XMEN) disease (3,4). Here, we explore these dual roles by examining cells from both healthy and MAGT1-deficient humans.Mg 2ϩ is the most abundant divalent cation in eukaryotic cells, with intracellular concentrations ranging from 15 to 20 mM depending on the cell type. Most Mg 2ϩ is tightly bound to cellular substituents, especially nucleic acids, nucleoside triphosphates, and enzymes. The unbound intracellular free Mg 2ϩ is estimated to be 0.4 -1.0 mM or ϳ1-5% of the total Mg 2ϩ concentration in the cell (5, 6), and because Mg 2ϩ is the biologically active form of Mg, these intracellular concentra-

The possibility of the pandemic spread of influenza viruses highlights the need for an effective cure for this life-threatening disease. Influenza A virus, belonging to a family of orthomyxoviruses, is a negative-strand RNA virus which encodes 11 viral proteins. A numbers of intracellular signaling pathways in the host cells interact with influenza the viral proteins, which affect various stages of viral infection and replication. In this study, we investigated how inhibition of Akt kinase activity impacts on influenza virus infection by using "Akt-in", a peptide Akt inhibitor. In PR8 influenza-infected A549 cells, Akt interacted with the NS1 (Non structural protein 1), and hence increased phosphorylation of Akt kinase activity and NS1. Treatment of cells with either "TCL1- or TCL1b-based Akt-in" efficiently suppressed Akt kinase activity while decreasing the levels of phosphorylated NS1; this, in turn, inhibited viral replication in a dose- and time-dependent manner. The inhibitory effect on viral replication appears to not be due to inhibition of the production of inflammatory cytokines, including IL-6 and IL-8, in the host cells. Inhibition of Akt kinase activity in the host cells inhibited the efficiency of viral entry, which is associated with decreased levels of phosphorylated glycogen synthase kinase 3, a substrate of Akt. Thus inhibition of Akt kinase activity in host cells may have therapeutic advantages for influenza virus infection by inhibiting viral entry and replication.

Mg2+ is required at micromolar concentrations as a cofactor for ATP, enzymatic reactions, and other biological processes. We show that decreased extracellular Mg2+ reduced intracellular Mg2+ levels and impaired the Ca2+ flux, activation marker up-regulation, and proliferation after T cell receptor (TCR) stimulation. Reduced Mg2+ specifically impairs TCR signal transduction by IL-2–inducible T cell kinase (ITK) due to a requirement for a regulatory Mg2+ in the catalytic pocket of ITK. We also show that altered catalytic efficiency by millimolar changes in free basal Mg2+ is an unrecognized but conserved feature of other serine/threonine and tyrosine kinases, suggesting a Mg2+ regulatory paradigm of kinase function. Finally, a reduced serum Mg2+ concentration in mice causes an impaired CD8+ T cell response to influenza A virus infection, reduces T cell activation, and exacerbates morbidity. Thus, Mg2+ directly regulates the active site of specific kinases during T cell responses, and maintaining a high serum Mg2+ concentration is important for antiviral immunity in otherwise healthy animals.

Serine–threonine kinase Akt (also known as PKB, protein kinase B), a core intracellular mediator of cell survival, is involved in various human cancers and has been suggested to play an important role in the regulation of autophagy in mammalian cells. Nonetheless, the physiological function of Akt in the lysosomes is currently unknown. We have reported previously that PtdIns(3)P-dependent lysosomal accumulation of the Akt–Phafin2 complex is a critical step for autophagy induction. Here, to characterize the molecular function of activated Akt in the lysosomes in the process of autophagy, we searched for the molecules that interact with the Akt complex at the lysosomes after induction of autophagy. By time-of-flight–mass spectrometry (TOF/MS) analysis, kinases of the VRK family, a unique serine–threonine family of kinases in the human kinome, were identified. VRK2 interacts with Akt1 and Akt2, but not with Akt3; the C terminus of Akt and the N terminus of VRK2 facilitate the interaction of Akt and VRK2 in mammalian cells. The kinase-dead form of VRK2A (KD VRK2A) failed to interact with Akt in coimmunoprecipitation assays. Bimolecular fluorescence complementation (BiFC) experiments showed that, in the lysosomes, Akt interacted with VRK2A but not with VRK2B or KD VRK2A. Immunofluorescent assays revealed that VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction. WT VRK2A, but not KD VRK2A or VRK2B, facilitated accumulation of phosphorylated Akt in the lysosomes. Downregulation of VRK2 abrogated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB; these events coincided to inhibition of autophagy induction. The VRK2–Akt complex is required for control of lysosomal size, acidification, bacterial degradation, and for viral replication. Moreover, lysosomal VRK2–Akt controls cellular proliferation and mitochondrial outer-membrane stabilization. Given the roles of autophagy in the pathogenesis of human cancer, the current study provides a novel insight into the oncogenic activity of VRK2–Akt complexes in the lysosomes via modulation of autophagy.

Protooncogene T-cell leukemia 1 (TCL1), which is implicated in human T-cell prolymphocytic leukemia (T-PLL), interacts with Akt and enhances its kinase activity, functioning as an Akt kinase co-activator. Two major isoforms of TCL1 Protooncogenes (TCL1 and TCL1b) are present adjacent to each other on human chromosome 14q.32. In human T-PLL, both TCL1 and TCL1b are activated by chromosomal translocation. Moreover, TCL1b-transgenic mice have never been created. Therefore, it remains unclear whether TCL1b itself, independent of TCL1, exhibits oncogenicity. In co-immunoprecipitation assays, both ectopic and endogenous TCL1b interacted with Akt. In in vitro Akt kinase assays, TCL1b enhanced Akt kinase activity in dose- and time-dependent manners. Bioinformatics approaches utilizing multiregression analysis, cluster analysis, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway mapping, Venn diagrams and Gene Ontology (GO) demonstrated that TCL1b showed highly homologous gene-induction signatures similar to Myr-Akt or TCL1. TCL1b exhibited oncogenicity in in vitro colony-transformation assay. Further, two independent lines of β-actin promoter-driven TCL1b-transgenic mice developed angiosarcoma on the intestinal tract. Angiosarcoma is a rare form of cancer in humans with poor prognosis. Using immunohistochemistry, 11 out of 13 human angiosarcoma samples were positively stained with both anti-TCL1b and anti-phospho-Akt antibodies. Consistently, in various cancer tissues, 69 out of 146 samples were positively stained with anti-TCL1b, out of which 46 were positively stained with anti-phospho-Akt antibodies. Moreover, TCL1b structure-based inhibitor ‘TCL1b-Akt-in' inhibited Akt kinase activity in in vitro kinase assays and PDGF (platelet-derived growth factor)-induced Akt kinase activities—in turn, ‘TCL1b-Akt-in' inhibited cellular proliferation of sarcoma. The current study disclosed TCL1b bears oncogenicity and hence serves as a novel therapeutic target for human neoplastic diseases.