TRIM16 Acts as an E3 Ubiquitin Ligase and Can Heterodimerize with Other TRIM Family Members

The TRIM family of proteins is distinguished by its tripartite motif (TRIM). Typically, TRIM proteins contain a RING finger domain, one or two B-box domains, a coiled-coil domain and the more variable C-terminal domains. TRIM16 does not have a RING domain but does harbour two B-box domains. Here we showed that TRIM16 homodimerized through its coiled-coil domain and heterodimerized with other TRIM family members; TRIM24, Promyelocytic leukaemia (PML) protein and Midline-1 (MID1). Although, TRIM16 has no classic RING domain, three-dimensional modelling of TRIM16 suggested that its B-box domains adopts RING-like folds leading to the hypothesis that TRIM16 acts as an ubiquitin ligase. Consistent with this hypothesis, we demonstrated that TRIM16, devoid of a classical RING domain had auto-polyubiquitination activity and acted as an E3 ubiquitin ligase in vivo and in vitro assays. Thus via its unique structure, TRIM16 possesses both heterodimerization function with other TRIM proteins and also has E3 ubiquitin ligase activity

MYCN promotes neuroblastoma malignancy by establishing a regulatory circuit with transcription factor AP4

Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4) has been reported to be a direct transcriptional target of MYC. We show for the first time that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome. siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells led to inhibition of cell proliferation and migration. Chromatin immunoprecipitation assay demonstrated that TFAP4 expression is positively regulated by MYCN. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study suggests a regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying potential therapeutic targets for aggressive forms of this disease

Characterising the role of PA2G4 and its interaction with MYCN in neuroblastoma progression

Neuroblastoma, an embryonal tumour of the sympathetic nervous system, is the most common solid tumour in childhood. MYCN oncogene amplification is found in one third of primary neuroblastoma at diagnosis, and correlates with poor prognosis. We identified Proliferation-associated protein 2G4 (PA2G4) as a binding partner for MYCN using co-immunoprecipitation and mass spectrometry. The long isoform of PA2G4 (p48) has a known oncogenic function, whereas the short isoform (p42) acts as a tumour suppressor. However, the role of PA2G4 in neuroblastoma and its link with MYCN is currently unknown. Using a panel of human neuroblastoma cell lines and patient tumour samples, we analysed the expression of PA2G4 by real-time PCR and Western blotting. We found that high expression of PA2G4 was a strong independent clinical predictor of poor survival and positively correlated with MYCN expression. Analysis of pre-cancerous ganglia cells from TH-MYCN neuroblastoma mouse model showed PA2G4 mRNA was expressed 8-fold higher in ganglia from TH-MYCN transgenic homozygous, compared to wild-type, mice. Chromatin immunoprecipitation showed MYCN binds to PA2G4 promoter to activate its transcription. Surprisingly, suppression of PA2G4 in neuroblastoma cell lines markedly reduced MYCN protein level. Cycloheximide-chase assay confirmed that PA2G4 increased MYCN protein stability, thus creating a positive forward feedback loop essential for maintaining mutual high expression. Suppression of PA2G4 down-regulated pMDM2 and pAKT, leading to an increase in p53 expression. Furthermore, PA2G4 knockdown reduced cell migration and colony formation; importantly expression of MYCN was required for the effect of PA2G4 on colony formation. Most significantly, overexpression of PA2G4 induced tumorigenesis in a non-tumorigenic cell line. Furthermore, a small molecule inhibitor of PA2G4, WS6, significantly decreased neuroblastoma cell growth and PA2G4 and MYCN protein levels in vitro, and delayed tumour growth in vivo. This research highlights the importance and underlying mechanisms of PA2G4 as an onco-factor in MYCN driven neuroblastoma. It identifies PA2G4 as a novel MYCN-binding protein which increases MYCN protein stability and acts as an oncogenic co-factor in a forward feedback expression loop with MYCN. It provides strong evidence that PA2G4 plays a critical role in tumorigenesis and a novel molecular target for the treatment of neuroblastoma

TRIM16 homodimerizes through its coiled-coil domain

<p>. (A) TRIM16-GFP domain deletion plasmids. (B) Co-transfection of TRIM16-GFP and TRIM16-myc-His in HEK293 cells and subsequent immunoprecipitation by anti-myc antibody (Ab) and Western blot with anti-GFP antibody. Whole cell extract (WCE) used as total input. (C) TRIM16 homodimerizes through its coiled-coil domain. GFP deletion mutants were co-transfected with the TRIM16-myc-His vector. Anti-GFP antibody was used to pull down proteins binding the GFP tagged proteins and the TRIM16-myc-His was used to detect self-association via its different tag (right panel). Transfection efficiency was confirmed (left panel). TRIM16-GFP mutants were efficiently pulled down (middle panel). * non-specific bands, # refer to text.</p

Amino acid sequence comparison of TRIM16 and MID1 and modeling of TRIM16 B-boxes.

<p>The conserved residues in the zinc binding regions are in bold underlined type and are; cysteine; C, histidine; H, alanine; A aspartic acid; D. (<b>A</b>) TRIM16 and MID1 share the zinc binding consensus sequence for B-box1. (B) TRIM16 and MID1 share the Zinc binding consensus sequence for B-box2. (C/D) Modeling of B-boxes reveals zinc binding capability. Superimposition of the alpha-carbon backbone of the B-boxes from the MDM1 NMR structure (purple) and the homology model of TRIM16 (blue-grey). These two structures overlay with an average root-mean-square deviation of 0.4 Å.</p

B-boxes are required for TRIM16’s E3 ligase activity.

<p>(A) TRIM16 <i>in vivo</i> polyubiquitination assay. In HEK293 cells, HA-Ub was co-transfected with various TRIM16-GFP domain deletion expression plasmids. The protein lysate was subjected to immunoprecipitation by GFP antibody, and subjected to Western blot and probed with anti-HA antibody for Ub (right panel) and anti-GFP antibody for TRIM16 (left and middle panel). Two exposure times are shown. GFP antibodies detect both un-ubiquitinated and polyubiquitinated forms of TRIM16. Polyubiquitinated smear is present in the sample transfected with wild-type TRIM16 and shown by anti-GFP and anti-HA antibodies. (B) <i>In vitro</i> ubiquitination assay with myc-His tagged TRIM16 together with a panel of E2 enzymes, showing activity with the UbcH5 family. (C) <i>In vitro</i> ubiquitination assay with full-length TRIM16, TRIM16 domain deletion mutants or empty vector showing extensive polyubiquitination with full-length TRIM16 as detected by Western blot with anti-myc antibodies. Numbers indicate protein size in kDa. (D) Recombinant TRIM16 (Abnova) was evaluated for E3 activity in the presence of recombinant E1, UbcH5b, and HA-Ub as indicated. The capacity to catalyse auto-ubiquitination <i>in vitro</i> was observed only in the presence of ZnCl₂ and ATP. Western Blot (lower panel) with TRIM16 antibody showed amount of TRIM16 protein in each lane.</p

TRIM16 can heterodimerize with MID1, TRIM24 and PML.

<p>(A) Schematic structures of TRIM proteins used in heterodimerization studies. (B) TRIM16 binds MID1. Co-transfection of MID1-GFP and TRIM16-myc-His in HEK293 cells and subsequent immunoprecipitation by anti-myc antibody and Western blot with anti-GFP antibody. (C) MID1 was pulled down via its GFP antibody and a Western blot was performed to detect TRIM16-myc-His in the immunoprecipitated protein complex. (D) Whole cell lysates (WCL) of HEK293 cells transfected with empty vector (EV) or TRIM16-GFP were immunopreciptated with anti-GFP antibody. An anti-PML antibody was used to detect PML as a binding partner of TRIM16. (E) Lysates containing both TRIM16-GFP and TRIM24-His proteins were immunopreciptated with anti-GFP antibody. Anti-His antibody was used to detect the presence of TRIM24 in the TRIM16-associated complex.</p

Aurora Kinase A inhibition enhances DNA damage and tumor cell death with 131I-MIBG therapy in high-risk neuroblastoma

BackgroundNeuroblastoma is the most common extra-cranial pediatric solid tumor.&nbsp;131I-metaiodobenzylguanidine (MIBG) is a targeted radiopharmaceutical highly specific for neuroblastoma tumors, providing potent radiotherapy to widely metastatic disease. Aurora kinase A (AURKA) plays a role in mitosis and stabilization of the MYCN protein in neuroblastoma. We aimed to study the impact of AURKA inhibitors on DNA damage and tumor cell death in combination with 131I-MIBG therapy in a pre-clinical model of high-risk neuroblastoma.ResultsUsing an in vivo model of high-risk neuroblastoma, we demonstrated a marked combinatorial effect of&nbsp;131I-MIBG and alisertib on tumor growth. In MYCN amplified cell lines, the combination of radiation and an AURKA A inhibitor increased DNA damage and apoptosis and decreased MYCN protein levels.ConclusionThe combination of AURKA inhibition with&nbsp;131I-MIBG treatment is active in resistant neuroblastoma models

Vaccination of cattle with a CpG oligodeoxynucleotide-formulated mycobacterial protein vaccine and Mycobacterium bovis BCG induces levels of protection against bovine tuberculosis superior to those induced by vaccination with BCG alone

The development of a subunit protein vaccine for bovine tuberculosis which could be used either in combination with Mycobacterium bovis BCG (to improve the efficacy of that vaccine) or alone would offer significant advantages over currently available strategies. A study was conducted with cattle to determine the protective efficacy of a strategy based on concurrent immunization with an M. bovis culture filtrate (CFP) vaccine and BCG compared to vaccination with either vaccine alone. One group of calves (10 animals per group) was vaccinated subcutaneously with CFP formulated with Emulsigen and combined with a CpG oligodeoxynucleotide (ODN). A second group was vaccinated with both the CFP vaccine and BCG injected at adjacent sites (CFP-BCG). One further group was vaccinated subcutaneously with BCG, while another group served as nonvaccinated control animals. Vaccination with CFP-BCG induced levels of antigen-specific gamma interferon (IFN-γ) and interleukin-2 (IL-2) in whole-blood cultures that were higher than those induced by vaccination with BCG alone. The combination of CFP and BCG did not enhance the production of antibodies to M. bovis CFP compared to vaccination with CFP alone. Vaccination with CFP alone led to delayed antigen-specific IFN-γ and IL-2 responses. Vaccination with CFP-BCG induced a high level of protection against an intratracheal challenge with virulent M. bovis, based on a significant enhancement of six pathological and microbiological parameters of protection compared with the nonvaccinated group. In contrast, vaccination with BCG alone induced a significant enhancement of protection in only one parameter, while CFP alone induced no protection. These results suggest that a combination of a CpG ODN-formulated protein vaccine and BCG offers better protection against bovine tuberculosis than does BCG alone