Smac mimetics activate the E3 ligase activity of cIAP1 protein by promoting RING domain dimerization

The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono- or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways.     

Dual-site regulation of MDM2 E3-ubiquitin ligase activity

The control of p53 ubiquitination by MDM2 provides a model system to define how an E3-ligase functions on a conformationally flexible substrate. The mechanism of MDM2-mediated ubiquitination of p53 has been analyzed by deconstructing, in vitro, the MDM2-dependent ubiquitination reaction. Surprisingly, ligands binding to the hydrophobic cleft of MDM2 do not inhibit its E3-ligase function. However, peptides from within the DNA binding domain of p53 that bind the acid domain of MDM2 inhibit ubiquitination of p53, localizing a motif that harbors a key ubiquitination signal. The binding of ligands to the N-terminal hydrophobic cleft of MDM2 reactivates, in vitro and in vivo, MDM2-catalyzed ubiquitination of p53F19A, a mutant p53 normally refractory to MDM2-catalyzed ubiquitination. We propose a model in which the interaction between the p53-BOX-I domain and the N terminus of MDM2 promotes conformational changes in MDM2 that stabilize acid-domain interactions with a ubiquitination signal in the DNA binding domain of the p53 tetramer.     

Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase

Tripartite motif (TRIM) proteins comprise a large family of RING‐type ubiquitin E3 ligases that regulate important biological processes. An emerging general model is that TRIMs form elongated antiparallel coiled‐coil dimers that prevent interaction of the two attendant RING domains. The RING domains themselves bind E2 conjugating enzymes as dimers, implying that an active TRIM ligase requires higher‐order oligomerization of the basal coiled‐coil dimers. Here, we report crystal structures of the TRIM23 RING domain in isolation and in complex with an E2–ubiquitin conjugate. Our results indicate that TRIM23 enzymatic activity requires RING dimerization, consistent with the general model of TRIM activation.     

The contribution of RING and B-box 2 domains to retroviral restriction mediated by monkey TRIM5alpha

TRIM5alpha is a cytoplasmic protein that mediates a post-entry block to infection by some retroviruses. TRIM5alpha contains a tripartite motif (TRIM), which includes RING, B-box 2, and coiled-coil domains, and a C-terminal B30.2 (SPRY) domain. We investigated the contribution of the RING and B-box 2 domains to the antiretroviral activity of rhesus monkey TRIM5alpha (TRIM5alpharh), which potently restricts infection by human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus of African green monkeys (SIVagm). Disruption of the RING domain caused mislocalization of TRIM5alpharh so that the cytoplasmic level of the protein was decreased compared with that of the wild-type protein. Nonetheless, partial ability to restrict HIV-1 and SIVagm was retained by the RING domain mutants. By contrast, although TRIM5alpharh mutants with disrupted B-box 2 domains were efficiently expressed and correctly localized to the cytoplasm, antiretroviral activity was absent. The B-box 2 mutants colocalized and associated with wild-type TRIM5alpharh and exerted dominant-negative effects on the antiretroviral activity of the wild-type protein. Taken together with other data, these results indicate that functionally defective TRIM5alpharh molecules that retain a coiled coil can act as dominant-negative inhibitors of wild-type TRIM5alpharh function. The RING domain of TRIM5alpharh is not absolutely required for retrovirus restriction but can influence cytoplasmic levels of the protein and thus indirectly alter function. The B-box 2 domain, by contrast, appears to be essential for efficient retrovirus restriction.     

Sphingosine-1-phosphate induced mTOR-activation is mediated by the E3-ubiquitin ligase PAM

The signaling pathways that are regulated by sphingosine-1-phosphate (S1P) and mammalian target of rapamycin (mTOR) modulate cell growth, mitogenesis and apoptosis in various cell types and are of major interest for the development of new cancer therapeutics. Previous reports show that S1P can cross-activate the mTOR pathway although the mechanisms that connect both pathways are still unknown. We found that S1P-treatment activates mTOR in several cancer cell lines and primary cells. The activation was independent of ERK, Akt and PI3-kinase, but instead was mediated by the E3 ubiquitin ligase Protein Associated with Myc (PAM). Increased intracellular PAM concentrations facilitated S1P- and insulin-induced mTOR activation as well as p70S6K and 4EBP1 phosphorylation while genetic deletion of PAM decreased S1P- and insulin-induced mTOR activation. PAM activated by facilitating the GDP/GTP-exchange of Rheb which is an activator of mTOR. In conclusion we show that PAM is a novel regulator of the mTOR pathway and that PAM may directly activate Rheb as a guanosine exchange factor (GEF).     

Solution structure of the E3 ligase HOIL-1 Ubl domain

The E3 ligases HOIL‐1 and parkin are each comprised of an N‐terminal ubiquitin‐like (Ubl) domain followed by a zinc‐binding region and C‐terminal RING–In‐between‐RING–RING domains. These two proteins, involved in the ubiquitin‐mediated degradation pathway, are the only two known E3 ligases to share this type of multidomain architecture. Further, the Ubl domain of both HOIL‐1 and parkin has been shown to interact with the S5a subunit of the 26S proteasome. The solution structure of the HOIL‐1 Ubl domain was solved using NMR spectroscopy to compare it with that of parkin to determine the structural elements responsible for S5a intermolecular interactions. The final ensemble of 20 structures had a β‐grasp Ubl‐fold with an overall backbone RMSD of 0.59 ± 0.10 Å in the structured regions between I55 and L131. HOIL‐1 had a unique extension of both β1 and β2 sheets compared to parkin and other Ubl domains, a result of a four‐residue insertion in this region. A similar 15‐residue hydrophobic core in the HOIL‐1 Ubl domain resulted in a comparable stability to the parkin Ubl, but significantly lower than that observed for ubiquitin. A comparison with parkin and other Ubl domains indicates that HOIL‐1 likely uses a conserved hydrophobic patch (W58, V102, Y127, Y129) found on the β1 face, the β3–β4 loop and β5, as well as a C‐terminal basic residue (R134) to recruit the S5a subunit as part of the ubiquitin‐mediated proteolysis pathway.     

Functional replacement of the RING, B-box 2, and coiled-coil domains of tripartite motif 5alpha (TRIM5alpha) by heterologous TRIM domains

Tripartite motif 5alpha (TRIM5alpha) restricts some retroviruses, including human immunodeficiency virus type 1 (HIV-1), from infecting the cells of particular species. TRIM5alpha is a member of the TRIM family of proteins, which contain RING, B-box, coiled-coil (CC), and, in some cases, B30.2(SPRY) domains. Here we investigated the abilities of domains from TRIM proteins (TRIM6, TRIM34, and TRIM21) that do not restrict HIV-1 infection to substitute for the domains of rhesus monkey TRIM5alpha (TRIM5alpha(rh)). The RING, B-box 2, and CC domains of the paralogous TRIM6 and TRIM34 proteins functionally replaced the corresponding TRIM5alpha(rh) domains, allowing HIV-1 restriction. By contrast, similar chimeras containing the components of TRIM21, a slightly more distant relative of TRIM5, did not restrict HIV-1 infection. The TRIM21 B-box 2 domain and its flanking linker regions contributed to the functional defectiveness of these chimeras. All of the chimeric proteins formed trimers. All of the chimeras that restricted HIV-1 infection bound the assembled HIV-1 capsid complexes. These results indicate that heterologous RING, B-box 2, and CC domains from related TRIM proteins can functionally substitute for TRIM5alpha(rh) domains.     

TRIM proteins regulate autophagy: TRIM5 is a selective autophagy receptor mediating HIV-1 restriction

The tripartite motif protein family (TRIM) constitutes a class of immune-regulated proteins with antiviral, immune, cancer, and other properties reminiscent of those ascribed to autophagy. We show that TRIMs have dual roles in autophagy: as regulators and as cargo receptors. As regulators, TRIMs nucleate the core autophagy machinery by acting as platforms that assemble ULK1 and BECN1 into a functional complex in preparation for autophagy. TRIMs also act as novel selective autophagy receptors as exemplified by TRIM5/TRIM5α, a known HIV-1 restriction factor with a hitherto poorly defined mode of action. TRIM5 recognizes and targets HIV-1 for autophagic destruction. TRIM5 interactions with mammalian Atg8 proteins are required for this effector function. This establishes TRIM family members as regulators of autophagy, explains the antiretroviral mechanism of TRIM5, and defines a new basis for selective autophagy.     

TRIM proteins regulate autophagy: TRIM5 is a selective autophagy receptor mediating HIV-1 restriction

The tripartite motif protein family (TRIM) constitutes a class of immune-regulated proteins with antiviral, immune, cancer, and other properties reminiscent of those ascribed to autophagy. We show that TRIMs have dual roles in autophagy: as regulators and as cargo receptors. As regulators, TRIMs nucleate the core autophagy machinery by acting as platforms that assemble ULK1 and BECN1 into a functional complex in preparation for autophagy. TRIMs also act as novel selective autophagy receptors as exemplified by TRIM5/TRIM5α, a known HIV-1 restriction factor with a hitherto poorly defined mode of action. TRIM5 recognizes and targets HIV-1 for autophagic destruction. TRIM5 interactions with mammalian Atg8 proteins are required for this effector function. This establishes TRIM family members as regulators of autophagy, explains the antiretroviral mechanism of TRIM5, and defines a new basis for selective autophagy.     

POSH2 is a RING finger E3 ligase with Rac1 binding activity through a partial CRIB domain

The Plenty of SH3 domains protein (POSH) is an E3 ligase and a scaffold in the JNK mediated apoptosis, linking Rac1 to downstream components.We here describe POSH2 which was identified from a p21-activated kinase 2 (PAK2) interactor screen. POSH2 is highly homologous with other members of the POSH family; it contains four Src homology 3 (SH3) domains and a RING finger domain which confers E3 ligase activity to the protein. In addition POSH2 contains an N-terminal extension which is conserved among its mammalian counterparts. POSH2 interacts with GTP-loaded Rac1. We have mapped this interaction to a previously unrecognized partial Cdc42/Rac1-interactive binding domain.

Structured summary

MINT-7987761: POSH1 (uniprotkb:Q9HAM2) physically interacts (MI:0915) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7987932: PAK2 (uniprotkb:Q13177) binds (MI:0407) to CDC42 (uniprotkb:Q07912) by solid phase assay (MI:0892)MINT-7987908: POSH1 (uniprotkb:Q9HAM2) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)MINT-7987880: POSH2 (uniprotkb:Q8TEJ3) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)MINT-7987734: POSH2 (uniprotkb:Q8TEJ3) physically interacts (MI:0915) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7987779, MINT-7987804, MINT-7987824, MINT-7987838, MINT-7987853: Rac1 (uniprotkb:P63000) physically interacts (MI:0915) with POSH2 (uniprotkb:Q8TEJ3) by anti tag coimmunoprecipitation (MI:0007)MINT-7987920: PAK2 (uniprotkb:Q13177) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)     

Modulation of retroviral restriction and proteasome inhibitor-resistant turnover by changes in the TRIM5alpha B-box 2 domain

An intact B-box 2 domain is essential for the antiretroviral activity of TRIM5alpha. We modeled the structure of the B-box 2 domain of TRIM5alpha based on the existing three-dimensional structure of the B-box 2 domain of human TRIM29. Using this model, we altered the residues predicted to be exposed on the surface of this globular structure. Most of the alanine substitutions in these residues exerted little effect on the antiretroviral activity of human TRIM5alphahu or rhesus monkey TRIM5alpharh. However, alteration of arginine 119 of TRIM5alphahu or the corresponding arginine 121 of TRIM5alpharh diminished the abilities of the proteins to restrict retroviral infection without affecting trimerization or recognition of the viral capsid. The abilities of these functionally defective TRIM5alpha proteins to accelerate the uncoating of the targeted retroviral capsid were abolished. Removal of the positively charged side chain from B-box 2 arginines 119/120/121 resulted in diminished proteasome-independent turnover of TRIM5alpha and the related restriction factor TRIMCyp. However, testing of an array of mutants revealed that the rapid turnover and retroviral restriction functions of this B-box 2 region are separable.     

The SCF-FBXW5 E3-ubiquitin ligase is regulated by PLK4 and targets HsSAS-6 to control centrosome duplication

Deregulated centrosome duplication can result in genetic instability and contribute to tumorigenesis. Here, we show that centrosome duplication is regulated by the activity of an E3-ubiquitin ligase that employs the F-box protein FBXW5 (ref. 3) as its targeting subunit. Depletion of endogenous FBXW5 or overexpression of an F-box-deleted mutant version results in centrosome overduplication and formation of multipolar spindles. We identify the centriolar protein HsSAS-6 (refs 4,5) as a critical substrate of the SCF-FBXW5 complex. FBXW5 binds HsSAS-6 and promotes its ubiquitylation in vivo. The activity of SCF-FBXW5 is in turn negatively regulated by Polo-like kinase 4 (PLK4), which phosphorylates FBXW5 at Ser 151 to suppress its ability to ubiquitylate HsSAS-6. FBXW5 is a cell-cycle-regulated protein with expression levels peaking at the G1/S transition. We show that FBXW5 levels are controlled by the anaphase-promoting (APC/C) complex, which targets FBXW5 for degradation during mitosis and G1, thereby helping to reset the centrosome duplication machinery. In summary, we show that a cell-cycle-regulated SCF complex is regulated by the kinase PLK4, and that this in turn restricts centrosome re-duplication through degradation of the centriolar protein HsSAS-6.     

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N‐terminal portion which comprises a canonical RING domain, one or two B‐box domains and a coiled‐coil region that mediates ligase dimerization. Self‐association via the coiled‐coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin‐loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full‐length protein. Our data reveal an unexpected diversity in the self‐association mechanism of TRIMs that might be crucial for their biological function.     

Species-specific variation in the B30.2(SPRY) domain of TRIM5alpha determines the potency of human immunodeficiency virus restriction

Retroviruses encounter dominant postentry restrictions in cells of particular species. Human immunodeficiency virus type 1 (HIV-1) is blocked in the cells of Old World monkeys by TRIM5alpha, a tripartite motif (TRIM) protein composed of RING, B-box 2, coiled-coil, and B30.2(SPRY) domains. Rhesus monkey TRIM5alpha (TRIM5alpha(rh)) more potently blocks HIV-1 infection than human TRIM5alpha (TRIM5alpha(hu)). Here, by studying chimeric TRIM5alpha proteins, we demonstrate that the major determinant of anti-HIV-1 potency is the B30.2(SPRY) domain. Analysis of species-specific variation in TRIM5alpha has identified three variable regions (v1, v2, and v3) within the B30.2 domain. The TRIM5alpha proteins of Old World primates exhibit expansion, duplication, and residue variation specifically in the v1 region. Replacement of three amino acids in the N terminus of the TRIM5alpha(hu) B30.2 v1 region with the corresponding TRIM5alpha(rh) residues resulted in a TRIM5alpha molecule that restricted HIV-1 nearly as efficiently as wild-type TRIM5alpha(rh). Surprisingly, a single-amino-acid change in this region of TRIM5alpha(hu) allowed potent restriction of simian immunodeficiency virus, a phenotype not observed for either wild-type TRIM5alpha(hu) or TRIM5alpha(rh). Some of the chimeric TRIM5alpha proteins that are >98% identical to the human protein yet mediate a strong restriction of HIV-1 infection may have therapeutic utility. These observations implicate the v1 variable region of the B30.2(SPRY) domain in TRIM5alpha(rh) antiviral potency.     

Determinants of the higher order association of the restriction factor TRIM5alpha and other tripartite motif (TRIM) proteins

Many tripartite motif (TRIM) proteins self-associate, forming dimers and higher order complexes. For example, dimers of TRIM5α, a host factor that restricts retrovirus infection, assemble into higher order arrays on the surface of the viral capsid, resulting in an increase in avidity. Here we show that the higher order association of different TRIM proteins exhibits a wide range of efficiencies. Homologous association (self-association) was more efficient than the heterologous association of different TRIM proteins, indicating that specificity determinants of higher order self-association exist. To investigate the structural determinants of higher order self-association, we studied TRIM mutants and chimeras. These studies revealed the following: 1) the RING domain contributes to the efficiency of higher order self-association, which enhances the binding of TRIM5α to the human immunodeficiency virus (HIV-1) capsid; 2) the RING and B-box 2 domains work together as a homologous unit to promote higher order association of dimers; 3) dimerization is probably required for efficient higher order self-association; 4) the Linker 2 region contributes to higher order self-association, independently of effects of Linker 2 changes on TRIM dimerization; and 5) for efficiently self-associating TRIM proteins, the B30.2(SPRY) domain is not required for higher order self-association. These results support a model in which both ends of the core TRIM dimer (RING-B-box 2 at one end and Linker 2 at the other) contribute to the formation of higher order arrays.