Uncoupling JAK2 V617F activation from cytokine-induced signalling by modulation of JH2 αC helix.

The mechanisms by which JAK2 is activated by the prevalent pseudokinase (JH2) V617F mutation in blood cancers remain elusive. Via structure-guided mutagenesis and transcriptional and functional assays, we identify a community of residues from the JH2 helix αC, SH2-JH2 linker and JH1 kinase domain that mediate V617F-induced activation. This circuit is broken by altering the charge of residues along the solvent-exposed face of the JH2 αC, which is predicted to interact with the SH2-JH2 linker and JH1. Mutations that remove negative charges or add positive charges, such as E596A/R, do not alter the JH2 V617F fold, as shown by the crystal structure of JH2 V617F E596A. Instead, they prevent kinase domain activation via modulation of the C-terminal residues of the SH2-JH2 linker. These results suggest strategies for selective V617F JAK2 inhibition, with preservation of wild-type function.

Regulation of Hematopoiesis and Methionine Homeostasis by mTORC1 Inhibitor NPRL2.

Nitrogen permease regulator-like 2 (NPRL2) is a component of a conserved complex that inhibits mTORC1 (mammalian Target Of Rapamycin Complex 1) in response to amino acid insufficiency. Here, we show that NPRL2 is required for mouse viability and that its absence significantly compromises fetal liver hematopoiesis in developing embryos. Moreover, NPRL2 KO embryos have significantly reduced methionine levels and exhibit phenotypes reminiscent of cobalamin (vitamin B12) deficiency. Consistent with this idea, NPRL2 KO liver and mouse embryonic fibroblasts (MEFs) show defective processing of the cobalamin-transport protein transcobalamin 2, along with impaired lysosomal acidification and lysosomal gene expression. NPRL2 KO MEFs exhibit a significant defect in the cobalamin-dependent synthesis of methionine from homocysteine, which can be rescued by supplementation with cyanocobalamin. Taken together, these findings demonstrate a role for NPRL2 and mTORC1 in the regulation of lysosomal-dependent cobalamin processing, methionine synthesis, and maintenance of cellular re-methylation potential, which are important during hematopoiesis.

Genome editing of mouse fibroblasts by homologous recombination for sustained secretion of PDGF-B and augmentation of wound healing.

BACKGROUND: Exogenous cytokines, such as platelet-derived growth factor (PDGF)-B, can augment wound healing, but sustained delivery to maintain therapeutic levels remains a problem. "Genome editing" is a new technology in which precise genome modifications are made within cells using engineered site-specific nucleases. Genome editing avoids many of the complications associated with traditional gene therapy and the use of viral vectors, including random integration, imprecise gene expression, and inadvertent oncogene activation. METHODS: This study demonstrates site-specific nuclease-mediated integration of a PDGF-B transgene into a predefined locus within the genome of primary mouse fibroblasts. Engineered fibroblasts were applied to splinted mouse wounds and evaluated after 14 days and 5 months for the retention of engineered fibroblasts, wound healing morphology, angiogenesis, and systemic PDGF-B expression. RESULTS: The application of engineered PDGF-B-expressing fibroblasts enhanced wound healing compared with controls. Low-level, constitutive expression of PDGF-B was achieved without detectable levels of systemic PDGF-B. The mechanism of improved wound healing is, at least in part, the result of increased wound vascularization, as the wounds treated with PDGF-B fibroblasts had a blood vessel density 2.5 times greater than controls. After 5 months, the engineered fibroblasts persisted in the wound bed. No adverse effects were detected from the application of these fibroblasts after 5 months as assessed by hematoxylin and eosin staining of wounds and by mouse necropsy. CONCLUSIONS: These data support that site-specific genome editing allows for sustained cell-based cytokine delivery. Furthermore, sustained release of PDGF-B increases the speed and quality of wound healing after a single application.

Structural basis for the prion-like MAVS filaments in antiviral innate immunity.

Mitochondrial antiviral signaling (MAVS) protein is required for innate immune responses against RNA viruses. In virus-infected cells MAVS forms prion-like aggregates to activate antiviral signaling cascades, but the underlying structural mechanism is unknown. Here we report cryo-electron microscopic structures of the helical filaments formed by both the N-terminal caspase activation and recruitment domain (CARD) of MAVS and a truncated MAVS lacking part of the proline-rich region and the C-terminal transmembrane domain. Both structures are left-handed three-stranded helical filaments, revealing specific interfaces between individual CARD subunits that are dictated by electrostatic interactions between neighboring strands and hydrophobic interactions within each strand. Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidate the structural mechanism of MAVS polymerization, and explain how an α-helical domain uses distinct chemical interactions to form self-perpetuating filaments. DOI: http://dx.doi.org/10.7554/eLife.01489.001.

The erythropoietin receptor is a downstream effector of Klotho-induced cytoprotection.

Although the role of the erythropoietin (EPO) receptor (EpoR) in erythropoiesis has been known for decades, its role in nonhematopoietic tissues is still not well defined. Klotho has been shown and EPo has been suggested to protect against acute ischemia-reperfusion injury in the kidney. Here we found in rat kidney and in a rat renal tubular epithelial cell line (NRK cells) EpoR transcript and antigen, and EpoR activity signified as EPo-induced phosphorylation of Jak2, ErK, Akt, and Stat5 indicating the presence of functional EpoR. Transgenic overexpression of Klotho or addition of exogenous recombinant Klotho increased kidney EpoR protein and transcript. In NRK cells, Klotho increased EpoR protein, enhanced EPo-triggered phosphorylation of Jak2 and Stat5, the nuclear translocation of phospho-Stat5, and protected NRK cells from hydrogen peroxide cytotoxicity. Knockdown of endogenous EpoR rendered NRK cells more vulnerable, and overexpression of EpoR more resistant to peroxide-induced cytotoxicity, indicating that EpoR mitigates oxidative damage. Knockdown of EpoR by siRNA abolished Epo-induced Jak2, and Stat5 phosphorylation, and blunted the protective effect of Klotho against peroxide-induced cytotoxicity. Thus in the kidney, EpoR and its activity are downstream effectors of Klotho enabling it to function as a cytoprotective protein against oxidative injury.

Advances in understanding the pathogenesis of primary familial and congenital polycythaemia.

Primary familial and congenital polycythemia (PFCP) is an autosomal-dominant proliferative disorder characterized by erythrocytosis and hypersensitivity of erythroid progenitors to erythropoietin (Epo). Several lines of evidence suggest a causal role of truncated erythropoietin receptor (EpoR) in this disease. In this review, we discuss PFCP in the context of erythrocytosis and EpoR signalling. We focus on recent studies describing mechanisms underlying Epo-dependent EpoR down-regulation. One mechanism depends on internalization mediated through the p85 regulatory subunit of the Phosphoinositide 3-Kinase, and the other utilizes ubiquitin-based proteasomal degradation. Truncated PFCP EpoRs are not properly down-regulated upon stimulation, underscoring the importance of these mechanisms in the pathogenesis of PFCP.

Identification of a VLDL-induced, FDNPVY-independent internalization mechanism for the LDLR.

The low-density lipoprotein (LDL) receptor (LDLR) binds to and internalizes lipoproteins that contain apolipoproteinB100 (apoB100) or apolipoproteinE (apoE). Internalization of the apoB100 lipoprotein ligand, LDL, requires the FDNPVY(807) sequence on the LDLR cytoplasmic domain, which binds to the endocytic machinery of coated pits. We show here that inactivation of the FDNPVY(807) sequence by mutation of Y807 to cysteine prevented the uptake of LDL; however, this mutation did not prevent LDLR-dependent uptake of the apoE lipoprotein ligand, beta-VLDL. Comparison of the surface localization of the LDLR-Y807C using LDLR-immunogold, LDL-gold and beta-VLDL-gold probes revealed enrichment of LDLR-Y807C-bound beta-VLDL in coated pits, suggesting that beta-VLDL binding promoted the internalization of the LDLR-Y807C. Consistent with this possibility, treatment with monensin, which traps internalized LDLR in endosomes, resulted in the loss of surface LDLR-Y807C only when beta-VLDL was present. Reconstitution experiments in which LDLR variants were introduced into LDLR-deficient cells showed that the HIC(818) sequence is involved in beta-VLDL uptake by the LDLR-Y807C. Together, these experiments demonstrate that the LDLR has a very low-density lipoprotein (VLDL)-induced, FDNPVY-independent internalization mechanism.