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Post-translational modification of proteins by ubiquitin is required for nearly all aspects of eukaryotic cell function. The numerous targets of ubiquitylation, and variety of ubiquitin modifications, are often likened to a code, where the ultimate messages are diverse responses to target ubiquitylation. E1, E2, and E3 multiprotein enzymatic assemblies modify specific targets and thus function as messengers. Recent advances in chemical and protein tools have revolutionized our ability to explore the ubiquitin system, through enabling new high-throughput screening methods, matching ubiquitylation enzymes with their cellular targets, revealing intricate allosteric mechanisms regulating ubiquitylating enzymes, facilitating structural revelation of transient assemblies determined by multivalent interactions, and providing new paradigms for inhibiting and redirecting ubiquitylation in vivo as new therapeutics. Here we discuss the development of methods that control, disrupt, and extract the flow of information across the ubiquitin system and have enabled elucidation of the underlying molecular and cellular biology., Competing Interests: Declaration of interests B.A.S. is on the Scientific Advisory Board of Interline Therapeutics, and is Adjunct Faculty at St. Jude Children's Research Hospital, and Honorary Professor at Technical University of Munich., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Ubiquitin-activating enzyme (E1) is the first enzyme of the ubiquitination pathway and is required to activate ubiquitin. E1 of Saccharomyces cerevisiae is a large multidomain monomeric protein. There are no studies available on the domains of yeast E1 as independent entities. Four domains of E1 namely, first catalytic cysteine half-domain (FCCH), four-helix bundle (4HB), second catalytic cysteine half-domain (SCCH) and ubiquitin fold domain (UFD) were characterised to understand their structural and functional independence vis-a-vis full length E1. Spectroscopic characterisation using circular dichroism and fluorescence suggested that these domains can act as independent folding units and attain native-like secondary structure. The structural features obtained with the peptides SCCH and FCCH of S. cerevisiae bear a high degree of structural similarity to the corresponding fragments of mouse from literature. Nearly 50% of the residues of the 4HB domain of the S. cerevisiae sample showed helical conformation. They displayed a high degree of conservation when compared with 4HB of mouse with respect to their identity and arrangement. The fragment UFD of yeast formed an α/β domain as in the whole protein and exhibited 45% homology with that of mouse, showing a similar arrangement of α and β elements in its secondary structure. Overexpression of the domains in vivo indicated that the SCCH domain and to some extent UFD apparently interfere with cellular functions such as survival under various stresses.

Ubiquitin (Ub) signaling plays a key regulatory role in nearly every aspect of eukaryotic biology and is initiated by E1 enzymes that activate and transfer Ub to E2 Ub-conjugating enzymes. Despite Ub E1's fundamental importance to the cell and its attractiveness as a target for therapeutic intervention in cancer and other diseases, its only available structural information is derived from yeast orthologs of human ubiquitin-like modifier-activating enzyme 1 (hUBA1). To illuminate structural differences between yeast and hUBA1 structures that might be exploited for the development of small-molecule therapeutics, we determined the first crystal structure of a hUBA1-Ub complex. Using structural analysis, molecular modeling, and biochemical analysis, we demonstrate that hUBA1 shares a conserved overall structure and mechanism with previously characterized yeast orthologs, but displays subtle structural differences, particularly within the active site. Computational analysis revealed four potential ligand-binding hot spots on the surface of hUBA1 that might serve as targets to inhibit hUBA1 at the level of Ub activation or E2 recruitment or that might potentially be used in approaches such as protein-targeting chimeric molecules. Taken together, our work enhances our understanding of the hUBA1 mechanism, provides an improved framework for the development of small-molecule inhibitors of UBA1, and serves as a stepping stone for structural studies that involve the enzymes of the human Ub system at the level of both E1 and E2., (© 2018 Lv et al.)

E1 enzymes for ubiquitin (Ub) and Ub-like modifiers (Ubls) harbor two catalytic activities that are required for Ub/Ubl activation: adenylation and thioester bond formation. Structural studies of the E1 for the Ubl s mall u biquitin-like mo difier (SUMO) revealed a single active site that is transformed by a conformational switch that toggles its competency for catalysis of these two distinct chemical reactions. Although the mechanisms of adenylation and thioester bond formation revealed by SUMO E1 structures are thought to be conserved in Ub E1, there is currently a lack of structural data supporting this hypothesis. Here, we present a structure of Schizosaccharomyces pombe Uba1 in which the second catalytic cysteine half-domain (SCCH domain) harboring the catalytic cysteine has undergone a 106° rotation that results in a completely different network of intramolecular interactions between the SCCH and adenylation domains and translocation of the catalytic cysteine 12 Å closer to the Ub C terminus compared with previous Uba1 structures. SCCH domain alternation is accompanied by conformational changes within the Uba1 adenylation domains that effectively disassemble the adenylation active site. Importantly, the structural and biochemical data suggest that domain alternation and remodeling of the adenylation active site are interconnected and are intrinsic structural features of Uba1 and that the overall structural basis for adenylation and thioester bond formation exhibited by SUMO E1 is indeed conserved in Ub E1. Finally, the mechanistic insights provided by the novel conformational snapshot of Uba1 presented in this study may guide efforts to develop small molecule inhibitors of this critically important enzyme that is an active target for anticancer therapeutics., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The reversible post-translational modification of proteins by ubiquitin and ubiquitin-like proteins regulates almost all cellular processes, by affecting protein degradation, localization, and complex formation. Deubiquitinases (DUBs) are proteases that remove ubiquitin modifications or cleave ubiquitin chains. Most DUBs are cysteine proteases, which makes them well suited for study by activity-based probes. These DUB probes report on deubiquitinase activity by reacting covalently with the active site in an enzyme-catalyzed manner. They have proven to be important tools to study DUB selectivity and proteolytic activity in different settings, to identify novel DUBs, and to characterize deubiquitinase inhibitors. Inspired by the efficacy of activity-based probes for DUBs, several groups have recently reported probes for the ubiquitin conjugation machinery (E1, E2, and E3 enzymes). Many of these enzymes, while not proteases, also posses active site cysteine residues and can be targeted by covalent probes. In this review, we will discuss how features of the probe (cysteine-reactive group, recognition element, and reporter tag) affect reactivity and suitability for certain experimental applications. We will also review the diverse applications of the current probes, and discuss the need for new probe types to study emerging aspects of ubiquitin biology., (© 2017 Federation of European Biochemical Societies.)

Ubiquitin (Ub) E1 initiates the Ub conjugation cascade by activating and transferring Ub to tens of different E2s. How Ub E1 cooperates with E2s that differ substantially in their predicted E1-interacting residues is unknown. Here, we report the structure of S. pombe Uba1 in complex with Ubc15, a Ub E2 with intrinsically low E1-E2 Ub thioester transfer activity. The structure reveals a distinct Ubc15 binding mode that substantially alters the network of interactions at the E1-E2 interface compared to the only other available Ub E1-E2 structure. Structure-function analysis reveals that the intrinsically low activity of Ubc15 largely results from the presence of an acidic residue at its N-terminal region. Notably, Ub E2 N termini are serine/threonine rich in many other Ub E2s, leading us to hypothesize that phosphorylation of these sites may serve as a novel negative regulatory mechanism of Ub E2 activity, which we demonstrate biochemically and in cell-based assays., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ubiquitin (Ub) is a multifunctional protein that mainly functions to tag proteins for selective degradation by the 26S proteasome. We cloned an Ub gene TaUb2 from wheat (Triticum aestivum L.) previously. To study the function of TaUB2 in chilling stress, sense and antisense Ub transgenic tobacco plants (Nicotiana tabacum L.), as well as wild type (WT) and vector control β-glucuronidase (T-GUS) plants, were used. Under stress, leaf wilting in sense plants was significantly less than in controls, but more severe in antisense plants. Meanwhile, the net photosynthetic rate (Pn) and the maximal photochemical efficiency of PSII (Fv/Fm) in sense plants were greater than controls, but lower in antisense plants during chilling stress and recovery. Less wilting in sense plants resulted from improved water status, which may be related to the accumulation of proline and solute sugar. Furthermore, as indicated by electrolyte leakage, membrane damage under stress was less in sense plants and more severe in antisense plants than controls. Consistent with electrolyte leakage, the malondialdehyde (MDA) content was less in sense plants, but more in antisense plants compared to controls. Meanwhile, the less accumulation of reactive oxygen species (ROS) and the greater antioxidant enzyme activity in sense plants implied the improved antioxidant competence by the overexpression of monoubiquitin gene Ta-Ub2 from wheat. We suggest that overexpressing Ub is a useful strategy to promote chilling tolerance. The improvement of ROS scavenging may be an important mechanism underlying the role of Ub in promoting plants tolerant to chilling stress., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Ubiquitination is a posttranslational modification of proteins that involves the covalent attachment of ubiquitin, either as a single moiety or as polymers. This process controls almost every cellular metabolic pathway through a variety of combinations of linkages. Mass spectrometry now allows high throughput approaches for the identification of the thousands of ubiquitinated proteins and of their ubiquitination sites. Despite major technological improvements in mass spectrometry in terms of sensitivity, resolution and acquisition speed, the use of efficient purification methods of ubiquitinated proteins prior to mass spectrometry analysis is critical to achieve an efficient characterization of the ubiquitome. This critical step is achieved using different approaches that possess advantages and pitfalls. Here, we discuss the limits that can be encountered when deciphering the ubiquitome. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The extracellular ubiquitin–proteasome system is involved in sperm binding to and/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is ubiquitinated and degraded by the sperm proteasome during the sperm penetration of the VC and that a 700-kDa ubiquitin-conjugating enzyme complex is released upon sperm activation on the VC, which is designated the "sperm reaction". However, the de novo function of ubiquitin-activating enzyme (UBA/E1) during fertilization is poorly understood. Here, we show that PYR-41, a UBA inhibitor, strongly inhibited the fertilization of H. roretzi. cDNA cloning of UBA1 and UBA6 from H. roretzi gonads was carried out, and their 3D protein structures were predicted to be very similar to those of human UBA1 and UBA6, respectively, based on AlphaFold2. These two genes were transcribed in the ovary and testis and other organs, among which the expression of both was highest in the ovary. Immunocytochemistry showed that these enzymes are localized on the sperm head around a mitochondrial region and the follicle cells surrounding the VC. These results led us to propose that HrUBA1, HrUBA6, or both in the sperm head mitochondrial region and follicle cells may be involved in the ubiquitination of HrVC70, which is responsible for the fertilization of H. roretzi. [ABSTRACT FROM AUTHOR]

The extracellular ubiquitin–proteasome system is involved in sperm binding to and/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is ubiquitinated and degraded by the sperm proteasome during the sperm penetration of the VC and that a 700-kDa ubiquitin-conjugating enzyme complex is released upon sperm activation on the VC, which is designated the “sperm reaction”. However, the de novo function of ubiquitin-activating enzyme (UBA/E1) during fertilization is poorly understood. Here, we show that PYR-41, a UBA inhibitor, strongly inhibited the fertilization of H. roretzi. cDNA cloning of UBA1 and UBA6 from H. roretzi gonads was carried out, and their 3D protein structures were predicted to be very similar to those of human UBA1 and UBA6, respectively, based on AlphaFold2. These two genes were transcribed in the ovary and testis and other organs, among which the expression of both was highest in the ovary. Immunocytochemistry showed that these enzymes are localized on the sperm head around a mitochondrial region and the follicle cells surrounding the VC. These results led us to propose that HrUBA1, HrUBA6, or both in the sperm head mitochondrial region and follicle cells may be involved in the ubiquitination of HrVC70, which is responsible for the fertilization of H. roretzi.

The ubiquitin (Ub) and Ub-like (Ubl) protein-conjugation cascade is initiated by E1 enzymes that catalyze Ub/Ubl activation through C-terminal adenylation, thioester bond formation with an E1 catalytic cysteine, and thioester bond transfer to Ub/Ubl E2 conjugating enzymes. Each of these reactions is accompanied by conformational changes of the E1 domain that contains the catalytic cysteine (Cys domain). Open conformations of the Cys domain are associated with adenylation and thioester transfer to E2s, while a closed conformation is associated with pyrophosphate release and thioester bond formation. Several structures are available for Ub E1s, but none has been reported in the open state before pyrophosphate release or in the closed state. Here, we describe the structures of Schizosaccharomyces pombe Ub E1 in these two states, captured using semisynthetic Ub probes. In the first, with a Ub-adenylate mimetic (Ub-AMSN) bound, the E1 is in an open conformation before release of pyrophosphate. In the second, with a Ub-vinylsulfonamide (Ub-AVSN) bound covalently to the catalytic cysteine, the E1 is in a closed conformation required for thioester bond formation. These structures provide further insight into Ub E1 adenylation and thioester bond formation. Conformational changes that accompany Cys-domain rotation are conserved for SUMO and Ub E1s, but changes in Ub E1 involve additional surfaces as mutational and biochemical analysis of residues within these surfaces alter Ub E1 activities. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV can be sensed by host innate immunity to induce expression of interferons (IFNs) and a number of antiviral effectors. In this study, we found HCV infection induced the expression of neuralized E3 ubiquitin protein ligase 3 (NEURL3), a putative E3 ligase, in a manner that requires the involvement of innate immune sensing but is independent of the IFN action. Furthermore, we showed that NEURL3 inhibited HCV infection while it had little effect on other RNA viruses, including Zika virus (ZIKV), dengue virus (DENV), and vesicular stomatitis virus (VSV). Mechanistic studies demonstrated that NEURL3 inhibited HCV assembly by directly binding HCV envelope glycoprotein E1 to interfere with the E1/E2 heterodimerization, an important prerequisite for virion morphogenesis. Finally, we showed that knockout of NEURL3 significantly enhanced HCV infection. In summary, we identified NEURL3 as a novel inducible antiviral host factor that suppresses HCV assembly. Our results not only shed new insight into how host innate immunity acts against HCV but also revealed a new important biological function for NEURL3. IMPORTANCE The exact biological function of NEURL3, a putative E3 ligase, remains largely unknown. In this study, we found that NEURL3 could be upregulated upon HCV infection in a manner dependent on pattern recognition receptor-mediated innate immune response. NEURL3 inhibits HCV assembly by directly binding viral E1 envelope glycoprotein to disrupt its interaction with E2, an action that requires its Neuralized homology repeat (NHR) domain but not the RING domain. Furthermore, we found that NEURL3 has a pangenotypic anti-HCV activity and interacts with E1 of genotypes 2a, 1b, 3a, and 6a but does not inhibit other closely related RNA viruses, such as ZIKV, DENV, and VSV. To our knowledge, our study is the first report to demonstrate that NEURL3 functions as an antiviral host factor. Our results not only shed new insight into how host innate immunity acts against HCV, but also revealed a new important biological function for NEURL3. [ABSTRACT FROM AUTHOR]

Atg8 is a unique ubiquitin-like protein that is covalently conjugated with a phosphatidylethanolamine through reactions similar to ubiquitination and plays essential roles in autophagy. Atg7 is the E1 enzyme for Atg8, and it activates the C-terminal Gly116 of Atg8 using ATP. Here, we report the crystal structure of Atg8 bound to the C-terminal domain of Atg7 in an unprecedented mode. Atg8 neither contacts with the central β-sheet nor binds to the catalytic site of Atg7, both of which were observed in previously reported Atg7–Atg8 structures. Instead, Atg8 binds to the C-terminal α-helix and crossover loop, thereby changing the autoinhibited conformation of the crossover loop observed in the free Atg7 structure into a short helix and a disordered loop. Mutational analyses suggested that this interaction mode is important for the activation reaction. We propose that Atg7 recognizes Atg8 through multiple steps, which would be necessary to induce a conformational change in Atg7 that is optimal for the activation reaction. [ABSTRACT FROM AUTHOR]

The regulation of protein fate by modification with the small ubiquitin-related modifier (SUMO) plays an essential and crucial role in most cellular pathways. Sumoylation is highly dynamic due to the opposing activities of SUMO conjugation and SUMO deconjugation. SUMO conjugation is performed by the hierarchical action of E1, E2 and E3 enzymes, while its deconjugation involves SUMO-specific proteases. In this review, we summarize and compare the mechanistic principles of how SUMO gets conjugated to its substrate. We focus on the interplay of the E1, E2 and E3 enzymes and discuss how specificity could be achieved given the limited number of conjugating enzymes and the thousands of substrates. [ABSTRACT FROM AUTHOR]

In our previous study, a Met-to-Ile substitution at amino acid 256 in the catalytic domain of Uba1 was determined in temperature-sensitive CHO-K1 mutant tsTM3 cells, which exhibited chromosomal instability and cell-cycle arrest in the S to G 2 phases with decreased DNA synthesis at the nonpermissive temperature, 39 °C. Mutant cells were also characterized by a significant decrease of Uba1 in the nucleus with decreased ubiquitination activity at 39 °C. Defects of ubiquitination activity in the nucleus resulted in an inappropriate balance between Cdt1 and geminin, a licensing factor of DNA replication and its inhibitor. In the present study, we found that the Cu(I)-catalyzed [3 + 2] cycloaddition (click) reaction inhibits the subsequent indirect immunolabeling of Cdt1 but allows for the detection of PCNA with nascent DNA. Using a procedure without the click reaction, we also demonstrated that Cdt1 remained close to active replication sites in tsTM3 cells at the nonpermissive temperature. Analysis of genome replication by DNA fiber spreading revealed that DNA synthesis continues for at least 10 h after incubation at 39 °C, suggesting that impaired ubiquitination in the nucleus, caused by the defect of Uba1, affected DNA replication only after a long delay. [ABSTRACT FROM AUTHOR]

In addition to its central roles in protein quality control, regulation of cell cycle, intracellular signaling, DNA damage response and transcription regulation, the ubiquitin-proteasome system (UPS) plays specific roles in the nervous system, where it contributes to precise connectivity through development, and later assures functionality by regulating a wide spectrum of neuron-specific cellular processes. Aberrations in this system have been implicated in the etiology of neurodevelopmental and neurodegenerative diseases. In this review, we provide an updated view on the UPS and highlight recent findings concerning its role in normal and diseased nervous systems. We discuss the advantages of the model organism Caenorhabditis elegans as a tool to unravel the major unsolved questions concerning this biochemical pathway and its involvement in nervous system function and dysfunction, and expose the new possibilities, using state-of-the-art techniques, to assess UPS function using this model system. [ABSTRACT FROM AUTHOR]

Abstract: V(D)J recombination, the process that rearranges gene segments to assemble mature antigen receptor genes, relies on a recombinase comprising the RAG1 and RAG2 proteins. RAG1 is a multi-functional enzyme including DNA binding and cleavage as well as ubiquitin ligase activities, all of which appear to contribute to its role in recombination. Here we demonstrate that components of the ubiquitin conjugation machinery and the 26S proteasome are required for an early step in V(D)J recombination. Inhibitors of the 26S proteasome and ubiquitin activating enzyme (E1) blocked both chromosomal and extra-chromosomal recombination when added 1h following transfection/induction, but they had no effect when added 16h later. There was no effect on expression of RAG1, and recombination did not require transit through the cell cycle, confirming that inhibition was not due to an indirect effect on cell cycle arrest or protein expression. Experiments in which RAG1 translation was blocked with cyclohexamide after 16h of expression indicated that many active recombination complexes were formed within this window, although recombination products continued to accumulate for 48h. These data suggest that ubiquitin-dependent degradation is an early step in complex assembly or activation, and are consistent with our previous hypothesis that degradation of a negative regulator is required to trigger recombination. [Copyright &y& Elsevier]

Abstract: Ubiquitination plays a key role in regulating the tumour suppressor p53. It targets p53 for degradation by the 26S proteasome. The ubiquitin pathway also regulates the activity and localisation of p53. Ubiquitination requires ubiquitin-activating and -conjugating enzymes and ubiquitin ligases. In addition, ubiquitination can be reversed by the action of deubiquitinating enzymes. Here we give an overview of the role of components of the ubiquitin–proteasome system in the regulation of p53 and review progress in targeting these proteins to activate wild-type p53 for the treatment of cancer. [Copyright &y& Elsevier]

Abstract: Ubiquitin activating enzyme (UAE, UBE1, or E1) and seven known homologous “E1s” initiate the conjugation pathways for ubiquitin and 16 other ubiquitin-like modifiers (ULMs) found in humans. The initial step catalyzed by E1s uses adenosine triphosphate (ATP) to adenylate the C terminus of the appropriate ULM and results in the production of inorganic pyrophosphate (PPi). The mechanism of these enzymes can be studied with assays that measure the rate of ULM-dependent ATP:PPi exchange. The traditional method follows the initial velocity of [32P]PPi incorporation into ATP by capturing the nucleotide on activated charcoal powder to separate it from excess [32P]PPi and then measuring [32P]ATP in a scintillation counter. We have modified the method by using charcoal paper to capture the nucleotide and a phosphorimager to quantify the [32P]ATP. The significant increase in throughput that these modifications provide is accomplished without any sacrifice in sensitivity or accuracy compared with the traditional method. To demonstrate this, we reproduce and extend the characterization of the NEDD8 activating enzyme. [Copyright &y& Elsevier]

Abstract: The configuration of the active site of E2 ligases, central enzymes in the ubiquitin/ubiquitin-like protein (Ub/Ubl) conjugation systems, has long puzzled researchers. Taking advantage of the wealth of newly available structures and sequences of E2s from diverse organisms, we performed a large-scale comparative analysis of these proteins. As a result we identified a previously under-appreciated diversity in the active site of these enzymes, in particular, the spatial location of the catalytic cysteine and a constellation of associated conserved residues that potentially contributes to catalysis. We observed structural innovations of differing magnitudes occurring in various families across the E2 fold that might correlate in part with differences in target interaction. A key finding was the independent emergence on multiple occasions of a polar residue, often a histidine, in the vicinity of the catalytic cysteine in different E2 families. We propose that these convergently emerging polar residues have a common function, such as in the stabilization of oxyanion holes during Ub/Ubl transfer and spatial localization of the Ub/Ubl tails in the active site. Thus, the E2 ligases represent a rare example in enzyme evolution of high structural diversity of the active site and position of the catalytic residue despite all characterized members catalyzing a similar reaction. Our studies also indicated certain evolutionarily conserved features in all active members of the E2 superfamily that stabilize the unusual flap-like structure in the fold. These features are likely to form a critical mechanical element of the fold required for catalysis. The results presented here could aid in new experiments to understand E2 catalysis. [Copyright &y& Elsevier]

Covalent attachment of ubiquitin-like proteins (UbIs) is a predominant mechanism for regulating protein function in eukaryotes. Several structurally related Ubls, such as ubiquitin, SUMO, NEDD8, and ISG15, modify a vast number of proteins, altering their functions in a variety of ways. UbI modifications can affect the target's half-life, subcellular localization, enzymatic activity, or ability to interact with protein or DNA partners. Generally, these diverse UbIs are covalently attached via their C termini to their targets by parallel, but specific, cascades involving three classes of enzymes known as El, E2, and E3. Structures are now available for many protein complexes in EI-E2-E3 cascades, revealing a series of modular building blocks and providing mechanistic insights into their functions. [ABSTRACT FROM AUTHOR]

Plant defences require a multitude of tightly regulated resistance responses. In Arabidopsis, the unique gain-of-function mutant suppressor of npr1-1 constitutive 1 ( snc1) carries a point mutation in a Resistance ( R)-gene, resulting in constitutive activation of defence responses without interaction with pathogens. This has allowed us to identify various downstream signalling components essential in multiple defence pathways. One mutant that suppresses snc1-mediated constitutive resistance is modifier of snc1 5 ( mos5), which carries a 15-bp deletion in UBA1, one of two ubiquitin-activating enzyme genes in Arabidopsis. A mutation in UBA2 does not suppress snc1, suggesting that these two genes are not equally required in Arabidopsis disease resistance. On the other hand, a mos5 uba2 double mutant is lethal, implying partial redundancy of the two homologues. Apart from affecting snc1-mediated resistance, mos5 also exhibits enhanced disease susceptibility to a virulent pathogen and is impaired in response to infection with avirulent bacteria carrying the protease elicitor AvrRpt2. The mos5 mutation in the C-terminus of UBA1 might affect binding affinity of the downstream ubiquitin-conjugating enzymes, thus perturbing ubiquitination of target proteins. Furthermore, SGT1b and RAR1, which are necessary for resistance conferred by the SNC1-related R-genes RPP4 and RPP5, are dispensable in snc1-mediated resistance. Our data reveal the definite requirement for the ubiquitination pathway in the activation and downstream signalling of several R-proteins. [ABSTRACT FROM AUTHOR]

Abstract: Ubiquitin is the founding member of a family of structurally conserved proteins that regulate a host of processes in eukaryotic cells. Ubiquitin and its relatives carry out their functions through covalent attachment to other cellular proteins, thereby changing the stability, localization, or activity of the target protein. This article reviews the basic biochemistry of these protein conjugation reactions, focusing on ubiquitin itself and emphasizing recent insights into mechanism and specificity. [Copyright &y& Elsevier]

Post-translational covalent attachment of ubiquitin and ubiquitin-like proteins (ubls) has emerged as a predominant cellular regulatory mechanism, with important roles in controlling cell division, signal transduction, embryonic development, endocytic trafficking and the immune response. Ubls function by remodeling the surface of their target proteins, changing their target's half-life, enzymatic activity, protein-protein interactions, subcellular localization or other properties. At least 10 different ubiquitin-like modifications exist in mammals, and attachment of different ubls to a target leads to different biological consequences. Ubl-conjugation cascades are initiated by activating enzymes, which also coordinate the ubls with their downstream pathways. A number of biochemical and structural studies have provided insights into the mechanism of ubl-activating enzymes and their roles in ubl conjugation cascades.Oncogene (2004) 23, 1958-1971. doi:10.1038/sj.onc.1207393 [ABSTRACT FROM AUTHOR]