N-degron E3 ubiquitin ligases recognize specific residues at the N-termini of substrates. Although molecular details of N-degron recognition are known for several E3 ligases, the range of N-terminal motifs that can bind a given E3 substrate binding domain remains unclear. Here, we discovered capacity of Gid4 and Gid10 substrate receptor subunits of yeast "GID"/human "CTLH" multiprotein E3 ligases to tightly bind a wide range of N-terminal residues whose recognition is determined in part by the downstream sequence context. Screening of phage displaying peptide libraries with exposed N-termini identified novel consensus motifs with non-Pro N-terminal residues binding Gid4 or Gid10 with high affinity. Structural data reveal that conformations of flexible loops in Gid4 and Gid10 complement sequences and folds of interacting peptides. Together with analysis of endogenous substrate degrons, the data show that degron identity, substrate domains harboring targeted lysines, and varying E3 ligase higher-order assemblies combinatorially determine efficiency of ubiquitylation and degradation.

The endoplasmic reticulum (ER) responds to changes in intracellular homeostasis through activation of the unfolded protein response (UPR). Yet, it is not known how UPR-signaling coordinates adaptation versus cell death. Previous studies suggested that signaling through PERK/ATF4 is required for cell death. We show that high levels of ER stress (i.e., ischemia-like conditions) induce transcription of the ubiquitin ligases Siah1/2 through the UPR transducers PERK/ATF4 and IRE1/sXBP1. In turn, Siah1/2 attenuates proline hydroxylation of ATF4, resulting in its stabilization, thereby augmenting ER stress output. Conversely, ATF4 activation is reduced upon Siah1/2 KD in cultured cells, which attenuates ER stress-induced cell death. Notably, Siah1a(+/-)::Siah2(-/-) mice subjected to neuronal ischemia exhibited smaller infarct volume and were protected from ischemia-induced death, compared with the wild type (WT) mice. In all, Siah1/2 constitutes an obligatory fine-tuning mechanism that predisposes cells to death under severe ER stress conditions.

Cullin RING ligases are multi-subunit complexes consisting of a cullin protein which forms a scaffold onto which the RING protein Rbx1/2 and substrate receptor subunits assemble. CAND1, which binds to cullins that are not conjugated with Nedd8 and not associated with substrate receptors, has been shown to function as a positive regulator of Cullin ligases in vivo. Two models have been proposed to explain this requirement: (i) CAND1 sequesters cullin proteins and thus prevents autoubiquitination of substrate receptors, and (ii) CAND1 is required to promote the exchange of bound substrate receptors. Using mammalian cells, we show that CAND1 is predominantly cytoplasmically localized and that cullins are the major CAND1 interacting proteins. However, only small amounts of CAND1 bind to Cul1 in cells, despite low basal levels of Cul1 neddylation and approximately equal cytoplasmic endogenous protein concentrations of CAND1 and Cul1. Compared to F-box protein substrate receptors, binding of CAND1 to Cul1 in vivo is weak. Furthermore, preventing binding of F-box substrate receptors to Cul1 does not increase CAND1 binding. In conclusion, our study suggests that CAND1 does not function by sequestering cullins in vivo to prevent substrate receptor autoubiquitination and is likely to regulate cullin RING ligase activity via alternative mechanisms.

Regulated choice between cell fate maintenance and differentiation provides decision points in development to progress toward more restricted cell fates or to maintain the current one. Caenorhabditis elegans embryogenesis follows an invariant cell lineage where cell fate is generally more restricted upon each cell division. EMS is a progenitor cell in the four-cell embryo that gives rise to the endomesoderm. We recently found that when ubiquitin-mediated protein degradation is compromised, the anterior daughter of EMS, namely MS, reiterates the EMS fate. This observation demonstrates an essential function of ubiquitin-mediated protein degradation in driving the progression of EMS-to-MS differentiation. Here we report a genome-wide screen of the ubiquitin pathway and extensive lineage analyses. The results suggest a broad role of E3 ligases in driving differentiation progression. First, we identified three substrate-binding proteins for two Cullin-RING ubiquitin ligase (CRL) E3 complexes that promote the progression from the EMS fate to MS, namely LIN-23/β-TrCP and FBXB-3 for the CRL1/SCF complex and ZYG-11/ZYG-11B for the CRL2 complex. Genetic analyses suggest these E3 ligases function through a multifunctional protein OMA-1 and the endomesoderm lineage specifier SKN-1 to drive differentiation. Second, we found that depletion of components of the CRL1/SCF complex induces fate reiteration in all major founder cell lineages. These data suggest that regulated choice between self-renewal and differentiation is widespread during C. elegans embryogenesis as in organisms with regulative development, and ubiquitin-mediated protein degradation drives the choice towards differentiation. Finally, bioinformatic analysis of time series gene expression data showed that expression of E3 genes is transiently enriched during time windows of developmental stage transitions. Transcription factors show similar enrichment, but not other classes of regulatory genes. Based on these findings we propose that ubiquitin-mediated protein degradation, like many transcription factors, function broadly as regulators driving developmental progression during embryogenesis in C. elegans.

SIZ1 is a small ubiquitin-related modifier (SUMO) E3 ligase that mediates post-translational SUMO modification of target proteins and thereby regulates developmental processes and hormonal and environmental stress responses in Arabidopsis. However, the role of SUMO E3 ligases in crop plants is largely unknown. Here, we identified and characterized two Glycine max (soybean) SUMO E3 ligases, GmSIZ1a and GmSIZ1b. Expression of GmSIZ1a and GmSIZ1b was induced in response to salicylic acid (SA), heat, and dehydration treatment, but not in response to cold, abscisic acid (ABA), and NaCl treatment. Although GmSIZ1a was expressed at higher levels than GmSIZ1b, both genes encoded proteins with SUMO E3 ligase activity in vivo. Heterologous expression of GmSIZ1a or GmSIZ1b rescued the mutant phenotype of Arabidopsis siz1-2, including dwarfism, constitutively activated expression of pathogen-related genes, and ABA-sensitive seed germination. Simultaneous downregulation of GmSIZ1a and GmSIZ1b (GmSIZ1a/b) using RNA interference (RNAi)-mediated gene silencing decreased heat shock-induced SUMO conjugation in soybean. Moreover, GmSIZ1RNAi plants exhibited reduced plant height and leaf size. However, unlike Arabidopsis siz1-2 mutant plants, flowering time and SA levels were not significantly altered in GmSIZ1RNAi plants. Taken together, our results indicate that GmSIZ1a and GmSIZ1b mediate SUMO modification and positively regulate vegetative growth in soybean.

Cadherin family proteins play a central role in epithelial and endothelial cell-cell adhesion. The dynamic regulation of cell adhesion is achieved in part through endocytic membrane trafficking pathways that modulate cadherin cell surface levels. Here, we define the role for various MARCH family ubiquitin ligases in the regulation of cadherin degradation. We find that MARCH2 selectively downregulates VE-cadherin, resulting in loss of adherens junction proteins at cell borders and a loss of endothelial barrier function. Interestingly, N-cadherin is refractory to MARCH ligase expression, demonstrating that different classical cadherin family proteins are differentially regulated by MARCH family ligases. Using chimeric cadherins, we find that the specificity of different MARCH family ligases for different cadherins is conferred by the cadherin transmembrane domain. Further, juxta-membrane lysine residues are required for cadherin degradation by MARCH proteins. These findings expand our understanding of cadherin regulation and highlight a new role for mammalian MARCH family ubiquitin ligases in differentially regulating cadherin turnover.

SARS-CoV-2 is responsible for the ongoing world-wide pandemic which has already taken more than two million lives. Effective treatments are urgently needed. The enzymatic activity of the HECT-E3 ligase family members has been implicated in the cell egression phase of deadly RNA viruses such as Ebola through direct interaction of its VP40 Protein. Here we report that HECT-E3 ligase family members such as NEDD4 and WWP1 interact with and ubiquitylate the SARS-CoV-2 Spike protein. Furthermore, we find that HECT family members are overexpressed in primary samples derived from COVID-19 infected patients and COVID-19 mouse models. Importantly, rare germline activating variants in the NEDD4 and WWP1 genes are associated with severe COVID-19 cases. Critically, I3C, a natural NEDD4 and WWP1 inhibitor from Brassicaceae, displays potent antiviral effects and inhibits viral egression. In conclusion, we identify the HECT family members of E3 ligases as likely novel biomarkers for COVID-19, as well as new potential targets of therapeutic strategy easily testable in clinical trials in view of the established well-tolerated nature of the Brassicaceae natural compounds.

To define the links between paramyxovirus budding and cellular ESCRT machinery, we previously identified angiomotin-like 1 (AMOTL1) in a screen for host factors that bind to the matrix (M) protein of parainfluenza virus 5 (PIV5). This protein harbors three L/PPXY sequences, allowing it to interact with WW domain containing proteins including NEDD4 family members. We hypothesize that paramyxoviruses use AMOTL1 as a linker to indirectly recruit the same NEDD4 ubiquitin ligases for budding that other enveloped viruses recruit directly through their PPXY late domains. In support of this hypothesis, we found that AMOTL1 could link together M proteins and NEDD4 family proteins in three-way co-IP experiments. Both PIV5 and mumps virus M proteins could be linked to the NEDD4 family proteins NEDD4-1, NEDD4L, and NEDL1, provided that AMOTL1 was co-expressed as a bridging protein. AMOT and AMOTL2 could not substitute for AMOTL1, as they lacked the ability to bind with paramyxovirus M proteins. Attachment of a PPXY late domain sequence to PIV5 M protein obviated the need for AMOTL1 as a linker between M and NEDD4 proteins. Together, these results suggest a novel host factor recruitment strategy for paramyxoviruses to achieve particle release.

Members of the potassium channel tetramerization domain (KCTD) family are soluble non-channel proteins that commonly function as Cullin3 (Cul3)-dependent E3 ligases. Solution studies of the N-terminal BTB domain have suggested that some KCTD family members may tetramerize similarly to the homologous tetramerization domain (T1) of the voltage-gated potassium (Kv) channels. However, available structures of KCTD1, KCTD5 and KCTD9 have demonstrated instead pentameric assemblies. To explore other phylogenetic clades within the KCTD family, we determined the crystal structures of the BTB domains of a further five human KCTD proteins revealing a rich variety of oligomerization architectures, including monomer (SHKBP1), a novel two-fold symmetric tetramer (KCTD10 and KCTD13), open pentamer (KCTD16) and closed pentamer (KCTD17). While these diverse geometries were confirmed by small-angle X-ray scattering (SAXS), only the pentameric forms were stable upon size-exclusion chromatography. With the exception of KCTD16, all proteins bound to Cul3 and were observed to reassemble in solution as 5 : 5 heterodecamers. SAXS data and structural modelling indicate that Cul3 may stabilize closed BTB pentamers by binding across their BTB-BTB interfaces. These extra interactions likely also allow KCTD proteins to bind Cul3 without the expected 3-box motif. Overall, these studies reveal the KCTD family BTB domain to be a highly versatile scaffold compatible with a range of oligomeric assemblies and geometries. This observed interface plasticity may support functional changes in regulation of this unusual E3 ligase family.

The ubiquitin-proteasome pathway (UPP) is a hallmark of the eukaryotic cell. In jawed vertebrates, it has been co-opted by the adaptive immune system, where proteasomal degradation produces endogenous peptides for major histocompatibility complex class I antigen presentation. However, proteolytic products are also necessary for the phylogenetically widespread innate immune system, as they often play a role as host defense peptides (HDPs), pivotal effectors against pathogens. Here, we report the identification of the arachnid HDP oligoventin, which shares homology to a core member of the UPP, E3 ubiquitin ligases. Oligoventin has broad antimicrobial activity and shows strong synergy with lysozymes. Using computational and phylogenetic approaches, we show high conservation of the oligoventin signature in HECT E3s. In silico simulation of HECT E3s self-proteolysis provides evidence that HDPs can be generated by fine-tuned 26S proteasomal degradation, and therefore are consistent with the hypothesis that oligoventin is a cryptic peptide released by the proteolytic processing of an Nedd4 E3 precursor protein. Finally, we compare the production of HDPs and endogenous antigens from orthologous HECT E3s by proteasomal degradation as a means of analyzing the UPP coupling to metazoan immunity. Our results highlight the functional plasticity of the UPP in innate and adaptive immune systems as a possibly recurrent mechanism to generate functionally diverse peptides.

Asparaginyl endopeptidases (AEPs) are a class of enzymes commonly associated with proteolysis in the maturation of seed storage proteins. However, a subset of AEPs work preferentially as peptide ligases, coupling release of a leaving group to formation of a new peptide bond. These "ligase-type" AEPs require only short recognition motifs to ligate a range of targets, making them useful tools in peptide and protein engineering for cyclisation of peptides or ligation of separate peptides into larger products. Here we report the recombinant expression, ligase activity and cyclisation kinetics of three new AEPs from the cyclotide producing plant Oldenlandia affinis with superior kinetics to the prototypical recombinant AEP ligase OaAEP1b. These AEPs work preferentially as ligases at both acidic and neutral pH and we term them "canonical AEP ligases" to distinguish them from other AEPs where activity preferences shift according to pH. We show that these ligases intrinsically favour ligation over hydrolysis, are highly efficient at cyclising two unrelated peptides and are compatible with organic co-solvents. Finally, we demonstrate the broad scope of recombinant AEPs in biotechnology by the backbone cyclisation of an intrinsically disordered protein, the 25 kDa malarial vaccine candidate Plasmodium falciparum merozoite surface protein 2 (MSP2).

Formins are actin-binding proteins that are key to maintaining the actin cytoskeleton in cells. However, molecular mechanisms controlling the stability of formin proteins in plants remain unknown. Here, we have identified six rice SIAH-type E3 ligases, named RIP1-6 (RMD Interacting Protein 1-6) respectively, with ubiquitination enzyme activity in vitro. All six proteins can form homo- and hetero-dimers with themselves, and hetero-dimers with type II formin RMD/OsFH5. In vivo assays showed that RIP1-6 proteins localize in the cytoplasm with a punctate distribution, and all of them interact with RMD to change its native diffuse cytoplasmic localization to match that of RIP1-6. To our surprise, degradation experiments revealed that RIP1, RIP5, and RIP6 decrease rather than increase the degradation rate of RMD. Genetic analyses revealed redundancy between these six genes; either single or double mutants did not show any obvious phenotypes. However, the sextuple rip1-6 mutant displayed dwarf height, wrinkled seeds and wider leaves that were similar to the previously reported rmd mutant, and defective microfilaments and increased flag leaf angles that were not reported in rmd mutant. Collectively, our study provides insights into the mechanisms determining formin protein stability in plants.

The pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which are thought to underlie tumor growth, treatment resistance, and recurrence. To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown disrupted the SOX2 gene network and inhibited both self-renewal capacity as well as in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance.

Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics.

Cellular cross-talk between ubiquitination and other posttranslational modifications contributes to the regulation of numerous processes. One example is ADP-ribosylation of the carboxyl terminus of ubiquitin by the E3 DTX3L/ADP-ribosyltransferase PARP9 heterodimer, but the mechanism remains elusive. Here, we show that independently of PARP9, the conserved carboxyl-terminal RING and DTC (Deltex carboxyl-terminal) domains of DTX3L and other human Deltex proteins (DTX1 to DTX4) catalyze ADP-ribosylation of ubiquitin's Gly76 Structural studies reveal a hitherto unknown function of the DTC domain in binding NAD+ Deltex RING domain recruits E2 thioesterified with ubiquitin and juxtaposes it with NAD+ bound to the DTC domain to facilitate ADP-ribosylation of ubiquitin. This ubiquitin modification prevents its activation but is reversed by the linkage nonspecific deubiquitinases. Our study provides mechanistic insights into ADP-ribosylation of ubiquitin by Deltex E3s and will enable future studies directed at understanding the increasingly complex network of ubiquitin cross-talk.

RING-between-RING (RBR) E3 ubiquitin ligases are implicated in various developmental processes, and mutations in genes encoding RBR proteins HHARI/ARIH1 and Parkin are associated with human diseases. Here we show by phylogenetic analysis that the ARI1 family has undergone a dramatic expansion within the Caenorhabditis clade in recent history, a characteristic shared by some genes involved in germline development. We then examined the effects of deleting all ARI1 family members in the nematode Caenorhabditis elegans, which to our knowledge represents the first complete knockout of ARI1 function in a metazoan. Hermaphrodites that lacked or had strongly reduced ARI1 activity had low fecundity and were partially defective in initiation of oocyte differentiation. We provide evidence that the C. elegans ARI1s likely function downstream or in parallel to FBF-1 and FBF-2, two closely related RNA-binding proteins that are required for the switch from spermatogenesis to oogenesis during late larval development. Previous studies have shown that the E2 enzymes UBC-18/UBCH7 and UBC-3/CDC34 can functionally collaborate with ARI1 family members. Our data indicated that UBC-18, but not UBC-3, specifically cooperates with the ARI1s in germline development. These findings provide new insights into the functions of RING-between-RING proteins and Ariadne E3s during development.

NleG homologues constitute the largest family of Type 3 effectors delivered by pathogenic E. coli, with fourteen members in the enterohaemorrhagic (EHEC) O157:H7 strain alone. Identified recently as part of the non-LEE-encoded (Nle) effector set, this family remained uncharacterised and shared no sequence homology to other proteins including those of known function. The C-terminal domain of NleG2-3 (residues 90 to 191) is the most conserved region in NleG proteins and was solved by NMR. Structural analysis of this structure revealed the presence of a RING finger/U-box motif. Functional assays demonstrated that NleG2-3 as well as NleG5-1, NleG6-2 and NleG9' family members exhibited a strong autoubiquitination activity in vitro; a characteristic usually expressed by eukaryotic ubiquitin E3 ligases. When screened for activity against a panel of 30 human E2 enzymes, the NleG2-3 and NleG5-1 homologues showed an identical profile with only UBE2E2, UBE2E3 and UBE2D2 enzymes supporting NleG activity. Fluorescence polarization analysis yielded a binding affinity constant of 56+/-2 microM for the UBE2D2/NleG5-1 interaction, a value comparable with previous studies on E2/E3 affinities. The UBE2D2 interaction interface on NleG2-3 defined by NMR chemical shift perturbation and mutagenesis was shown to be generally similar to that characterised for human RING finger ubiquitin ligases. The alanine substitutions of UBE2D2 residues Arg5 and Lys63, critical for activation of eukaryotic E3 ligases, also significantly decreased both NleG binding and autoubiquitination activity. These results demonstrate that bacteria-encoded NleG effectors are E3 ubiquitin ligases analogous to RING finger and U-box enzymes in eukaryotes.

Seven in absentia homologs (SIAHs) comprise a family of highly conserved E3 ubiquitin ligases that play an important role in regulating signalling pathways in tumorigenesis, including the DNA damage repair and hypoxia response pathways. SIAH1 and SIAH2 have been found to function as a tumour repressor and a proto-oncogene, respectively, despite the high sequence identity of their substrate binding domains (SBDs). Ubiquitin-specific protease USP19 is a deubiquitinase that forms a complex with SIAHs and counteracts the ligase function. Much effort has been made to find selective inhibitors of the SIAHs E3 ligases. Menadione was reported to inhibit SIAH2 specifically.

Montbretins are rare specialized metabolites found in montbretia (Crocosmia x crocosmiiflora) corms. Montbretin A (MbA) is of particular interest as a novel therapeutic for type-2 diabetes and obesity. There is no scalable production system for this complex acylated flavonol glycoside. MbA biosynthesis has been reconstructed in Nicotiana benthamiana using montbretia genes for the assembly of MbA from its various different building blocks. However, in addition to smaller amounts of MbA, the therapeutically inactive montbretin B (MbB) was the major product of this metabolic engineering effort. MbA and MbB differ in a single hydroxyl group of their acyl side chains, which are derived from caffeoyl-CoA and coumaroyl-CoA, respectively. Biosynthesis of both MbA and MbB also require coumaroyl-CoA for the formation of the myricetin core. Caffeoyl-CoA and coumaroyl-CoA are formed in the central phenylpropanoid pathway by acyl activating enzymes (AAEs) known as 4-coumaroyl-CoA ligases (4CLs). Here we investigated a small family of montbretia AAEs and 4CLs, and their possible contribution to montbretin biosynthesis.