Functional analysis of Cullin 3 E3 ligases in tumorigenesis

Cullin 3-RING ligases (CRL3) play pivotal roles in the regulation of various physiological and pathological processes, including neoplastic events. The substrate adaptors of CRL3 typically contain a BTB domain that mediates the interaction between Cullin 3 and target substrates to promote their ubiquitination and subsequent degradation. The biological implications of CRL3 adaptor proteins have been well described where they have been found to play a role as either an oncogene, tumor suppressor, or can mediate either of these effects in a context-dependent manner. Among the extensively studied CRL3-based E3 ligases, the role of the adaptor protein SPOP (speckle type BTB/POZ protein) in tumorigenesis appears to be tissue or cellular context dependent. Specifically, SPOP acts as a tumor suppressor via destabilizing downstream oncoproteins in many malignancies, especially in prostate cancer. However, SPOP has largely an oncogenic role in kidney cancer. Keap1, another well-characterized CRL3 adaptor protein, likely serves as a tumor suppressor within diverse malignancies, mainly due to its specific turnover of its downstream oncogenic substrate, NRF2 (nuclear factor erythroid 2-related factor 2). In accordance with the physiological role the various CRL3 adaptors exhibit, several pharmacological agents have been developed to disrupt its E3 ligase activity, therefore blocking its potential oncogenic activity to mitigate tumorigenesis.     

Cereblon versus VHL: Hijacking E3 ligases against each other using PROTACs

The von Hippel-Lindau (VHL) and cereblon (CRBN) proteins are substrate recognition subunits of two ubiquitously expressed and biologically important Cullin RING E3 ubiquitin ligase complexes. VHL and CRBN are also the two most popular E3 ligases being recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a target protein. Using homo-PROTACs, VHL and CRBN have been independently dimerized to induce their own degradation. Here we report the design, synthesis and cellular activity of VHL-CRBN hetero-dimerizing PROTACs featuring diverse conjugation patterns. We found that the most active compound 14a induced potent, rapid and profound preferential degradation of CRBN over VHL in cancer cell lines. At lower concentrations, weaker degradation of VHL was instead observed. This work demonstrates proof of concept of designing PROTACs to hijack different E3 ligases against each other, and highlights a powerful and generalizable proximity-induced strategy to achieve E3 ligase knockdown.     

Drosophila Cand1 regulates Cullin3-dependent E3 ligases by affecting the neddylation of Cullin3 and by controlling the stability of Cullin3 and adaptor protein

Cullin-RING ubiquitin ligases (CRLs), which comprise the largest class of E3 ligases, regulate diverse cellular processes by targeting numerous proteins. Conjugation of the ubiquitin-like protein Nedd8 with Cullin activates CRLs. Cullin-associated and neddylation-dissociated 1 (Cand1) is known to negatively regulate CRL activity by sequestering unneddylated Cullin1 (Cul1) in biochemical studies. However, genetic studies of Arabidopsis have shown that Cand1 is required for optimal CRL activity. To elucidate the regulation of CRLs by Cand1, we analyzed a Cand1 mutant in Drosophila. Loss of Cand1 causes accumulation of neddylated Cullin3 (Cul3) and stabilizes the Cul3 adaptor protein HIB. In addition, the Cand1 mutation stimulates protein degradation of Cubitus interruptus (Ci), suggesting that Cul3-RING ligase activity is enhanced by the loss of Cand1. However, the loss of Cand1 fails to repress the accumulation of Ci in Nedd8^AN015 or CSN5^null mutant clones. Although Cand1 is able to bind both Cul1 and Cul3, mutation of Cand1 suppresses only the accumulation of Cul3 induced by the dAPP-BP1 mutation defective in the neddylation pathway, and this effect is attenuated by inhibition of proteasome function. Furthermore, overexpression of Cand1 stabilizes the Cul3 protein when the neddylation pathway is partially suppressed. These data indicate that Cand1 stabilizes unneddylated Cul3 by preventing proteasomal degradation. Here, we propose that binding of Cand1 to unneddylated Cul3 causes a shift in the equilibrium away from the neddylation of Cul3 that is required for the degradation of substrate by CRLs, and protects unneddylated Cul3 from proteasomal degradation. Cand1 regulates Cul3-mediated E3 ligase activity not only by acting on the neddylation of Cul3, but also by controlling the stability of the adaptor protein and unneddylated Cul3.     

The E3 ubiquitin ligase Cullin 4A regulates meiotic progression in mouse spermatogenesis

The Cullin-RING ubiquitin-ligase CRL4 controls cell cycle and DNA damage checkpoint response and ensures genomic integrity. Inactivation of the Cul4 component of the CRL4 E3 ligase complex in Caenorhabditis elegans by RNA interference results in massive mitotic DNA re-replication in the blast cells, largely due to failed degradation of the DNA licensing protein, CDT-1, and premature spermatogenesis. Here we show that inactivation of Cul4a by gene-targeting in mice only affected male but not female fertility. This male infertility phenotype resulted from a combination of decreased spermatozoa number, reduced sperm motility and defective acrosome formation. Agenesis of the mutant germ cells was accompanied by increased cell death in pachytene/diplotene cells with markedly elevated levels of phospho-p53 and CDT-1. Despite apparent normal assembly of synaptonemal complexes and DNA double strand break repair, dissociation of MLH1, a component of the late recombination nodule, was delayed in Cul4a−/− diplotene spermatocytes, which potentially led to subsequent disruptions in meiosis II and spermiogenesis. Together, our study revealed an indispensable role for Cul4a during male germ cell meiosis.     

The rise of covalent proteolysis targeting chimeras

Targeted protein degradation offers several advantages over direct inhibition of protein activity and is gaining increasing interest in chemical biology and drug discovery. Proteolysis targeting chimeras (PROTACs) in particular are enjoying widespread application. However, PROTACs, which recruit an E3 ligase for degradation of a target protein, still suffer from certain challenges. These include a limited selection for E3 ligases on the one hand and the requirement for potent target binding on the other hand. Both issues restrict the target scope available for PROTACs. Degraders that covalently engage the target protein or the E3 ligase can potentially expand the pool of both targets and E3 ligases. Moreover, they may offer additional advantages by improving the kinetics of ternary complex formation or by endowing additional selectivity to the degrader. Here, we review the recent progress in the emerging field of covalent PROTACs.     

Targeting Cullin-RING ligases by MLN4924 induces autophagy via modulating the HIF1-REDD1-TSC1-mTORC1-DEPTOR axis

MLN4924, a newly discovered small molecule inhibitor of NEDD8-activating enzyme (NAE), inactivates Cullin-RING E3 ubiquitin Ligases (CRLs) by blocking cullin neddylation. As a result, MLN4924 causes accumulation of several key substrates of CRLs and effectively suppresses tumor cell growth by inducing apoptosis and senescence. However, the role of MLN4924 in induction of autophagy and its biological significance are totally unknown. Here we showed that MLN4924 effectively induces autophagy in both time- and dose-dependent manners in multiple human cancer lines, indicating a general phenomenon. Mechanistically, by inactivating CRLs, MLN4924 causes accumulation of DEPTOR and HIF1α. The siRNA knockdown and gene KO studies showed that DEPTOR and the HIF1-REDD1-TSC1 axis are responsible for MLN4924-induced autophagy via inhibiting mTORC1. Biologically, autophagy is a survival signal to tumor cells, and blockage of autophagy via siRNA knockdown, gene KO and small molecule inhibitor remarkably enhanced MLN4924-induced apoptosis. Our study reveals an uncharacterized mechanism of MLN4924 action and provides the proof-of-concept evidence for strategic drug combination of MLN4924 with an autophagy inhibitor for maximal killing of tumor cells via enhancing apoptosis.     

Cullin-based ubiquitin ligases: Cul3-BTB complexes join the family

Cullin-based E3 ligases target substrates for ubiquitin-dependent degradation by the 26S proteasome. The SCF (Skp1-Cul1-F-box) and ECS (ElonginC-Cul2-SOCS box) complexes are so far the best-characterized cullin-based ligases. Their atomic structure has been solved recently, and several substrates have been described in different organisms. In addition to Cul1 and Cul2, higher eucaryotic genomes encode for three other cullins: Cul3, Cul4, and Cul5. Recent results have shed light on the molecular composition and function of Cul3-based E3 ligases. In these complexes, BTB-domain-containing proteins may bridge the cullin to the substrate in a single polypeptide, while Skp1/F-box or ElonginC/SOCS heterodimers fulfill this function in the SCF and ECS complexes. BTB-containing proteins are evolutionary conserved and involved in diverse biological processes, but their function has not previously been linked to ubiquitin-dependent degradation. In this review, we present these new findings and compare the composition of Cul3-based ligases to the well-defined SCF and ECS ligases.     

Structural modeling of protein ensembles between E3 RING ligases and SARS-CoV-2: The role of zinc binding domains

BackgroundThe ubiquitin system is a modification process with many different cellular functions including immune signaling and antiviral functions. E3 ubiquitin ligases are enzymes that recruit an E2 ubiquitin-conjugating enzyme bound to ubiquitin in order to catalyze the transfer of ubiquitin from the E2 to a protein substrate. The RING E3s, the most abundant type of ubiquitin ligases, are characterized by a zinc (II)-binding domain called RING (Really Interesting New Gene). Viral replication requires modifying and hijacking key cellular pathways within host cells such as cellular ubiquitination. There are well-established examples where a viral proteins bind to RING E3s, redirecting them to degrade otherwise long-lived host proteins or inhibiting E3’s ubiquitination activity. Recently, three binary interactions between SARS-CoV-2 proteins and innate human immune signaling Ε3 RING ligases: NSP15-RNF41, ORF3a-TRIM59 and NSP9-MIB1 have been experimentally established.MethodsIn this work, we have investigated the mode of the previous experimentally supported NSP15-RNF41, ORF3a,-TRIM59 and NSP9-MIB1 binary interactions by in silico methodologies intending to provide structural insights of E3-virus interplay that can help identify potential inhibitors that could block SARS-CoV-2 infection of immune cells.ConclusionIn silico methodologies have shown that the above human E3 ligases interact with viral partners through their Zn(II) binding domains. This RING mediated formation of stable SARS-CoV-2-E3 complexes indicates a critical structural role of RING domains in immune system disruption by SARS-CoV-2-infection.Data AvailabilityThe data used to support the findings of this research are included within the article and are labeled with references.     

The CRL4DCAF1 cullin‐RING ubiquitin ligase is activated following a switch in oligomerization state

The cullin‐4‐based RING‐type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the ˜29 CRL4 substrate receptors reported, the DDB1‐ and CUL4‐associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4^DCAF1 ligase. In the 8.4 Å cryo‐EM map of CRL4^DCAF1, four CUL4‐RBX1‐DDB1‐DCAF1 protomers are organized into two dimeric sub‐assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C‐terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4^DCAF1 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin‐conjugating enzymes. Upon CRL4^DCAF1 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization‐deficient CRL4^DCAF1 mutant has higher ubiquitin ligase activity compared to the wild‐type. This study identifies a novel mechanism by which unneddylated and substrate‐free CUL4 ligases can be maintained in an inactive state.     

Characterization of the Cullin7 E3 ubiquitin ligase--heterodimerization of cullin substrate receptors as a novel mechanism to regulate cullin E3 ligase activity

Cul1 and Cul7 are cullin E3 ubiquitin ligase scaffold proteins. Cul1 is known to form a complex with the RING domain protein Rbx1 and one of approximately 70 different F-box proteins. F-box proteins function as substrate receptor subunits and recruit numerous substrates for poly-ubiquitination. Similarly to Cul1, Cul7 interacts with Rbx1, however, only one F-box protein, Fbxw8, has been shown to bind to Cul7. To date only few Cul7 E3 ubiquitin ligase substrates, including cyclin D1, IRS-1 and GRASP65, have been reported, and using Fbxw8 affinity purification, we were unable to identify additional substrate proteins. Here we provide evidence for a model in which Cul7-Rbx1 can promote the ubiquitination of Cul1 substrates by forming high order complexes with Cul1-Rbx1. Binding of Cul1-Rbx1 to Cul7-Rbx1 is mediated via heterodimerization of Fbxw8 with other F-box proteins which function to recruit substrates into the E3 ligase complex. The formation of this high order complex is likely to increase polyubiquitination efficiency.     

CYP3A4 ubiquitination by gp78 (the tumor autocrine motility factor receptor, AMFR) and CHIP E3 ligases

Human liver CYP3A4 is an endoplasmic reticulum (ER)-anchored hemoprotein responsible for the metabolism of >50% of clinically prescribed drugs. After heterologous expression in Saccharomyces cerevisiae, it is degraded via the ubiquitin (Ub)-dependent 26S proteasomal pathway that utilizes Ubc7p/Cue1p, but none of the canonical Ub-ligases (E3s) Hrd1p/Hrd3p, Doa10p, and Rsp5p involved in ER-associated degradation (ERAD). To identify an Ub-ligase capable of ubiquitinating CYP3A4, we examined various in vitro reconstituted mammalian E3 systems, using purified and functionally characterized recombinant components. Of these, the cytosolic domain of the ER-protein gp78, also known as the tumor autocrine motility factor receptor (AMFR), an UBC7-dependent polytopic RING-finger E3, effectively ubiquitinated CYP3A4 in vitro, as did the UbcH5a-dependent cytosolic E3 CHIP. CYP3A4 immunoprecipitation coupled with anti-Ub immunoblotting analyses confirmed its ubiquitination in these reconstituted systems. Thus, both UBC7/gp78 and UbcH5a/CHIP may be involved in CYP3A4 ERAD, although their relative physiological contribution remains to be established.     

Allosteric auto‐inhibition and activation of the Nedd4 family E3 ligase Itch

The Nedd4 family E3 ligases are key regulators of cell growth and proliferation and are often misregulated in human cancers and other diseases. The ligase activities of Nedd4 E3s are tightly controlled via auto‐inhibition. However, the molecular mechanism underlying Nedd4 E3 auto‐inhibition and activation is poorly understood. Here, we show that the WW domains proceeding the catalytic HECT domain play an inhibitory role by binding directly to HECT in the Nedd4 E3 family member Itch. Our structural and biochemical analyses of Itch reveal that the WW2 domain and a following linker allosterically lock HECT in an inactive state inhibiting E2‐E3 transthiolation. Binding of the Ndfip1 adaptor or JNK1‐mediated phosphorylation relieves the auto‐inhibition of Itch in a WW2‐dependent manner. Aberrant activation of Itch leads to migration defects of cortical neurons during development. Our study provides a new mechanism governing the regulation of Itch.     

E3 ubiquitin ligases as regulators of membrane protein trafficking and degradation

Ubiquitination is a regulated post-translational modification that conjugates ubiquitin (Ub) to lysine residues of target proteins and determines their intracellular fate. The canonical role of ubiquitination is to mediate degradation by the proteasome of short-lived cytoplasmic proteins that carry a single, polymeric chain of Ub on a specific lysine residue. However, protein modification by Ub has much broader and diverse functions involved in a myriad of cellular processes. Monoubiquitination, at one or multiple lysine residues of transmembrane proteins, influences their stability, protein-protein recognition, activity and intracellular localization. In these processes, Ub functions as an internalization signal that sends the modified substrate to the endocytic/sorting compartments, followed by recycling to the plasma membrane or degradation in the lysosome. E3 ligases play a pivotal role in ubiquitination, because they recognize the acceptor protein and hence dictate the high specificity of the reaction. The multitude of E3s present in nature suggests their nonredundant mode of action and the need for their controlled regulation. Here we give a short account of E3 ligases that specifically modify and regulate membrane proteins. We emphasize the intricate network of interacting proteins that contribute to the substrate-E3 recognition and determine the substrate's cellular fate.     

The prolific ATL family of RING-H2 ubiquitin ligases

An abundant class of E3 ubiquitin ligases encodes the RING-finger domain. The RING finger binds to the E2 ubiquitin-conjugating enzyme and brings together both the E2 and substrate. It is predicted that 477 RING finger E3 ligases exist in Arabidopsis thaliana. A particular family among them, named Arabidopsis Tóxicos en Levadura (ATL), consists of 91 members that contain the RING-H2 variation and a hydrophobic domain located at the N-terminal end. Transmembrane E3 ligases are important in several biological processes. For instance, some transmembrane RING finger E3 ligases are main participants in the endoplasmic reticulum-associated degradation pathway that targets misfolded proteins. Functional analysis of a number of ATLs has shown that some of them regulate distinct pathways in plants. Several ATLs have been shown to participate in defense responses, while others play a role in the regulation of the carbon/nitrogen response during post-germinative seedling growth transition, in the regulation of cell death during root development, in endosperm development, or in the transition to flowering under short day conditions. The ATL family has also been instrumental in evolution studies for showing how gene families are expanded in plant genomes.     

Molecular recognition of Cullin3 by KCTDs: Insights from experimental and computational investigations

Recent investigations have highlighted a key role of the proteins of the KCTD (K-potassium channel tetramerization domain containing proteins) family in several fundamental biological processes. Despite the growing importance of KCTDs, our current understanding of their biophysical and structural properties is very limited. Biochemical characterizations of these proteins have shown that most of them act as substrate adaptor in E3 ligases during protein ubiquitination. Here we present a characterization of the KCTD5-Cullin3 interactions which are mediated by the KCTD5 BTB domain. Isothermal titration calorimetry experiments reveal that KCTD5 avidly binds the Cullin3 (Cul3). The complex presents a 5:5 stoichiometry and a dissociation constant of 59 nM. Molecular modeling and molecular dynamics simulations clearly indicate that the two proteins form a stable (KCTD5–Cul3)₅ pinwheel-shaped heterodecamer in which two distinct KCTD5 subunits cooperate in the binding of each cullin chain. Molecular dynamics simulations indicate that different types of interactions contribute to the stability of the assembly. Interestingly, residues involved in Cul3 recognitions are conserved in the KCTD5 orthologs and paralogs implicated in important biological processes. These residues are also rather well preserved in most of the other KCTD proteins. By using molecular modeling techniques, the entire ubiquitination system including the E3 ligase, the E2 conjugating enzyme and ubiquitin was generated. The analysis of the molecular architecture of this complex machinery provides insights into the ubiquitination processes which involve E3 ligases with a high structural complexity.     

The emerging family of CULLIN3‐RING ubiquitin ligases (CRL3s): cellular functions and disease implications

Protein ubiquitylation is a post‐translational modification that controls all aspects of eukaryotic cell functionality, and its defective regulation is manifested in various human diseases. The ubiquitylation process requires a set of enzymes, of which the ubiquitin ligases (E3s) are the substrate recognition components. Modular CULLIN‐RING ubiquitin ligases (CRLs) are the most prevalent class of E3s, comprising hundreds of distinct CRL complexes with the potential to recruit as many and even more protein substrates. Best understood at both structural and functional levels are CRL1 or SCF (SKP1/CUL1/F‐box protein) complexes, representing the founding member of this class of multimeric E3s. Another CRL subfamily, called CRL3, is composed of the molecular scaffold CULLIN3 and the RING protein RBX1, in combination with one of numerous BTB domain proteins acting as substrate adaptors. Recent work has firmly established CRL3s as major regulators of different cellular and developmental processes as well as stress responses in both metazoans and higher plants. In humans, functional alterations of CRL3s have been associated with various pathologies, including metabolic disorders, muscle, and nerve degeneration, as well as cancer. In this review, we summarize recent discoveries on the function of CRL3s in both metazoans and plants, and discuss their mode of regulation and specificities.     

RGLG3 and RGLG4, novel ubiquitin ligases modulating jasmonate signaling

JAs are important hormones for plant development and defense, and JA signaling is regulated by diverse mechanisms. We have recently identified two RING-type ubiquitin ligases, RGLG3 and RGLG4, as essential JA signaling regulators. In this addendum, we discuss some characters of RGLG3 and RGLG4, which further support their important roles in JA pathway. RGLG3 and RGLG4 didn’t interact with known key factors of the core JA pathway, rather, it might target on unknown protein that negatively regulated JA signaling. RGLG3 and RGLG4 expression was suppressed by SA treatment in an NPR1-independent manner, and rglg3 rglg4 moderated SA-inhibited JA-responsive PDF1.2 expression, suggesting RGLG3 and RGLG4 took roles in SA-JA antagonism. RGLG3 and RGLG4 could be important players of a regulatory network and coordinated diverse signals to modulate JA signaling.