Ubiquitin removal in the TGF-β pathway

The transforming growth factor (TGF-β) pathway is regulated by ubiquitin-mediated proteolysis at different levels. Two studies now identify deubiquitinating enzymes (DUBs) for the TGF-β type I receptor. Both ubiquitin-specific peptidase-4 (USP4) and -15 (USP15) extend the life of activated receptors against the negative pressure of receptor-ubiquitinating complexes, but through distinct modes of action.     

Exploring the Structural Mechanism of Covalently Bound E3 Ubiquitin Ligase: Catalytic or Allosteric Inhibition?

Covalent inhibition has recently gained a resurgence of interest in several drug discovery areas. The expansion of this approach is based on evidence elucidating the selectivity and potency of covalent inhibitors when bound to particular amino acids of a biological target. The Nedd4-1, an E3 ubiquitin ligase, is characterized by two covalent binding sites, of which catalytic Cys^cat and allosteric Cys^allo are enclosed. This enzyme has demonstrated inhibition at both the above-mentioned binding sites; however, a detailed molecular understanding of the structural mechanism of inhibition upon Cys^cat and Cys^allo binding remains vague. This prompted us to provide the first account of investigating the preferential covalent binding mode and the underlying structural and molecular dynamic implications. Based on the molecular dynamic analyses, it was evident that although both catalytic and allosteric covalent binding led to greater stability of the enzyme, a preferential covalent mechanism of inhibition was seen in the allosteric-targeted system. This was supported by a more favorable binding energy in the allosteric site compared to the catalytic site, in addition to the larger number of residue interactions and stabilizing hydrogen bonds occurring in the allosteric covalent bound complex. The fundamental dynamic analysis presented in this report compliments, as well as adds to previous experimental findings, thus leading to a crucial understanding of the structural mechanism by which Nedd4-1 is inhibited. The findings from this study may assist in the design of more target-specific Nedd4-1 covalent inhibitors exploring the surface-exposed cysteine residues.     

Ubiquitin and its kin: how close are the family ties?

Modification of proteins by the covalent attachment of ubiquitin is known to target them for degradation by proteasomes. Several proteins have been discovered recently that are related to ubiquitin or function similarly. Some of these proteins act as modifiers; others bear ubiquitin-like domains embedded in their polypeptide chain but do not form conjugates with cellular proteins. Ubiquitin-like proteins mediate an impressive range of cellular functions, including cell-cycle progression, DNA repair and apoptosis. Recent discoveries endorse the view that, in many cases, the function of the relatives of ubiquitin is linked to the ubiquitin pathway.     

Ubiquitin in motion: structural studies of the ubiquitin-conjugating enzyme∼ubiquitin conjugate

Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for the transfer of Ub to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2～Ub conjugate. Therefore, full characterization of the structure and dynamics of E2～Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2～Ub conjugates of two human enzymes, UbcH5c～Ub and Ubc13～Ub, in solution as determined by nuclear magnetic resonance and small-angle X-ray scattering. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: the UbcH5c～Ub conjugate populates an array of extended conformations, and the population of Ubc13～Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces helix 2 of Ubc13. We propose that the varied conformations adopted by Ub represent available binding modes of the E2～Ub species and thus provide insight into the diverse E2～Ub protein interactome, particularly with regard to interaction with Ub ligases.     

Ubiquitin Transfer from the E2 Perspective: Why is UbcH5 So Promiscuous?

Protein ubiquitination is a regulatory process that influences nearly every aspect of eukaryotic cell biology. Pathways that range from cell-cycle progression and differentiation to DNA repair to vesicle budding all rely on regulated modification of target proteins by ubiquitin. Target proteins can be tagged by a single molecule of ubiquitin or modified by ubiquitin polymers that can vary in length and linkage specificity, and these variations influence how ubiquitination signals are interpreted. Surprisingly, little is understood regarding mechanisms of protein ubiquitination and how poly-ubiquitin chains are synthesized. Simple models to explain ubiquitin transfer have dominated the literature, but recent work suggests basic assumptions as to how proteins assemble to facilitate protein ubiquitination and poly-ubiquitin chain synthesis should be re-examined. This is particularly necessary for understanding the roles played by E2 ubiquitin-conjugating enzymes, a central protein component in all ubiquitin transfer reactions. In particular, UbcH5, a canonical E2 protein that is active in a broad number of in vitro ubiquitin transfer reactions, is capable of binding ubiquitin non-covalently on a surface distinct from its active site. This unique property allows activated UbcH5~Ub complexes to self-assemble and has a profound influence on poly-ubiquitin chain synthesis.     

Ubiquitin — protein conjugates

Summary The data available at present indicates there are three distinct functions of ubiquitin, two of which are related to protein conjugation. The first of these has been extensively studied by our laboratory and others interested in nucleosomes and changes in chromatin states. The ubiquitin-histone (Ub-2A, Ub-2B) conjugation reaction now appears to be a very dynamic process. In the deconjugation (lyase) reaction, both the histone 2A and the ubiquitin are left intact and in a form which makes possible ready reconjugation. Accordingly, this may be a mechanism for moment-to-moment control of the genome.The second function in which ubiquitin is conjugated involves proteolytic activity. This activity is correlated with protein turnover. In this process, the ubiquitin-protein conjugate apparently serves as a signal for the protease cleavage of the protein. The released ubiquitin is also intact and is probably available for reconjugation.In the third function, ubiquitin was suggested to serve as hormone. The studies thus far have been carried out primarily on induction of T- and B-lymphocytes, reduction or delay of Coombs' positivity and reduction of spleen weight. The precise physiological role of this reported function is still unclear, particularly because the ubiquitin used was probably not the physiologically active form.     

RNF-121 Is an Endoplasmic Reticulum-Membrane E3 Ubiquitin Ligase Involved in the Regulation of β-Integrin

We report on the characterization of RNF-121, an evolutionarily conserved E3 ligase RING finger protein that is expressed in the endoplasmic reticulum (ER) of various cells and tissues in Caenorhabditis elegans. Inactivation of RNF-121 induced an elevation in BiP expression and increased the sensitivity of worms to ER stress. Genetic analysis placed RNF-121 downstream of the unfolded protein response (UPR) regulator protein kinase-like endoplasmic reticulum kinase (PERK). We identify PAT-3::GFP, the β subunit of the heterodimeric integrin receptors, as an RNF-121 substrate; whereas induction of RNF-121 expression reduced the level of PAT-3::GFP in the gonad distal tip cells, inhibition of RNF-121 led to the accumulation of stably bound PAT-3::GFP inclusions. Correspondingly, overexpression of RNF-121 during early stages of gonad development led to aberrations in germline development and gonad migration that overlap with those observed after PAT-3 inactivation. The formation of these gonad abnormalities required functional ER-associated degradation (ERAD) machinery. Our findings identify RNF-121 as an ER-anchored ubiquitin ligase that plays a specific role in the ERAD pathway by linking it to the regulation of the cell adhesion integrin receptors.     

Measuring Activity in the Ubiquitin–Proteasome System: From Large Scale Discoveries to Single Cells Analysis

The ubiquitin–proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS has provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington’s disease. These reporters, usually consisting of a recognition sequence fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes. This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, a recent study is presented highlighting the development of a novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples.     

Ubiquitin及其功能的研究进展

Ubiquitin是广泛存在于真核细胞的热稳定多肽,由76个氨基酸残基组成。Ubiquitin在进化上显示出高度保守性。从酵母、植物到哺乳动物,仅有3个氨基酸残基不同。它在介导细胞内蛋白质降解、转录的调节以及应激反应中发挥重要作用。文中主要对近年来在ubiquitin结构、基因及其功能方面的研究进展进行综述。     

Oxidative Stress, NF-κB and the Ubiquitin Proteasomal Pathway in the Pathology of Calpainopathy

The neuromuscular disorder, calpainopathy (LGMD 2A), is a major muscular dystrophy classified under limb girdle muscular dystrophies. Genetic mutations of the enzyme calpain 3 cause LGMD 2A. Calpainopathy is phenotypically observed as progressive muscle wasting and weakness. Pathomechanisms of muscle wasting of calpainopathy remain poorly understood. Oxidative stress, NF-κB and the ubiquitin proteasomal pathway underlie the pathology of several muscle wasting conditions but their role in calpainopathic dystrophy is not known. Oxidative and nitrosative stress, the source of reactive oxygen species, NF-κB signaling and protein ubiquitinylation were studied in 15 calpainopathic and 8 healthy control human muscle biopsies. Oxidative stress and NF-κB/IKK β signaling were increased in calpainopathic muscle and may contribute to increased protein ubiquitinylation and muscle protein loss. Preventing oxidative stress or inhibition of NF-κB signaling could be considered for treatment of LGMD 2A.     

What Was the Set of Ubiquitin and Ubiquitin-Like Conjugating Enzymes in the Eukaryote Common Ancestor?

Ubiquitin (Ub)-conjugating enzymes (E2) are key enzymes in ubiquitination or Ub-like modifications of proteins. We searched for all proteins belonging to the E2 enzyme super-family in seven species (Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Arabidopsis thaliana) to identify families and to reconstruct each family’s phylogeny. Our phylogenetic analysis of 207 genes led us to define 17 E2 families, with 37 E2 genes, in the human genome. The subdivision of E2 into four classes did not correspond to the phylogenetic tree. The sequence signature HPN (histidine–proline–asparagine), followed by a tryptophan residue at 16 (up to 29) amino acids, was highly conserved. When present, the active cysteine was found 7 to 8 amino acids from the C-terminal end of HPN. The secondary structures were characterized by a canonical alpha/beta fold. Only family 10 deviated from the common organization because the proteins were devoid of enzymatic activity. Family 7 had an insertion between beta strands 1 and 2; families 3, 5 and 14 had an insertion between the active cysteine and the conserved tryptophan. The three-dimensional data of these proteins highlight a strong structural conservation of the core domain. Our analysis shows that the primitive eukaryote ancestor possessed a diversified set of E2 enzymes, thus emphasizing the importance of the Ub pathway. This comprehensive overview of E2 enzymes emphasizes the diversity and evolution of this superfamily and helps clarify the nomenclature and true orthologies. A better understanding of the functions of these enzymes is necessary to decipher several human diseases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Selective Proteasomal Degradation of the B′β Subunit of Protein Phosphatase 2A by the E3 Ubiquitin Ligase Adaptor Kelch-like 15

Protein phosphatase 2A (PP2A), a ubiquitous and pleiotropic regulator of intracellular signaling, is composed of a core dimer (AC) bound to a variable (B) regulatory subunit. PP2A is an enzyme family of dozens of heterotrimers with different subcellular locations and cellular substrates dictated by the B subunit. B′β is a brain-specific PP2A regulatory subunit that mediates dephosphorylation of Ca²⁺/calmodulin-dependent protein kinase II and tyrosine hydroxylase. Unbiased proteomic screens for B′β interactors identified Cullin3 (Cul3), a scaffolding component of E3 ubiquitin ligase complexes, and the previously uncharacterized Kelch-like 15 (KLHL15). KLHL15 is one of ∼40 Kelch-like proteins, many of which have been identified as adaptors for the recruitment of substrates to Cul3-based E3 ubiquitin ligases. Here, we report that KLHL15-Cul3 specifically targets B′β to promote turnover of the PP2A subunit by ubiquitylation and proteasomal degradation. Comparison of KLHL15 and B′β tissue expression profiles suggests that the E3 ligase adaptor contributes to selective expression of the PP2A/B′β holoenzyme in the brain. We mapped KLHL15 residues critical for homodimerization as well as interaction with Cul3 and B′β. Explaining PP2A subunit selectivity, the divergent N terminus of B′β was found necessary and sufficient for KLHL15-mediated degradation, with Tyr-52 having an obligatory role. Although KLHL15 can interact with the PP2A/B′β heterotrimer, it only degrades B′β, thus promoting exchange with other regulatory subunits. E3 ligase adaptor-mediated control of PP2A holoenzyme composition thereby adds another layer of regulation to cellular dephosphorylation events.     

Structures of REV1 UBM2 Domain Complex with Ubiquitin and with a Small-Molecule that Inhibits the REV1 UBM2–Ubiquitin Interaction

REV1 is a DNA damage tolerance protein and encodes two ubiquitin-binding motifs (UBM1 and UBM2) that are essential for REV1 functions in cell survival under DNA-damaging stress. Here we report the first solution and X-ray crystal structures of REV1 UBM2 and its complex with ubiquitin, respectively. Furthermore, we have identified the first small-molecule compound, MLAF50, that directly binds to REV1 UBM2. In the heteronuclear single quantum coherence NMR experiments, peaks of UBM2 but not of UBM1 are significantly shifted by the addition of ubiquitin, which agrees to the observation that REV1 UBM2 but not UBM1 is required for DNA damage tolerance. REV1 UBM2 interacts with hydrophobic residues of ubiquitin such as L8 and L73. NMR data suggest that MLAF50 binds to the same residues of REV1 UBM2 that interact with ubiquitin, indicating that MLAF50 can compete with the REV1 UBM2–ubiquitin interaction orthosterically. Indeed, MLAF50 inhibited the interaction of REV1 UBM2 with ubiquitin and prevented chromatin localization of REV1 induced by cisplatin in U2OS cells. Our results structurally validate REV1 UBM2 as a target of a small-molecule inhibitor and demonstrate a new avenue to targeting ubiquitination-mediated protein interactions with a chemical tool.     

A U-Box E3 Ubiquitin Ligase,PUB20, Interacts with the Arabidopsis G-Protein β Subunit,AGB1

An Arabidopsis U-box E3 ubiquitin ligase Plant U-box 20 (PUB20; alternatively called AtCMPG1) was identified as a possible interactor of the Arabidopsis G-protein β subunit, AGB1, by yeast two-hybrid screening. A bimolecular fluorescence complementation (BiFC) assay showed that PUB20 interacted with AGB1 in the nuclei and the cytosol. The expression levels of PUB20 and its closest homolog, PUB21 were stable under many conditions. GUS driven by the PUB20 promoter was active in anthers, pollen, premature seeds and receptacles and GUS driven by the PUB21 promoter was active in anthers and funiculi. PUB20 was found to have autoubiquitination activity in vitro.     

Filamins but Not Janus Kinases Are Substrates of the ASB2α Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells

The ASB2α protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hematopoietic differentiation and is proposed to exert its effects by regulating the turnover of specific proteins. Three ASB2α substrates have been described so far: the actin-binding protein filamins, the Mixed Lineage Leukemia protein, and the Janus kinases 2 and 3. To determine the degradation of which substrate drives ASB2α biological effects is crucial for the understanding of ASB2α functions in hematopoiesis. Here, we show that neither endogenous nor exogenously expressed ASB2α induces degradation of JAK proteins in hematopoietic cells. Furthermore, we performed molecular modeling to generate the first structural model of an E3 ubiquitin ligase complex of an ASB protein bound to one of its substrates.