AG490 and PF431396 Sensitive Tyrosine Kinase Control the Population Heterogeneity of Basal STAT1 Activity in Ube1l Deficient Cells

A population often contains distinct sub-populations, thereby increasing the complexity of the overall heterogeneity. However, the cellular origin and biological relevance of sub-populations in cell population have not been clearly identified. Here we demonstrated the novel roles of ISGylation, which is an IFN-induced post-translational modification, controlling heterogeneity at the population level in cultured adherent cells. Without UBE1L, an E1 enzyme of ISGylation, mouse embryonic fibroblasts (MEF) exhibited low viral resistance despite high STAT1 and ISG expression compared with the wild-type MEF. We observe that Ube1l(-/-) MEF populations consist of two behaviorally distinguishable sub-populations with distinct basal STAT1 activity, while wild-type MEF populations are unimodal. This population heterogeneity in Ube1l knock-out cells was perturbed by tyrosine kinase inhibitors, AG490 and PF431396. In contrast, the neutralization of type I IFN did not affect population heterogeneity. Based on these results, we concluded that UBE1L functions to adjust basal immunological states with the regulation of population heterogeneity.111Ysciescopu

Ubiquitin-like modifier FAT10 attenuates RIG-I mediated antiviral signaling by segregating activated RIG-I from its signaling platform

RIG-I is a key cytosolic RNA sensor that mediates innate immune defense against RNA virus. Aberrant RIG-I activity leads to severe pathological states such as autosomal dominant multi-system disorder, inflammatory myophathies and dermatomyositis. Therefore, identification of regulators that ensure efficient defense without harmful immune-pathology is particularly critical to deal with RIG-I-associated diseases. Here, we presented the inflammatory inducible FAT10 as a novel negative regulator of RIG-Imediated inflammatory response. In various cell lines, FAT10 protein is undetectable unless it is induced by pro-inflammatory cytokines. FAT10 non-covalently associated with the 2CARD domain of RIG-I, and inhibited viral RNA-induced IRF3 and NF-kappa B activation through modulating the RIG-I protein solubility. We further demonstrated that FAT10 was recruited to RIG-I-TRIM25 to form an inhibitory complex where FAT10 was stabilized by E3 ligase TRIM25. As the result, FAT10 inhibited the antiviral stress granules formation contains RIG-I and sequestered the active RIG-I away from the mitochondria. Our study presented a novel mechanism to dampen RIG-I activity. Highly accumulated FAT10 is observed in various cancers with pro-inflammatory environment, therefore, our finding which uncovered the suppressive effect of the accumulated FAT10 during virus-mediated inflammatory response may also provide molecular clue to understand the carcinogenesis related with infection and inflammation.11109Ysciescopu

Mutations in DDX58, which Encodes RIG-I, Cause Atypical Singleton-Merten Syndrome

Singleton-Merten syndrome (SMS) is an autosomal-dominant multi-system disorder characterized by dental dysplasia, aortic calcification, skeletal abnormalities, glaucoma, psoriasis, and other conditions. Despite an apparent autosomal-dominant pattern of inheritance, the genetic background of SMS and information about its phenotypic heterogeneity remain unknown. Recently, we found a family affected by glaucoma, aortic calcification, and skeletal abnormalities. Unlike subjects with classic SMS, affected individuals showed normal dentition, suggesting atypical SMS. To identify genetic causes of the disease, we performed exome sequencing in this family and identified a variant (c.1118A>C [p.Glu373Ala]) of DDX58, whose protein product is also known as RIG-I. Further analysis of DDX58 in 100 individuals with congenital glaucoma identified another variant (c.803G>T [p.Cys268Phe]) in a family who harbored neither dental anomalies nor aortic calcification but who suffered from glaucoma and skeletal abnormalities. Cys268 and Glu373 residues of DDX58 belong to ATP-binding motifs I and II, respectively, and these residues are predicted to be located closer to the ADP and RNA molecules than other nonpathogenic missense variants by protein structure analysis. Functional assays revealed that DDX58 alterations confer constitutive activation and thus lead to increased interferon (IFN) activity and IFN-stimulated gene expression. In addition, when we transduced primary human trabecular meshwork cells with c.803G>T (p.Cys268Phe) and c.1118A>C (p.Glu373Ala) mutants, cytopathic effects and a significant decrease in cell number were observed. Taken together, our results demonstrate that DDX58 mutations cause atypical SMS manifesting with variable expression of glaucoma, aortic calcification, and skeletal abnormalities without dental anomalies

Mutations in DDX58, which Encodes RIG-I, Cause Atypical Singleton-Merten Syndrome

Singleton-Merten syndrome (SMS) is an autosomal-dominant multi-system disorder characterized by dental dysplasia, aortic calcification, skeletal abnormalities, glaucoma, psoriasis, and other conditions. Despite an apparent autosomal-dominant pattern of inheritance, the genetic background of SMS and information about its phenotypic heterogeneity remain unknown. Recently, we found a family affected by glaucoma, aortic calcification, and skeletal abnormalities. Unlike subjects with classic SMS, affected individuals showed normal dentition, suggesting atypical SMS. To identify genetic causes of the disease, we performed exome sequencing in this family and identified a variant (c.1118A>C [p.GLu373Ala]) of DDX58, whose protein product is also known as RIG-I. Further analysis of DDX58 in 100 individuals with congenital glaucoma identified another variant (c.803G>T [p.Cys268Phe]) in a family who harbored neither dental anomalies nor aortic calcification but who suffered from glaucoma and skeletal abnormalities. Cys268 and Glu373 residues of DDX58 belong to ATP-binding motifs I and II, respectively, and these residues are predicted to be located closer to the ADP and RNA molecules than other nonpathogenic missense variants by protein structure analysis. Functional assays revealed that DDX58 alterations confer constitutive activation and thus lead to increased interferon (IFN) activity and IFN-stimulated gene expression. In addition, when we transduced primary human trabecular meshwork cells with c.803G>T (p.Cys268Phe) and c.1118A>C (p.Glu373A1a) mutants, cytopathic effects and a significant decrease in cell number were observed. Taken together, our results demonstrate that DDX58 mutations cause atypical SMS manifesting with variable expression of glaucoma, aortic calcification, and skeletal abnormalities without dental anomalies.X116452Ysciescopu

A protein-kinase, IFN-inducible double-stranded RNA dependent inhibitor and repressor of p58 (PRKRIR) enhances type I IFN-mediated antiviral response through the stability control of RIG-I protein

The cellular RIG-I-like receptor (RLR) senses pathogenic RNA molecular patterns and transmits signals for type I interferon (IFN) production. It acts as a center for antiviral responses, and large numbers of RIG-I (retinoic acid inducible gene-I) interacting proteins are identified as signaling regulators. In the present study, we report PRKRIR, a negative regulator of PKR inhibitor, as a novel RIG-I interacting protein. In HEK293FT cells, PRKRIR synergistically enhances type I IFN production mediated by a signal activated- or constitutively active form of RIG-I. The C-terminal domain of the PRKRIR was required for physical interaction and the signal augmentation. The PRKRIR blocks poly-ubiquitination and protein degradation of RIG-I, thereby increasing cellular levels of RIG-I proteins. Furthermore, overexpression of PRKRIR, along with a signal activated- or constitutively active form of RIG-I, efficiently inhibits virus replication in the infected host. In conclusion, PRKRIR provides a novel positive regulator controlling the RIG-I-IFN production system through protein stability control. (C) 2011 Elsevier Inc. All rights reserved.X1133sciescopu

The <i>Ube1l</i>-deficient MEFs are more susceptible to viruses with higher STAT1 activity.

<p>(A) Immunoblot assay for measuring expression of indicated genes in <i>Ube1l</i>^<i>+/+</i> and <i>Ube1l</i>^<i>-/-</i> MEF with or without IFNβ (100 U/ml, 16 hour). (B) Immunoblot assay for measuring STAT1 activity (phosphorylation of Tyr701 on STAT1) in in <i>Ube1l</i>^<i>+/+</i> and <i>Ube1l</i>^<i>-/-</i> MEFs with or without IFNβ (100 U/ml, 30 min). (C) Relative mRNA expression of the indicated genes in basal <i>Ube1l</i>^<i>+/+</i> and <i>Ube1l</i>^<i>-/-</i> MEFs. <i>Left</i>–Using real-time quantitative PCR, normalizing with <i>Gapdh</i>. <i>Right</i>–Representative agarose gel image using conventional reverse-transcription PCR. Numbers beside image indicate the size of PCR product (base pairs). (D) Intracellular viral RNA normalizing with <i>Gapdh</i> in <i>Ube1l</i>^<i>+/+</i> and <i>Ube1l</i>^<i>-/-</i> MEFs infected with influenza A virus (10 TCID50) for the indicated time. (E) Relative amounts of intracellular viral RNA normalizing with <i>Gapdh</i> in <i>Ube1l</i>^+/+ and <i>Ube1l</i>^<i>-/-</i> MEFs infected with Sendai virus (10 TCID50) for indicated time. (A-B) Numbers beside blot images indicate the molecular weight (kDa). (C-E) Mean and standard error obtained from three independent experiments, unpaired t test, * p<0.05, ** p<0.01, ***p<0.001.</p

Effect of AG490 and PF431396 inhibitor on basal STAT1 activity and ISG expression.

<p>(A) Immunoblot assay for measuring basal STAT1 activity and expression of indicated genes in MEFs treated with AG490 (50 μM, 16 h). (B) Relative mRNA expression of the indicated genes were measured in MEFs treated with AG490 (50 μM, 16hr). (C) Immunoblot assay for measuring basal STAT1 activity and expression of indicated genes in MEFs treated with PF431396 (10 μM, indicated time). Numbers above the blot denotes treatment time in hours. (D) Relative mRNA expression of the indicated genes were measured in MEFs treated with PF431396 (15 μM, 12 h). (A, C) Numbers beside blot images indicate the molecular weight (kDa). (B, D) <i>Top</i>–Using real-time quantitative PCR, normalizing with <i>Gapdh</i>. Mean and standard error obtained from three independent experiments, unpaired t test, * p<0.05, ** p<0.01, ***p<0.001. <i>Bottom</i>–Representative agarose gel image using conventional reverse-transcription PCR. Numbers beside image indicate the size of PCR product (base pairs).</p

AG490 and PF431396 Sensitive Tyrosine Kinase Control the Population Heterogeneity of Basal STAT1 Activity in <i>Ube1l</i> Deficient Cells

<div><p>A population often contains distinct sub-populations, thereby increasing the complexity of the overall heterogeneity. However, the cellular origin and biological relevance of sub-populations in cell population have not been clearly identified. Here we demonstrated the novel roles of ISGylation, which is an IFN-induced post-translational modification, controlling heterogeneity at the population level in cultured adherent cells. Without UBE1L, an E1 enzyme of ISGylation, mouse embryonic fibroblasts (MEF) exhibited low viral resistance despite high STAT1 and ISG expression compared with the wild-type MEF. We observe that <i>Ube1l</i>^{<i>−/−</i>} MEF populations consist of two behaviorally distinguishable sub-populations with distinct basal STAT1 activity, while wild-type MEF populations are unimodal. This population heterogeneity in <i>Ube1l</i> knock-out cells was perturbed by tyrosine kinase inhibitors, AG490 and PF431396. In contrast, the neutralization of type I IFN did not affect population heterogeneity. Based on these results, we concluded that UBE1L functions to adjust basal immunological states with the regulation of population heterogeneity.</p></div

Bimodality in <i>Ube1l</i> deficient MEF populations are not mediated by basal type I IFN signaling.

<p>(A) FACS analysis of surface H-2Kb expression on MEFs treated with a neutralizing antibody for IFNAR1 (5 μg/ml) or the isotype control. (B) <i>Left—</i>FACS analysis of surface H-2Kb expression in MEFs transduced with the control or mouse <i>Ifnar1</i> shRNA lentivirus. <i>Right–</i>Relative expression of <i>Ifnar1</i> mRNA normalizing with <i>Gapdh</i> in control or <i>Ifnar1</i> shRNA lentivirus treated MEF. (C-D) <i>Ube1l</i>^<i>+/+</i> (C) and <i>Ube1l</i>^<i>-/-</i> (D) were treated with 10uM of Jak Inhibitor 1 (Jak Inh 1) for 4 hours followed by additional 12 hours of IFNβ (100U/ml) treatment. Samples were harvested and measure the H-2Kb expression with FACS.</p

<i>Ube1l</i>-deficient population harbors sub-populations with distinct surface H-2Kb expression.

<p> (A) FACS analysis of surface H-2Kb expression on <i>Ube1l</i>^<i>+/+</i> MEFs with indicated amount of IFNβ for 16hour. (B) FACS analysis of surface H-2Kb expression on <i>Ube1l</i>^<i>+/+</i> and MEFs, with 100U/ml of IFNβ for indicated time. (C) FACS analysis of surface H-2Kb expression on <i>Ube1l</i>^<i>-/-</i> MEFs with indicated amount of IFNβ for 16hour. (D) FACS analysis of surface H-2Kb expression on <i>Ube1l</i>^<i>-/-</i> and MEFs, with 100U/ml of IFNβ for indicated time. (E) With or without the treatment of IFNβ (100U/ml, 16 h), <i>Ube1l</i>^<i>+/+</i> and <i>Ube1l</i>^<i>-/-</i> MEFs were harvested and permeabilized followed by FACS analysis for the whole cell H-2Kb expression. (F) <i>Top</i>—FACS analysis of surface H-2Kb expression on <i>Ube1l</i>^<i>+/+</i> and <i>Ube1l</i>^<i>-/-</i> MEFs transduced with the control or <i>HA-hUBE1L</i>-lentivirus. <i>Bottom</i>—Immunoblot for confirming HA-UBE1L expression. Numbers beside blot images indicate the molecular weight (kDa).</p