Ubiquitin Ligases: Structure, Function, and Regulation.

Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation. At the end of a three-enzyme cascade, ubiquitin ligases mediate the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to specific substrate proteins. Early investigations of E3s of the RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) types shed light on their enzymatic activities, general architectures, and substrate degron-binding modes. Recent studies have provided deeper mechanistic insights into their catalysis, activation, and regulation. In this review, we summarize the current progress in structure-function studies of ubiquitin ligases as well as exciting new discoveries of novel classes of E3s and diverse substrate recognition mechanisms. Our increased understanding of ubiquitin ligase function and regulation has provided the rationale for developing E3-targeting therapeutics for the treatment of human diseases.

Timer-based proteomic profiling of the ubiquitin-proteasome system reveals a substrate receptor of the GID ubiquitin ligase.

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.

UbiBrowser 2.0: a comprehensive resource for proteome-wide known and predicted ubiquitin ligase/deubiquitinase-substrate interactions in eukaryotic species.

As an important post-translational modification, ubiquitination mediates ∼80% of protein degradation in eukaryotes. The degree of protein ubiquitination is tightly determined by the delicate balance between specific ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase-mediated deubiquitination. In 2017, we developed UbiBrowser 1.0, which is an integrated database for predicted human proteome-wide E3-substrate interactions. Here, to meet the urgent requirement of proteome-wide E3/deubiquitinase-substrate interactions (ESIs/DSIs) in multiple organisms, we updated UbiBrowser to version 2.0 (http://ubibrowser.ncpsb.org.cn). Using an improved protocol, we collected 4068/967 known ESIs/DSIs by manual curation, and we predicted about 2.2 million highly confident ESIs/DSIs in 39 organisms, with >210-fold increase in total data volume. In addition, we made several new features in the updated version: (i) it allows exploring proteins' upstream E3 ligases and deubiquitinases simultaneously; (ii) it has significantly increased species coverage; (iii) it presents a uniform confidence scoring system to rank predicted ESIs/DSIs. To facilitate the usage of UbiBrowser 2.0, we also redesigned the web interface for exploring these known and predicted ESIs/DSIs, and added functions of 'Browse', 'Download' and 'Application Programming Interface'. We believe that UbiBrowser 2.0, as a discovery tool, will contribute to the study of protein ubiquitination and the development of drug targets for complex diseases.

Substrate recognition by TRIM and TRIM-like proteins in innate immunity.

TRIM (Tripartite motif) and TRIM-like proteins have emerged as an important class of E3 ligases in innate immunity. Their functions range from activation or regulation of innate immune signaling pathway to direct detection and restriction of pathogens. Despite the importance, molecular mechanisms for many TRIM/TRIM-like proteins remain poorly characterized, in part due to challenges of identifying their substrates. In this review, we discuss several TRIM/TRIM-like proteins in RNA sensing pathways and viral restriction functions. We focus on those containing PRY-SPRY, the domain most frequently used for substrate recognition, and discuss emerging mechanisms that are commonly utilized by several TRIM/TRIM-like proteins to tightly control their interaction with the substrates.

The role of E3 ubiquitin ligases in synapse function in the healthy and diseased brain.

Ubiquitination is a key posttranslational modification for the controlled protein degradation and proteostasis. The substrate specificity is determined by a family of E3 ubiquitin ligases, which are encoded by more than 600 genes in the mammalian genome. Gain- or loss-of-function of a number of E3 genes results in neurodegeneration or neurodevelopmental disorders, affecting synapse function. This implies that the specific ubiquitination of synaptic substrates are of crucial importance for the normal neuronal network. In this review, we will summarize the history, current topics, and challenges in the field of ubiquitination-dependent regulations of synaptogenesis and synaptic transmission.

iUUCD 2.0: an update with rich annotations for ubiquitin and ubiquitin-like conjugations.

Here, we described the updated database iUUCD 2.0 (http://iuucd.biocuckoo.org/) for ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), ubiquitin-protein ligases (E3s), deubiquitinating enzymes (DUBs), ubiquitin/ubiquitin-like binding domains (UBDs) and ubiquitin-like domains (ULDs), which act as key regulators in modulating ubiquitin and ubiquitin-like (UB/UBL) conjugations. In total, iUUCD 2.0 contained 136 512 UB/UBL regulators, including 1230 E1s, 5636 E2s, 93 343 E3s, 9548 DUBs, 30 173 UBDs and 11 099 ULDs in 148 eukaryotic species. In particular, we provided rich annotations for regulators of eight model organisms, especially in humans, by compiling and integrating the knowledge from nearly 70 widely used public databases that cover cancer mutations, single nucleotide polymorphisms (SNPs), mRNA expression, DNA and RNA elements, protein-protein interactions, protein 3D structures, disease-associated information, drug-target relations, post-translational modifications, DNA methylation and protein expression/proteomics. Compared with our previously developed UUCD 1.0 (∼0.41 GB), iUUCD 2.0 has a size of ∼32.1 GB of data with a >75-fold increase in data volume. We anticipate that iUUCD 2.0 can be a more useful resource for further study of UB/UBL conjugations.

Classification of barley U-box E3 ligases and their expression patterns in response to drought and pathogen stresses.

BACKGROUND: Controlled turnover of proteins as mediated by the ubiquitin proteasome system (UPS) is an important element in plant defense against environmental and pathogen stresses. E3 ligases play a central role in subjecting proteins to hydrolysis by the UPS. Recently, it has been demonstrated that a specific class of E3 ligases termed the U-box ligases are directly associated with the defense mechanisms against abiotic and biotic stresses in several plants. However, no studies on U-box E3 ligases have been performed in one of the important staple crops, barley. RESULTS: In this study, we identified 67 putative U-box E3 ligases from the barley genome and expressed sequence tags (ESTs). Similar to Arabidopsis and rice U-box E3 ligases, most of barley U-box E3 ligases possess evolutionary well-conserved domain organizations. Based on the domain compositions and arrangements, the barley U-box proteins were classified into eight different classes. Along with this new classification, we refined the previously reported classifications of U-box E3 ligase genes in Arabidopsis and rice. Furthermore, we investigated the expression profile of 67 U-box E3 ligase genes in response to drought stress and pathogen infection. We observed that many U-box E3 ligase genes were specifically up-and-down regulated by drought stress or by fungal infection, implying their possible roles of some U-box E3 ligase genes in the stress responses. CONCLUSION: This study reports the classification of U-box E3 ligases in barley and their expression profiles against drought stress and pathogen infection. Therefore, the classification and expression profiling of barley U-box genes can be used as a platform to functionally define the stress-related E3 ligases in barley.

Emerging RNA-binding roles in the TRIM family of ubiquitin ligases.

TRIM proteins constitute a large, diverse and ancient protein family which play a key role in processes including cellular differentiation, autophagy, apoptosis, DNA repair, and tumour suppression. Mostly known and studied through the lens of their ubiquitination activity as E3 ligases, it has recently emerged that many of these proteins are involved in direct RNA binding through their NHL or PRY/SPRY domains. We summarise the current knowledge concerning the mechanism of RNA binding by TRIM proteins and its biological role. We discuss how RNA-binding relates to their previously described functions such as E3 ubiquitin ligase activity, and we will consider the potential role of enrichment in membrane-less organelles.

Regulation of the p53 Family Proteins by the Ubiquitin Proteasomal Pathway.

The tumor suppressor p53 and its homologues, p63 and p73, play a pivotal role in the regulation of the DNA damage response, cellular homeostasis, development, aging, and metabolism. A number of mouse studies have shown that a genetic defect in the p53 family could lead to spontaneous tumor development, embryonic lethality, or severe tissue abnormality, indicating that the activity of the p53 family must be tightly regulated to maintain normal cellular functions. While the p53 family members are regulated at the level of gene expression as well as post-translational modification, they are also controlled at the level of protein stability through the ubiquitin proteasomal pathway. Over the last 20 years, many ubiquitin E3 ligases have been discovered that directly promote protein degradation of p53, p63, and p73 in vitro and in vivo. Here, we provide an overview of such E3 ligases and discuss their roles and functions.

Structural mechanisms of HECT-type ubiquitin ligases.

Ubiquitin ligases (E3 enzymes) transfer ubiquitin from ubiquitin-conjugating (E2) enzymes to target proteins. By determining the selection of target proteins, modification sites on those target proteins, and the types of ubiquitin modifications that are formed, E3 enzymes are key specificity factors in ubiquitin signaling. Here, I summarize our knowledge of the structural mechanisms in the HECT E3 subfamily, many members of which play important roles in human disease. I discuss interactions of the conserved HECT domain with E2 enzymes, ubiquitin and target proteins, as well as macromolecular interactions with regulatory functions. While we understand individual steps in the catalytic cycle of HECT E3 enzymes on a structural level, this review also highlights key aspects that have yet to be elucidated. For instance, it remains unclear how diverse target proteins are presented to the catalytic center and how certain HECT E3 enzymes achieve specificity in ubiquitin linkage formation. The structural and functional properties of the N-terminal regions of HECT E3 enzymes that likely act as signaling hubs are also largely unknown. Structural insights into these aspects may open up routes for a therapeutic intervention with specific HECT E3 functions in distinct pathophysiological settings.

TRIM-NHL proteins in development and disease.

TRIM-NHL proteins are key regulators of developmental transitions, for example promoting differentiation, while inhibiting cell growth and proliferation, in stem and progenitor cells. Abnormalities in these proteins have been also associated with human diseases, particularly affecting muscular and neuronal functions, making them potential targets for therapeutic intervention. The purpose of this review is to provide a systematic and comprehensive summary on the most studied TRIM-NHL proteins, highlighting examples where connections were established between structural features, molecular functions and biological outcomes.

Phylogenetic analysis of the SINA/SIAH ubiquitin E3 ligase family in Metazoa.

BACKGROUND: The RAS signaling pathway is a pivotal developmental pathway that controls many fundamental biological processes including cell proliferation, differentiation, movement and apoptosis. Drosophila Seven-IN-Absentia (SINA) is a ubiquitin E3 ligase that is the most downstream signaling "gatekeeper" whose biological activity is essential for proper RAS signal transduction. Vertebrate SINA homologs (SIAHs) share a high degree of amino acid identity with that of Drosophila SINA. SINA/SIAH is the most conserved signaling component in the canonical EGFR/RAS/RAF/MAPK signal transduction pathway. RESULTS: Vertebrate SIAH1, 2, and 3 are the three orthologs to invertebrate SINA protein. SINA and SIAH1 orthologs are found in all major taxa of metazoans. These proteins have four conserved functional domains, known as RING (Really Interesting New Gene), SZF (SIAH-type zinc finger), SBS (substrate binding site) and DIMER (Dimerization). In addition to the siah1 gene, most vertebrates encode two additional siah genes (siah2 and siah3) in their genomes. Vertebrate SIAH2 has a highly divergent and extended N-terminal sequence, while its RING, SZF, SBS and DIMER domains maintain high amino acid identity/similarity to that of SIAH1. But unlike vertebrate SIAH1 and SIAH2, SIAH3 lacks a functional RING domain, suggesting that SIAH3 may be an inactive E3 ligase. The SIAH3 subtree exhibits a high degree of amino acid divergence when compared to the SIAH1 and SIAH2 subtrees. We find that SIAH1 and SIAH2 are expressed in all human epithelial cell lines examined thus far, while SIAH3 is only expressed in a limited subset of cancer cell lines. CONCLUSION: Through phylogenetic analyses of metazoan SINA and SIAH E3 ligases, we identified many invariant and divergent amino acid residues, as well as the evolutionarily conserved functional motifs in this medically relevant gene family. Our phylomedicinal study of this unique metazoan SINA/SIAH protein family has provided invaluable evolution-based support towards future effort to design logical, potent, and durable anti-SIAH-based anticancer strategies against oncogenic K-RAS-driven metastatic human cancers. Thus, this method of evolutionary study should be of interest in cancer biology.

UUCD: a family-based database of ubiquitin and ubiquitin-like conjugation.

In this work, we developed a family-based database of UUCD (http://uucd.biocuckoo.org) for ubiquitin and ubiquitin-like conjugation, which is one of the most important post-translational modifications responsible for regulating a variety of cellular processes, through a similar E1 (ubiquitin-activating enzyme)-E2 (ubiquitin-conjugating enzyme)-E3 (ubiquitin-protein ligase) enzyme thioester cascade. Although extensive experimental efforts have been taken, an integrative data resource is still not available. From the scientific literature, 26 E1s, 105 E2s, 1003 E3s and 148 deubiquitination enzymes (DUBs) were collected and classified into 1, 3, 19 and 7 families, respectively. To computationally characterize potential enzymes in eukaryotes, we constructed 1, 1, 15 and 6 hidden Markov model (HMM) profiles for E1s, E2s, E3s and DUBs at the family level, separately. Moreover, the ortholog searches were conducted for E3 and DUB families without HMM profiles. Then the UUCD database was developed with 738 E1s, 2937 E2s, 46 631 E3s and 6647 DUBs of 70 eukaryotic species. The detailed annotations and classifications were also provided. The online service of UUCD was implemented in PHP + MySQL + JavaScript + Perl.

The fate of tandemly duplicated genes assessed by the expression analysis of a group of Arabidopsis thaliana RING-H2 ubiquitin ligase genes of the ATL family.

Gene duplication events exert key functions on gene innovations during the evolution of the eukaryotic genomes. A large portion of the total gene content in plants arose from tandem duplications events, which often result in paralog genes with high sequence identity. Ubiquitin ligases or E3 enzymes are components of the ubiquitin proteasome system that function during the transfer of the ubiquitin molecule to the substrate. In plants, several E3s have expanded in their genomes as multigene families. To gain insight into the consequences of gene duplications on the expansion and diversification of E3s, we examined the evolutionary basis of a cluster of six genes, duplC-ATLs, which arose from segmental and tandem duplication events in Brassicaceae. The assessment of the expression suggested two patterns that are supported by lineage. While retention of expression domains was observed, an apparent absence or reduction of expression was also inferred. We found that two duplC-ATL genes underwent pseudogenization and that, in one case, gene expression is probably regained. Our findings provide insights into the evolution of gene families in plants, defining key events on the expansion of the Arabidopsis Tóxicos en Levadura family of E3 ligases.

A human ubiquitin conjugating enzyme (E2)-HECT E3 ligase structure-function screen.

Here we describe a systematic structure-function analysis of the human ubiquitin (Ub) E2 conjugating proteins, consisting of the determination of 15 new high-resolution three-dimensional structures of E2 catalytic domains, and autoubiquitylation assays for 26 Ub-loading E2s screened against a panel of nine different HECT (homologous to E6-AP carboxyl terminus) E3 ligase domains. Integration of our structural and biochemical data revealed several E2 surface properties associated with Ub chain building activity; (1) net positive or neutral E2 charge, (2) an "acidic trough" located near the catalytic Cys, surrounded by an extensive basic region, and (3) similarity to the previously described HECT binding signature in UBE2L3 (UbcH7). Mass spectrometry was used to characterize the autoubiquitylation products of a number of functional E2-HECT pairs, and demonstrated that HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. Our data set represents the first comprehensive analysis of E2-HECT E3 interactions, and thus provides a framework for better understanding the molecular mechanisms of ubiquitylation.

A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity.

Despite the sequencing of the human and mouse genomes, few genetic mechanisms for protecting against autoimmune disease are currently known. Here we systematically screen the mouse genome for autoimmune regulators to isolate a mouse strain, sanroque, with severe autoimmune disease resulting from a single recessive defect in a previously unknown mechanism for repressing antibody responses to self. The sanroque mutation acts within mature T cells to cause formation of excessive numbers of follicular helper T cells and germinal centres. The mutation disrupts a repressor of ICOS, an essential co-stimulatory receptor for follicular T cells, and results in excessive production of the cytokine interleukin-21. sanroque mice fail to repress diabetes-causing T cells, and develop high titres of autoantibodies and a pattern of pathology consistent with lupus. The causative mutation is in a gene of previously unknown function, roquin (Rc3h1), which encodes a highly conserved member of the RING-type ubiquitin ligase protein family. The Roquin protein is distinguished by the presence of a CCCH zinc-finger found in RNA-binding proteins, and localization to cytosolic RNA granules implicated in regulating messenger RNA translation and stability.

Functional analysis of tomato CHIP ubiquitin E3 ligase in heat tolerance.

Plants have evolved genetic and physiological mechanisms to mitigate the adverse effects of high temperature. CARBOXYL TERMINUS OF THE HSC70-INTERACTING PROTEINS (CHIP) is a conserved chaperone-dependent ubiquitin E3 ligase that targets misfolded proteins. Here, we report functional analysis of the SlCHIP gene from tomato (Solanum lycopersicum) in heat tolerance. SlCHIP encodes a CHIP protein with three tandem tetracopeptide repeat (TPR) motifs and a C-terminal U box domain. Phylogenetic analysis of CHIP homologs from animals, spore-bearing and seed plants revealed a tree topology similar to the evolutionary tree of the organisms. Expression of SlCHIP was induced under high temperature and was also responsive to plant stress hormones. Silencing of SlCHIP in tomato reduced heat tolerance based on increased heat stress symptoms, reduced photosynthetic activity, elevated electrolyte leakage and accumulation of insoluble protein aggregates. The accumulated protein aggregates in SlCHIP-silenced plants were still highly ubiquitinated, suggesting involvement of other E3 ligases in ubiquitination. SlCHIP restored the heat tolerance of Arabidopsis chip mutant to the wild type levels. These results indicate that tomato SlCHIP plays a critical role in heat stress responses most likely by targeting degradation of misfolded proteins that are generated during heat stress.

Elucidating the Antiviral Mechanism of Different MARCH Factors.

The membrane-associated RING-CH (MARCH) proteins belong to a family of E3 ubiquitin ligases, whose main function is to remove transmembrane proteins from the plasma membrane. Recent work has shown that the human MARCH1, 2, and 8 are antiretroviral factors that target the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins by reducing their incorporation in the budding virions. Nevertheless, the dearth of information regarding the antiviral mechanism of this family of proteins necessitates further examination. In this study, using both the human MARCH proteins and their mouse homologues, we provide a comprehensive analysis of the antiretroviral mechanism of this family of proteins. Moreover, we show that human MARCH proteins restrict to various degrees the envelope glycoproteins of a diverse number of viruses. This report sheds light on the important antiviral function of MARCH proteins and their significance in cell intrinsic immunity.IMPORTANCE This study examines the mechanism utilized by different MARCH proteins to restrict retrovirus infection. MARCH proteins block the incorporation of envelope glycoproteins to the budding virions. In this report, by comparing the human and mouse MARCH genes and using murine leukemia virus (MLV) and HIV-1, we identify differences in the mechanism of restriction among MARCH proteins. Furthermore, we perform a comprehensive analysis on a number of envelope glycoproteins and show that MARCH proteins have broad antiviral functions.

Ancient origin of animal U-box ubiquitin ligases.

BACKGROUND: The patterns of emergence and diversification of the families of ubiquitin ligases provide insights about the evolution of the eukaryotic ubiquitination system. U-box ubiquitin ligases (UULs) are proteins characterized by containing a peculiar protein domain known as U box. In this study, the origin of the animal UUL genes is described. RESULTS: Phylogenetic and structural data indicate that six of the seven main UUL-encoding genes found in humans (UBE4A, UBE4B, UIP5, PRP19, CHIP and CYC4) were already present in the ancestor of all current metazoans and the seventh (WDSUB1) is found in placozoans, cnidarians and bilaterians. The fact that only 4-5 genes orthologous to the human ones are present in the choanoflagellate Monosiga brevicollis suggests that several animal-specific cooptions of the U box to generate new genes occurred. Significantly, Monosiga contains five additional UUL genes that are not present in animals. One of them is also present in distantly-related protozoans. Along animal evolution, losses of UUL-encoding genes are rare, except in nematodes, which lack three of them. These general patterns are highly congruent with those found for other two families (RBR, HECT) of ubiquitin ligases. CONCLUSIONS: Finding that the patterns of emergence, diversification and loss of three unrelated families of ubiquitin ligases (RBR, HECT and U-box) are parallel indicates that there are underlying, linage-specific evolutionary forces shaping the complexity of the animal ubiquitin system.