The poxviral RING protein p28 is a ubiquitin ligase that targets ubiquitin to viral replication factories

The poxviral RING protein p28 is a virulence factor whose molecular function is unknown. Many cellular RING-containing proteins act as ubiquitin ligases (RING-E3s) connecting selected substrate proteins to the ubiquitination machinery. Here we demonstrate that vaccinia virus p28 and its homologue in myxoma virus, M143R, can mediate the formation of polyubiquitin conjugates, while RING mutants of both p28 and M143R cannot. Furthermore, p28 is ubiquitinated in vivo and ubiquitin colocalizes with p28 to virus factories independently of an intact RING domain. These results implicate the ubiquitin system in poxviral virulence.     

Cullin 4b-RING ubiquitin ligase targets IRGM1 to regulate Wnt signaling and intestinal homeostasis

Hierarchical organization of intestine relies on the self-renewal and tightly regulated differentiation of intestinal stem cells (ISCs). Although signals like Wnt are known to sustain the continued intestinal renewal by maintaining ISCs activity and lineage commitment, molecular mechanisms underlying ISCs ‘stemness’ and supportive niche have not been well understood. Here, we found that CUL4B-RING ubiquitin ligase (CRL4B) regulates intestinal homeostasis by targeting immunity-related GTPase family M member 1 (IRGM1) for proteasomal degradation. CUL4B was mainly expressed at ISCs zone. Deletion of Cul4b led to reduced self-renewal of ISCs and a decreased lineage differentiation towards secretory progenitors through downregulated Wnt signals. Besides, Cul4b-null mice exhibited impaired Paneth cells number and structure. Mechanistically, CRL4B complex were associated with WD40 proteins and targeted IRGM1 at K270 for ubiquitination and proteosomal degradation. Impaired intestinal function caused by CUL4B deletion was rescued by down-regulation of its substrate IRGM1. Our results identified CUL4B as a novel regulator of ISCs and revealed a new 26 S proteasome degradation mechanism in intestine self-renewal and lineage commitment. Subject terms: Development, Ubiquitin ligases     

Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase

The Saccharomyces cerevisiae high-affinity copper transporter, Ctr1p, mediates cellular uptake of Cu(I). We report that when copper (50 microm CuSO(4)) is added to the growth medium of copper-starved cells, Ctr1p is rapidly internalized by endocytosis, delivered to the lumen of the lysosome-like vacuole and slowly degraded by vacuolar proteases. Through analysis of the trafficking and degradation of Ctr1p mutants, two lysine residues in the C-terminal cytoplasmic tail of Ctr1p, Lys340 and Lys345, were found to be critical for copper-dependent endocytosis and degradation. In response to copper addition, Ctr1p was found to be ubiquitylated and a mutation in the Rsp5 ubiquitin ligase largely abolished ubiquitylation, endocytosis and degradation. In a strain lacking the Rsp5p accessory factors Bul1p and Bul2p, endocytosis and degradation of Ctr1p-green fluorescent protein were substantially diminished. Surprisingly, a Ctr1p mutant that lacks Lys340 and Lys345 was still ubiquitylated in a copper-dependent manner, indicating that ubiquitylation of Ctr1p on other sites is insufficient to drive copper-dependent endocytosis and degradation. This study demonstrates that copper regulates turnover of Ctr1p by stimulating Rsp5p-dependent endocytosis and degradation of Ctr1p in the vacuole.     

HOIL‐1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation

HOIL‐1, a component of the linear ubiquitin chain assembly complex (LUBAC), ubiquitylates serine and threonine residues in proteins by esterification. Here, we report that mice expressing an E3 ligase‐inactive HOIL‐1[C458S] mutant accumulate polyglucosan in brain, heart and other organs, indicating that HOIL‐1’s E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL‐1 monoubiquitylates glycogen and α1:4‐linked maltoheptaose in vitro and identify the C6 hydroxyl moiety of glucose as the site of ester‐linked ubiquitylation. The monoubiquitylation of maltoheptaose was accelerated > 100‐fold by the interaction of Met1‐linked or Lys63‐linked ubiquitin oligomers with the RBR domain of HOIL‐1. HOIL‐1 also transferred pre‐formed ubiquitin oligomers to maltoheptaose en bloc, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL‐1, bound to unbranched and infrequently branched glucose polymers in vitro, but not to highly branched mammalian glycogen, suggesting a potential function in targeting HOIL‐1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal from cells, preventing precipitation as polyglucosan.     

PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity

PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin’s ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.     

HECT ubiquitin ligase Smurf1 targets the tumor suppressor ING2 for ubiquitination and degradation

Inhibitor of growth 2 (ING2) gene encodes a candidate tumor suppressor and is frequently reduced in many tumors. However, the mechanisms underlying the regulation of ING2, in particular its protein stability, are 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 ING2 for poly-ubiquitination and proteasomal degradation. Intriguingly, the ING2 binding domain in Smurf1 was mapped to the catalytic HECT domain. Furthermore, the C-terminal PHD domain of ING2 was required for Smurf1-mediated degradation. This study provided the first evidence that the stability of ING2 could be regulated by ubiquitin-mediated degradation.

Structured summary

MINT-7894271: ING2 (uniprotkb:Q9H160) binds (MI:0407) to Smurf1 (uniprotkb:Q9HCE7) by pull-down (MI:0096)MINT-7894319, MINT-7894339: ING2 (uniprotkb:Q9H160) physically interacts (MI:0915) with Smurf1 (uniprotkb:Q9HCE7) by anti tag co-immunoprecipitation (MI:0007)MINT-7894301: Smurf1 (uniprotkb:Q9HCE7) physically interacts (MI:0915) with ING2 (uniprotkb:Q9H160) by anti bait co-immunoprecipitation (MI:0006)MINT-7894358: ING1b (uniprotkb:Q9UK53-2) physically interacts (MI:0915) with Smurf1 (uniprotkb:Q9HCE7) by anti tag co-immunoprecipitation (MI:0007)MINT-7894249: ING2 (uniprotkb:Q9H160) physically interacts (MI:0915) with ubiquitin (uniprotkb:P62988) by anti tag co-immunoprecipitation (MI:0007)     

Lif1p targets the DNA ligase Lig4p to sites of DNA double-strand breaks

DNA ligases catalyse the joining of DNA single- and double-strand breaks. Saccharomyces cerevisiae Cdc9p is a homologue of mammalian DNA ligase I and is required for DNA replication, recombination and single-strand break repair. The other yeast ligase, Lig4p/Dnl4p, is a homologue of mammalian DNA ligase IV, and functions in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair [1] [2] [3] [4]. Lig4p interacts with Lif1p, the yeast homologue of the human ligase IV-associated protein, XRCC4 [5]. This interaction takes place through the carboxy-terminal domain of Lig4p and is required for Lig4p stability. We show that the carboxy-terminal interaction region of Lig4p is necessary for NHEJ but, when fused to Cdc9p, is insufficient to confer NHEJ function to Cdc9p. Also, Lif1p stimulates the in vitro catalytic activity of Lig4p in adenylation and DNA ligation. Nevertheless, Lig4p is inactive in NHEJ in the absence of Lif1p in vivo, even when Lig4p is stably expressed. We show that Lif1p binds DNA in vitro and, through in vivo cross-linking and chromatin immuno precipitation assays, demonstrate that it targets Lig4p to chromosomal DNA double-strand breaks. Furthermore, this targeting requires another key NHEJ protein, Ku.     

The CUL7 E3 ubiquitin ligase targets insulin receptor substrate 1 for ubiquitin-dependent degradation

Recent genetic studies have documented a pivotal growth-regulatory role played by the Cullin 7 (CUL7) E3 ubiquitin ligase complex containing the Fbw8-substrate-targeting subunit, Skp1, and the ROC1 RING finger protein. In this report, we identified insulin receptor substrate 1 (IRS-1), a critical mediator of the insulin/insulin-like growth factor 1 signaling, as a proteolytic target of the CUL7 E3 ligase in a manner that depends on mammalian target of rapamycin and the p70 S6 kinase activities. Interestingly, while embryonic fibroblasts of Cul7-/- mice were found to accumulate IRS-1 and exhibit increased activation of IRS-1's downstream Akt and MEK/ERK pathways, these null cells grew poorly and displayed phenotypes reminiscent of those associated with oncogene-induced senescence. Taken together, our findings demonstrate a key role for the CUL7 E3 in targeting IRS-1 for degradation, a process that may contribute to the regulation of cellular senescence.     

Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation

In eukaryotes, endoplasmic reticulum-associated degradation (ERAD) functions in cellular quality control and regulation of normal ER-resident proteins. ERAD proceeds by the ubiquitin-proteasome pathway, in which the covalent attachment of ubiquitin to proteins targets them for proteasomal degradation. Ubiquitin-protein ligases (E3s) play a crucial role in this process by recognizing target proteins and initiating their ubiquitination. Here we show that Hrd1p, which is identical to Der3p, is an E3 for ERAD. Hrd1p is required for the degradation and ubiquitination of several ERAD substrates and physically associates with relevant ubiquitin-conjugating enzymes (E2s). A soluble Hrd1 fusion protein shows E3 activity in vitro - catalysing the ubiquitination of itself and test proteins. In this capacity, Hrd1p has an apparent preference for misfolded proteins. We also show that Hrd1p functions as an E3 in vivo, using only Ubc7p or Ubc1p to specifically program the ubiquitination of ERAD substrates.     

Cullin 4B protein ubiquitin ligase targets peroxiredoxin III for degradation

Cullin 4B (CUL4B) is a scaffold protein that assembles cullin-RING ubiquitin ligase (E3) complexes. Recent studies have revealed that germ-line mutations in CUL4B can cause mental retardation, short stature, and many other abnormalities in humans. Identifying specific CUL4B substrates will help to better understand the physiological functions of CUL4B. Here, we report the identification of peroxiredoxin III (PrxIII) as a novel substrate of the CUL4B ubiquitin ligase complex. Two-dimensional gel electrophoresis coupled with mass spectrometry showed that PrxIII was among the proteins up-regulated in cells after RNAi-mediated CUL4B depletion. The impaired degradation of PrxIII observed in CUL4B knockdown cells was confirmed by Western blot. We further demonstrated that DDB1 and ROC1 in the DDB1-CUL4B-ROC1 complex are also indispensable for the proteolysis of PrxIII. In addition, the degradation of PrxIII is independent of CUL4A, a cullin family member closely related to CUL4B. In vitro and in vivo ubiquitination assays revealed that CUL4B promoted the polyubiquitination of PrxIII. Furthermore, we observed a significant decrease in cellular reactive oxygen species (ROS) production in CUL4B-silenced cells, which was associated with increased resistance to hypoxia and H(2)O(2)-induced apoptosis. These findings are discussed with regard to the known function of PrxIII as a ROS scavenger and the high endogenous ROS levels required for neural stem cell proliferation. Together, our study has identified a specific target substrate of CUL4B ubiquitin ligase that may have significant implications for the pathogenesis observed in patients with mutations in CUL4B.     

Determinants of RING-E2 fidelity for Hrd1p, a membrane-anchored ubiquitin ligase

A critical aspect of E3 ubiquitin ligase function is the selection of a particular E2 ubiquitin-conjugating enzyme to accomplish ubiquitination of a substrate. We examined the requirements for correct E2-E3 specificity in the RING-H2 ubiquitin ligase Hrd1p, an ER-localized protein known to use primarily Ubc7p for its function. Versions of Hrd1p containing the RING motif from homologous E3s were unable to carry out Hrd1p function, revealing a requirement for the specific Hrd1p RING motif in vivo. An in vitro assay revealed that these RING motifs were sufficient to function as ubiquitin ligases, but that they did not display the E2 specificity predicted from in vivo results. We further refined the in vitro assay of Hrd1p function by demanding not only ubiquitin ligase activity, but also specific activity that recapitulated both the E2 specificity and RING selectivity observed in vivo. Doing so revealed that correct E2 engagement by Hrd1p required the presence of portions of the Hrd1p soluble cytoplasmic domain outside the RING motif, the placement of the Hrd1p ubiquitin ligase in the ER membrane, and presentation of Ubc7p in the cytosolic context. We confirmed that these conditions supported the ubiquitination of Hrd1p itself, and the transfer of ubiquitin to the prototype substrate Hmg2p-GFP, validating Hrd1p self-ubiquitination as a viable assay of ligase function.     

Tuning BRCA1 and BARD1 activity to investigate RING ubiquitin ligase mechanisms

The tumor‐suppressor protein BRCA1 works with BARD1 to catalyze the transfer of ubiquitin onto protein substrates. The N‐terminal regions of BRCA1 and BARD1 that contain their RING domains are responsible for dimerization and ubiquitin ligase activity. This activity is a common feature among hundreds of human RING domain‐containing proteins. RING domains bind and activate E2 ubiquitin‐conjugating enzymes to promote ubiquitin transfer to substrates. We show that the identity of residues at specific positions in the RING domain can tune activity levels up or down. We report substitutions that create a structurally intact BRCA1/BARD1 heterodimer that is inactive in vitro with all E2 enzymes. Other substitutions in BRCA1 or BARD1 RING domains result in hyperactivity, revealing that both proteins have evolved attenuated activity. Loss of attenuation results in decreased product specificity, providing a rationale for why nature has tuned BRCA1 activity. The ability to tune BRCA1 provides powerful tools for understanding its biological functions and provides a basis to assess mechanisms for rescuing the activity of cancer‐associated variations. Beyond the applicability to BRCA1, we show the identity of residues at tuning positions that can be used to predict and modulate the activity of an unrelated RING E3 ligase. These findings provide valuable insights into understanding the mechanism and function of RING E3 ligases like BRCA1.     

Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase

Ubiquitin ligases (E3) select proteins for ubiquitylation, a modification that directs altered subcellular trafficking and/or degradation of the target protein. HECT domain E3 ligases not only recognize, but also directly catalyze, ligation of ubiquitin to their protein substrates. The crystal structure of the HECT domain of the human ubiquitin ligase WWP1/AIP5 maintains a two-lobed structure like the HECT domain of the human ubiquitin ligase E6AP. While the individual N and C lobes of WWP1 possess very similar folds to those of E6AP, the organization of the two lobes relative to one another is different from E6AP due to a rotation about a polypeptide hinge linking the N and C lobes. Mutational analyses suggest that a range of conformations achieved by rotation about this hinge region is essential for catalytic activity.     

A phosphorylated form of Mel-18 targets the Ring1B histone H2A ubiquitin ligase to chromatin

Recent studies have shown that PRC1-like Polycomb repressor complexes monoubiquity-late chromatin on histone H2A at lysine residue 119. Here we have analyzed the function of the polycomb protein Mel-18. Using affinity-tagged human MEL-18, we identify a polycomb-like complex, melPRC1, containing the core PRC1 proteins, RING1/2, HPH2, and CBX8. We show that, in ES cells, melPRC1 can functionally substitute for other PRC1-like complexes in Hox gene repression. A reconstituted subcomplex containing only Ring1B and Mel-18 functions as an efficient ubiquitin E3 ligase. This complex ubiquitylates free histone substrates nonspecifically but is highly specific for histone H2A lysine 119 in the context of nucleosomes. Mutational analysis demonstrates that while Ring1B is required for E3 function, Mel-18 directs this activity to H2A lysine 119 in chromatin. Moreover, this substrate-targeting function of Mel-18 is dependent on its prior phosphorylation at multiple residues, providing a direct link between chromatin modification and cell signaling pathways.     

Shigella ubiquitin ligase IpaH7.8 targets gasdermin D for degradation to prevent pyroptosis and enable infection

1. Download : Download high-res image (201KB)
2. Download : Download full-size image

     

Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4

Cse4 is a variant of histone H3 that is incorporated into a single nucleosome at each centromere in budding yeast. We have discovered an E3 ubiquitin ligase, called Psh1, which controls the cellular level of Cse4 via ubiquitylation and proteolysis. The activity of Psh1 is dependent on both its RING and zinc finger domains. We demonstrate the specificity of the ubiquitylation activity of Psh1 toward Cse4 in vitro and map the sites of ubiquitylation. Mutation of key lysines prevents ubiquitylation of Cse4 by Psh1 in vitro and stabilizes Cse4 in vivo. While deletion of Psh1 stabilizes Cse4, elimination of the Cse4-specific chaperone Scm3 destabilizes Cse4, and the addition of Scm3 to the Psh1-Cse4 ubiquitylation reaction prevents Cse4 ubiquitylation, together suggesting Scm3 may protect Cse4 from ubiquitylation. Without Psh1, Cse4 overexpression is toxic and Cse4 is found at ectopic locations. Our results suggest Psh1 functions to prevent the mislocalization of Cse4.