p53 degradation by a coronavirus papain-like protease suppresses type I interferon signaling.

Infection by human coronaviruses is usually characterized by rampant viral replication and severe immunopathology in host cells. Recently, the coronavirus papain-like proteases (PLPs) have been identified as suppressors of the innate immune response. However, the molecular mechanism of this inhibition remains unclear. Here, we provide evidence that PLP2, a catalytic domain of the nonstructural protein 3 of human coronavirus NL63 (HCoV-NL63), deubiquitinates and stabilizes the cellular oncoprotein MDM2 and induces the proteasomal degradation of p53. Meanwhile, we identify IRF7 (interferon regulatory factor 7) as a bona fide target gene of p53 to mediate the p53-directed production of type I interferon and the innate immune response. By promoting p53 degradation, PLP2 inhibits the p53-mediated antiviral response and apoptosis to ensure viral growth in infected cells. Thus, our study reveals that coronavirus engages PLPs to escape from the innate antiviral response of the host by inhibiting p53-IRF7-IFNß signaling.

SCFFBXL¹5 regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1.

Smad ubiquitination regulatory factor 1 (Smurf1), an homologous to E6AP C-terminus (HECT)-type E3 ubiquitin ligase, performs a crucial role in the regulation of the bone morphogenetic protein (BMP) signalling pathway in both embryonic development and bone remodelling. How the stability and activity of Smurf1 are negatively regulated remains largely unclear. Here, we report that F-box and LRR domain-containing protein 15 (FBXL15), an F-box protein of the FBXL family, forms an Skp1-Cullin1-F-box protein-Roc1 (SCF)(FBXL15) ubiquitin ligase complex and targets Smurf1 for ubiquitination and proteasomal degradation. FBXL15, through its leucine-rich repeat domain, specifically recognizes the large subdomain within the N-lobe of the Smurf1 HECT domain and promotes the ubiquitination of Smurf1 on K355 and K357 within the WW-HECT linker region. In this way, FBXL15 positively regulates BMP signalling in mammalian cells. Knockdown of fbxl15 expression in zebrafish embryos by specific antisense morpholinos causes embryonic dorsalization phenocoping BMP-deficient mutants. Injection of FBXL15 siRNAs into rat bone tissues leads to a significant loss of bone mass and decrease in bone mineral density. Collectively, our results demonstrate that Smurf1 stability is suppressed by SCF(FBXL15)-mediated ubiquitination and that FBXL15 is a key regulator of BMP signalling during embryonic development and adult bone formation.

The HECT type ubiquitin ligase NEDL2 is degraded by anaphase-promoting complex/cyclosome (APC/C)-Cdh1, and its tight regulation maintains the metaphase to anaphase transition.

NEDD4-like ubiquitin ligase 2 (NEDL2) is a HECT type ubiquitin ligase. NEDL2 enhances p73 transcriptional activity and degrades ATR kinase in lamin misexpressed cells. Compared with the important functions of other HECT type ubiquitin ligase, there is less study concerning the function and regulation of NEDL2. Using primary antibody immunoprecipitation and mass spectrometry, we identify a list of potential proteins that are putative NEDL2-interacting proteins. The candidate list contains many of mitotic proteins, especially including several subunits of anaphase-promoting complex/cyclosome (APC/C) and Cdh1, an activator of APC/C. Cdh1 can interact with NEDL2 in vivo and in vitro. Cdh1 recognizes one of the NEDL2 destruction boxes (R(740)GSL(743)) and targets it for degradation in an APC/C-dependent manner during mitotic exit. Overexpression of Cdh1 reduces the protein level of NEDL2, whereas knockdown of Cdh1 increases the protein level of NEDL2 but has no effect on the NEDL2 mRNA level. NEDL2 associates with mitotic spindles, and its protein level reaches a maximum in mitosis. The function of NEDL2 during mitosis is essential because NEDL2 depletion prolongs metaphase, and overexpression of NEDL2 induces chromosomal lagging. Elevated expression of NEDL2 protein and mRNA are both found in colon cancer and cervix cancer. We conclude that NEDL2 is a novel substrate of APC/C-Cdh1 as cells exit mitosis and functions as a regulator of the metaphase to anaphase transition. Its overexpression may contribute to tumorigenesis.

Apak competes with p53 for direct binding to intron 1 of p53AIP1 to regulate apoptosis.

The KRAB-type zinc-finger protein Apak was recently identified as a negative regulator of p53-mediated apoptosis. However, the mechanism of this selective regulation is not fully understood. Here, we show that Apak recognizes the TCTTN2−30TTGT consensus sequence through its zinc-fingers. This sequence is specifically found in intron 1 of the proapoptotic p53 target gene p53AIP1 and largely overlaps with the p53-binding sequence. Apak competes with p53 for binding to this site to inhibit p53AIP1 expression. Upon DNA damage, Apak dissociates from the DNA, which abolishes its inhibitory effect on p53-mediated apoptosis.

CKIP-1 couples Smurf1 ubiquitin ligase with Rpt6 subunit of proteasome to promote substrate degradation.

CKIP-1 is an activator of the Smurf1 ubiquitin ligase acting to promote the ubiquitylation of Smad5 and MEKK2. The mechanisms involved in the recognition and degradation of these substrates by the proteasome remain unclear. Here, we show that CKIP-1, through its leucine zipper, interacts directly with the Rpt6 ATPase of the 19S regulatory particle of the proteasome. CKIP-1 mediates the Smurf1-Rpt6 interaction and delivers the ubiquitylated substrates to the proteasome. Depletion of CKIP-1 reduces the degradation of Smurf1 and its substrates by Rpt6. These findings reveal an unexpected adaptor role of CKIP-1 in coupling the ubiquitin ligase and the proteasome.

Identification of UHRF1/2 as new N-methylpurine DNA glycosylase-interacting proteins.

N-methylpurine DNA glycosylase (MPG), a DNA repair enzyme, functions in the DNA base excision repair (BER) pathway. Aberrant over-expression of MPG in various cancers suggests an important role of MPG in carcinogenesis. Identification of MPG-interacting proteins will help to dissect the molecular link between MPG and cancer development. In the present study, using immunoprecipitation coupled with mass spectrometry (IP/MS), we screened ubiquitin-like, containing PHD and RING finger domains 1 (UHRF1), an essential protein required for the maintenance of DNA methylation, as a MPG-interacting protein. Endogenous co-immunoprecipitation assay in cancer cells confirmed that UHRF1 interacted with MPG in a p53 status-independent manner. Confocal microscopy showed that endogenous MPG and UHRF1 were co-localized in the nucleoplasm. Furthermore, co-immunoprecipitation assay indicated that UHRF2, the homolog of UHRF1, could also interact with MPG. These results show that MPG and the UHRF family of proteins interact, thus providing a functional linkage between MPG and UHRF1/2.

Integrated analysis of genomics and proteomics reveals that CKIP-1 is a novel macrophage migration regulator.

Casein kinase-2 interacting protein-1 (CKIP-1) has been identified to play an important role in cell morphology, differentiation and apoptosis. However, the role of CKIP-1 in other cellular processes is still unknown. Here we investigated transcriptome profiles of WT and CKIP-1-deficient mouse embryonic fibroblasts (MEFs), and found that innate immunity and cell migration related pathways were significantly correlated with CKIP-1 expression. As macrophage is a key cell type in innate immunity, we then used murine macrophage RAW264.7 cells to discover CKIP-1 interacting proteins by immunoprecipitation/mass spectrometry (IP/MS). Analysis of these proteins revealed migration related pathways were enriched. Further experiments indicated that knockdown of CKIP-1 in RAW264.7 cells resulted in impaired cell migration. Our study suggests that CKIP-1 is a novel regulator of macrophage migration.

Pivotal role of the C2 domain of the Smurf1 ubiquitin ligase in substrate selection.

The C2-WW-HECT-type ubiquitin ligases Smurf1 and Smurf2 play a critical role in embryogenesis and adult bone homeostasis via regulation of bone morphogenetic protein, Wnt, and RhoA signaling pathways. The intramolecular interaction between C2 and HECT domains autoinhibits the ligase activity of Smurf2. However, the role of the Smurf1 C2 domain remains elusive. Here, we show that the C2-HECT autoinhibition mechanism is not observed in Smurf1, and instead its C2 domain functions in substrate selection. The Smurf1 C2 domain exerts a key role in localization to the plasma membrane and endows Smurf1 with differential activity toward RhoA versus Smad5 and Runx2. Crystal structure analysis reveals that the Smurf1 C2 domain possesses a typical anti-parallel ß-sandwich fold. Examination of the sulfate-binding site analysis reveals two key lysine residues, Lys-28 and Lys-85, within the C2 domain that are important for Smurf1 localization at the plasma membrane, regulation on cell migration, and robust ligase activity toward RhoA, which further supports a Ca(2+)-independent localization mechanism for Smurf1. These findings demonstrate a previously unidentified role of the Smurf1 C2 domain in substrate selection and cellular localization.

The E3 ligase Smurf1 regulates Wolfram syndrome protein stability at the endoplasmic reticulum.

The HECT-type ubiquitin ligase (E3) Smad ubiquitination regulatory factor 1 (Smurf1) targets various substrates, including Smad1/5, RhoA, Prickle 1, MEKK2, and JunB for degradation and thereby regulates adult bone formation and embryonic development. Here, we identify the endoplasmic reticulum (ER)-localized Wolfram syndrome protein (WFS1) as a specific degradation substrate of Smurf1. Mutations in the WFS1 gene cause Wolfram syndrome, an autosomal recessive disorder characterized by diabetes mellitus and optic atrophy. WFS1 negatively regulates the ER stress response, and WFS1 deficiency in mice increases ER stress and triggers apoptosis. We show that Smurf1 interacts with WFS1 at the ER and promotes the ubiquitination and proteasomal degradation of WFS1. A C-terminal luminal region in WFS1, including residues 667-700, is involved in this degradation. Wild-type WFS1 as well as a subset of WFS1 mutants that include this degron region are susceptible to Smurf1-mediated degradation. By contrast, pathophysiological deletion mutants of WFS1 lacking the degron, such as W648X, Y660X, and Q667X, are resistant to degradation by Smurf1. Depletion of Smurf1 by RNA interference results in increased WFS1 and decreased ATF6α levels. Furthermore, we show that ER stress induces Smurf1 degradation and WFS1 up-regulation. These findings reveal for the first time that Smurf1 targets an ER-localized protein for degradation and that Smurf1 is regulated by ER stress.

Murine calvaria-derived progenitor cells express high levels of osterix and lose their adipogenic capacity.

Though the mouse is the most widely used biomedical animal model, it is difficult to isolate murine mesenchymal stem cells (MSCs) from the bone marrow because of contamination by hematopoietic cells. The murine compact bone tissue of long bones is considered a novel and reliable source of MSCs with low hematopoietic cell contamination. We investigated whether the murine compact bone of the calvaria would be a promising source of MSCs due to its low bone marrow content. We isolated cells from both long bones and the calvaria using the same method. Although they shared morphological features and surface antigens similar to those of long bone-derived MSCs, the calvaria-derived cells highly expressed the osteogenic transcription factor osterix, lost their adipogenic capacity and gained a higher osteogenic capacity. These findings suggest that the cells that migrated from the calvaria were progenitor cells rather than MSCs and that the differentiation fate of mesenchymal stem/progenitor cells existing in different murine compact bone deposits is already committed.

Smad ubiquitylation regulatory factor 1/2 (Smurf1/2) promotes p53 degradation by stabilizing the E3 ligase MDM2.

The tumor suppressor p53 protein is tightly regulated by a ubiquitin-proteasomal degradation mechanism. Several E3 ubiquitin ligases, including MDM2 (mouse double minute 2), have been reported to play an essential role in the regulation of p53 stability. However, it remains unclear how the activity of these E3 ligases is regulated. Here, we show that the HECT-type E3 ligase Smurf1/2 (Smad ubiquitylation regulatory factor 1/2) promotes p53 degradation by enhancing the activity of the E3 ligase MDM2. We provide evidence that the role of Smurf1/2 on the p53 stability is not dependent on the E3 activity of Smurf1/2 but rather is dependent on the activity of MDM2. We find that Smurf1/2 stabilizes MDM2 by enhancing the heterodimerization of MDM2 with MDMX, during which Smurf1/2 interacts with MDM2 and MDMX. We finally provide evidence that Smurf1/2 regulates apoptosis through p53. To our knowledge, this is the first report to demonstrate that Smurf1/2 functions as a factor to stabilize MDM2 protein rather than as a direct E3 ligase in regulation of p53 degradation.

ATM-mediated NuSAP phosphorylation induces mitotic arrest.

NuSAP is a microtubule-associated protein that plays an important role in spindle assembly. NuSAP deficiency in mice leads to early embryonic lethality. Spindle assembly in NuSAP-deficient cells is highly inefficient and chromosomes remain dispersed in the mitotic cytoplasm. ATM is a key kinase that phosphorylates a series of substrates to mediate G1/S control. However, the role of ATM at the G2/M phase is not well understood. Here we demonstrate that ectopic expression of NuSAP lead to mitotic arrest observably dependent on the kinase activity of ATM. When endogenous ATM was depleted or its kinase activity was inhibited, NuSAP could not cause mitotic arrest. We further show ATM interacts with NuSAP and phosphorylates NuSAP on Ser124. The phosphorylation and interaction occur specifically at G2/M-phase. Collectively, our work has uncovered an ATM-dependent checkpoint pathway that prevents mitotic progression by targeting a microtubule-associated protein, NuSAP.

Smurf1 ubiquitin ligase targets Kruppel-like factor KLF2 for ubiquitination and degradation in human lung cancer H1299 cells.

Krüppel-like factor 2 (KLF2) has been demonstrated to be essential for normal lung development, erythroid differentiation, T-cell differentiation, migration and homing. However, the mechanisms underlying the regulation of KLF2, in particular its responsible E3 ligase is still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets KLF2 for poly-ubiquitination and proteasomal degradation specifically in lung cancer H1299 cells. The catalytic ligase activity of Smurf1 is required for it to regulate KLF2. Consequently, Smurf1 represses the transcriptional factor activity of KLF2 and regulates the expression its downstream genes such as CD62L and Wee1. This study provided the first evidence that Smurf1 functions as an E3 ligase to promote the ubiquitination and proteasomal degradation of KLF2.

Differential regulation of Apak by various DNA damage signals.

The tumor suppressor p53 lies at the center of a protein-signaling network that responds to many types of stress, and p53 activation leads to cell cycle arrest or apoptosis. We recently identified ATM and p53-associated KZNF protein (Apak) as a negative regulator of p53-mediated apoptosis. After treatment of cells with methyl methanesulfonate (MMS), Apak is phosphorylated by ATM kinase and dissociates from p53, resulting in p53 activation and induction of apoptosis. However, the mechanism by which Apak is regulated in response to other types of DNA damage signals remains unclear. Here, we show that four of seven types of DNA damage signals we examined (induction by etoposide, doxorubicin, camptothecin and cisplatin treatment) resulted in significant Apak phosphorylation and dissociation of Apak from p53, releasing the inhibition of p53 transcriptional activity. In contrast, Apak was not phosphorylated at Ser68 after 5-fluorouracil or alpha-lipoic acid treatment and persistently inhibited p53 activity. These findings provide evidence that the Apak-p53 interaction is regulated differentially by various DNA damage signals.

Ubiquitin ligase Smurf1 targets TRAF family proteins for ubiquitination and degradation.

The HECT-type E3 Smad ubiquitination regulation factor 1 (Smurf1) functions in regulation of cell polarity and bone homeostasis by targeting Smads, Runx2, RhoA and MEKK2 for ubiquitination and degradation. In a yeast two-hybrid screening, we identified TNF receptor-associated factor 4 (TRAF4) as a candidate substrate and was further validated. The PY motifs of TRAF4 mediated the interaction with the second WW domain of Smurf1. Overexpression of Smurf1 reduced the protein levels of TRAF4 dependent of its E3 activity and the proteasome. Further, we showed that all six members of TRAF family could be ubiquitinated by Smurf1. Consequently, Smurf1 interfered with the functions of TRAFs in NF-kappaB signaling under stimulation or not. These results suggested a new role of Smurf1 in inflammation and immunity through controlling the degradation of TRAFs.

C-type lectin LSECtin interacts with DC-SIGNR and is involved in hepatitis C virus binding.

Hepatitis C virus (HCV) is a major cause of liver disease. However, the detailed mechanism underlying hepatocyte infection with HCV is not yet completely understood. We previously identified a novel C-type lectin--LSECtin predominantly expressed on liver sinusoidal endothelial cells. Here we demonstrate that LSECtin can interact with two HCV receptors, DC-SIGNR and CD81, through its central ectodomain. Furthermore, cells expressing LSECtin specifically can be attached by the naturally occurring HCV in the sera of infected individuals. This binding was found to be mediated by the HCV E2 glycoprotein and could be efficiently inhibited by EGTA but not by mannan treatment. The present study suggests that LSECtin interaction with DC-SIGNR might contribute to HCV binding to liver sinusoidal endothelial cells.

E3 ubiquitin ligase SIAH1 mediates ubiquitination and degradation of TRB3.

Tribbles 3 homolog (TRB3) is recently identified as a scaffold-like regulator of various signal transducers and has been implicated in several processes including insulin signaling, NF-kappaB signaling, lipid metabolism and BMP signaling. To further understand cellular mechanisms of TRB3 regulation, we performed a yeast two-hybrid screen to identify novel TRB3 interacting proteins and totally obtained ten in-frame fused preys. Candidate interactions were validated by co-immunoprecipitation assays in mammalian cells. We further characterized the identified proteins sorted by Gene Ontology Annotation. Its interaction with the E3 ubiquitin ligase SIAH1 was further investigated. SIAH1 could interact with TRB3 both in vitro and in vivo. Importantly, SIAH1 targeted TRB3 for proteasome-dependent degradation. Cotransfection of SIAH1 could withdraw up-regulation of TGF-beta signaling by TRB3, suggesting SIAH1-induced degradation of TRB3 represents a potential regulatory mechanism for TGF-beta signaling.

Histone methyltransferase protein SETD2 interacts with p53 and selectively regulates its downstream genes.

SETD2 (SET domain containing protein 2) is a histone H3K36 trimethyltransferase protein that associates with hyperphosphorylated RNA polymerase II and involves in transcriptional elongation. However, whether and how SETD2 is implicated in the specific regulation of gene transcription remains unknown. Here we show that SETD2 could interact with p53 and selectively regulate the transcription factor activity of p53. The interaction was dependent of C-terminal region of SETD2, which contains the SET and WW domains, and the N-terminal transactivation domain (residues 1-45) of p53. Overexpression of SETD2 upregulated the expression levels of a subset of p53 targets including puma, noxa, p53AIP1, fas, p21, tsp1, huntingtin, but downregulated that of hdm2. In contrast, it had no significant effect on those of 14-3-3sigma, gadd45 and pig3. Consistently, knockdown of endogenous SETD2 expression by RNA interference resulted in converse effects as expected. In p53-deficient H1299 cells, SETD2 lost the ability to regulate these gene expression except hdm2, indicating the dependence of p53. Furthermore, we demonstrated that SETD2 downregulated hdm2 expression by targeting its P2 promoter and then enhanced p53 protein stability. Collectively, these findings suggest that the histone methyltransferase SETD2 could selectively regulate the transcription of subset genes via cooperation with the transcription factor p53.

Ceap/BLOS2 interacts with BRD7 and selectively inhibits its transcription-suppressing effect on cellular proliferation-associated genes.

The centrosome associated protein Ceap-16 (also termed BLOS2) can accelerate the proliferation of mouse fibroblast NIH3T3 cells, which mechanism remains unclear. Here we identified tumor suppressor candidate BRD7 (bromodomain containing protein 7), which could negatively regulate cell proliferation and growth, as a novel Ceap-16-interacting protein. Ceap-16 and BRD7 interacted with each other both in vitro and in vivo. The C-terminus of BRD7 and the central region of Ceap-16 mediated the interaction. Through this binding, Ceap-16 could translocate from cytoplasm to the nucleus where it selectively inhibited the transcriptional suppression activity of BRD7 towards certain target genes including E2F3 and cyclin A. Moreover, Ceap-16, BRD7 and histone H3/H4 could form a complex and Ceap-16 did not compete with BRD7 binding to histones. These findings suggest a novel function for Ceap-16 in the transcriptional regulation through associating with BRD7.

JAB1 accelerates mitochondrial apoptosis by interaction with proapoptotic BclGs.

The Bcl-2 family of proteins is the key regulators of cell apoptosis at the mitochondria level. The BH3-only pro-apoptotic member BclGs was unique among the family due to its highly specific expression in human testis and has been demonstrated to induce apoptosis dependent on the BH3 domain. However, the molecular mechanism of BclGs-induced apoptosis remains unclear. Here we show that overexpression of BclGs could induce Bax expression upregulation and translocation to mitochondria, cytochrome c release and activation of caspase-3. Moreover, we identified JAB1 as a novel BclGs-specific binding protein through a yeast two-hybrid screening in a human testis cDNA library. BclGs interacts with JAB1 both in vitro and in vivo. N-terminal region of BclGs (aa 1-67) was required for the interaction. Importantly, JAB1 and BclGs co-expression synergistically induces apoptosis. JAB1 could compete with Bcl-XL/Bcl-2 to bind to BclGs; thus, promote the apoptosis. RNAi-mediated knock-down of JAB1 results in the reduced proapoptotic activity of BclGs. Taken together, our results provided the first evidence that JAB1 is involved in the regulation of mitochondrial apoptotic pathway through specific interaction with BclGs.