The emerging family of CULLIN3-RING ubiquitin ligases (CRL3s): cellular functions and disease implications
2013; Springer Nature; Volume: 32; Issue: 17 Linguagem: Inglês
10.1038/emboj.2013.173
ISSN1460-2075
AutoresPascal Genschik, Izabela Sumara, Esther Lechner,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoReview2 August 2013free access The emerging family of CULLIN3-RING ubiquitin ligases (CRL3s): cellular functions and disease implications Pascal Genschik Corresponding Author Pascal Genschik Unité Propre de Recherche 2357, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Conventionné avec l'Université de Strasbourg, Strasbourg, France Search for more papers by this author Izabela Sumara Izabela Sumara Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France Search for more papers by this author Esther Lechner Esther Lechner Unité Propre de Recherche 2357, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Conventionné avec l'Université de Strasbourg, Strasbourg, France Search for more papers by this author Pascal Genschik Corresponding Author Pascal Genschik Unité Propre de Recherche 2357, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Conventionné avec l'Université de Strasbourg, Strasbourg, France Search for more papers by this author Izabela Sumara Izabela Sumara Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France Search for more papers by this author Esther Lechner Esther Lechner Unité Propre de Recherche 2357, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Conventionné avec l'Université de Strasbourg, Strasbourg, France Search for more papers by this author Author Information Pascal Genschik 1, Izabela Sumara2 and Esther Lechner1 1Unité Propre de Recherche 2357, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Conventionné avec l'Université de Strasbourg, Strasbourg, France 2Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France *Corresponding author. Institut de Biologie Moléculaire des Plantes (CNRS), 12 rue du Général Zimmer, 67084 Strasbourg, France. Tel.:+33 3 67 15 53 96; Fax:+33 3 88 61 44 42; E-mail: [email protected] The EMBO Journal (2013)32:2307-2320https://doi.org/10.1038/emboj.2013.173 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Glossary ABA abscisic acid adRP autosomal dominant Retinitis Pigmentosa BTB/POZ Bric-a-brac, Tramtrack and Broad Complex/Pox virus and Zinc finger CAND1 Cullin-Associated and Neddylation-Disassociated 1 Ci Cubitus Interruptus CPC Chromosomal Passenger Complex CRL CULLIN-RING Ubiquitin Ligase DCN1 Defective in Cullin Neddylation 1 E2 ubiquitin-conjugating enzyme E3 ubiquitin ligase FBP F-box protein IFN Interferon MATH Meprin and TRAF homology NAE Nedd8-Activating Enzyme NPH3 Nonphototropic Hypocotyl 3 Nrf2 NF-E2-related factor 2 PID protein interaction domain PLK1 Polo-Like Kinase 1 SA Salicylic Acid SAC Spindle Assembly Checkpoint SAR Systemic Acquired Resistance SCF SKP1/CUL1/F-box protein TPR Tetratrico Peptide Repeat UPS Ubiquitin/Proteasome System Introduction Regulation of protein stability by the ubiquitin/proteasome system (UPS) participates in a broad range of physiologically and developmentally controlled processes in all eukaryotes (Ciechanover et al, 2000; Smalle and Vierstra, 2004). A critical step in this pathway involves ubiquitin ligases (also known as E3 enzymes or E3s), which facilitate the transfer of ubiquitin moieties to substrate proteins, as a preparative step for their degradation by the 26S proteasome. Several hundred different E3s have been identified in metazoan and plant genomes, based on specific, commonly shared structural motifs. Among them, CULLIN-RING ubiquitin ligases (CRLs) are the most prevalent class (Petroski and Deshaies, 2005; Hua and Vierstra, 2011). CRLs are multimeric E3s, in which one particular CULLIN protein serves as a molecular scaffold linking up the catalytic module, composed of a RING finger domain protein and a ubiquitin-conjugating (or E2) enzyme, to a specific substrate recognition module, which physically interacts with target proteins. Among the CRL family, the founding member is the SCF (SKP1/CUL1/F-box protein (FBP)) complex (Figure 1A), which employs one of 68 (human) or 700 (Arabidopsis thaliana) FBPs for substrate recognition (Gagne et al, 2002; Jin et al, 2004). Beside CUL1, eukaryotic genomes encode additional cullins (CUL2, CUL3, CUL4, CUL5, and CUL7) (Gieffers et al, 2000; Sarikas et al, 2008) that have likewise been found to form protein complexes with E3 activities, modifying a variety of substrates by using distinct sets of adaptor modules. Figure 1.Structural organization of SCF/CRL1 and the CRL3 complexes. (A) The SCF/CRL1 and the CRL3 complexes share a similar catalytic core module composed of the scaffold proteins CUL1 and CUL3, respectively, and the RING finger protein RBX1 (also known as Hrt1 or ROC1). Single-subunit BTB domain proteins bridge CUL3-RBX1 to substrates, while this function requires an SKP1/FBP heterodimer in SCF/CRL1. Substrate recognition is governed by an independent protein–protein interaction domain (PID) found in most of the FBPs and CUL3-interacting BTB domain proteins. (B) Non-exhaustive list of protein domains is commonly found associated with the BTB domain in CRL3 adaptors. MATH and Ankyrin domains occur in both metazoans and higher plants, while other domains are specific to either kingdom. BTB-KELCH; BTB-WD40; BTB-T1-Kv (voltage-gated potassium channel T1); BTB-Rho (Ras homology); BTB-bZip (basic leucine Zipper); BTB-MATH (Meprin and TRAF homology); BTB-ANKYRIN repeat; BTB-NPH3 (non-phototropic hypocotyl 3); BTB-TPR (Tetratrico Peptide Repeat); BTB-ARM (Armadillo); BTB-TAZ (Transcriptional Adaptor Zinc finger); BTB-PENT (Pentapeptide). Download figure Download PowerPoint Recent research has firmly established CUL3 as the molecular scaffold of a major class of CRLs controlling different developmental and stress responses (Table I) as well as human pathologies (Table II). CUL3 is a highly conserved CULLIN family member present in the genomes of all eukaryotes. In C. elegans, CUL3 loss-of-function leads to a defect of cytokinesis in single-cell embryos (Kurz et al, 2002), and the deletion of this gene in mouse produces an arrest during early embryogenesis (Singer et al, 1999). In the model plant Arabidopsis thaliana, disruption of the two related CUL3A and CUL3B genes also causes embryo lethality, affecting both embryo pattern formation and endosperm development (Figueroa et al, 2005; Thomann et al, 2005; Gingerich et al, 2007). In contrast to this situation in multicellular organisms, the function of CUL3 orthologues is not essential in either budding or fission yeasts (Geyer et al, 2003; Michel et al, 2003). Table 1. List of functional CUL3-based ubiquitin ligases and their substrates in different organisms Organisms Name Protein domains Substrates Function Reference Mammals KLHL9/13/21 BTB-Kelch Aurora Ba Mitotic progression Sumara et al (2007) and Maerki et al (2009) KLHDC5 BTB-Kelch p60/katanin Mitotic spindle formation Cummings et al (2009) BACURD BTBb RhoA Actin cytoskeleton structure Chen et al (2009b) KLHL12 BTB-Kelch Dsh Wnt/β-catenin signalling Angers et al (2006) Sec31a Collagen secretion Jin et al (2012) KEAP1/KLHL19 BTB-Kelch Nrf2 Oxidative stress response Cullinan et al (2004), Kobayashi et al (2004) and Zhang et al (2004) IKKβ NF-κB signalling Lee et al (2009) KLHL20 BTB-Kelch PML Hypoxia response Yuan et al (2011) DAPK IFN-induced cell death Lee et al (2010) KLHL22 BTB-Kelch PLK1a Chromosome segregation Beck et al (2013) KLHL25 BTB-Kelch 4E-BP1 Translational homeostasis Yanagiya et al (2012) KLHL8 BTB-Kelch Rapsyn Neurotransmitter signalling Nam et al (2009) KLHL3 BTB-Kelch WNK1, WNK4 Blood pressure regulation Ohta et al (2013) and Wakabayashi et al (2013) SPOP MATH-BTB Gli2/Gli3 Hedgehog signalling Chen et al (2009a) Daxx Transcription, apoptosis Kwon et al (2006) SRC-3 Transcription by nuclear receptors Li et al (2011) PIPKIIβa Phosphoinositide signalling Bunce et al (2008) BMI1aMacroH2Aa Epigenetic silencing Hernandez-Munoz et al (2005) D. rerio Btbd6a BTB-PHR Plzf Neurogenesis Sobieszczuk et al (2010) D. melanogaster HIB/SPOP MATH-BTB Ci/Gli Hedgehog signalling Zhang et al (2006) Puckered TNF-mediated JNK signalling Liu et al (2009) C. elegans KEL-8 BTB-Kelch RPY-1 Neurotransmitter signalling Schaefer and Rongo (2006) and Nam et al (2009) MEL-26 MATH-BTB MEI-1 Microtubule reorganization Pintard et al (2003b) A. thaliana ETO1/EOL1/EOL2 BTB-TPR Type-2 ACSs Ethylene biosynthesis Christians et al (2009), Wang et al (2004) and Thomann et al (2009) NPH3 BTB-NPH3 PHOT1 Phototropism Roberts et al (2011) NPR3/NPR4 BTB-Ank-repeat NPR1 Systemic acquired resistance (SAR) Fu et al (2012) BPM1-6 MATH-BTB AtHB6 ABA response Lechner et al (2011) WRI1 Fatty acid metabolism Chen et al (2013) a BTB-associated domains and substrates supported by strong in vivo evidence are also given. a Substrates that may not be degraded and whose ubiquitylation may serve non-proteolytic functions. b BACURD contains 180 C-terminal residues with no recognizable sequence motif. Table 2. List of mutations detected in CUL3 and BTB substrate-specific adaptors in patients suffering from indicated diseases Name Disease Mutation Domain Effect Reference CUL3 Pseudohypoaldosteronism type II (PHAII)/Gordon's syndrome/hypertension Delection aa 403–459 Segment between BTB-binding and RING binding domains Loss of KLHL3 binding Boyden et al (2012) and Wakabayashi et al (2013) KLHL3 PHAII/Gordon's syndrome/hypertension Numerous recessive and dominant mutationsaA77EM78VE85AC164FQ309RR384QL387PS410LS432NR528HR528CN529K BTBBACKKelch Loss of CUL3 bindingLoss of CUL3 bindingLoss of substrate binding Boyden et al (2012), Louis-Dit-Picard et al (2012), Ohta et al (2013) and Wakabayashi et al (2013) KLHL7 Autosomal dominant Retinitis Pigmentosa (adRP)/Blindness A153TA153V BACK Loss of CUL3 bindingLower E3 ligase activity Kigoshi et al (2011) KLHL9 Distal myopathy/skeletal muscle atrophy L95F BTB Reduction in CUL3 binding Cirak et al (2010) KBTBD13 Nemaline myopathy (NEM) R248SaK390Na R408Ca Kelch Predicted disruption of β propeller structure Sambuughin et al (2012) Gigaxonin Giant axonal neuropathy GAN/neuropathy of peripheral nerves and central nervous system R15SaS52GaS79LaV82FaR138HaR269QaR293XL309RaC393XaW401XaQ483Xa E486KaR545CaC570Ya BTBBACKKelch Predicted loss of CUL3 bindingLoss of substrate binding Bomont et al (2000) and Ding et al (2002) KCTD7 Epilepsy, progressive myoclonic 3 (EPM3)Neuronal ceroid lipofuscinosis (NCL) R99XaR184C BTB Protein truncationLoss of CUL3 binding Van Bogaert et al (2007) and Staropoli et al (2012) Keap1 Lung cancer R272CG333SG364CL413RR415GG430C BACKKelch Inhibition of E3 ligase activity but not binding to CUL3Loss of substrate binding Padmanabhan et al (2006), Singh et al (2006) and Ohta et al (2008) a Detected mutations, of which predicted effects were not confirmed experimentally. At the structural level, CUL3 interacts with BTB/POZ (for ‘Bric-a-brac, Tramtrack and Broad Complex/Pox virus and Zinc finger’, hereafter referred to simply as BTB) domain proteins, which function as substrate-specific adaptors (Furukawa et al, 2003; Xu et al, 2003; Pintard et al, 2003b). They bind CUL3 via the BTB domain, and commonly direct substrate specificity through an independent additional protein–protein interaction domain (PID) (Figure 1A), thus uniting the functions of the SKP1/FBP heterodimer in SCF/CRL1 complexes in a single polypeptide. Sequence analyses have so far identified over a dozen different protein domains that are associated, sometimes in combinations, with the BTB domain (Stogios et al, 2005). Some of these are widely distributed throughout eukaryotic genomes (such as the Meprin and TRAF homology (MATH) domain), while others are specific to either metazoans (e.g., the Kelch domain) or plants (e.g., the BTB-non-phototropic hypocotyl 3 (NPH3) domain; Figure 1B). It should be noted that only a subset of all BTB domain proteins actually serve as CRL3 adaptors, and they are set apart from the large fraction of zinc-finger BTB proteins by the presence of an additional paired helical structure (called 3-box motif) positioned C-terminal to the BTB domain, which fulfils an important function in CRL3s assembly analogous to the F-box and SOCS box motifs of other Cullin-based E3s (Zhuang et al, 2009). It is noteworthy that the number of BTB proteins—and thus potential CRL3s—varies a lot between organisms. The human genome encodes nearly 200 BTB domain proteins, (Stogios et al, 2005), although those lacking the 3-box structures may not be engaged in functional CRL3 complexes, while about 80 BTB proteins have been identified in A. thaliana (Dieterle et al, 2005; Figueroa et al, 2005; Gingerich et al, 2005) and even fewer in D. melanogaster. This contrasts the situation for the SCF/CRL1 complexes, where the large number of FBPs in plants indicates increased versatility (Gagne et al, 2002). However, as will be illustrated below, recent research indicates that this does not mean that CRL3s are of minor importance in plants. Biological processes involving CRL3s in metazoans A key regulator of basic cellular functions in metazoans The ubiquitin/proteasome system is a major regulator of the cell cycle in all eukaryotes, targeting dozens of regulatory proteins for degradation and thus ensuring irreversible cell-cycle stage transitions (Mocciaro and Rape, 2012). While the key E3s for this are the anaphase promoting complex/cyclosome (APC/C) and SCF complexes, more recently CRL3s also entered into the game. In mammalian cells, CRL3s play an essential function in the progression of mitosis and completion of cytokinesis via ubiquitination of Aurora B kinase and thereby preventing chromosomal passenger complex (CPC) accumulation on mitotic chromosomes (Sumara et al, 2007). Aurora B is poly-ubiquitylated on mitotic chromosomes during prometaphase, in a manner dependent on the Kelch-BTB proteins KLHL9 and KLHL13. Rather than triggering its degradation by the proteasome, Aurora B polyubiquitylation during mitosis however seems to serve as a signal for its extraction from chromosomes. During anaphase, another BTB protein, KLHL21, was shown to mono-ubiquitylate Aurora B on microtubules of the spindle midzone (Maerki et al, 2009). Similarly, a CUL3-KLHL22 E3 ligase complex mono-ubiquitylates Polo-like kinase 1 (PLK1) to remove it from kinetochores after chromosomes have achieved bi-orientiation in metaphase (Beck et al, 2013), with this non-degradative PLK1 ubiquitylation being necessary for spindle assembly checkpoint (SAC) silencing and mitotic chromosome segregation. Thus, CUL3 recruits various BTB-containing proteins to target cell-cycle kinases, and possibly also other cell-cycle regulators, at distinct subcellular localizations and at different steps of mitosis. Moreover, the fact that CRL3s not only target substrates for proteasomal degradation, but also reversibly regulate processes by controlling subcellular localization and possibly even the activity and/or interactions of substrates in space and time, significantly expands the versatility and potential roles of CRL3s. One of the first identified CRL3 substrate adaptors is the well-characterized nematode MATH-BTB protein MEL-26 (reviewed in Pintard et al, 2004). MEL-26 recruits the microtubule-severing katanin protein MEI-1, which is required for meiotic spindle formation but thereafter undergoes rapid CRL3-dependent degradation prior to the onset of mitotic divisions, when its persistence would lead to small and misoriented mitotic spindles. This mechanism appears to be conserved in metazoans, as the mammalian katanin catalytic subunit is also degraded via CRL3-mediated ubiquitylation, involving the Kelch repeat-containing BTB adaptor protein KLHDC5 (Cummings et al, 2009). In addition to microtubule dynamics, CRL3 regulation also affects the actin cytoskeleton (Chen et al, 2009b). Here, the BTB protein BACURD, which does not contain a known recognizable substrate recognition motif in its C-terminal region, mediates the turnover of the small GTPase RhoA that controls the organization of actin cytoskeleton structure. Failure to degrade RhoA leads to abnormal stress fibres and inhibits the migration capabilities of mammalian cells. Protein trafficking pathways CUL3 and its BTB adaptor protein KLHL12 are important regulators of embryonic stem (ES) cell morphology, by affecting the deposition of the extracellular matrix component collagen, which is essential in all metazoans and important for ES cell division (Jin et al, 2012). Similarly to KLHL22, KLHL12 promotes mono-ubiquitylation of its target SEC31, a coat protein of COPII vesicles, and this allows the formation of enlarged COPII vesicle structures required for exocytosis and deposition of rigid, rod-shaped collagen molecules. In addition to secretion, CUL3 has also been implicated in the regulation of late endosome maturation, although the BTB adaptor proteins and their substrates involved in this process remain to be identified (Huotari et al, 2012). Transcription in developmental signalling In Drosophila, morphogens such as Hedgehog (Hh) have key roles in developmental processes. A pivotal mediator of Hh signalling, the transcription factor Cubitus Interruptus (Ci), needs to be specifically expressed in and sometimes restricted to specific tissues during development. One way to achieve this is targeted proteolysis, as illustrated by the MATH-BTB protein HIB/SPOP, which is expressed in the Drosophila eye disc posterior to the morphogenic furrow and that promotes Ci degradation to ensure normal eye development (Zhang et al, 2006). This process appears to be conserved in metazoans, as the mammalian SPOP homologue serves as a CRL3 adaptor for degradation of Gli2 and Gli3, two Gli transcription factors homologous to Drosophila Ci (Chen et al, 2009a). Importantly, the work on SPOP CRL3s defines mechanistically how SPOP interacts with its substrates to control transcriptional outputs (Chen et al, 2009a; Zhuang et al, 2009). In vertebrates, another important signalling protein targeted by a CRL3 complex is Dishevelled (Dsh) (Angers et al, 2006), which constitutes a critical node in cell differentiation/proliferation decisions via the Wnt/β-catenin signalling pathway. Therefore, Dsh protein levels need to be tightly regulated for normal embryonic development, and this is achieved through Wnt signal-dependent Dsh interaction with the BTB-Kelch protein KLHL12, leading to Dsh degradation. Transcription in stress responses One of the best-understood CRL3 roles in mammalian cells lies in the Keap1-Nrf2 (NF-E2-related factor 2) stress response pathway (for a recent in-depth review, see Taguchi et al, 2011). Nrf2 is a major transcriptional activator that induces expression of numerous protective genes in response to oxidative stress. Under normal growth conditions (i.e., in the absence of cellular stress), the BTB-Kelch substrate adaptor Keap1 triggers Nrf2 ubiquitin-dependent degradation by the proteasome in the cytoplasm (Cullinan et al, 2004; Kobayashi et al, 2004; Zhang et al, 2004). However, several cysteine residues in Keap1 can react with electrophiles produced during stress, which negatively affects CUL3-Keap1 ubiquitin E3 ligase activity (Dinkova-Kostova et al, 2002; Wakabayashi et al, 2004) (Figure 2). Upon oxidative stress, Nrf2 is therefore free to translocate into the nucleus and bind to the anti-oxidant responsive elements (AREs) in the promoter regions of its target genes. Besides Nrf2, Keap1 also recognizes other target proteins, such as the oncogenic kinase IKKβ (Lee et al, 2009) (discussed in more detail below). Figure 2.Mode of regulation of CRL3 activity and substrate recognition. (A) Nrf2 is constitutively targeted for Keap1-dependent degradation under normal conditions. In response to oxidative stress, oxidative modifications (denoted as (e), electrophile) on Keap1 impair its activity and result in Nrf2 stabilization. (B) In plant immunity, the transcription coactivator NPR1 is regulated at several levels. In unchallenged cells, NPR1 is predominantly sequestered in the cytoplasm in an oligomeric form through redox-sensitive intermolecular disulphide bonds. Upon pathogen infection, salicylic acid (SA) signals lead to alterations in reduction potential and partially relieves NPR1 to enter the nucleus. High SA concentrations immediately at sites of infection promote its binding to the BTB protein NPR3 and enhance NPR3–NPR1 interaction and subsequent NPR1 degradation, thereby favouring programmed cell death. Lower SA levels in neighbouring cells are insufficient to trigger NPR3-mediated NPR1 ubiquitylation, enabling NPR1 to accumulate and establish systemic acquired resistance (SAR). See text for details. Download figure Download PowerPoint Other levels of gene expression control CRL3s can also control gene expression at other levels than transcriptional activation. In mammalian cells, the CRL3 adaptor SPOP mediates ubiquitylation of the Polycomb group protein BMI1 and the variant histone MacroH2A1 (Hernandez-Munoz et al, 2005), apparently affecting their function in a non-proteolytic fashion. CRL3-SPOP function is required for proper MacroH2A1 localization to the inactive X chromosome, and might thus be actively involved in the epigenetic silencing process that leads to X inactivation. Further downstream in the process of gene expression, CRL3 was recently implicated in the control of translational homeostasis in mammals (Yanagiya et al, 2012). Here, the BTB adaptor KLHL25 promotes degradation of 4E-BP1, a protein that acts as a repressor of translation initiation. 4E-BP1 is only targeted when it is hypophosphorylated and therefore unable to interact with the mRNA cap-binding protein eIF4E, providing a means to control the levels of translation to maintain cellular homeostasis. Cell death In mammalian cells, CUL3 and the adaptor KLHL20 ubiquitylate the death-associated protein kinase (DAPK), an apoptosis mediator involved in interferon (IFN)-induced cell death as well as in response to a variety of other stimuli (Lee et al, 2010). Interestingly, this process is controlled at the level of sequestration of the CRL3 adaptor, whereby IFN induction leads to KLHL20 sequestration in promyelocytic leukaemia (PML) nuclear bodies, thus disrupting its interaction with DAPK and stabilizing the kinase (Lee et al, 2010). The MATH-BTB protein SPOP is also involved in various apoptotic pathways. In Drosophila, SPOP mediates degradation of the Jun kinase phosphatase Puckered (Puc), which is required for apoptosis depending on the tumour necrosis factor (TNF) Eiger during embryonic segmentation (Liu et al, 2009). Human SPOP is involved in the turnover of the death-associated protein DAXX, an anti-apoptotic regulator (Kwon et al, 2006). An unexpected mechanism of CUL3 action is exemplified by its role in vertebrate caspase activation (Jin et al, 2009). CUL3-dependent ubiquitylation of caspase-8 does not lead to its degradation, but instead promotes its stabilization and thus apoptosis induction. Moreover, CUL3 directly associates with caspase-8 and may not require a BTB-domain adaptor protein, although the presence of an as-yet unidentified copurified adaptor protein cannot fully be ruled out at this stage. Caspase-8 can also be targeted by an unrelated E3, TRAF2, for polyubiquitylation and proteasomal degradation, in this case to shut off cell-extrinsic apoptosis (Gonzalvez et al, 2012). CULLIN3-RING ligases in human disease Given the importance of CRL3s in controlling different cellular and developmental processes, it is perhaps of little surprise that they are also linked to the pathology of various human diseases, including metabolic disorders, muscle and nerve degeneration, but also neoplastic diseases. In this regard, gene dosage alterations and expression regulation of CRL3 complex components appear to be the major underlying pathophysiological mechanisms. In addition, elaborate sequencing approaches and database-mining efforts identified a number of specific mutations in patients suffering from several diseases (Table II). This information helps to understand CRL3 pathways at the molecular level and may in the future even allow their targeting via new therapeutic approaches. Metabolic diseases Recent exome sequencing approaches identified numerous recessive and dominant mutations in CUL3 and KLHL3 genes in patients suffering from type II pseudohypoaldosteronism (PHAII) or Gordon's syndrome, a rare disease featuring hypertension due to misbalance between renal salt reabsorption and electrolyte excretion (Boyden et al, 2012; Louis-Dit-Picard et al, 2012). Previously, mutations in WNK (‘with no lysine’) kinases have been correlated with this pathological condition (Wilson et al, 2003). Interestingly, 9 of 16 dominant mutations were found to cluster within the Kelch propeller of KLHL3 and in the vicinity of the other sites implicated in direct substrate binding, suggesting that KLHL3 mutations may abrogate binding and ubiquitylation of targets normally required for modulation of renal salt K+ and H+ handling in response to physiological challenge (Boyden et al, 2012). This notion gains support from recent studies presenting evidence that WNK kinase isoforms may be the critical CUL3-KLHL3 ubiquitylation targets (Ohta et al, 2013; Shibata et al, 2013; Wakabayashi et al, 2013). Disease-causing mutations in KLHL3 abolish interactions with either CUL3 or WNK kinases, and conversely disease mutations within acidic motifs in WNK1 and WNK4 disrupt interaction with KLHL3 (Ohta et al, 2013; Wakabayashi et al, 2013). The CUL3-RhoBTB1 E3 ligase has also been implicated in hypertension and vascular smooth muscle function, via its regulation of PPARγ and RhoA/Rho-kinase pathways (Pelham et al, 2012), and further support for the importance of CUL3 in blood vessel homeostasis comes from the role of the CUL3–BAZF complex in regulating angiogenesis via Notch signalling (Ohnuki et al, 2012). Finally, CUL3-SPOP controls the stability of the pancreatic duodenal homeobox 1 (Pdx1) transcription factor, and thereby affects pancreatic β cell function in glucose homeostasis (Claiborn et al, 2010). Thus, the CRL3 system emerges as an important regulator of metabolic homeostasis, perhaps by regulating responses to specific stress signals. Dystrophies Causative mutations for autosomal dominant Retinitis Pigmentosa (adRP), a heritable form of progressive retinal dystrophy that results in blindness and visual field loss, have been identified in the KLHL7 gene (Kigoshi et al, 2011). While not affecting KLHL7 dimerization, the resulting substitutions of a conserved alanine residue (A153T and A153V) in the KLHL7 BACK domain disrupt interaction with CUL3, consistent with the recently established structural requirement for the BACK domain in CUL3 complex assembly (Canning et al, 2013). As E3 ligase activity was strongly reduced upon mutation of this residue (Kigoshi et al, 2011), adaptor protein interaction with CUL3 but not adaptor protein dimerization status appears to determine CUL3 activity (see below). Similarly, Leucine 95 mutation (L95F) of KLHL9, found in patients suffering from a form of distal myopathy of skeletal muscles, results in reduced interaction with CUL3, although with less pronounced effects (Cirak et al, 2010). In Nemaline myopathy (NEM), one of the most common congenital myopathies, dominant mutations have been identified in the KBTBD13 protein (Sambuughin et al, 2010), later found to be a component of a functional CRL3 complex (Sambuughin et al, 2012). The substitutions R248S, K390N, and R408C are located within the β-sheets of the highly conserved second and fifth Kelch repeats and are predicted to disrupt th
Referência(s)