Xiaomei Tong

Ndfip1 Negatively Regulates RIG-I–Dependent Immune Signaling by Enhancing E3 Ligase Smurf1-Mediated MAVS Degradation

Ndfip1 functions as both a recruiter and an activator of multiple HECT domain E3 ubiquitin ligases of the Nedd4 family. In this study, we demonstrate that Ndfip1 is involved in the ubiquitin-mediated degradation of mitochondrial antiviral signaling (MAVS), which is a key adaptor protein in RIG-I–like receptor–mediated immune signaling. We found that overexpression of Ndfip1 severely impaired MAVS and Sendai virus–mediated activation of IFN-stimulated response element, NF-κB, IFN-β promoter, and polyinosinic-polycytidylic acid or influenza virus RNA–stimulated IRF-3 phosphorylation, as well as the transcription of IFN-β. This functional interaction was confirmed by knockdown of Ndfip1, which facilitated MAVS-mediated downstream signaling and elevated MAVS protein levels. Further analysis indicated that Ndfip1 enhances both self-ubiquitination of HECT domain-containing E3 ubiquitin ligase Smurf1 and its interaction with MAVS, and eventually promotes MAVS degradation. In addition, the activation of IFN-β by MAVS, influenza virus RNA, polyinosinic-polycytidylic acid, and Sendai virus was enhanced in Ndfip1 knockdown cells. These results reveal that Ndfip1 is a potent inhibitor of MAVS-mediated antiviral response.

Heat Shock Protein 70 Inhibits the Activity of Influenza A Virus Ribonucleoprotein and Blocks the Replication of Virus In Vitro and In Vivo

Background Heat shock protein 70 (Hsp70) was identified as a cellular interaction partner of the influenza virus ribonucleoprotein (RNP) complex. The biological significance of the interaction between Hsp70 and RNP has not been fully investigated. Principal Findings Here we demonstrated that Hsp70 was involved in the regulation of influenza A viral transcription and replication. It was found that Hsp70 was associated with viral RNP by directly interacting with the PB1 and PB2 subunits, and the ATPase domain of Hsp70 was required for the association. Immunofluorescence analysis showed that Hsp70 was translocated from the cytoplasm into the nucleus in infected cells. Then we found that Hsp70 negatively regulated the expression of viral proteins in infected cells. Real-time PCR analysis revealed that the transcription and replication of all eight viral segments were significantly reduced in Hsp70 overexpressed cells and greatly increased as Hsp70 was knocked down by RNA interference. Luciferase assay showed that overexpression of Hsp70 could inhibit the viral RNP activity on both vRNA and cRNA promoters. Biochemical analysis demonstrated that Hsp70 interfered with the integrity of RNP. Furthermore, delivered Hsp70 could inhibit the replication of influenza A virus in mice. Significance Our study indicated that Hsp70 interacted with PB1 and PB2 of RNP and could interfere with the integrity of RNP and block the virus replication in vitro and in vivo possibly through disrupting the binding of viral polymerase with viral RNA.

Phosphorylation of MCM3 Protein by Cyclin E/Cyclin-dependent Kinase 2 (Cdk2) Regulates Its Function in Cell Cycle

Background: Cyclin E/Cdk2 is a protein kinase in cell cycle regulation. Results: MCM3 is a novel substrate of cyclin E/Cdk2, which phosphorylates MCM3 at the site of Thr-722 and regulates its loading onto chromatin. Conclusion: Excess of MCM3 loading onto DNA will activate the checkpoint pathway. Significance: We revealed the novel role of MCM3 regulated by cyclin E/Cdk2 in controlling the S phase checkpoint. MCM2–7 proteins form a stable heterohexamer with DNA helicase activity functioning in the DNA replication of eukaryotic cells. The MCM2–7 complex is loaded onto chromatin in a cell cycle-dependent manner. The phosphorylation of MCM2–7 proteins contributes to the formation of the MCM2–7 complex. However, the regulation of specific MCM phosphorylation still needs to be elucidated. In this study, we demonstrate that MCM3 is a substrate of cyclin E/Cdk2 and can be phosphorylated by cyclin E/Cdk2 at Thr-722. We find that the MCM3 T722A mutant binds chromatin much less efficiently when compared with wild type MCM3, suggesting that this phosphorylation site is involved in MCM3 loading onto chromatin. Interestingly, overexpression of MCM3, but not MCM3 T722A mutant, inhibits the S phase entry, whereas it does not affect the exit from mitosis. Knockdown of MCM3 does not affect S phase entry and progression, indicating that a small fraction of MCM3 is sufficient for normal S phase completion. These results suggest that excess accumulation of MCM3 protein onto chromatin may inhibit DNA replication. Other studies indicate that excess of MCM3 up-regulates the phosphorylation of CHK1 Ser-345 and CDK2 Thr-14. These data reveal that the phosphorylation of MCM3 contributes to its function in controlling the S phase checkpoint of cell cycle in addition to the regulation of formation of the MCM2–7 complex.

Phosphorylation of Minichromosome Maintenance Protein 7 (MCM7) by Cyclin/Cyclin-dependent Kinase Affects Its Function in Cell Cycle Regulation

Background: Cyclin/CDKs play an important role in cell cycle regulation. Results: MCM7 can be phosphorylated at Serine-121 by cyclin E/Cdk2 and cyclin B/Cdk1. Excess of phosphorylated MCM7 blocks S phase entry, and its phosphorylation is essential for proper mitotic exit. Conclusion: Cyclin/CDKs can phosphorylate MCM7 to regulate cell cycle progression. Significance: We revealed the novel function of MCM7 on checkpoint activation and mitotic exit regulated by cyclin/Cdks. MCM7 is one of the subunits of the MCM2–7 complex that plays a critical role in DNA replication initiation and cell proliferation of eukaryotic cells. After forming the pre-replication complex (pre-RC) with other components, the MCM2–7 complex is activated by DDK/cyclin-dependent kinase to initiate DNA replication. Each subunit of the MCM2–7 complex functions differently under regulation of various kinases on the specific site, which needs to be investigated in detail. In this study, we demonstrated that MCM7 is a substrate of cyclin E/Cdk2 and can be phosphorylated on Ser-121. We found that the distribution of MCM7-S121A is different from wild-type MCM7 and that the MCM7-S121A mutant is much less efficient to form a pre-RC complex with MCM3/MCM5/cdc45 compared with wild-type MCM7. By using the Tet-On inducible HeLa cell line, we revealed that overexpression of wild-type MCM7 but not MCM7-S121A can block S phase entry, suggesting that an excess of the pre-RC complex may activate the cell cycle checkpoint. Further analysis indicates that the Chk1 pathway is activated in MCM7-overexpressed cells in a p53-dependent manner. We performed experiments with the human normal cell line HL-7702 and also observed that overexpression of MCM7 can cause S phase block through checkpoint activation. In addition, we found that MCM7 could also be phosphorylated by cyclin B/Cdk1 on Ser-121 both in vitro and in vivo. Furthermore, overexpression of MCM7-S121A causes an obvious M phase exit delay, which suggests that phosphorylation of MCM7 on Ser-121 in M phase is very important for a proper mitotic exit. These data suggest that the phosphorylation of MCM7 on Ser-121 by cyclin/Cdks is involved in preventing DNA rereplication as well as in regulation of the mitotic exit.

Tetraspanin 6 (TSPAN6) negatively regulates retinoic acid-inducible gene I-like receptor-mediated immune signaling in a ubiquitination-dependent manner.

Background: The RIG-I-mediated signaling pathway is important for the antiviral immune response. Results: TSPAN6 inhibits the formation of the MAVS-centered signalosome. Conclusion: TSPAN6 negatively regulates the RLR-mediated signaling pathway. Significance: We revealed the function of TSPAN6 in regulating the RLR-mediated innate immune response. The recognition between retinoic acid-inducible gene I-like receptors (RLRs) and viral RNA triggers an intracellular cascade of signaling to induce the expression of type I IFNs. Both positive and negative regulation of the RLR signaling pathway are important for the host antiviral immune response. Here, we demonstrate that the tetraspanin protein TSPAN6 inhibits RLR signaling by affecting the formation of the adaptor MAVS (mitochondrial antiviral signaling)-centered signalosome. We found that overexpression of TSPAN6 impaired RLR-mediated activation of IFN-stimulated response element, NF-κB, and IFN-β promoters, whereas knockdown of TSPAN6 enhanced the RLR-mediated signaling pathway. Interestingly, as the RLR pathway was activated, TSPAN6 underwent Lys-63-linked ubiquitination, which promoted its association with MAVS. The interaction of TSPAN6 and MAVS interfered with the recruitment of RLR downstream molecules TRAF3, MITA, and IRF3 to MAVS. Further study revealed that the first transmembrane domain of TSPAN6 is critical for its ubiquitination and association with MAVS as well as its inhibitory effect on RLR signaling. We concluded that TSPAN6 functions as a negative regulator of the RLR pathway by interacting with MAVS in a ubiquitination-dependent manner.

Ankrd17 positively regulates RIG‐I‐like receptor (RLR)‐mediated immune signaling

Retinoic acid‐inducible gene‐I (RIG‐I)‐like receptors (RLRs), such as RIG‐I, melanoma differentiation‐associated gene 5 (MDA5), and virus‐induced signaling adaptor (VISA), are intracellular molecules that sense diverse viral RNAs and trigger immune responses. In this study, we demonstrate that the ankyrin repeat protein ankrd17 interacts with RIG‐I, MDA5, and VISA and upregulates RLR‐mediated immune signaling. Overexpression of ankrd17 enhances RLR‐mediated activation of IRF‐3 and NF‐κB and upregulates the transcription of IFN‐β. It also promotes RLR signaling in response to poly (I:C), influenza virus RNA, and Sendai virus. Consistently, knockdown of ankrd17 impairs RLR signaling. Furthermore, we demonstrate that ankrd17 enhances the interaction of RIG‐I and MDA5 with VISA; the ankyrin repeat domain of ankrd17 is required for its interaction with RIG‐I as well as for its function in regulating the RLR pathway. Taken together, our results indicate that ankrd17 is a positive regulator of the RLR signaling pathway.

Casein Kinase 1γ1 Inhibits the RIG-I/TLR Signaling Pathway through Phosphorylating p65 and Promoting Its Degradation

The casein kinase 1 (CK1) plays an important role in various biological processes by phosphorylating its target proteins. In this study, we demonstrate that CK1γ1 inhibits RNA virus–mediated activation of retinoic acid–inducible gene I (RIG-I) signaling by affecting the stability of NF-κB subunit p65. First, we found that ectopic expression of CK1γ1 inhibits RIG-I pathway–mediated activation of IFN-β, whereas knockdown of CK1γ1 potentiates the activation of IFN-β and NF-κB induced by Sendai virus (SeV). We then revealed that CK1γ1 interacts with p65 and specifically enhances its phosphorylation at Ser536 induced by SeV. By using an in vitro kinase assay, we confirmed that CK1γ1 can phosphorylate p65 at Ser536. We also showed that the kinase dead mutants CK1γ1K73A and CK1γ1N169A did not inhibit SeV-induced activation of IFN-β and NF-κB, suggesting that the kinase activity of CK1γ1 is critical for its inhibitory effect on RIG-I signaling. Additionally, we found that CK1γ1 also has the similar effect on TLR signaling. Further analysis indicated that CK1γ1 phosphorylates p65 and consequently promotes its degradation by ubiquitin E3 ligases CUL2 and COMMD1. These results revealed a novel negative regulatory manner of CK1γ1 on innate immune signaling.

Phosphorylation of Right Open Reading Frame 2 (Rio2) Protein Kinase by Polo-like Kinase 1 Regulates Mitotic Progression

Background: Rio2 is a protein kinase and involved in ribosomal subunit maturation. Results: Rio2 is a novel substrate of Plk1. Overexpression of Rio2 causes a prolonged mitotic exit whereas knockdown of Rio2 accelerates mitotic progression. Conclusion: Plk1-dependent phosphorylation of Rio2 regulates the timing of the metaphase-anaphase transition. Significance: We unveiled the novel role of Rio2 and its phosphorylation by Plk1 in regulating mitotic progression. Polo-like kinase 1 (Plk1) plays essential roles during multiple stages of mitosis by phosphorylating a number of substrates. Here, we report that the atypical protein kinase Rio2 is a novel substrate of Plk1 and can be phosphorylated by Plk1 at Ser-335, Ser-380, and Ser-548. Overexpression of Rio2 causes a prolonged mitotic exit whereas knockdown of Rio2 accelerates mitotic progression, suggesting that Rio2 is required for the proper mitotic progression. Overexpression of phospho-mimicking mutant Rio2 S3D but not the nonphosphorylatable mutant Rio2 S3A displays a profile similar to that of wild-type Rio2. These results indicate that the phosphorylation status of Rio2 correlates with its function in mitosis. Furthermore, time-lapse imaging data show that overexpression of Rio2 but not Rio2 S3A results in a slowed metaphase-anaphase transition. Collectively, these findings strongly indicate that the Plk1-mediated phosphorylation of Rio2 regulates metaphase-anaphase transition during mitotic progression.

Cyclin D1 promotes cell cycle progression through enhancing NDR1/2 kinase activity independent of cyclin-dependent kinase 4.

Background: Cyclin D1/Cdk4 and NDR1/2 are critical kinases in cell cycle regulation. Results: We demonstrate that cyclin D1 interacts with NDR1 and NDR2 and enhances their kinase activity. Conclusion: Cyclin D1 promotes cell cycle progression partly through up-regulating NDR1/2 activity. Significance: We revealed the novel function of cyclin D1 in cell cycle progression in a Cdk4-independent manner. Cyclin/cyclin-dependent kinases (Cdks) are critical protein kinases in regulating cell cycle progression. Among them, cyclin D1/Cdk4 exerts its function mainly in the G1 phase. By using the tandem affinity purification tag approach, we identified a set of proteins interacting with Cdk4, including NDR1/2. Interestingly, confirming the interactions between NDR1/2 and cyclin D1/Cdk4, we observed that NDR1/2 interacted with cyclin D1 independent of Cdk4, but NDR1/2 and cyclin D1/Cdk4 did not phosphorylate each other. In addition, we found that NDR1/2 did not affect the kinase activity of cyclin D1/Cdk4 upon phosphorylation of GST-Rb. However, cyclin D1 but not Cdk4 promoted the kinase activity of NDR1/2. We also demonstrated that cyclin D1 K112E, which could not bind Cdk4, enhanced the kinase activity of NDR1/2. To test whether cyclin D1 promotes G1/S transition though enhancing NDR1/2 kinase activity, we performed flow cytometry analysis using cyclin D1 and cyclin D1 K112E Tet-On inducible cell lines. The data show that both cyclin D1 and cyclin D1 K112E promoted G1/S transition. Importantly, knockdown of NDR1/2 almost completely abolished the function of cyclin D1 K112E in promoting G1/S transition. Consistently, we found that the protein level of p21 was reduced in cells overexpressing cyclin D1 K112E but not when NDR1/2 was knocked down. Taken together, these results reveal a novel function of cyclin D1 in promoting cell cycle progression by enhancing NDR kinase activity independent of Cdk4.

The complement C1qA enhances retinoic acid‐inducible gene‐I‐mediated immune signalling

The cellular innate immune response is essential for recognizing and defending against viral infection. Retinoic acid‐inducible gene‐I (RIG‐I) and virus‐induced signaling adaptor (VISA) mediated immune signalling is critically involved in RNA‐virus‐induced innate immune responses. Here we demonstrate that the complement C1qA interacts with different RIG‐I pathway components and enhances RIG‐I‐VISA‐mediated signalling pathway as well as TBK1‐mediated activation of interferon‐β (IFN‐β) promoter. Our data show that over‐expression of C1qA up‐regulates RIG‐I‐mediated activation of IFN‐stimulated responsive element (ISRE) and nuclear factor‐κB reporters and IFN‐β transcription, but not IFN regulatory factor‐3‐mediated and inhibitor of κB kinase‐mediated activation of ISRE and nuclear factor‐κB promoter. In addition, C1qA can counteract the function of the C1q receptor gC1qR in RIG‐I‐mediated signalling. Our results reveal the important role of complement C1qA in the innate immune response.