Targeted substrate degradation by an engineered double RING ubiquitin ligase

Recognition of the substrates by ubiquitin ligases is crucial for substrate specificity in the ubiquitin-proteasome proteolytic pathway. In the present study, we designed a double RING finger ubiquitin ligase to direct the ubiquitin machinery to a specific substrate. The engineered ligase contains the RING finger domains of both BRCA1 and BARD1 linked to a substrate recognition site PCNA, which is known to interact with cyclin-dependent kinase inhibitor p57. The double RING finger ubiquitin ligase formed a homo-oligomer complex and exhibited significant ligase activity. Co-transfection of the ligase reduced the expression of transfected p57 to the background level in a proteasome-dependent manner and restored the colony formation ability of U2OS cells that is otherwise inhibited by overexpressed p57. The results indicate the ability of the engineered double RING ubiquitin ligase to target the intended substrate. By redesigning the substrate recognition site, expression of engineered double RING ubiquitin ligases may provide a useful tool for removing many different gene products at the protein level.     

BARD1 regulates BRCA1 apoptotic function by a mechanism involving nuclear retention

BRCA1 is involved in maintaining genomic integrity and, as a regulator of the G2/M checkpoint, contributes to DNA repair and cell survival. The overexpression of BRCA1 elicits diverse cellular responses including apoptosis due to the stimulation of specific signaling pathways. BRCA1 is normally regulated by protein turnover, but is stabilized by BARD1 which can recruit BRCA1 to the nucleus to form a ubiquitin E3 ligase complex involved in DNA repair or cell survival. Here, we identify BARD1 as a regulator of BRCA1-dependent apoptosis. Using transfected MCF-7 breast cancer cells, we found that BRCA1-induced apoptosis was independent of p53 and was stimulated by BRCA1 nuclear export. Conversely, BARD1 reduced BRCA1-dependent apoptosis by a mechanism involving nuclear sequestration. Regulation of apoptosis by BARD1 was reduced by BRCA1 cancer mutations that disrupt Ub ligase function. Transfection of BRCA1 N-terminal peptides that disrupted the cellular BRCA1-BARD1 interaction caused a loss of nuclear BRCA1 that correlated with increased apoptosis in single cell assays, but did not alter localization or expression of endogenous BARD1. Reducing BARD1 levels by siRNA caused a small increase in apoptosis. Our findings identify a novel apoptosis inhibitory function of BARD1 and suggest that nuclear retention of BRCA1-BARD1 complexes contributes to both DNA repair and cell survival.     

Mutational analysis of Mdm2 C-terminal tail suggests an evolutionarily conserved role of its length in Mdm2 activity toward p53 and indicates structural differences between Mdm2 homodimers and Mdm2/MdmX heterodimers

Mdm2 can mediate p53 ubiquitylation and degradation either in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer. The ubiquitin ligase activity of these complexes resides mainly in their respective RING finger domains and also requires adjacent C-terminal tails. So far, structural studies have failed to show significant differences between Mdm2 RING homodimers and Mdm2/MdmX RING heterodimers. Here, we report that not only the primary amino acid sequence, but also the length of the C-terminal tail of Mdm2 is highly conserved through evolution and plays an important role in Mdm2 activity toward p53. Mdm2 mutants with extended C termini do not ubiquitylate p53 despite being capable of forming Mdm2 homodimers through both RING-acidic domain and RING-RING interactions. All extended mutants also retained the ability to interact with MdmX, and this interaction led to reactivation of their E3 ubiquitin ligase activity. In contrast, only a subset of extended Mdm2 mutants was activated by the interaction with Mdm2 RING domain, suggesting that Mdm2 homodimers and Mdm2/MdmX heterodimers may not be structurally and functionally fully equivalent.     

泛素连接酶E3

蛋白质的泛素化修饰具有高度的特异性,它参与调节细胞内许多的生理活动。蛋白质的泛素化修饰涉及一系列的酶参与反应,包括泛素激活酶E1、结合酶E2以及连接酶E3。而其中泛素连接酶E3对靶蛋白的特异性识别起关键作用。泛素连接酶E3主要由HECT结构域家族、RING结构域家族和U-box结构域家族组成。现对泛素连接酶E3的分类、结构及其对靶蛋白的识别机制等进行综述。     

TRIM44 interacts with and stabilizes terf, a TRIM ubiquitin E3 ligase

Terf/TRIM17 is a member of the TRIM family of proteins, which is characterized by the RING finger, B-box, and coiled-coil domains. In the present study, we found that terf interacts with TRIM44. Terf underwent ubiquitination in vitro in the presence of the E2 enzyme UbcH6; this suggests that terf exhibits E3 ubiquitin ligase activity. It was also found that terf was conjugated with polyubiquitin chains and stabilized by the proteasome inhibitor in mammalian cells; this suggested that terf rendered itself susceptible to proteasomal degradation through polyubiquitination. We also found that TRIM44 inhibited ubiquitination of terf, and thus stabilized the protein. The N-terminal region of TRIM44 contains a zinc-finger domain found in ubiquitin hydrolases (ZF UBP) and ubiquitin specific proteases (USPs). Thus, we proposed that TRIM44 may function as a new class of the “USP-like-TRIM” which regulates the activity of associated TRIM proteins.     

Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins

The function of small ubiquitin-like modifier (SUMO)-binding proteins is key to understanding how SUMOylation regulates cellular processes. We identified two related Schizosaccharomyces pombe proteins, Rfp1 and Rfp2, each having an N-terminal SUMO-interacting motif (SIM) and a C-terminal RING-finger domain. Genetic analysis shows that Rfp1 and Rfp2 have redundant functions; together, they are essential for cell growth and genome stability. Mammalian RNF4, an active ubiquitin E3 ligase, is an orthologue of Rfp1/Rfp2. Rfp1 and Rfp2 lack E3 activity but recruit Slx8, an active RING-finger ubiquitin ligase, through a RING-RING interaction, to form a functional E3. RNF4 complements the growth and genomic stability defects of rfp1rfp2, slx8, and rfp1rfp2slx8 mutant cells. Both the Rfp-Slx8 complex and RNF4 specifically ubiquitylate artificial SUMO-containing substrates in vitro in a SUMO binding-dependent manner. SUMOylated proteins accumulate in rfp1rfp2 double-null cells, suggesting that Rfp/Slx8 proteins may promote ubiquitin-dependent degradation of SUMOylated targets. Hence, we describe a family of SIM-containing RING-finger proteins that potentially regulates eukaryotic genome stability through linking SUMO-interaction with ubiquitin conjugation.     

Regulating the Regulators: Recent Revelations in the Control of E3 Ubiquitin Ligases

Since its discovery as a post-translational signal for protein degradation, our understanding of ubiquitin (Ub) has vastly evolved. Today, we recognize that the role of Ub signaling is expansive and encompasses diverse processes including cell division, the DNA damage response, cellular immune signaling, and even organismal development. With such a wide range of functions comes a wide range of regulatory mechanisms that control the activity of the ubiquitylation machinery. Ub attachment to substrates occurs through the sequential action of three classes of enzymes, E1s, E2s, and E3s. In humans, there are 2 E1s, ∼35 E2s, and hundreds of E3s that work to attach Ub to thousands of cellular substrates. Regulation of ubiquitylation can occur at each stage of the stepwise Ub transfer process, and substrates can also impact their own modification. Recent studies have revealed elegant mechanisms that have evolved to control the activity of the enzymes involved. In this minireview, we highlight recent discoveries that define some of the various mechanisms by which the activities of E3-Ub ligases are regulated.     

Molecular Dynamics Reveal the Essential Role of Linker Motions in the Function of Cullin-RING E3 Ligases

Tagging proteins by polyubiquitin is a key step in protein degradation. Cullin-RING E3 ubiquitin ligases facilitate ubiquitin transfer from the E2-conjugating enzyme to the substrate, yet crystallography indicates a large distance between the E2 and the substrate, raising the question of how this distance is bridged in the ubiquitin transfer reaction. Here, we demonstrate that the linker motions in the substrate binding proteins can allosterically shorten this distance to facilitate this crucial ubiquitin transfer step and increase this distance to allow polyubiquitination. We performed molecular dynamics simulations for five substrate binding proteins, Skp2, Fbw7, β-TrCP1, Cdc4, and pVHL, in two forms: bound to their substrates and bound to both substrate and adaptor. The adaptor connects the substrate binding proteins to the cullin. In the bound-to-both forms of all cases, we observed rotations of the substrate binding domain, shortening the gap between the tip of the substrate peptide and the E2 active site by 7-12 Å compared with the crystal structures. Overall, together with our earlier simulations of the unbound forms and the bound-to-adaptor forms, the emerging picture is that the maximum distance of 51-73 Å between the substrate binding domain and the E2 active site in the modeled unbound forms of these five proteins shrinks to a minimum of 39-49 Å in the bound-to-both forms. This large distance range, the result of allosterically controlled linker motions, facilitates ubiquitin transfer and polyubiquitination and as such argues that the cullin-RING E3 ubiquitin ligase is under conformational control. We further observed that substrate binding proteins with multiple substrate acceptor lysines have a larger distance range between the substrate and the E2 as compared with β-TrCP1, with only one acceptor lysine.     

泛素化在植物抗病中的作用

泛素化作为植物体内一种广泛存在的调控细胞反应的机制,参与调控植物抗病反应。本文综述了泛素化系统在植物抗病反应中的功能及作用机制,重点介绍了CRLs型E3泛素连接酶和RING/U-box型E3泛素连接酶如何参与调控植物抗病信号途径,以及病原物通过效应蛋白和毒性因子调控植物抗病性的分子机理,为阐明植物抗病机理和植物病害防治方法提供参考。     

K6非典型泛素化修饰研究进展

泛素化是一种存在于真核生物内的蛋白质翻译后修饰,介导了蛋白质的特异性降解与信号转导,参与了诸多生命过程的调控进而影响着机体方方面面的功能。泛素化网络的紊乱和失衡是导致人类严重疾病的重要原因。泛素分子可以形成8种不同拓扑结构的同质泛素链,其丰度和功能差别巨大。目前,丰度较高的K48及K63经典泛素链的修饰底物较多、功能研究相对充分,而其他非典型泛素链的含量低、研究相对较少,但是诸多证据表明非典型泛素链在细胞内发挥着重要的调节功能。K6泛素链是一种重要的非典型泛素链,与K48链相似,具有紧密的空间结构。目前研究发现K6泛素链在DNA损伤修复、线粒体质量控制等过程中发挥重要调节功能,在肿瘤发生、发展以及帕金森疾病的致病过程中有着重要的作用。目前,由于缺乏特异性的K6泛素链抗体和有效的富集手段,导致K6泛素链修饰的底物、调控机制研究相对较少,诸多调控过程和功能有待进一步深入研究。本文系统综述了K6非典型泛素链的结构特征、调控机制以及相关的生物学功能与疾病,为K6泛素链的功能研究提供参考。     

Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4‐2 using proteome arrays

Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2–4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4‐1) and Nedd4L (Nedd4‐2), but their global substrate recognition or differences in substrate specificity are unknown. We performed in vitro ubiquitylation and binding assays of human Nedd4‐1 and Nedd4‐2, and rat‐Nedd4‐1, using protein microarrays spotted with ～8200 human proteins. Top hits (substrates) for the ubiquitylation and binding assays mostly contain PY motifs. Although several substrates were recognized by both Nedd4‐1 and Nedd4‐2, others were specific to only one, with several Tyr kinases preferred by Nedd4‐1 and some ion channels by Nedd4‐2; this was subsequently validated in vivo. Accordingly, Nedd4‐1 knockdown or knockout in cells led to sustained signalling via some of its substrate Tyr kinases (e.g. FGFR), suggesting Nedd4‐1 suppresses their signalling. These results demonstrate the feasibility of identifying substrates and deciphering substrate specificity of mammalian E3 ligases.     

Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B

RING proteins constitute the largest class of E3 ubiquitin ligases. Unlike most RINGs, AO7 (RNF25) binds the E2 ubiquitin-conjugating enzyme, UbcH5B (UBE2D2), with strikingly high affinity. We have defined, by co-crystallization, the distinctive means by which AO7 binds UbcH5B. AO7 contains a structurally unique UbcH5B binding region (U5BR) that is connected by an 11-amino acid linker to its RING domain, forming a clamp surrounding the E2. The U5BR interacts extensively with a region of UbcH5B that is distinct from both the active site and the RING-interacting region, referred to as the backside of the E2. An apparent paradox is that the high-affinity binding of the AO7 clamp to UbcH5B, which is dependent on the U5BR, decreases the rate of ubiquitination. We establish that this is a consequence of blocking the stimulatory, non-covalent, binding of ubiquitin to the backside of UbcH5B. Interestingly, when non-covalent backside ubiquitin binding cannot occur, the AO7 clamp now enhances the rate of ubiquitination. The high-affinity binding of the AO7 clamp to UbcH5B has also allowed for the co-crystallization of previously described and functionally important RING mutants at the RING-E2 interface. We show that mutations having marked effects on function only minimally affect the intermolecular interactions between the AO7 RING and UbcH5B, establishing a high degree of complexity in activation through the RING-E2 interface.     

Ubiquitin ligase Kf-1 is involved in the endoplasmic reticulum-associated degradation pathway

Kf-1 was first identified as a gene showing enhanced expression in the cerebral cortex of a sporadic Alzheimer’s disease patient. To date, however, the functional properties of Kf-1 protein remain unknown. In this study, immunohistochemical analysis showed that Kf-1 immunoreactivity was detected in rat hippocampus and cerebral cortex neurons. Interestingly, it was colocalized with endoplasmic reticulum (ER) marker. To investigate the specific function of Kf-1 protein, we generated Myc tagged wild type Kf-1 (Myc-Kf-1WT) and RING finger domain deletion mutant of Kf-1 (Myc-Kf-1ΔR), and then transfected in HEK293 cells. Myc-Kf-1WT displayed a reticular pattern typical of ER localization, with large perinuclear aggregates and colocalized with ER marker, calnexin. Myc-Kf-1WT facilitated ubiquitination of endogenous proteins, whereas Myc-Kf-1ΔR did not show ubiquitin ligase activity. In addition, we found that Kf-1 interacted with components of the ER-associated degradation (ERAD) pathway, including Derlin-1 and VCP. Taken together, these properties suggest that Kf-1 is an ER ubiquitin ligase involved in the ERAD pathway.