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Bdelloid rotifers are part of the restricted circle of multicellular animals that can withstand a wide range of genotoxic stresses at any stage of their life cycle. In this study, bdelloid rotifer Adineta vaga is used as a model to decipher the molecular basis of their extreme tolerance. Proteomic analysis shows that a specific DNA ligase, different from those usually involved in DNA repair in eukaryotes, is strongly over-represented upon ionizing radiation. A phylogenetic analysis reveals its orthology to prokaryotic DNA ligase E, and its horizontal acquisition by bdelloid rotifers and plausibly other eukaryotes. The fungus Mortierella verticillata, having a single copy of this DNA Ligase E homolog, also exhibits an increased radiation tolerance with an over-expression of this DNA ligase E following X-ray exposure. We also provide evidence that A. vaga ligase E is a major contributor of DNA breaks ligation activity, which is a common step of all important DNA repair pathways. Consistently, its heterologous expression in human cell lines significantly improves their radio-tolerance. Overall, this study highlights the potential of horizontal gene transfers in eukaryotes, and their contribution to the adaptation to extreme conditions.

Regulated degradation of proteins by the 26S proteasome plays important roles in maintenance and signalling in eukaryotic cells. Proteins are marked for degradation by the action of E3 ligases that site-specifically modify their substrates by adding chains of ubiquitin. Innate immune signalling in plants is deeply reliant on the ubiquitin-26S proteasome system. While progress has been made in understanding substrate ubiquitination during plant immunity, how these substrates are processed upon arrival at the proteasome remains unclear. Here we show that specific members of the HECT domain-containing family of ubiquitin protein ligases (UPL) play important roles in proteasomal substrate processing during plant immunity. Mutations in UPL1, UPL3 and UPL5 significantly diminished immune responses activated by the immune hormone salicylic acid (SA). In depth analyses of upl3 mutants indicated that these plants were impaired in reprogramming of nearly the entire SA-induced transcriptome and failed to establish immunity against a hemi-biotrophic pathogen. UPL3 was found to physically interact with the regulatory particle of the proteasome and with other ubiquitin-26S proteasome pathway components. In agreement, we demonstrate that UPL3 enabled proteasomes to form polyubiquitin chains, thereby regulating total cellular polyubiquitination levels. Taken together, our findings suggest that proteasome-associated ubiquitin ligase activity of UPL3 promotes proteasomal processivity and is indispensable for development of plant immunity.

In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4 CRBN ) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4 CRBN . Here we present crystal structures of the DDB1–CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4 CRBN and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4 CRBN . Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4 CRBN while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins. The crystal structures of thalidomide and its derivatives bound to the E3 ligase subcomplex DDB1–CRBN are shown; these drugs are found to have dual functions, interfering with the binding of certain cellular substrates to the E3 ligase but promoting the binding of others, thereby modulating the degradation of cellular proteins. Thalidomide's dual mechanism of action Introduced in Europe in 1957 as a mild sedative, thalidomide was widely used in pregnant women as a treatment for morning sickness. This led to the birth of thousands of children with multiple defects and the drug was withdrawn in 1962. Since then thalidomide and its derivatives have emerged as effective treatments for the cancer multiple myeloma and the associated disorder 5q-dysplasia. The primary teratogenic target of thalidomide is cereblon (CRBN), part of E3 ubiquitin ligase complex CUL4–RBX1–DDB1–CRBN (CRL4 CRBN ). Here, Nicolas Thomä and colleagues present the crystal structure of DDB1–CRBN E3 ubiquitin ligase bound to thalidomide and to the related drugs lenalidomide and pomalidomide. The structure establishes the molecular mechanism underlying CRBN's enantioselective action. Further structure–function analysis reveals that these drugs have dual functions, interfering with the binding of certain cellular substrates to the E3 ligase but promoting the binding of others, thereby modulating the degradation of cellular proteins.

Although serum from patients with Parkinson's disease contains elevated levels of numerous pro-inflammatory cytokines including IL-6, TNF, IL-1β, and IFNγ, whether inflammation contributes to or is a consequence of neuronal loss remains unknown . Mutations in parkin, an E3 ubiquitin ligase, and PINK1, a ubiquitin kinase, cause early onset Parkinson's disease . Both PINK1 and parkin function within the same biochemical pathway and remove damaged mitochondria from cells in culture and in animal models via mitophagy, a selective form of autophagy . The in vivo role of mitophagy, however, is unclear, partly because mice that lack either PINK1 or parkin have no substantial Parkinson's-disease-relevant phenotypes . Mitochondrial stress can lead to the release of damage-associated molecular patterns (DAMPs) that can activate innate immunity , suggesting that mitophagy may mitigate inflammation. Here we report a strong inflammatory phenotype in both Prkn and Pink1 mice following exhaustive exercise and in Prkn ;mutator mice, which accumulate mutations in mitochondrial DNA (mtDNA) . Inflammation resulting from either exhaustive exercise or mtDNA mutation is completely rescued by concurrent loss of STING, a central regulator of the type I interferon response to cytosolic DNA . The loss of dopaminergic neurons from the substantia nigra pars compacta and the motor defect observed in aged Prkn ;mutator mice are also rescued by loss of STING, suggesting that inflammation facilitates this phenotype. Humans with mono- and biallelic PRKN mutations also display elevated cytokines. These results support a role for PINK1- and parkin-mediated mitophagy in restraining innate immunity.

Half a century ago, thalidomide was widely prescribed to pregnant women as a sedative but was found to be teratogenic, causing multiple birth defects. Today, thalidomide is still used in the treatment of leprosy and multiple myeloma, although how it causes limb malformation and other developmental defects is unknown. Here, we identified cereblon (CRBN) as a thalidomide-binding protein. CRBN forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1) and Cul4A that is important for limb outgrowth and expression of the fibroblast growth factor Fgf8 in zebrafish and chicks. Thalidomide initiates its teratogenic effects by binding to CRBN and inhibiting the associated ubiquitin ligase activity. This study reveals a basis for thalidomide teratogenicity and may contribute to the development of new thalidomide derivatives without teratogenic activity.

Skeletal muscle atrophy is caused by various conditions, including aging, disuse related to a sedentary lifestyle and lack of physical activity, and cachexia. Our insufficient understanding of the molecular mechanism underlying muscle atrophy limits the targets for the development of effective pharmacologic treatments and preventions. Here, we identified Krüppel-like factor 5 (KLF5), a zinc-finger transcription factor, as a key mediator of the early muscle atrophy program. KLF5 was up-regulated in atrophying myotubes as an early response to dexamethasone or simulated microgravity in vitro. Skeletal muscle–selective deletion of Klf5 significantly attenuated muscle atrophy induced by mechanical unloading in mice. Transcriptome- and genome-wide chromatin accessibility analyses revealed that KLF5 regulates atrophy-related programs, including metabolic changes and E3-ubiquitin ligase-mediated proteolysis, in coordination with Foxo1. The synthetic retinoic acid receptor agonist Am80, a KLF5 inhibitor, suppressed both dexamethasone- and microgravity-induced muscle atrophy in vitro and oral Am80 ameliorated disuse– and dexamethasone-induced atrophy in mice. Moreover, in three independent sets of transcriptomic data from human skeletal muscle, KLF5 expression significantly increased with age and the presence of sarcopenia and correlated positively with the expression of the atrophy-related ubiquitin ligase genes FBXO32 and TRIM63. These findings demonstrate that KLF5 is a key transcriptional regulator mediating muscle atrophy and that pharmacological intervention with Am80 is a potentially preventive treatment.

Ubiquitination is one of the most prevalent protein posttranslational modifications. Here, we show that E3 ligase Nedd4l positively regulates antiviral immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3. Deficiency of Nedd4l significantly impairs type I interferon and proinflammatory cytokine production induced by virus infection both in vitro and in vivo. Nedd4l deficiency inhibits virus-induced ubiquitination of TRAF3, the binding between TRAF3 and TBK1, and subsequent phosphorylation of TBK1 and IRF3. Nedd4l directly interacts with TRAF3 and catalyzes K29-linked ubiquitination of Cys56 and Cys124, two cysteines that constitute zinc fingers, resulting in enhanced association between TRAF3 and E3 ligases, cIAP1/2 and HECTD3, and also increased K48/K63-linked ubiquitination of TRAF3. Mutation of Cys56 and Cys124 diminishes Nedd4l-catalyzed K29-linked ubiquitination, but enhances association between TRAF3 and the E3 ligases, supporting Nedd4l promotes type I interferon production in response to virus by catalyzing ubiquitination of the cysteines in TRAF3. Ubiquitination is a prevalent post translational modification. Here, the authors show a pivotal role for Nedd4l in the regulation of antiviral immunity via promotion of ubiquitination of TRAF3 and go on to show disruption of Nedd4l both in vitro and in vivo perturbs the antiviral immune response.

The post-translational modification of proteins regulates many biological processes. Their dysfunction relates to diseases. Ubiquitination is one of the post-translational modifications that target lysine residue and regulate many cellular processes. Three enzymes are required for achieving the ubiquitination reaction: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). E3s play a pivotal role in selecting substrates. Many structural studies have been conducted to reveal the molecular mechanism of the ubiquitination reaction. Recently, the structure of PCAF_N, a newly categorized E3 ligase, was reported. We present a review of the recent progress toward the structural understanding of E3 ligases.

Eukaryotic cell biology depends on cullin–RING E3 ligase (CRL)-catalysed protein ubiquitylation 1 , which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD8 2 – 6 . However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1 β-TRCP promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin–RING proteins. A cryo-electron microscopy structure provides insights into the activation of cullin–RING E3 ligases by NEDD8 and the consequent catalysis of ubiquitylation reactions.

E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates 1 , 2 . However, rather than functioning individually, many neddylated cullin–RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family—which together account for nearly half of all ubiquitin ligases in humans—form E3–E3 super-assemblies 3 – 7 . Here, by studying CRLs in the SKP1–CUL1–F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3–E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3–E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3–E3 super-assembly may therefore underlie widespread ubiquitylation. Cryo-electron microscopy of neddylated SCF-family ligases interacting with the RBR-type E3 ligase ARIH1 reveals the steps through which E3–E3 super-assemblies ubiquitylate a diverse set of substrates presented on F-box proteins.