A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination

Protein ubiquitination is a powerful regulatory modification that influences nearly every aspect of eukaryotic cell biology. The general pathway for ubiquitin (Ub) modification requires the sequential activities of a Ub-activating enzyme (E1), a Ub transfer enzyme (E2), and a Ub ligase (E3). The E2 must recognize both the E1 and a cognate E3 in addition to carrying activated Ub. These central functions are performed by a topologically conserved alpha/beta-fold core domain of approximately 150 residues shared by all E2s. However, as presented herein, the UbcH5 family of E2s can also bind Ub noncovalently on a surface well removed from the E2 active site. We present the solution structure of the UbcH5c/Ub noncovalent complex and demonstrate that this noncovalent interaction permits self-assembly of activated UbcH5c approximately Ub molecules. Self-assembly has profound consequences for the processive formation of polyubiquitin (poly-Ub) chains in ubiquitination reactions directed by the breast and ovarian cancer tumor susceptibility protein BRCA1.     

Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity

Upon Mycobacterium tuberculosis (Mtb) infection, protein kinase G (PknG), a eukaryotic‐type serine‐threonine protein kinase (STPK), is secreted into host macrophages to promote intracellular survival of the pathogen. However, the mechanisms underlying this PknG–host interaction remain unclear. Here, we demonstrate that PknG serves both as a ubiquitin‐activating enzyme (E1) and a ubiquitin ligase (E3) to trigger the ubiquitination and degradation of tumor necrosis factor receptor‐associated factor 2 (TRAF2) and TGF‐β‐activated kinase 1 (TAK1), thereby inhibiting the activation of NF‐κB signaling and host innate responses. PknG promotes the attachment of ubiquitin (Ub) to the ubiquitin‐conjugating enzyme (E2) UbcH7 via an isopeptide bond (UbcH7 K82‐Ub), rather than the usual C86‐Ub thiol‐ester bond. PknG induces the discharge of Ub from UbcH7 by acting as an isopeptidase, before attaching Ub to its substrates. These results demonstrate that PknG acts as an unusual ubiquitinating enzyme to remove key components of the innate immunity system, thus providing a potential target for tuberculosis treatment.     

Identification and characterization of RING-finger ubiquitin ligase UBR7 in mammalian spermatozoa

The ubiquitin-proteasome system (UPS) controls intracellular protein turnover in a substrate-specific manner via E3-type ubiquitin ligases. Mammalian fertilization and particularly sperm penetration through the oocyte vitelline coat, the zona pellucida (ZP), is regulated by UPS. We use an extrinsic substrate of the proteasome-dependent ubiquitin-fusion degradation pathway, the mutant ubiquitin UBB⁺¹, to provide evidence that an E3-type ligase activity exists in sperm-acrosomal fractions. Protein electrophoresis gels from such de novo ubiquitination experiments contained a unique protein band identified by tandem mass spectrometry as being similar to ubiquitin ligase UBR7 (alternative name: C14ORF130). Corresponding mRNA was amplified from boar testis and several variants of the UBR7 protein were detected in boar, mouse and human sperm extracts by Western blotting. Genomic analysis indicated a high degree of evolutionary conservation, remarkably constant purifying selection and conserved testis expression of the UBR7 gene. By immunofluorescence, UBR7 was localized to the spermatid acrosomal cap and sperm acrosome, in addition to hotspots of proteasomal activity in spermatids, such as the cytoplasmic lobe, caudal manchette, nucleus and centrosome. During fertilization, UBR7 remained with the ZP-bound acrosomal shroud following acrosomal exocytosis. Thus, UBR7 is present in the acrosomal cap of round spermatids and within the acrosomal matrix of mature boar spermatozoa. These data provide the first evidence of ubiquitin ligase activity in mammalian spermatozoa and indicate UBR7 involvement in spermiogenesis.     

En bloc transfer of polyubiquitin chains to PCNA in vitro is mediated by two different human E2�CE3 pairs

Post-replication DNA repair in eukaryotes is regulated by ubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination catalyzed by RAD6–RAD18 (an E2–E3 complex) stimulates translesion DNA synthesis, whereas polyubiquitination, promoted by additional factors such as MMS2–UBC13 (a UEV–E2 complex) and HLTF (an E3 ligase), leads to template switching in humans. Here, using an in vitro ubiquitination reaction system reconstituted with purified human proteins, we demonstrated that PCNA is polyubiquitinated predominantly via en bloc transfer of a pre-formed ubiquitin (Ub) chain rather than by extension of the Ub chain on monoubiquitinated PCNA. Our results support a model in which HLTF forms a thiol-linked Ub chain on UBC13 (UBC13∼Ub_n) and then transfers the chain to RAD6∼Ub, forming RAD6∼Ub_n+1. The resultant Ub chain is subsequently transferred to PCNA by RAD18. Thus, template switching may be promoted under certain circumstances in which both RAD18 and HLTF are coordinately recruited to sites of stalled replication.     

The F-box Protein FBXO25 Promotes the Proteasome-dependent Degradation of ELK-1 Protein

FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens.     

The Protein Stability of Axin,a Negative Regulator of Wnt Signaling,Is Regulated by Smad Ubiquitination Regulatory Factor 2 (Smurf2)

Axin is a negative regulator of Wnt/β-catenin signaling via regulating the level of β-catenin, which is a key effector molecule. Therefore, controlling the level of Axin is a critical step for the regulation of Wnt/β-catenin signaling. It has been shown that ubiquitination-mediated proteasomal degradation may play a critical role in the regulation of Axin; however, the E3 ubiquitin ligase(s), which attaches ubiquitin to a target protein in combination with an E2 ubiquitin-conjugating enzyme, for Axin has not yet been identified. Here, we show that Smurf2 is an E3 ubiquitin ligase for Axin. Transient expression of Smurf2 down-regulated the level of Axin and increased the ubiquitination of Axin. Conversely, shRNA specific to Smurf2 blocked Axin ubiquitination. Essential domains of Axin responsible for Smurf2 interaction as well as Smurf2-mediated down-regulation and ubiquitination were identified. In vitro ubiquitination assays followed by analysis using mass spectroscopy revealed that Smurf2 specifically ubiquitinylated Lys⁵⁰⁵ of Axin and that the Axin(K505R) mutant resisted degradation. Knockdown of endogenous Smurf2 increased the level of endogenous Axin and resulted in reduced β-catenin/Tcf reporter activity. Overall, our data strongly suggest that Smurf2 is a genuine E3 ligase for Axin.     

Physiological functions of FBW7 in cancer and metabolism

FBW7 is one of the most well characterized F-box proteins that serve as substrate recognition subunits of SCF (Skp1-Cullin 1-F-box proteins) E3 ubiquitin ligase complexes. SCF^FBW7 plays key roles in regulating cell cycle progression, differentiation, and stem cell maintenance largely through targeting a broad range of oncogenic substrates for proteasome-dependent degradation. The identification of an increasing number of FBW7 substrates for ubiquitination, and intensive in vitro and in vivo studies have revealed a network of signaling components controlled by FBW7 that contributes to metabolic regulation as well as its tumor suppressor role. Here we mainly focus on recent findings that highlight a critical role for FBW7 in cancer and metabolism.     

Regulation of the Polycomb protein RING1B ubiquitination by USP7

The E3 ubiquitin ligase RING1B plays an important role in Polycomb-mediated gene silencing by monoubiquitinating histone H2A. Both the activity and stability of RING1B are controlled by ubiquitination in two distinct manners. Self ubiquitination of RING1B generates K6, K27 and K48-based mixed polyubiquitin chain, and is required for its activity as a ligase. On the other hand, its proteasomal degradation is mediated by another ligase; E6-AP catalyzes the formation of K48-based chains. Since these two modes of ubiquitination target the same lysine residues and are therefore mutually exclusive, an important mode of regulation of RING1B should be at the level of deubiquitination. Here we identify USP7 as a deubiquitinating enzyme that regulates the ubiquitination state of RING1B. RING1B interacts with USP7, which is mediated in part by its RING domain. In addition, USP7 was found in a complex with other Polycomb proteins, suggesting a broad role in regulating these complexes. Although, USP7 directly and specifically deubiquitinates RING1B in vitro and in vivo, it does not discriminate between the activating and proteolysis-targeting modes of ubiquitination, and therefore has a stabilizing effect on RING1B.     

Modeling the CRL4A ligase complex to predict target protein ubiquitination induced by cereblon-recruiting PROTACs

PROteolysis TArgeting Chimeras (PROTACs) are hetero-bifunctional small molecules that can simultaneously recruit target proteins and E3 ligases to form a ternary complex, promoting target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities and enabling targeting of previously “undruggable” proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC-induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by cereblon (CRBN)-based PROTACs by leveraging available structural information of the CRL4A ligase complex (CRBN/DDB1/CUL4A/Rbx1/NEDD8/E2/Ub). We generated ternary complex ensembles with Rosetta, modeled multiple CRL4A ligase complex conformations, and predicted ubiquitination efficiency by separating the ternary ensemble into productive and unproductive complexes based on the proximity of the ubiquitin to accessible lysines on the target protein. We validated our CRL4A ligase complex models with published ternary complex structures and additionally employed our modeling workflow to predict ubiquitination efficiencies and sites of a series of cyclin-dependent kinases (CDKs) after treatment with TL12–186, a pan-kinase PROTAC. Our predictions are consistent with CDK ubiquitination and site-directed mutagenesis of specific CDK lysine residues as measured using a NanoBRET ubiquitination assay in HEK293 cells. This work structurally links PROTAC-induced ternary formation and ubiquitination, representing an important step toward prediction of target “degradability.”     

Induced TRIM21 ISGylation by IFN-β enhances p62 ubiquitination to prevent its autophagosome targeting

The tripartite motif-containing protein 21 (TRIM21) plays important roles in autophagy and innate immunity. Here, we found that HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), as an interferon-stimulated gene 15 (ISG15) E3 ligase, catalyzes the ISGylation of TRIM21 at the Lys260 and Lys279 residues. Moreover, IFN-β also induces TRIM21 ISGylation at multiple lysine residues, thereby enhancing its E3 ligase activity for K63-linkage-specific ubiquitination and resulting in increased levels of TRIM21 and p62 K63-linked ubiquitination. The K63-linked ubiquitination of p62 at Lys7 prevents its self-oligomerization and targeting to the autophagosome. Taken together, our study suggests that the ISGylation of TRIM21 plays a vital role in regulating self-oligomerization and localization of p62 in the autophagy induced by IFN-β.Subject terms: Proteins, Autophagy, Innate immunity, Post-translational modifications     

The effects of Sp7/Osterix gene silencing in the chondroprogenitor cell line, ATDC5

Ubiquitin is a small polypeptide and ubiquitination is the post-translational modification by ubiquitin protein, resulting in degradation of target proteins by the 26S proteasome complex. Here, we found that E3 ubiquitin ligase SINAT5, an Arabidopsis homologue of the Drosophila SINA RING-finger protein, interacts directly with LHY, a component of the circadian oscillator, and DET1, a negative regulator of light-regulated gene expression. We also found that SINAT5 has E3 ubiquitination activity for LHY but not for DET1. Interestingly, LHY ubiquitination by SINAT5 was inhibited by DET1. Late flowering of sinat5 mutants indicates that flowering time can be controlled by DET1 through regulation of LHY stability by SINAT5.     

RNF220, an E3 ubiquitin ligase that targets Sin3B for ubiquitination

Modification of proteins by ubiquitination plays important roles in various cellular processes. During this process, the target specificity is determined by ubiquitin ligases. Here we identify RNF220 (RING finger protein 220) as a novel ubiquitin ligase for Sin3B. As a conserved RING protein, RNF220 can bind E2 and mediate auto-ubiquitination of itself. Through a yeast two-hybrid screen, we isolated Sin3B as one of its targets, which is a scaffold protein of the Sin3/HDAC (histone deacetylase) corepressor complex. RNF220 specifically interacts with Sin3B both in vitro and in vivo. Sin3B can be regulated by the ubiquitin-proteasome system. Co-expression of RNF220 promotes the ubiquitination and proteasomal degradation of Sin3B. Taken together, these results reveal a new mechanism for regulating the Sin3/HDAC complex.     

The F-box protein SHORT PRIMARY ROOT modulates primary root meristem activity by targeting SEUSS-LIKE protein for degradation in rice

Root meristem activity is essential for root morphogenesis and adaptation, but the molecular mechanism regulating root meristem activity is not fully understood. Here, we identify an F-box family E3 ubiquitin ligase named SHORT PRIMARY ROOT (SHPR) that regulates primary root (PR) meristem activity and cell proliferation in rice. SHPR loss-of-function mutations impair PR elongation in rice. SHPR is involved in the formation of an SCF complex with the Oryza sativa SKP1-like protein OSK1/20. We show that SHPR interacts with Oryza sativa SEUSS-LIKE (OsSLK) in the nucleus and is required for OsSLK polyubiquitination and degradation by the ubiquitin 26S-proteasome system (UPS). Transgenic plants overexpressing OsSLK display a shorter PR phenotype, which is similar to the SHPR loss-of-function mutants. Genetic analysis suggests that SHPR promotes PR elongation in an OsSLK-dependent manner. Collectively, our study establishes SHPR as an E3 ubiquitin ligase that targets OsSLK for degradation, and uncovers a protein ubiquitination pathway as a mechanism for modulating root meristem activity in rice.     

The RING-Finger E3 Ubiquitin Ligase COP1 SUPPRESSOR1 Negatively Regulates COP1 Abundance in Maintaining COP1 Homeostasis in Dark-Grown Arabidopsis Seedlings

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) functions as an E3 ubiquitin ligase in both plants and animals. In dark-grown Arabidopsis thaliana seedlings, COP1 targets photomorphogenesis-promoting factors for degradation to repress photomorphogenesis. Little is known, however, about how COP1 itself is regulated. Here, we identify COP1 SUPPRESSOR1 (CSU1), a RING-finger E3 ubiquitin ligase, as a regulator of COP1. Genetic evidence demonstrates that csu1 mutations suppress cop1-6 phenotypes completely in the dark. Furthermore, CSU1 colocalizes with COP1 in nuclear speckles and negatively regulates COP1 protein accumulation in darkness. CSU1 can ubiquitinate COP1 in vitro and is essential for COP1 ubiquitination in vivo. Therefore, we conclude that CSU1 plays a major role in maintaining COP1 homeostasis by targeting COP1 for ubiquitination and degradation in dark-grown seedlings.     

Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP

The E3 ubiquitin ligase CHIP is involved in protein triage, serving as a co-chaperone for refolding as well as catalyzing ubiquitination of substrates. CHIP functions with both the stress induced Hsp70 and constitutive Hsc70 chaperones, and also plays a role in maintaining their balance in the cell. When the chaperones carry no client proteins, CHIP catalyzes their polyubiquitination and subsequent proteasomal degradation. Although Hsp70 and Hsc70 are highly homologous in sequence and similar in structure, CHIP mediated ubiquitination promotes degradation of Hsp70 with a higher efficiency than for Hsc70. Here we report a detailed and systematic investigation to characterize if there are significant differences in the CHIP in vitro ubiquitination of human Hsp70 and Hsc70. Proteomic analysis by mass spectrometry revealed that only 12 of 39 detectable lysine residues were ubiquitinated by UbcH5a in Hsp70 and only 16 of 45 in Hsc70. The only conserved lysine identified as ubiquitinated in one but not the other heat shock protein was K159 in Hsc70. Ubiquitination assays with K-R ubiquitin mutants showed that multiple Ub chain types are formed and that the distribution is different for Hsp70 versus Hsc70. CHIP ubiquitination with the E2 enzyme Ube2W is predominantly directed to the N-terminal amine of the substrate; however, some internal lysine modifications were also detected. Together, our results provide a detailed view of the differences in CHIP ubiquitination of these two very similar proteins, and show a clear example where substantial differences in ubiquitination can be generated by a single E3 ligase in response to not only different E2 enzymes but subtle differences in the substrate.     

Reconstitution of the plant ubiquitination cascade in bacteria using a synthetic biology approach

Ubiquitination modulates nearly all aspects of plant life. Here, we reconstituted the Arabidopsis thaliana ubiquitination cascade in Escherichia coli using a synthetic biology approach. In this system, plant proteins are expressed and then immediately participate in ubiquitination reactions within E. coli cells. Additionally, the purification of individual ubiquitination components prior to setting up the ubiquitination reactions is omitted. To establish the reconstituted system, we co‐expressed Arabidopsis ubiquitin (Ub) and ubiquitination substrates with E1, E2 and E3 enzymes in E. coli using the Duet expression vectors. The functionality of the system was evaluated by examining the auto‐ubiquitination of a RING (really interesting new gene)‐type E3 ligase AIP2 and the ubiquitination of its substrate ABI3. Our results demonstrated the fidelity and specificity of this system. In addition, we applied this system to assess a subset of Arabidopsis E2s in Ub chain formation using E2 conjugation assays. Affinity‐tagged Ub allowed efficient purification of Ub conjugates in milligram quantities. Consistent with previous reports, distinct roles of various E2s in Ub chain assembly were also observed in this bacterial system. Therefore, this reconstituted system has multiple advantages, and it can be used to screen for targets of E3 ligases or to study plant ubiquitination in detail.     

E2 conjugating enzyme selectivity and requirements for function of the E3 ubiquitin ligase CHIP

The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2～Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2～Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2～Ub conjugate is derived from other molecular determinants.     

F-box proteins: the key to protein degradation

Summary The eukaryotic protein degradation pathway involves the ubiquitin (Ub) modification of substrates targeted for degradation by the 26S proteasome. The addition of Ub, a process called ubiquitination, is mediated by enzymes including the E3 Ub ligases which transfer the Ub to targeted substrates. A major type of E3 Ub ligases, the SCF (Skp–Cullin–F-box) complex, is composed of four major components: Skp1, Cul1/Cdc53, Roc1/Rbx1/Hrt1, and an F-box protein. The F-box component of the SCF machineries is responsible for recognizing different substrates for ubiquitination. Interaction with components of the SCF complex is mediated through the F-box motif of the F-box protein while it associates with phosphorylated substrates through its second protein–protein interaction motif such as Trp–Asp (WD) repeats or leucine-rich repeats (LRRs). By targeting diverse substrates, F-box proteins exert controls over stability of proteins and regulate the mechanisms for a wide-range of cellular processes. Here we discuss the importance of F-box proteins by providing a general overview and examples of how F-box proteins function in various cellular settings such as tissue development, cell proliferation, and cell death, in the modeling organism Drosophila.     

植物泛素/26S蛋白酶体途径研究进展

泛素/26S蛋白酶体途径是最重要的,有高度选择性的蛋白质降解途径,由泛素激活酶、泛素结合酶、泛素蛋白连接酶和26S蛋白酶体组成,参与调控植物生长发育的多个方面。泛素蛋白酶体途径参与植物体内的众多生理过程,如植物激素信号,光形态建成、自交不亲和反应和细胞周期等。本文就泛素/26S蛋白酶体途径以及在植物生长发育中的作用的研究近况做一综述。