Endo‐lysosomal sorting of G‐protein‐coupled receptors by ubiquitin: Diverse pathways for G‐protein‐coupled receptor destruction and beyond

Ubiquitin is covalently attached to substrate proteins in the form of a single ubiquitin moiety or polyubiquitin chains and has been generally linked to protein degradation, however, distinct types of ubiquitin linkages are also used to control other critical cellular processes like cell signaling. Over forty mammalian G protein‐coupled receptors (GPCRs) have been reported to be ubiquitinated, but despite the diverse and rich complexity of GPCR signaling, ubiquitin has been largely ascribed to receptor degradation. Indeed, GPCR ubiquitination targets the receptors for degradation by lysosome, which is mediated by the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery, and the proteasome. This has led to the view that ubiquitin and ESCRTs primarily function as the signal to target GPCRs for destruction. Contrary to this conventional view, studies indicate that ubiquitination of certain GPCRs and canonical ubiquitin‐binding ESCRTs are not required for receptor degradation and revealed that diverse and complex pathways exist to regulate endo‐lysosomal sorting of GPCRs. In other studies, GPCR ubiquitination has been shown to drive signaling and not receptor degradation and further revealed novel insight into the mechanisms by which GPCRs trigger the activity of the ubiquitination machinery. Here, we discuss the diverse pathways by which ubiquitin controls GPCR endo‐lysosomal sorting and beyond.      

Pathway choice between proteasomal and autophagic degradation

Efficient degradation of abnormal or aggregated proteins is crucial to protect the cell against proteotoxic stress. Selective targeting and disposal of such proteins usually occurs in a ubiquitin-dependent manner by proteasomes and macroautophagy/autophagy. Whereas proteasomes are efficient in degrading abnormal soluble proteins, protein aggregates are typically targeted for degradation by autophagic vesicles. Both processes require ubiquitin-binding receptors, which are targeted to proteasomes via ubiquitin-like domains or to phagophores (the precursors to autophagosomes) via Atg8/LC3 binding motifs, respectively. The use of substrate modification by ubiquitin in both pathways raised the question of how degradative pathway choice is achieved. In contrast to previous models, proposing different types of ubiquitin linkages for substrate targeting, we find that pathway choice is a late event largely determined by the oligomeric state of the receptors. Monomeric proteasome receptors bind soluble substrates more efficiently due to their higher affinity for ubiquitin. Upon substrate aggregation, autophagy receptors with lower ubiquitin binding affinity gain the upper hand due to higher avidity achieved by receptor bundling. Thus, our work suggests that ubiquitination is a shared signal of an adaptive protein quality control system, which targets substrates for the optimal proteolytic pathway.     

Rad8Rad5/Mms2–Ubc13 ubiquitin ligase complex controls translesion synthesis in fission yeast

Many DNA lesions cause pausing of replication forks at lesion sites; thus, generating gaps in the daughter strands that are filled‐in by post‐replication repair (PRR) pathways. In Saccharomyces cerevisiae, PRR involves translesion synthesis (TLS) mediated by Polη or Polζ, or Rad5‐dependent gap filling through a poorly characterized error‐free mechanism. We have developed an assay to monitor error‐free and mutagenic TLS across single DNA lesions in Schizosaccharomyces pombe. For both main UV photolesions, we have delineated a major error‐free pathway mediated by a distinct combination of TLS polymerases. Surprisingly, these TLS pathways require enzymes needed for poly‐ubiquitination of proliferating cell nuclear antigen (PCNA) as well as those required for mono‐ubiquitination. For pathways that require several TLS polymerases the poly‐ubiquitin chains of PCNA may facilitate their recruitment through specific interactions with their multiple ubiquitin‐binding motifs. These error‐free TLS pathways may at least partially account for the previously described poly‐ubiquitination‐dependent error‐free branch of PRR. This work highlights major differences in the control of lesion tolerance pathways between S. pombe and S. cerevisiae despite the homologous sets of PRR genes these organisms share.     

An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana benthamiana

The ubiquitination proteasome pathway has been demonstrated to regulate all plant developmental and signaling processes. E3 ligase/substrate‐specific interactions and ubiquitination play important roles in this pathway. However, due to technical limitations only a few instances of E3 ligase–substrate binding and protein ubiquitination in plants have been directly evidenced. An efficient in vivo and in vitro ubiquitination assay was developed for analysis of protein ubiquitination reactions by agroinfiltration expression of both substrates and E3 ligases in Nicotiana benthamiana. Using a detailed analysis of the well‐known E3 ligase COP1 and its substrate HY5, we demonstrated that this assay allows for fast and reliable detection of the specific interaction between the substrate and the E3 ligase, as well as the effects of MG132 and substrate ubiquitination and degradation. We were able to differentiate between the original and ubiquitinated forms of the substrate in vivo with antibodies to ubiquitin or to the target protein. We also demonstrated that the substrate and E3 ligase proteins expressed by agroinfiltration can be applied to analyze ubiquitination in in vivo or in vitro reactions. In addition, we optimized the conditions for different types of substrate and E3 ligase expression by supplementation with the gene‐silencing suppressor p19 and by time‐courses of sample collection. Finally, by testing different protein extraction buffers, we found that different types of buffer should be used for different ubiquitination analyses. This method should be adaptable to other protein modification studies.     

Nonspecific yet decisive: Ubiquitination can affect the native‐state dynamics of the modified protein

Ubiquitination is one of the most common post‐translational modifications of proteins, and mediates regulated protein degradation among other cellular processes. A fundamental question regarding the mechanism of protein ubiquitination is whether and how ubiquitin affects the biophysical nature of the modified protein. For some systems, it was shown that the position of ubiquitin within the attachment site is quite flexible and ubiquitin does not specifically interact with its substrate. Nevertheless, it was revealed that polyubiquitination can decrease the thermal stability of the modified protein in a site‐specific manner because of alterations of the thermodynamic properties of the folded and unfolded states. In this study, we used detailed atomistic simulations to focus on the molecular effects of ubiquitination on the native structure of the modified protein. As a model, we used Ubc7, which is an E2 enzyme whose in vivo ubiquitination process is well characterized and known to lead to degradation. We found that, despite the lack of specific direct interactions between the ubiquitin moiety and Ubc7, ubiquitination decreases the conformational flexibility of certain regions of the substrate Ubc7 protein, which reduces its entropy and thus destabilizes it. The strongest destabilizing effect was observed for systems in which Lys48‐linked tetra‐ubiquitin was attached to sites used for in vivo degradation. These results reveal how changes in the configurational entropy of the folded state may modulate the stability of the protein's native state. Overall, our results imply that ubiquitination can modify the biophysical properties of the attached protein in the folded state and that, in some proteins, different ubiquitination sites will lead to different biophysical outcomes. We propose that this destabilizing effect of polyubiquitin on the substrate is linked to the functions carried out by the modification, and in particular, regulatory control of protein half‐life through proteasomal degradation.     

Lysine 63‐linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome

Recruitment of substrates to the 26S proteasome usually requires covalent attachment of the Lys48‐linked polyubiquitin chain. In contrast, modifications with the Lys63‐linked polyubiquitin chain and/or monomeric ubiquitin are generally thought to function in proteasome‐independent cellular processes. Nevertheless, the ubiquitin chain‐type specificity for the proteasomal targeting is still poorly understood, especially in vivo. Using mass spectrometry, we found that Rsp5, a ubiquitin‐ligase in budding yeast, catalyzes the formation of Lys63‐linked ubiquitin chains in vitro. Interestingly, the 26S proteasome degraded well the Lys63‐linked ubiquitinated substrate in vitro. To examine whether Lys63‐linked ubiquitination serves in degradation in vivo, we investigated the ubiquitination of Mga2‐p120, a substrate of Rsp5. The polyubiquitinated p120 contained relatively high levels of Lys63‐linkages, and the Lys63‐linked chains were sufficient for the proteasome‐binding and subsequent p120‐processing. In addition, Lys63‐linked chains as well as Lys48‐linked chains were detected in the 26S proteasome‐bound polyubiquitinated proteins. These results raise the possibility that Lys63‐linked ubiquitin chain also serves as a targeting signal for the 26S proteaseome in vivo.     

Perturbation of host ubiquitin systems by plant pathogen/pest effector proteins

Microbial pathogens and pests of animals and plants secrete effector proteins into host cells, altering cellular physiology to the benefit of the invading parasite. Research in the past decade has delivered significant new insights into the molecular mechanisms of how these effector proteins function, with a particular focus on modulation of host immunity‐related pathways. One host system that has emerged as a common target of effectors is the ubiquitination system in which substrate proteins are post‐translationally modified by covalent conjugation with the small protein ubiquitin. This modification, typically via isopeptide bond formation through a lysine side chain of ubiquitin, can result in target degradation, relocalization, altered activity or affect protein–protein interactions. In this review, I focus primarily on how effector proteins from bacterial and filamentous pathogens of plants and pests perturb host ubiquitination pathways that ultimately include the 26S proteasome. The activities of these effectors, in how they affect ubiquitin pathways in plants, reveal how pathogens have evolved to identify and exploit weaknesses in this system that deliver increased pathogen fitness.     

DCAFs, the missing link of the CUL4-DDB1 ubiquitin ligase

The CUL4-DDB1 ubiquitin ligase regulates cell proliferation, survival, DNA repair, and genomic integrity through targeted ubiquitination of key regulators, yet the substrate receptors that dictate the specificity of this ubiquitination machinery have been largely unknown. Recent work identified a family of DDB1 and CUL4-associated factors (DCAFs) as substrate receptors, implicating a broad spectrum of cellular processes regulated by CUL4-DDB1.     

LUBAC介导RabGEF1的线性泛素化修饰

目的 验证RabGEF1 (Rab guanine nucleotide exchange factor 1)为线性泛素化修饰的新底物。方法 在pEF6/MycHis C载体中克隆人RabGEF1基因。通过免疫共沉淀（Co-IP）实验验证RabGEF1和HOIP的相互作用。利用GST-pulldown实验探索RabGEFl与HOIP相互作用结构域。通过免疫荧光实验验证RabGEF1和HOIP的相互作用与亚细胞定位。应用体内泛素化实验检测RabGEF1的线性泛素化修饰。通过NTA-His泛素化实验进一步明确RabGEF1能够发生线性泛素化修饰。将RabGEF1的泛素化蛋白质样品进行质谱分析，根据质谱结果提示的RabGEF1泛素化位点构建赖氨酸位点突变质粒，进一步在体内泛素化实验中验证RabGEF1的线性泛素化修饰位点。结果 RabGEF1与HOIP存在相互作用，且HOIP通过ZF-NEF结构域与RabGEF1发生直接相互作用。RabGEF1与HOIP共同定位于细胞质。LUBAC介导RabGEF1发生线性泛素化修饰依赖于LUBAC酶活性，RabGEF1泛素化修饰位点为K158。结论 Rab...     

The RING finger of c-Cbl mediates desensitization of the epidermal growth factor receptor.

Ligand-induced activation of surface receptors, including the epidermal growth factor receptor (EGFR), is followed by a desensitization process involving endocytosis and receptor degradation. c-Cbl, a tyrosine phosphorylation substrate shared by several signaling pathways, accelerates desensitization by recruiting EGFR and increasing receptor polyubiquitination. Here we demonstrate that the RING type zinc finger of c-Cbl is essential for ubiquitination and subsequent desensitization of EGFR. Mutagenesis of a single cysteine residue impaired the ability of c-Cbl to enhance both down-regulation and ubiquitination of EGFR in living cells, although the mutant retained binding to the activated receptor. Consequently, the mutant form of c-Cbl acquired a dominant inhibitory function and lost the ability to inhibit signaling downstream to EGFR. In vitro reconstitution of EGFR ubiquitination implies that the RING finger plays an essential direct role in ubiquitin ligation. Our results attribute to the RING finger of c-Cbl a causative role in endocytic sorting of EGFR and desensitization of signal transduction.     

Mechanism of cullin3 e3 ubiquitin ligase dimerization

Cullin E3 ligases are the largest family of ubiquitin ligases with diverse cellular functions. One of seven cullin proteins serves as a scaffold protein for the assembly of the multisubunit ubiquitin ligase complex. Cullin binds the RING domain protein Rbx1/Rbx2 via its C-terminus and a cullin-specific substrate adaptor protein via its N-terminus. In the Cul3 ubiquitin ligase complex, Cul3 substrate receptors contain a BTB/POZ domain. Several studies have established that Cul3-based E3 ubiquitin ligases exist in a dimeric state which is required for binding of a number of substrates and has been suggested to promote ubiquitin transfer. In two different models, Cul3 has been proposed to dimerize either via BTB/POZ domain dependent substrate receptor homodimerization or via direct interaction between two Cul3 proteins that is mediated by Nedd8 modification of one of the dimerization partners. In this study, we show that the majority of the Cul3 proteins in cells exist as dimers or multimers and that Cul3 self-association is mediated via the Cul3 N-terminus while the Cul3 C-terminus is not required. Furthermore, we show that Cul3 self-association is independent of its modification with Nedd8. Our results provide evidence for BTB substrate receptor dependent Cul3 dimerization which is likely to play an important role in promoting substrate ubiquitination.     

A novel route for F-box protein-mediated ubiquitination links CHIP to glycoprotein quality control

In SCF (Skp1/Cullin/F-box protein) ubiquitin ligases, substrate specificity is conferred by a diverse array of F-box proteins. Only in fully assembled SCF complexes, it is believed, can substrates bound to F-box proteins become ubiquitinated. Here we show that Fbx2, a brain-enriched F-box protein implicated in the ubiquitination of glycoproteins discarded from the endoplasmic reticulum, binds the co-chaperone/ubiquitin ligase CHIP (C terminus of Hsc-70-interacting protein) through a unique N-terminal PEST domain in Fbx2. CHIP facilitates the ubiquitination and degradation of Fbx2-bound glycoproteins, including unassembled NMDA receptor subunits. These findings indicate that CHIP acts with Fbx2 in a novel ubiquitination pathway that links CHIP to glycoprotein quality control in neurons. In addition, they expand the repertoire of pathways by which F-box proteins can regulate ubiquitination and suggest a new role for PEST domains as a protein interaction motif.     

Emerging Roles and Research Tools of Atypical Ubiquitination

Ubiquitination is a posttranslational modification characterized by the covalent attachment of ubiquitin molecules to protein substrates. The ubiquitination modification process is reversible, dynamic, and involved in the regulation of various biological processes, such as autophagy, inflammatory responses, and DNA damage responses. The forms of ubiquitin modification are very diverse, incorporating either a single ubiquitin molecule or a complicated ubiquitin polymer, and different types of ubiquitination usually elicit corresponding cellular responses. The development of research tools and strategies has afforded more detailed insight into atypical ubiquitin signaling pathways that were previously poorly understood. Here, an update on the understanding of atypical ubiquitin chain signaling pathways is provided and the recent development of representative research tools for ubiquitin systems is discussed. In addition, the future challenges in ubiquitin research are reflected on and summarized.     

The Mechanism of Ubiquitination in the Cullin-RING E3 Ligase Machinery: Conformational Control of Substrate Orientation

In cullin-RING E3 ubiquitin ligases, substrate binding proteins, such as VHL-box, SOCS-box or the F-box proteins, recruit substrates for ubiquitination, accurately positioning and orienting the substrates for ubiquitin transfer. Yet, how the E3 machinery precisely positions the substrate is unknown. Here, we simulated nine substrate binding proteins: Skp2, Fbw7, β-TrCP1, Cdc4, Fbs1, TIR1, pVHL, SOCS2, and SOCS4, in the unbound form and bound to Skp1, ASK1 or Elongin C. All nine proteins have two domains: one binds to the substrate; the other to E3 ligase modules Skp1/ASK1/Elongin C. We discovered that in all cases the flexible inter-domain linker serves as a hinge, rotating the substrate binding domain, optimally and accurately positioning it for ubiquitin transfer. We observed a conserved proline in the linker of all nine proteins. In all cases, the prolines pucker substantially and the pucker is associated with the backbone rotation toward the E2/ubiquitin. We further observed that the linker flexibility could be regulated allosterically by binding events associated with either domain. We conclude that the flexible linker in the substrate binding proteins orients the substrate for the ubiquitin transfer. Our findings provide a mechanism for ubiquitination and polyubiquitination, illustrating that these processes are under conformational control.     

In vivo relevance of substrate recognition function of major Arabidopsis ubiquitin receptors

Ubiquitylation marks proteins for destruction by the 26S proteasome. These signals are deciphered and targeted by distinct direct and indirect pathways involving a set of evolutionarily conserved ubiquitin receptors. Although biochemical and structural studies have revealed the mechanistic complexity of these substrate recognition pathways, conclusive evidence of the in vivo relevance of their substrate recognition function is currently not available. We recently showed that the structural elements involved in substrate recognition are not responsible for the important roles of the ubiquitin receptor RPN10 in vegetative and reproductive growth or for the abundance of the two-capped proteasomes (RP2-CP). Moreover, Arabidopsis plants subjected to severe knockdown or knockout any of the major ubiquitin receptors displayed wild-type phenotypes. Our results clearly suggest a functional redundancy of the major Arabidopsis ubiquitin receptors, and this evolved multiplicity is probably used to secure the substrates delivery. Based on the reduced abundance of RP2-CP in rpn10-2 and a role of RPN10 in lid-base association, a structural role of RPN10 in 26S proteasome stability is likely to be more relevant in vivo. Further efforts using structural and functional analyses in higher-order mutants to identify the specific biological functions of substrate recognition for the major Arabidopsis ubiquitin receptors are described here.     

KCTD10 Biology: An Adaptor for the Ubiquitin E3 Complex Meets Multiple Substrates

Protein ubiquitination constitutes a post-translational modification mediated by ubiquitin ligases whereby ubiquitinated substrates are degraded through the proteasomal or lysosomal pathways, or acquire novel molecular functions according to their “ubiquitin codes.” Dysfunction of the ubiquitination process in cells causes various diseases such as cancers along with neurodegenerative, auto-immune/inflammatory, and metabolic diseases. KCTD10 functions as a substrate recognition receptor for cullin-3 (CUL3), a scaffold protein in RING-type ubiquitin ligase complexes. Recently, studies by ourselves and others have identified new substrates that are ubiquitinated by the CUL3/KCTD10 ubiquitin ligase complex. Moreover, the type of polyubiquitination (e.g., K27-, K48-, or K63-chain) of various substrates (e.g., RhoB, CEP97, EIF3D, and TRIF) mediated by KCTD10 underlies its divergent roles in endothelial barrier formation, primary cilium formation, plasma membrane dynamics, cell proliferation, and immune response. Here, the physiological functions of KCTD10 are summarized and potential mechanisms are proposed.     

Diversity of bacterial manipulation of the host ubiquitin pathways

Ubiquitination is generally considered as a eukaryotic protein modification, which is catalysed by a three‐enzyme cascade and is reversed by deubiquitinating enzymes. Ubiquitination directs protein degradation and regulates cell signalling, thereby plays key roles in many cellular processes including immune response, vesicle trafficking and cell cycle. Bacterial pathogens inject a series of virulent proteins, named effectors, into the host cells. Increasing evidence suggests that many effectors hijack the host ubiquitin pathways to benefit bacterial infection. This review summarizes the known functions and mechanisms of effectors from human bacterial pathogens including enteropathogenic Escherichia coli, Salmonella, Shigella, Chlamydia and Legionella, highlighting the diversity in their mechanisms for manipulating the host ubiquitin pathways. Many effectors adopt the molecular mimicry strategy to harbour similar structures or functional motifs with those of the host E3 ligases and deubiquitinases. On the other hand, a few of effectors evolve novel structures or new enzymatic activities to modulate various steps of the host ubiquitin pathways. The diversity in the mechanisms enhances the efficient exploitation of the host ubiquitination signalling by bacteria.     

Delivery of ubiquitinated substrates to protein-unfolding machines

Recent work has shown that ubiquitination leads to recognition of target proteins by diverse ubiquitin receptors. One family of receptors delivers the ubiquitinated proteins to the proteasome resulting in ATP-dependent substrate unfolding and proteolysis. A related family of ubiquitin-binding proteins seems to recruit ubiquitinated proteins to Cdc48, an ATPase ring complex that can also unfold proteins. Some targets seem to dock at Cdc48 before the proteasome does, in an ordered pathway. The intimate interplay between the proteasome and Cdc48, mediated in part by loosely associated ubiquitin receptors, has important functions in cellular regulation.     

Substrate properties of ubiquitin carboxyl-terminally derived peptide probes for protein ubiquitination

Protein ubiquitination is a widespread protein posttranslational modification in eukaryotes that regulates essentially every aspect of cellular processes. The attachment of ubiquitin to a protein substrate is accomplished through an enzymatic cascade involving the actions of an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). There are more than 600 E3 ligases estimated to exist in the human genome that regulate the targeting specificity of protein ubiquitination. To understand the dynamic role of protein ubiquitination in biological processes, robust tools need to be developed which can be employed to establish the substrate specificity of each of these E3 ligases. In this report, we show that the ubiquitin carboxyl-terminally derived peptide probes can serve as modest ubiquitin surrogates for the ubiquitination pathway. In the E1-catalyzed probe adenylation assay, peptide probe 3 with a RLRGG recognition sequence exhibited the highest activity, with the k cat/ K 1/2 determined to be 1.1 x 10 (4) M (-1) s (-1), roughly 470-fold lower than that of ubiquitin. The rate of transfer from the E1 peptide probe thioesters to E2 showed clear sequence dependency, with peptide probe 4 with an LRLRGG recognition sequence showed the fastest rate ( t 1/2 = 0.9 min), essentially identical to that of ubiquitin ( t 1/2 = 0.8 min) under our assay conditions. Furthermore, peptide probes 4 and 8 also exhibited the selective, parkin-mediated labeling of tubulins in a semipurified tubulin-parkin complex. Finally, these carboxyl-terminally derived peptide probes were shown to label the ubiquitination substrates in fraction II of the rabbit reticulocyte lysate with an efficiency parallel to their substrate properties. The selective use of these ubiquitin carboxyl-terminally derived peptide probes by the ubiquitination pathway suggests that perhaps more potent peptide ubiquitination probes based on the ubiquitin C-terminal scaffold can be developed through additional structural optimization.     

Distinct Ubiquitination Cascades Act on the Peroxisomal Targeting Signal Type 2 Co‐receptor Pex18p

Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by a peroxisomal targeting sequence (PTS) and ferry them to the peroxisomal membrane. Subsequently, the cargo is translocated into the peroxisomal lumen, whereas the receptor is released to the cytosol for further rounds of protein import. This cycle is controlled by the ubiquitination status of the receptor, which is best understood for the PTS1‐receptor. While polyubiquitination of PTS‐receptors results in their proteasomal degradation, the monoubiquitinated PTS‐receptors are exported to the cytosol and recycled for further rounds of protein import. Here, we describe the identification of two ubiquitination cascades acting on the PTS2 co‐receptor Pex18p. Using in vivo and in vitro approaches, we demonstrate that the polyubiquitination of Pex18p requires the ubiquitin‐conjugating enzyme (E2) Ubc4p, which cooperates with the RING (really interesting new gene)‐type ubiquitin‐protein ligases (E3) Pex2p as well as Pex10p. Monoubiquitination of Pex18p depends on the E2 enzyme Pex4p (Ubc10p), which functions in concert with the E3 enzymes Pex12p and Pex10p. Our findings for the PTS2‐pathway complement the data on PTS1‐receptor ubiquitination and add up to a unified concept of the ubiquitin‐based regulation of peroxisomal import .