Human Guanylate Binding Proteins Potentiate the Anti-Chlamydia Effects of Interferon-γ

Chlamydiae are obligate intracellular pathogens that are sensitive to pro-inflammatory cytokine interferon-γ. IFN-γ-inducible murine p47 GTPases have been demonstrated to function in resistance to chlamydia infection in vivo and in vitro. Because the human genome does not encode IFN-γ-inducible homologues of these proteins, the significance of the p47 GTPase findings to chlamydia pathogenesis in humans is unclear. Here we report a pair of IFN-γ-inducible proteins, the human guanylate binding proteins (hGBPs) 1 and 2 that potentiate the anti-chlamydial properties of IFN-γ. hGBP1 and 2 localize to the inclusion membrane, and their anti-chlamydial functions required the GTPase domain. Alone, hGBP1 or 2 have mild, but statistically significant and reproducible negative effects on the growth of Chlamydia trachomatis, whilst having potent anti-chlamydial activity in conjunction with treatment with a sub-inhibitory concentration of IFN-γ. Thus, hGBPs appear to potentiate the anti-chlamydial effects of IFN-γ. Indeed, depletion of hGBP1 and 2 in cells treated with IFN-γ led to an increase in inclusion size, indicative of better growth. Interestingly, chlamydia species/strains harboring the full-length version of the putative cytotoxin gene, which has been suggested to confer resistance to IFN-γ was not affected by hGBP overexpression. These findings identify the guanylate binding proteins as potentiators of IFN-γ inhibition of C. trachomatis growth, and may be the targets of the chlamydial cytotoxin.     

Ebola virus VP24 proteins inhibit the interaction of NPI-1 subfamily karyopherin alpha proteins with activated STAT1

The Zaire ebolavirus protein VP24 was previously demonstrated to inhibit alpha/beta interferon (IFN-α/β)- and IFN-γ-induced nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1) and to inhibit IFN-α/β- and IFN-γ-induced gene expression. These properties correlated with the ability of VP24 to interact with the nuclear localization signal receptor for PY-STAT1, karyopherin α1. Here, VP24 is demonstrated to interact not only with overexpressed but also with endogenous karyopherin α1. Mutational analysis demonstrated that VP24 binds within the PY-STAT1 binding region located in the C terminus of karyopherin α1. In addition, VP24 was found to inhibit PY-STAT1 binding to both overexpressed and endogenous karyopherin α1. We assessed the binding of both PY-STAT1 and the VP24 proteins from Zaire, mouse-adapted Zaire, and Reston Ebola viruses for interaction with all six members of the human karyopherin α family. We found, in contrast to previous studies, that PY-STAT1 can interact not only with karyopherin α1 but also with karyopherins α5 and α6, which together comprise the NPI-1 subfamily of karyopherin αs. Similarly, all three VP24s bound and inhibited PY-STAT1 interaction with karyopherins α1, α5, and α6. Consistent with their ability to inhibit the karyopherin-PY-STAT1 interaction, Zaire, mouse-adapted Zaire, and Reston Ebola virus VP24s displayed similar capacities to inhibit IFN-β-induced gene expression in human and mouse cells. These findings suggest that VP24 inhibits interaction of PY-STAT1 with karyopherins α1, α5, or α6 by binding within the PY-STAT1 binding region of the karyopherins and that this function is conserved among the VP24 proteins of different Ebola virus species.     

βArrestin1 Regulates the Guanine Nucleotide Exchange Factor RasGRF2 Expression and the Small GTPase Rac-mediated Formation of Membrane Protrusion and Cell Motility

βArrestin proteins shuttle between the cytosol and nucleus and have been shown to regulate G protein-coupled receptor signaling, actin remodeling, and gene expression. Here, we tested the hypothesis that βarrestin1 regulates actin remodeling and cell migration through the small GTPase Rac. Depletion of βarrestin1 promotes Rac activation, leading to the formation of multipolar protrusions and increased cell circularity, and overexpression of a dominant negative form of Rac reverses these morphological changes. Small interfering RNA library screen identifies RasGRF2 as a target of βarrestin1. RasGRF2 gene and protein expression levels are elevated following depletion of βarrestin1, and the consequent activation of Rac results in dephosphorylation of cofilin that can promote actin polymerization and formation of multipolar protrusions, thereby retarding cell migration and invasion. Together, these results suggest that βarrestin1 regulates rasgrf2 gene expression and Rac activation to affect membrane protrusion and cell migration and invasion.     

Anti-CD70 Immunocytokines for Exploitation of Interferon-γ-Induced RIP1-Dependent Necrosis in Renal Cell Carcinoma

Metastatic renal cell carcinoma (RCC) is an incurable disease in clear need of new therapeutic interventions. In early-phase clinical trials, the cytokine IFN-γ showed promise as a biotherapeutic for advanced RCC, but subsequent trials were less promising. These trials, however, focused on the indirect immunomodulatory properties of IFN-γ, and its direct anti-tumor effects, including its ability to kill tumor cells, remains mostly unexploited. We have previously shown that IFN-γ induces RIP1 kinase-dependent necrosis in cells lacking NF-κB survival signaling. RCC cells display basally-elevated NF-κB activity, and inhibiting NF-κB in these cells, for example by using the small-molecule proteasome blocker bortezomib, sensitizes them to RIP1-dependent necrotic death following exposure to IFN-γ. While these observations suggest that IFN-γ-mediated direct tumoricidal activity will have therapeutic benefit in RCC, they cannot be effectively exploited unless IFN-γ is targeted to tumor cells in vivo. Here, we describe the generation and characterization of two novel ‘immunocytokine’ chimeric proteins, in which either human or murine IFN-γ is fused to an antibody targeting the putative metastatic RCC biomarker CD70. These immunocytokines display high levels of species-specific IFN-γ activity and selective binding to CD70 on human RCC cells. Importantly, the IFN-γ immunocytokines function as well as native IFN-γ in inducing RIP1-dependent necrosis in RCC cells, when deployed in the presence of bortezomib. These results provide a foundation for the in vivo exploitation of IFN-γ-driven tumoricidal activity in RCC.     

A Highlights from MBoC Selection: αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.     

IFN-γ Mediates the Rejection of Haematopoietic Stem Cells in IFN-γR1-Deficient Hosts

Background

Interferon-γ receptor 1 (IFN-γR1) deficiency is a life-threatening inherited disorder, conferring predisposition to mycobacterial diseases. Haematopoietic stem cell transplantation (HSCT) is the only curative treatment available, but is hampered by a very high rate of graft rejection, even with intra-familial HLA-identical transplants. This high rejection rate is not seen in any other congenital disorders and remains unexplained. We studied the underlying mechanism in a mouse model of HSCT for IFN-γR1 deficiency.

Methods and Findings

We demonstrated that HSCT with cells from a syngenic C57BL/6 Ifngr1 ^+/+ donor engrafted well and restored anti-mycobacterial immunity in naive, non-infected C57BL/6 Ifngr1 ^−/− recipients. However, Ifngr1 ^−/− mice previously infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) rejected HSCT. Like infected IFN-γR1-deficient humans, infected Ifngr1 ^−/− mice displayed very high serum IFN-γ levels before HSCT. The administration of a recombinant IFN-γ-expressing AAV vector to Ifngr1 ^−/− naive recipients also resulted in HSCT graft rejection. Transplantation was successful in Ifngr1 ^−/− × Ifng ^−/− double-mutant mice, even after BCG infection. Finally, efficient antibody-mediated IFN-γ depletion in infected Ifngr1 ^−/− mice in vivo allowed subsequent engraftment.

Conclusions

High serum IFN-γ concentration is both necessary and sufficient for graft rejection in IFN-γR1-deficient mice, inhibiting the development of heterologous, IFN-γR1-expressing, haematopoietic cell lineages. These results confirm that IFN-γ is an anti-haematopoietic cytokine in vivo. They also pave the way for HSCT management in IFN-γR1-deficient patients through IFN-γ depletion from the blood. They further raise the possibility that depleting IFN-γ may improve engraftment in other settings, such as HSCT from a haplo-identical or unrelated donor.     

ASB2α, an E3 Ubiquitin Ligase Specificity Subunit,Regulates Cell Spreading and Triggers Proteasomal Degradation of Filamins by Targeting the Filamin Calponin Homology 1 Domain

Filamins are actin-binding and cross-linking proteins that organize the actin cytoskeleton and anchor transmembrane proteins to the cytoskeleton and scaffold signaling pathways. During hematopoietic cell differentiation, transient expression of ASB2α, the specificity subunit of an E3-ubiquitin ligase complex, triggers acute proteasomal degradation of filamins. This led to the proposal that ASB2α regulates hematopoietic cell differentiation by modulating cell adhesion, spreading, and actin remodeling through targeted degradation of filamins. Here, we show that the calponin homology domain 1 (CH1), within the filamin A (FLNa) actin-binding domain, is the minimal fragment sufficient for ASB2α-mediated degradation. Combining an in-depth flow cytometry analysis with mutagenesis of lysine residues within CH1, we find that arginine substitution at each of a cluster of three lysines (Lys-42, Lys-43, and Lys-135) renders FLNa resistant to ASB2α-mediated degradation without altering ASB2α binding. These lysines lie within previously predicted actin-binding sites, and the ASB2α-resistant filamin mutant is defective in targeting to F-actin-rich structures in cells. However, by swapping CH1 with that of α-actinin1, which is resistant to ASB2α-mediated degradation, we generated an ASB2α-resistant chimeric FLNa with normal subcellular localization. Notably, this chimera fully rescues the impaired cell spreading induced by ASB2α expression. Our data therefore reveal ubiquitin acceptor sites in FLNa and establish that ASB2α-mediated effects on cell spreading are due to loss of filamins.     

Structural and Biochemical Basis for the Inhibitory Effect of Liprin-α3 on Mouse Diaphanous 1 (mDia1) Function

Diaphanous-related formins are eukaryotic actin nucleation factors regulated by an autoinhibitory interaction between the N-terminal RhoGTPase-binding domain (mDia_N) and the C-terminal Diaphanous-autoregulatory domain (DAD). Although the activation of formins by Rho proteins is well characterized, its inactivation is only marginally understood. Recently, liprin-α3 was shown to interact with mDia1. Overexpression of liprin-α3 resulted in a reduction of the cellular actin filament content. The molecular mechanisms of how liprin-α3 exerts this effect and counteracts mDia1 activation by RhoA are unknown. Here, we functionally and structurally define a minimal liprin-α3 core region, sufficient to recapitulate the liprin-α3 determined mDia1-respective cellular functions. We show that liprin-α3 alters the interaction kinetics and thermodynamics of mDia_N with RhoA·GTP and DAD. RhoA displaces liprin-α3 allosterically, whereas DAD competes with liprin-α3 for a highly overlapping binding site on mDia_N. Liprin-α3 regulates actin polymerization by lowering the regulatory potency of RhoA and DAD on mDia_N. We present a model of a mechanistically unexplored and new aspect of mDia_N regulation by liprin-α3.     

Insulin Secretion Induced by Glucose-dependent Insulinotropic Polypeptide Requires Phosphatidylinositol 3-Kinase γ in Rodent and Human β-Cells

PI3Kγ, a G-protein-coupled type 1B phosphoinositol 3-kinase, exhibits a basal glucose-independent activity in β-cells and can be activated by the glucose-dependent insulinotropic polypeptide (GIP). We therefore investigated the role of the PI3Kγ catalytic subunit (p110γ) in insulin secretion and β-cell exocytosis stimulated by GIP. We inhibited p110γ with AS604850 (1 μmol/liter) or knocked it down using an shRNA adenovirus or siRNA duplex in mouse and human islets and β-cells. Inhibition of PI3Kγ blunted the exocytotic and insulinotropic response to GIP receptor activation, whereas responses to the glucagon-like peptide-1 or the glucagon-like peptide-1 receptor agonist exendin-4 were unchanged. Downstream, we find that GIP, much like glucose stimulation, activates the small GTPase protein Rac1 to induce actin remodeling. Inhibition of PI3Kγ blocked these effects of GIP. Although exendin-4 could also stimulate actin remodeling, this was not prevented by p110γ inhibition. Finally, forced actin depolymerization with latrunculin B restored the exocytotic and secretory responses to GIP during PI3Kγ inhibition, demonstrating that the loss of GIP-induced actin depolymerization was indeed limiting insulin exocytosis.     

IFN-γ Directly Controls IL-33 Protein Level through a STAT1- and LMP2-dependent Mechanism

IL-33 contributes to disease processes in association with Th1 and Th2 phenotypes. IL-33 mRNA is rapidly regulated, but the fate of synthesized IL-33 protein is unknown. To understand the interplay among IL-33, IFN-γ, and IL-4 proteins, recombinant replication-deficient adenoviruses were produced and used for dual expression of IL-33 and IFN-γ or IL-33 and IL-4. The effects of such dual gene delivery were compared with the effects of similar expression of each of these cytokines alone. In lung fibroblast culture, co-expression of IL-33 and IFN-γ resulted in suppression of the levels of both proteins, whereas co-expression of IL-33 and IL-4 led to mutual elevation. In vivo, co-expression of IL-33 and IFN-γ in the lungs led to attenuation of IL-33 protein levels. Purified IFN-γ also attenuated IL-33 protein in fibroblast culture, suggesting that IFN-γ controls IL-33 protein degradation. Specific inhibition of caspase-1, -3, and -8 had minimal effect on IFN-γ-driven IL-33 protein down-regulation. Pharmacological inhibition, siRNA-mediated silencing, or gene deficiency of STAT1 potently up-regulated IL-33 protein expression levels and attenuated the down-regulating effect of IFN-γ on IL-33. Stimulation with IFN-γ strongly elevated the levels of the LMP2 proteasome subunit, known for its role in IFN-γ-regulated antigen processing. siRNA-mediated silencing of LMP2 expression abrogated the effect of IFN-γ on IL-33. Thus, IFN-γ, IL-4, and IL-33 are engaged in a complex interplay. The down-regulation of IL-33 protein levels by IFN-γ in pulmonary fibroblasts and in the lungs in vivo occurs through STAT1 and non-canonical use of the LMP2 proteasome subunit in a caspase-independent fashion.     

IQ Domain GTPase-Activating Protein 1 is Involved in Shear Stress-Induced Progenitor-Derived Endothelial Cell Alignment

Shear stress is one of mechanical constraints which are exerted by blood flow on endothelial cells (ECs). To adapt to shear stress, ECs align in the direction of flow through adherens junction (AJ) remodeling. However, mechanisms regulating ECs alignment under shear stress are poorly understood. The scaffold protein IQ domain GTPase activating protein 1 (IQGAP1) is a scaffold protein which couples cell signaling to the actin and microtubule cytoskeletons and is involved in cell migration and adhesion. IQGAP1 also plays a role in AJ organization in epithelial cells. In this study, we investigated the potential IQGAP1 involvement in the endothelial cells alignment under shear stress. Progenitor-derived endothelial cells (PDECs), transfected (or not) with IQGAP1 small interfering RNA, were exposed to a laminar shear stress (1.2 N/m²) and AJ proteins (VE-cadherin and β-catenin) and IQGAP1 were labeled by immunofluorescence. We show that IQGAP1 is essential for ECs alignment under shear stress. We studied the role of IQGAP1 in AJs remodeling of PDECs exposed to shear stress by studying cell localization and IQGAP1 interactions with VE-cadherin and β-catenin by immunofluorescence and Proximity Ligation Assays. In static conditions, IQGAP1 interacts with VE-cadherin but not with β-catenin at the cell membrane. Under shear stress, IQGAP1 lost its interaction from VE-cadherin to β-catenin. This “switch” was concomitant with the loss of β-catenin/VE-cadherin interaction at the cell membrane. This work shows that IQGAP1 is essential to ECs alignment under shear stress and that AJ remodeling represents one of the mechanisms involved. These results provide a new approach to understand ECs alignment under to shear stress.     

The 3' untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1gamma and HAX-1

Previously, we have shown that the vimentin 3′ untranslated region (3′UTR) contains a highly conserved region, which is sufficient for the perinuclear localization of a reporter mRNA. This region was shown to specifically bind protein(s) by band shift analyses. UV-cross-linking studies suggest these proteins are 46- and 35-kDa in mass. Here, we have used this sequence as ‘bait’ to isolate RNA binding proteins using the yeast three-hybrid method. This technique relies on a functional assay detecting bona fide RNA–protein interaction in vivo. Three cDNA isolates, HAX-1, eEF-1γ and hRIP, code for proteins of a size consistent with in vitro cross- linking studies. In all cases, recombinant proteins were capable of binding RNA in vitro. Although hRIP is thought to be a general mRNA binding protein, this represents an unreported activity for eEF-1γ and HAX-1. Moreover, HAX-1 binding appears to be specific to vimentin’s 3′UTR. Both in vivo synthesized eEF-1γ and HAX-1 proteins were ‘pulled out’ of HeLa whole cell extracts by binding to a RNA affinity column comprised of vimentin’s 3′UTR. Moreover, size-fractionation of extracts results in the separation of large complexes containing either eEF-1γ or HAX-1. Thus, in addition to their known functions, both eEF-1γ and HAX-1 are RNA binding proteins, which suggests new roles in mRNA translation and/or perinuclear localization.     

GAR22β regulates cell migration,sperm motility,and axoneme structure

Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22β), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22β^−/− Sertoli cells moved faster than wild-type cells. In addition, GAR22β^−/− cells showed a more prominent focal adhesion turnover. GAR22β overexpression or its reexpression in GAR22β^−/− cells reduced cell motility and focal adhesion turnover. GAR22β–actin interaction was stronger than GAR22β–microtubule interaction, resulting in GAR22β localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22β interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22β–EB1 interaction was required for the ability of GAR22β to modulate cell motility. We found that GAR22β is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22β as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes.