Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012;7(5):e37470.
doi: 10.1371/journal.pone.0037470. Epub 2012 May 21.

TRIM16 acts as an E3 ubiquitin ligase and can heterodimerize with other TRIM family members

Jessica L Bell  1 , Alena Malyukova, Jessica K Holien, Jessica Koach, Michael W Parker, Maria Kavallaris, Glenn M Marshall, Belamy B Cheung
Affiliations

TRIM16 acts as an E3 ubiquitin ligase and can heterodimerize with other TRIM family members

Jessica L Bell et al. PLoS One. 2012.

Abstract

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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. TRIM16 homodimerizes through its coiled-coil domain
. (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.
Figure 2
Figure 2. TRIM16 can heterodimerize with MID1, TRIM24 and PML.
(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.
Figure 3
Figure 3. Amino acid sequence comparison of TRIM16 and MID1 and modeling of TRIM16 B-boxes.
The conserved residues in the zinc binding regions are in bold underlined type and are; cysteine; C, histidine; H, alanine; A aspartic acid; D. (A) 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.
Figure 4
Figure 4. TRIM16 is an unstable protein regulated by the ubiquitin-proteasome system.
(A) In vivo degradation assay was performed with cells being incubated with and without 30 µM MG132 for 4 hours. Lysates were subjected to Western blot analysis of endogenous TRIM16 antibody. (B) The liable nature of TRIM16 was further evaluated by inhibition of protein synthesis using 100 µg/ml cycloheximide. At the specified time points the cells were harvested and the protein were extracted for analysis by Western blots.
Figure 5
Figure 5. B-boxes are required for TRIM16’s E3 ligase activity.
(A) TRIM16 in vivo 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) In vitro ubiquitination assay with myc-His tagged TRIM16 together with a panel of E2 enzymes, showing activity with the UbcH5 family. (C) In vitro 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 in vitro 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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Joazeiro CA, Weissman AM. RING finger proteins: mediators of ubiquitin ligase activity. Cell. 2000;102:549–552. - PubMed
    1. Meroni G, Diez-Roux G, Meroni G, Diez-Roux G. TRIM/RBCC, a novel class of ‘single protein RING finger’ E3 ubiquitin ligases. Bioessays. 2005;27:1147–1157. - PubMed
    1. Cheung BB, Bell J, Raif A, Bohlken A, Yan J, et al. The estrogen-responsive B box protein is a novel regulator of the retinoid signal. J Biol Chem. 2006;281:18246–18256. - PubMed
    1. Raif A, Marshall GM, Bell JL, Koach J, Tan O, et al. The estrogen-responsive B box protein (EBBP) restores retinoid sensitivity in retinoid-resistant cancer cells via effects on histone acetylation. Cancer Lett. 2009;277:82–90. - PubMed
    1. Marshall GM, Bell JL, Koach J, Tan O, Kim P, et al. TRIM16 acts as a tumour suppressor by inhibitory effects on cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells. Oncogene. 2010;29:6172–6183. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources