# Cullin 5-RING E3 ubiquitin ligases, new therapeutic targets?

Isabelle Lamsoul, Sandrine Uttenweiler-Joseph, Christel Moog-Lutz, Pierre Lutz

► To cite this version:

Isabelle Lamsoul, Sandrine Uttenweiler-Joseph, Christel Moog-Lutz, Pierre Lutz. Cullin 5-RING E3 ubiquitin ligases, new therapeutic targets?. Biochimie, 2016, 122, pp.339-347. 10.1016/j.biochi.2015.08.003. hal-02406392

HAL Id: hal-02406392

https://hal.science/hal-02406392

Submitted on 8 Feb 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Cullin 5-RING E3 ubiquitin ligases, new therapeutic targets?

Isabelle Lamsoul<sup>a, b</sup>, Sandrine Uttenweiler-Joseph<sup>a, b</sup>, Christel Moog-Lutz<sup>a, b</sup>, Pierre G. Lutz<sup>a, b, *</sup>

Ubiquitylation is a reversible post-translational modification of proteins that controls a myriad of functions and cellular processes. It occurs through the sequential action of three distinct enzymes. E3 ubiquitin ligases (E3s) play the role of conductors of the ubiquitylation pathway making them attractive therapeutic targets. This review is dedicated to the largest family of multimeric E3s, the Cullin-RING E3 (CRL) family and more specifically to cullin 5 based CRLs that remains poorly characterized.

*Abbreviations:* ASB, ankyrin repeat-containing proteins with a SOCS box; β-Trcp, β-transducin repeat-containing protein; BTB, bric-a-brac; CIS1, cytokine-inducible Src homology 2-containing protein; CRL, Cullin-RING E3 ubiquitin Ligase; CAND1, cullin-associated and neddylation-dissociated 1; CSN, COP9 signalosome complex; DCAF, DDB1- and CUL4-associated factor homolog; DCNL, defective in cullin neddylation protein 1-like proteins; DCN1, defective in cullin neddylation protein 1; DDB1, DNA damage-binding protein 1; DUB, deubiquitylating enzyme; Elo, Elongin B; EloC, Elongin C; E1, E1 ubiquitin-activating enzyme; E2, E2 ubiquitin-conjugating enzyme; E3, E3 ubiquitin ligase; FAK, Focal Adhesion Kinase; Fbw7, F-box WD40 repeat-containing protein 7; HECT, Homologous to E6-associated protein carboxyl terminus; HIV-1, human immunodeficiency virus-1; IRS, Insulin Receptor Substrate; JAK2, Janus Kinase 2; KSHV, Kaposi's sarcoma-associated herpes virus; LANA, latency-associated nuclear antigen; MAL, Myeloid differentiation primary-response gene 88 -Adaptor-Like protein; MLL, Mixed Lineage Leukemia; MuHV-4, murid herpes virus-4; NAE, NEDD8-activating enzyme; Mre11, meiotic recombination 11; NEDD8, Neural precursor cell expressed developmentally down-regulated protein 8; RING, really interesting new gene; RBX, RING box protein; ROC, regulator of cullins; SKP1, S-phase kinase-associated protein 1; Skp2, S-phase kinase-associated protein 2; SOCS, suppressors of cytokine signaling; SPSB, SPRY domain-containing proteins with a SOCS box; TNF-R2, tumor necrosis factor receptor 2; VHL, von Hippel-Lindau; WSB, WD40 repeat-containing protein with a SOCS box; Vif, viral infectivity factor.

<footnote><sup>a</sup> CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, BP 64182, F-31077 Toulouse, France</footnote>

<footnote><sup>b</sup> Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France</footnote>

<footnote>* Corresponding author. Institut de Pharmacologie et de Biologie Structurale, UMR 5089 CNRS/UPS, BP64182, 205 Route de Narbonne, F-31077 Toulouse, France.</footnote>

<footnote>*E-mail address:* Pierre.Lutz@ipbs.fr (P.G. Lutz).</footnote>
A

<figure><img src="image_2.png" /></figure>

B

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** CRL5 in ubiquitin-dependent protein degradation. (A) Ubiquitin (Ub) is first activated in an ATP-dependent reaction that leads to the formation of a thioester intermediate that involves the carboxy-terminal glycine residue of ubiquitin and the active site cysteine residue on the ubiquitin-activating enzyme E1. Ubiquitin is then transferred to the active site cysteine of a ubiquitin-conjugating enzyme E2. In the case of RING-finger E3s, activated ubiquitin is transferred directly from the E2 to the substrate. After multiple rounds, Lys48-linked ubiquitin chains are formed. These represent the canonical recognition motif for proteasomal degradation of the protein substrate. (B) In the CRL5 family, CUL5 can assemble with the RING subunit RBX2 and with numerous specificity subunits (also called substrate receptor, substrate recognition module or substrate targeting protein) involved in the recruitment of specific targets via adaptors (EloB and EloC). RBX2 is involved in the binding of the E2 enzyme that allows substrate ubiquitylation and is required for the covalent attachment of NEDD8 to a lysine residue of CUL5. NEDD8 ligation to CUL5 is enhanced following N-terminal acetylation (Ac) of the E2 NEDD8-conjugating enzyme. When CUL5 is post-translationally modified by NEDD8 (neddylation), activation of the CRL5 occurs. CUL5 inactivation occurs through NEDD8 removal by deneddylation by the CSN.</figcaption></figure>

Protein ubiquitylation is a reversible post-translational modification that has proteolytic and non-proteolytic roles thereby regulating a wide spectrum of biological functions. As such, defects in the ubiquitin pathway can contribute to disease pathogenesis providing the rationale for drug discovery targeting this pathway.

**1. The ubiquitylation cascade**

The ubiquitin molecule is first activated through an ATP-dependent reaction by forming a high-energy thioester bond between the carboxy-terminal glycine residue of ubiquitin and the catalytic cysteine of an E1. Activated ubiquitin is then transferred
to the active site cysteine of an E2, where a second high-energy
thioester linkage is formed. In the last step, an E3 drives the
transfer of ubiquitin to a protein substrate, forming an isopeptide
bond between the ubiquitin carboxy-terminal glycine residue and
substrate lysine residue or forming a peptide bond with the
amino-terminus of the substrate (Fig. 1A) [1]. Indeed, E3s can be
divided into three main classes: (i) N-end rule E3s, (ii) E3s of the
HECT (Homologous to E6-associated protein Carboxyl Terminus)
domain family that function as ubiquitin carriers and (iii) E3s of
the RING (Really Interesting New Gene) domain family. RING-E3s
do not have inherent catalytic activity but use the RING structural
motif to recruit and direct a ubiquitin-charged E2 towards specific
substrates. RING-domain E3s can be further divided into two
subclasses: monomeric E3s, in which a unique protein harbors the
RING-finger motif and the substrate binding domain, and multi-
meric E3s in which RING-finger and substrate binding domains are
assembled together from different individual proteins [2]. Some
protein substrates can be ubiquitylated with only a single ubiq-
uitin molecule on a single lysine residue (monoubiquitylation) or
on multiple lysine residues (multiubiquitylation). After multiple
runs, linear and branched polyubiquitin chains can be formed
(polyubiquitylation) on one or more of the seven lysines of ubiq-
uitin (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63). Depend-
ing on the nature of ubiquitin attachment and the type of linkage
of the polyubiquitin chain, the fate of ubiquitylated proteins varies
[3]. For example, the Lys48-linked chains represent the canonical
recognition motif for degradation of the protein substrate by the
proteasome, whereas the K63-linked chains or mono-
ubiquitylation have non-degradative functions and alter protein
function and subcellular localization.

2. Cullin-RING E3 ubiquitin ligases

CRLs belong to the largest family of E3s and are responsible for
as much as 20% of all ubiquitylated proteins within cells [4] con-
trolling a myriad of functions. Cullins (CUL1, CUL2, CUL3, CUL4a,
CUL4b, CUL5, CUL7 and CUL9 in mammals) are molecular scaffolds
that assemble distinct subunits: (i) a RING finger protein, (ii) a
substrate recognition module containing an adaptor and a speci-
ficity subunit dedicated to the recruitment of specific target(s). In
the case of CRL3 complexes, the adaptor and specificity subunit are
within the same polypeptide. More precisely, the carboxy-terminus
of cullins can bind to a RING finger protein, RBX1 or RBX2 (RING box
protein 1 or 2), also called ROC 1 and 2 (regulator of cullins),
respectively. RBX2 uniquely associates with CUL5, whereas RBX1
binds to the other cullins. The RING domain of RBX1/2 recruits a
ubiquitin-loaded E2 to mediate ubiquitin conjugation. Cullin
amino-terminus binds to distinct adaptor(s) and to a specificity
subunit to recruit protein targets. Indeed, many specificity subunits
are involved in the recruitment of protein targets to be ubiquity-
lated: 69 F-box proteins for CRL1 [5], 80 proteins with a SOCS
(Suppressors Of Cytokine Signalling)-box or a VHL (von Hippel-
Lindau)-box for CRL2/5 [6], 180 BTB (bric-a-brac) proteins for
CRL3 [7], and 90 DCAF (DDB1- and CUL4-associated factor homo-
log) for CRL4A/B [8]. The specificity subunits of CRL1, CRL2, CRL5
and CRL7 use adaptors, a BTB fold protein, the S-phase kinase-
associated protein 1 (SKP1) or the elongin B (EloB)-elongin C
(EloC) complex, to interact with the cullin. The adaptor subunits for
CUL4A/B are composed of DDB1 (DNA Damage-Binding protein 1)
and members of the DCAF (DDB1 CUL4 Associated Factor) family.

Although cullins 2 and 5 share identical adaptors (EloB and EloC), they direct the assembly of distinct E3 complexes: CRL2 with the VHL-box proteins and CRL5 with SOCS-box proteins as specificity subunits (Fig. 1B). These proteins can bind to the Elongin BC complex through a conserved EloC-binding sequence motif (S,T,P,A)

LXXX(C,S,A)XXX(L,V,I,A), named BC-box [9–11]. Further studies
demonstrated that the BC-box is flanked by a CUL2 box (consensus
sequence: ΦΡΧΧΦΧΧΧΦ) or a CUL5 box (consensus sequence:
ΦΧΧLPΦΡΧΧΦΧΧ(Y,F)L) that determine binding specificity for
CUL2-RBX1 and CUL5-RBX2 modules, respectively [11,12].

From an evolutionary perspective, yeast genomes encode three
cullins (CUL1, CUL3 and CUL8 in *Saccharomyces cerevisiae* and CUL1,
CUL3 and CUL4 in *Schizosaccharomyces pombe*). Five cullins are
expressed in *Drosophila* (CUL1 to CUL5), six in *Caenorhabditis ele-
gans* (CUL1-CUL6) and seven in mammals, indicating that cullins
are ancient genes [13].

**3. Regulation of cullin-RING E3 ubiquitin ligases**

CRLs are subject to regulation by several mechanisms. Activation of CRLs occurs when cullins are post-translationally modified by the ubiquitin-like protein NEDD8 (Neural precursor cell Expressed Developmentally Down-regulated protein 8) [14,15]. Conversely, inactivation occurs through NEDD8 removal by deneddylation by the COP9 signalosome complex (CSN) [16,17]. Hence, unneddylated cullins are in an inactive state while neddylated cullins are in an active state. NEDD8 is a protein that is covalently linked to a lysine residue in a C-terminal winged-helix motif in cullins (Lys 724 in CUL5) through an E1 NEDD8-activating enzyme (NAE) – E2 NEDD8-conjugating enzyme cascade. NEDD8 E3 ligases catalyze the transfer of NEDD8 to the protein substrate. Among them, RBX1 and RBX2 are involved in the neddylation of cullins [18,19] resulting in the activation of ubiquitin transfer activity by favoring alternative conformations of the cullin C-terminal domain which place the RING domain of RBX1 or RBX2 in a different orientation [20,21]. This is likely to promote the sequential transfer of ubiquitin molecules to form polyubiquitin chains on substrates by multiple catalytic cycles [22]. Full activation of CRL5 requires acetylation of one of the two E2 NEDD8-conjugating enzymes (Ubc12 or UBE2F) at its N-terminus [23]. Indeed, Ubc12/UBE2F acetylation is important for stimulating neddylation of the CUL5-RBX2 module [19]. Cullin neddylation is enhanced by DCNLs (defective in cullin neddylation protein 1-like proteins) such as DCN1 [24]. In fact, DCN1 increases the recruitment of Ubc12 to the CUL1-RBX1 module and favors the orientation of the RBX1–Ubc12–NEDD8 complex for optimal NEDD8 transfer [25,26]. Although the CSN catalyzes the reaction that removes NEDD8 from cullin, the CSN contributes to maintaining CRL function [17,27]. The CSN is a multi-protein complex whose organization resembles that of the 26S proteasome lid [28]. The subunit 5 [CSN5; also known as c-Jun activation domain-binding protein-1 (Jab1)] harbors a regulated zinc-dependent isopeptidase activity [17,29]. In fact, CRLs undergo cycles of neddylation and neddylation that are required for the CRLs to exert their activity (Fig. 1B).

CRLs are also regulated at the level of their assembly. As
mentioned above, many specificity subunits can assemble with the
same adaptors and Cullin-RBX1/2 modules. Importantly, of a given
abundance of a specificity subunit dictates the assembly of a given
CRL [30]. In this context, it is not surprising that some of them
accumulate at particular cellular or developmental stages such as
specific phases of the cell cycle (e.g. Skp2 for S-phase kinase-
associated protein 2, Fbw7 for F-box WD40 repeat-containing
protein 7, and β-Trcp for β-transducin repeat-containing protein
[31]) or differentiation commitment (e.g. ASB2α (Ankyrin repeat-
containing proteins with a SOCS-Box 2α) and ASB2β proteins
[32–34]). Assembly of CRLs is also dependent upon CAND1 (cullin-
associated and neddylation-dissociated 1) that acts as a specificity
subunit exchange factor [35–37]. Although this has been well-
studied for CRL1, CUL4B and CUL5 were also found to associate
with CAND1 [30]. This mechanism of regulation of CRL assembly
<table><caption>Fig. 2. CRL5 and corresponding substrates.</caption><thead><tr><th>Specificity subunits</th><th>Substrate(s)</th><th>Pathways</th><th>Links or potential links to diseases</th></tr></thead><tbody><tr><td colspan="4"><strong>SH2 domain-containing proteins with a SOCS-box (SOCS)</strong></td></tr><tr><td>CIS</td><td>erythropoietin receptor</td><td>cell signalling</td><td></td></tr><tr><td rowspan="7">SOCS1</td><td>p65/RelA</td><td>transcription</td><td></td></tr><tr><td>FAK</td><td>cell signalling</td><td></td></tr><tr><td>IRS1, IRS2</td><td>cell signalling</td><td>insulin resistance syndromes, diabetes</td></tr><tr><td>JAK2</td><td>cell signalling</td><td></td></tr><tr><td>MAL</td><td>cell signalling</td><td></td></tr><tr><td>TEL-JAK2</td><td>cell signalling</td><td></td></tr><tr><td>Vav</td><td>cell signalling</td><td>leukemia</td></tr><tr><td>SOCS3</td><td>CD33 receptor</td><td>cell signalling</td><td></td></tr><tr><td>SOCS6</td><td>cKit receptor</td><td>cell signalling</td><td></td></tr><tr><td colspan="4"><strong>Ankyrin repeat-containing proteins with a SOCS-box (ASB)</strong></td></tr><tr><td>ASB2α</td><td>FLNa, FLNb, FLNc</td><td>cell motility, cell differentiation</td><td>leukemia</td></tr><tr><td>ASB2β</td><td>MLL</td><td>epigenetic regulation</td><td></td></tr><tr><td>ASB3</td><td>FLNb</td><td>cell differentiation</td><td></td></tr><tr><td>ASB4</td><td>TNF-R2</td><td>cell signalling</td><td></td></tr><tr><td rowspan="2">ASB9</td><td>IRS4</td><td>cell signalling,</td><td>preeclampsia</td></tr><tr><td>ID2</td><td>transcription, cell differentiation</td><td></td></tr><tr><td>ASB11</td><td>Delta A</td><td>metabolism</td><td></td></tr><tr><td colspan="4"><strong>SPRY domain-containing proteins with a SOCS-box (SPSB)</strong></td></tr><tr><td>SPSB1</td><td>iNOS</td><td>innate host response</td><td>chronic and persistent infections</td></tr><tr><td></td><td>TGF-β type II receptor</td><td>cell signalling</td><td></td></tr><tr><td>SPSB2</td><td>iNOS</td><td>innate host response</td><td>chronic and persistent infections</td></tr><tr><td>SPSB4</td><td>iNOS</td><td>innate host response</td><td>chronic and persistent infections</td></tr><tr><td colspan="4"><strong>WD40 repeat-containing proteins with a SOCS-box (WSB)</strong></td></tr><tr><td>WSB1</td><td>HIPK2</td><td>cell signalling</td><td></td></tr><tr><td></td><td>D2</td><td>metabolism</td><td></td></tr><tr><td colspan="4"><strong>Other SOCS-box proteins</strong></td></tr><tr><td>Elongin A</td><td>Rpb1</td><td>transcription</td><td></td></tr><tr><td colspan="4"><strong>Viral proteins with a SOCS-box</strong></td></tr><tr><td>HIV-1 Vif</td><td>APOBEC3F</td><td>antiviral activity</td><td>viral infections</td></tr><tr><td></td><td>APOBEC3G</td><td>antiviral activity</td><td></td></tr><tr><td rowspan="5">E4orf6/E1B55K</td><td>p53</td><td>cell cycle, apoptosis</td><td>viral infections</td></tr><tr><td>Mre1</td><td>DNA repair, recombination</td><td></td></tr><tr><td>DNA ligase IV</td><td>DNA repair, cell signalling</td><td></td></tr><tr><td>Integrin α3</td><td>cell motility</td><td></td></tr><tr><td>Rep52</td><td>viral DNA replication</td><td></td></tr><tr><td>GAM1</td><td>SAE1</td><td>sumoylation</td><td>viral infections</td></tr><tr><td>BLZF1</td><td>p53</td><td>cell cycle, apoptosis</td><td>viral infections</td></tr><tr><td>LANA</td><td>p53</td><td>cell cycle, apoptosis</td><td>viral infections</td></tr><tr><td></td><td>VHL</td><td>ubiquitylation</td><td></td></tr><tr><td>MuHV-4 ORF73</td><td>p65/RelA</td><td>transcription</td><td>viral infections</td></tr></tbody></table>

may be important when the levels of a specificity subunit and/or its substrates changed drastically during specific biological processes to build new CRLs.

## 4. CRL5 and SOCS-box proteins: targeting proteins for polyubiquitylation and subsequent degradation by the proteasome

The SOCS-box protein family comprises several members that are defined by the domain associated with the SOCS-box and which

is involved in substrate recognition: SH2 domain for SOCS proteins (SOCS1-7, CIS1 for Cytokine-Inducible Src homology 2-containing protein), SPRY domain for SPSB (SPRY domain-containing proteins with a SOCS-Box) proteins (SPSB-4), WD40 domain for WSD (WD40 repeat-containing protein with a SOCS-Box) proteins (WSB1 and 2), Ras domain for Rab-40A, Rab-40B and Rab-40C, and ankyrin domains for ASB proteins (ASB1 to 18) [38]. As mentioned above, SOCS-box proteins interact with the Elongin BC complex and the CUL5/RBX2 module to form an E3 ubiquitin ligase complex. It is now well-established that viral proteins such as the human
immunodeficiency virus-1 (HIV-1) viral infectivity factor (Vif) [39], Adenovirus proteins E4orf6 and E1B55K [40], Latency-Associated Nuclear Antigen (LANA) from Kaposi's sarcoma-associated herpes virus (KSHV) [41], can hijack CRL5 complexes to trigger degradation of host proteins. Surprisingly, despite the number of SOCS-box proteins, their substrates remain poorly identified. This is probably due to the weak affinity of substrates for their requisite specificity subunit and because of the labile nature of the substrate complexed with the specificity subunit [42]. Furthermore, it should be mentioned that most experiments have been carried out by overexpression of the SOCS-box protein and/or its substrate candidate, often in non-physiological models. Only CRL5 with known substrates are described below (Fig. 2).

## 4.1. SH2 domain-containing proteins with a SOCS-box (SOCS)

Cytokine-inducible SH2 domain containing protein (CIS) mediates proteasomal degradation of the erythropoietin receptor [43]. SOCS1 is involved in the ubiquitylation and subsequent degradation of Vav [44], the NF-kappaB family member p65/RelA [45], Focal Adhesion Kinase FAK [46] and Myeloid differentiation primary-response gene 88 -Adaptor-Like protein MAL [47]. The Janus Kinase 2 (JAK2) and the TEL-JAK2 oncofusion protein are also substrates of SOCS1 [48–50]. Importantly, inhibitory effect of SOCS1 on TEL-JAK2 transformation of primary mouse hematopoietic cells in vivo was demonstrated indicating that modulation of SOCS1 E3 activity may be therapeutically useful for treatment of hematologic malignances induced by tyrosine kinase fusions [49]. Insulin Receptor Substrates IRS1 and IRS2 are also targeted for degradation by SOCS1 [51]. Therefore, compounds that would protect IRS1 and IRS2 from SOCS1-mediated degradation might have value for the treatment of the insulin resistance syndromes and diabetes. SOCS3 is involved in the degradation of the CD33 receptor [52]. SOCS6 ubiquitylates the c-KIT receptor and the Src kinase Lck leading to their degradation [53,54].

## 4.2. Ankyrin repeat-containing proteins with a SOCS-box (ASB)

We have largely contributed to the understanding of the mechanisms of action of the largest family of SOCS-box containing proteins since the demonstration that ASB2α is the specificity subunit of a CRL5 complex [55]. We further showed that ASB2α drives polyubiquitylation and degradation by the proteasome of the actin-binding protein filaments A, B and C [56–58]. In contrast, filamin B but not filaments A and C, is a substrate of the second ASB2 isoform, ASB2β [33,59]. Although the levels of JAK2 and JAK3 were shown to be affected by ASB2α in overexpression experiments [60,61], neither endogenous nor exogenously expressed ASB2α induces degradation of JAK proteins in hematopoietic cells [62]. The MLL (Mixed Lineage Leukemia) protein was also shown to be degraded by ASB2α [63]. Importantly, the MLL-AF9 fusion protein resulting from the translocation between the MLL and AF9 genes escape from ASB2α-mediated degradation suggesting that ASB2α mis-function may participate in MLL fusion mediated leukemia development [63]. The ASB2 gene is also dysregulated at the transcriptional level in human leukemia. Indeed, ASB2 is a transcriptional target of three acute myeloid leukemia oncoproteins, PML-RARα, PLZF-RARα and AML1-ETO, that act as transcriptional repressors, suggesting that ASB2 repression may participate in the leukemogenesis process [32,64–66]. ASB3 was shown to trigger ubiquitylation of the tumour necrosis factor receptor 2 (TNF-R2) leading to its proteasomal degradation [67]. ASB4 mediates ubiquitylation and degradation of the adaptor molecule IRS4 [68]. ASB4 regulates trophoblast cell differentiation into placental vasculature through the degradation of the transcriptional regulator ID2

(Inhibitor of DNA binding 2) and loss of ASB4 in pregnant mice resulted in vascular dysfunction of the placenta, phenocopying women with preeclampsia [69]. ASB9 regulates creatine kinase B turnover [70]. ASB11 is able to specifically ubiquitylate and degrade Delta A thereby regulating the canonical Delta-Notch signaling pathway [71,72].

## 4.3. SPRY domain-containing proteins with a SOCS-box (SPSB)

SPSB1 enhances polyubiquitylation and degradation of the TGF-β type II receptor [73]. Inducible nitric oxide synthase (iNOS) is a substrate of SPSB1, SPSB2 and SPSB4 proteins [74,75]. SPSB2 knockout macrophages showed prolonged iNOS expression, resulting in enhanced killing of Leishmania major parasites. Therefore, disruption of the SPSB-iNOS interaction may be beneficial in chronic and persistent infections such as leishmaniasis [74]. Expression of SPSB1 is also linked to cancer. Indeed, SPSB1 expression correlated with c-MET activity in human breast cancers and with increased risk of relapse in breast cancer patients [76].

## 4.4. WD40 repeat-containing proteins with a SOCS-box (WSB)

WSB1 regulates ubiquitylation and degradation of the HIPK2 kinase [77] and of the thyroid-hormone-activating type 2 iodothyronine deiodinase (D2) [78].

## 4.5. Other SOCS-box proteins

Elongin A can assemble in a CRL5 complex to drive ubiquitylation and degradation of Rpb1 following UV irradiation [79].

## 4.6. Viral proteins with a SOCS-box

The Vif protein of HIV-1 is the specificity subunit of a CRL5 [80] that polyubiquitylates the cellular intrinsic restriction factors APOBEC3F [81] and APOBEC3G [39]. The E4orf6 and E1B55K proteins from human adenovirus type 5 can assemble in an active CRL5 complex [82–84] that recognize different proteins to be degraded: p53 [83,84], meiotic recombination 11 (Mre11) [85], DNA ligase IV [86], integrin α3 [87] and adeno-associated virus type 5 Rep52 and capsid proteins [88]. The Gam1 protein of the avian adenovirus CELO (chicken embryo lethal orphan) promotes ubiquitylation and degradation of the SUMO E1 subunit SAE1 [89]. Unexpectedly, Gam1 can assemble in a CRL2 or a CRL5 complex [89]. The transcriptional transactivator of the Epstein-Barr virus BZLF1 can bind to CUL2 and CUL5 leading to p53 degradation [90,91]. The LANA protein from KSHV induces the degradation of VHL and p53 [41]. The ORF73 protein encoded by the Murid Herpes Virus-4 (MuHV-4) targets p65/RelA to proteasomal degradation [92]. Through polyubiquitylation and proteasomal degradation of host proteins, all these viral proteins create a more amenable cellular environment for viral propagation.

# 5. Targeting CRL5 for therapeutic interventions

Proteasome inhibition has proved to be of therapeutic utility [93]. A small molecule called CC0651 is a specific inhibitor of the human E2 enzyme Cdc34A that inhibits the proliferation of human cancer cell lines [94]. Indeed, CC0651 stabilizes the normally low-affinity interaction between the E2 and ubiquitin at the donor site thereby inhibiting E2 catalytic activity [95]. The E2 enzymatic step in the ubiquitylation cascade was therefore proposed to be susceptible to small-molecule intervention. While these approaches affected steps common to many E3s, the strategy of modulating the activity of E3 ubiquitin ligases appears to be more
specific. Given the role of CRLs in many biological pathways, CRL dysfunction is likely to be involved in the development of diseases. Because CRL5s are hijacked by viruses, the inhibition of their activity holds also promise as an antiviral therapeutic.

MLN4924 is a small molecule that binds covalently to NAE thereby blocking its enzymatic activity and inhibiting cullin neddylation [4,96]. MLN4924 is currently in clinical trials and showed clinical activity in some solid tumor and hematologic malignancies. Indeed, this compound inhibits tumor cell growth by inducing apoptosis in diffuse large B-cell lymphoma models [97], in chronic lymphocytic leukemia B cells [98], in acute myeloid leukemia [99], in multiple myeloma [100], in Ewing sarcoma [101], or by inducing irreversible senescence [102]. MLN4924 is also cytotoxic to KSHV-infected lymphoma cells showing that inhibition of neddylation represents a novel approach for the treatment of KSHV-associated malignancies [103]. Importantly, MLN4924 induces autophagy in several human cancer lines [104,105]. This constitutes an overall survival signal for tumor cells. Consequently, abrogation of autophagy enhanced MLN4924-induced apoptosis and growth suppression suggesting that combination of MLN4924 with autophagy inhibitors may enhance therapeutic efficacy of MLN4924 [105]. Furthermore, MLN4924 induces autophagy and apoptosis to suppress liver cancer cell growth in a xenograft mouse model indicating that MLN4924 exhibits an antitumor activity in vivo [104]. Interestingly, MLN4924 was shown to be an effective radiosensitizing agent in breast cancer cell lines [106] and in pancreatic cancer both in vitro and in vivo [107]. A synergic cytotoxic effect between cisplatin and MLN4924 in both platinum-sensitive and platinum-resistant ovarian cancer cells was also reported [108,109]. This has been furthered to other DNA-damaging agents such as mitomycin C, cytarabine, SN-38 and gemcitabine [110,111]. Altogether, these studies highlight the therapeutic potential of MLN4924 in preclinical cancer models and provide a strong rationale for clinical investigations of antitumor agent and MLN4924 combinations in cancer therapy. However, not surprisingly, heterozygous mutations in the NAEβ (UBA3) subunit of NAE in cell lines and xenograft models of cancer can be observed following selection pressure with MLN4924 [112,113]. Therefore, second-generation NAE inhibitors may be developed to overcome resistance to MLN4924 mediated by mutations in NAEβ.

Since E3s determine the specificity of protein substrates they represent an important class of potential drug targets for pharmaceutical intervention. Through the degradation of several substrates, CRL1 control a multitude of processes including cell cycle progression and their dysregulation is implicated in many malignancies. Most studies have focused on the development of anticancer therapeutics by targeting CRL1 and some compound are currently at the preclinical stage of development [114,115]. Indeed, effective modulation of the activity of CRL5 for therapeutic means will benefit from our understanding of the structural architecture of CRL5 complexes and of the molecular mechanisms of substrate binding to specificity subunits. Indeed, crystal structures of components of CRL5 have been solved: SOCS2-EloB-EloC [116], SOCS4-EloB-EloC [117], SOCS2-EloB-EloC-CUL5 [118], ASB9-EloB-EloC-Cul5 [119,120] and HIV-1 Vif-CBFβ-CUL5-EloB-EloC [121]. Using the crystallographic structures of the SOCS2-EloB-EloC [116] and CUL1-RBX1-SKP1-SKP2 [122] complexes, we modeled the ASB2-α-EloB-EloC-CUL5-RBX2 E3 complex bound to an E2 enzyme [62]. Small molecule inhibitors of E3s often act by disrupting the interface between the specificity subunit and its substrate. Because CRLs may have multiple specific substrates, identification of the substrate whose degradation drives the biological functions of the CRL is crucial for targeting approaches. In this context, it is crucial to determine whether the ubiquitin ligase activity of the E3 drives polyubiquitylation and degradation of the substrate candidate in

physiological relevant settings. Indeed, selectivity of an E3 for a given substrate may be cell-type specific, dependent upon subcellular localization, timely regulated (e.g. cell cycle progression, differentiation, developmental stages, embryogenesis vs adult life, ageing ...) or regulated by extracellular signals from the microenvironment. Furthermore, deciphering the structural determinants for recognition of the substrates by the specificity subunit is also key step for the identification of targetable interfaces and for the design and/or optimization of inhibitors.

Despite the large number of CRL5, their specific substrates remain poorly identified (Fig. 2) and many remain with no established substrates. This has somehow limited the development of compounds targeting CRL5. One of them, the small molecule RN-18 antagonizes HIV-1 Vif function and inhibits viral replication only in the presence of APOBEC3G without affecting general ubiquitin-mediated protein degradation [123]. The HIV-1 Vif-APOBEC3G axis can also be modulated using the small inhibitor VEC-5 through inhibition of the binding of EloC to Vif [124]. Through inhibition of the degradation of the host antiviral factor APOBEC3G, these compounds made the proof-of-principle that blocking the activity of hijacked E3s could lead to the development of novel antiviral therapies.

## 6. Conclusion

Thanks to their high specificity for substrates, CRL5 represent promising drug targets. The understanding of the biological roles of CRL, the identification of their specific substrates and mechanism of recruitment, as well as the knowledge of CRL assembly and regulation will undoubtedly contribute to additional new drug targets.

## Acknowledgments

We apologize to our colleagues whose work we were not able to cover in this review because of space constraints. Research in our laboratory related to the subject of this review was supported by the Centre National de la Recherche Scientifique, the University of Toulouse, the Agence Nationale de la Recherche, the Comité Midi-Pyrénées de la Ligue contre le Cancer, the Fondation ARC, the Association Française contre les Myopathies, the Fondation pour la Recherche Médicale, the association Centpoursanglavie and the RITC Foundation.

## References

[1] A. Hershko, A. Ciechanover, The ubiquitin system, Annu. Rev. Biochem. 67 (1998) 425-479.

[2] M.B. Metzger, V.A. Hristova, A.M. Weissman, HECT and RING finger families of E3 ubiquitin ligases at a glance, J. Cell Sci. 125 (2012) 531-537.

[3] D. Komander, M. Rape, The ubiquitin code, Annu. Rev. Biochem. 81 (2012) 203-229.

[4] T.A. Soucy, P.G. Smith, M.A. Milhollen, A.J. Berger, J.M. Gavin, S. Adhikari, J.E. Brownell, K.E. Burke, D.P. Cardin, S. Critchley, C.A. Cullis, A. Doucette, J.J. Garnsey, J.L. Gaulin, R.E. Gershman, A.R. Lublinsky, A. McDonald, H. Mizutani, U. Narayanan, E.J. Olhava, S. Peluso, M. Rezaei, M.D. Sintchak, T. Talreja, M.P. Thomas, T. Traore, S. Vyskocil, G.S. Weatherhead, J. Yu, J. Zhang, L.R. Dick, C.F. Claiborne, M. Rolfe, J.B. Bolen, S.P. Langston, An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer, Nature 458 (2009) 732-736.

[5] J. Jin, T. Cardozo, R.C. Lovering, S.J. Elledge, M. Pagano, J.W. Harper, Systematic analysis and nomenclature of mammalian F-box proteins, Genes Dev. 18 (2004) 2573-2580.

[6] E.M. Linossi, S.E. Nicholson, The SOCS box-adapting proteins for ubiquitination and proteasomal degradation, IUBMB Life 64 (2012) 316-323.

[7] P.J. Stogios, G.S. Downs, J.J. Jauhal, S.K. Nandra, G.G. Prive, Sequence and structural analysis of BTB domain proteins, Genome Biol. 6 (2005) R82.

[8] Y.J. He, C.M. McCall, J. Hu, Y. Zeng, Y. Xiong, DDB1 functions as a linker to recruit receptor WD40 proteins to CUL4-ROC1 ubiquitin ligases, Genes Dev. 20 (2006) 2949-2954.

[9] A. Kibel, O. Iliopoulos, J.A. DeCaprio, W.G. Kaelin Jr., Binding of the von Hippel-Lindau tumor suppressor protein to Elongin B and C, Science 269
(1995) 1444–1446.

[10] T. Kishida, T.M. Stackhouse, F. Chen, M.I. Lerman, B. Zbar, Cellular proteins that bind the von Hippel-Lindau disease gene product: mapping of binding domains and the effect of missense mutations, Cancer Res. 55 (1995) 4544–4548.

[11] T. Kamura, S. Sato, D. Haque, L. Liu, W.G. Kaelin Jr., R.C. Conaway, J.W. Conaway, The Elongin BC complex interacts with the conserved SOCS-box motif present in members of the SOCS, ras, WD-40 repeat, and ankyrin repeat families, Genes Dev. 12 (1998) 3872–3881.

[12] N. Mahrour, W.B. Redwine, L. Florens, S.K. Swanson, S. Martin-Brown, W.D. Bradford, K. Staehling-Hampton, M.P. Washburn, R.C. Conaway, J.W. Conaway, Characterization of cullin-box sequences that direct recruitment of CUL2-RBX1 and CUL5-RBX2 modules to Elongin BC-based ubiquitin ligases, J. Biol. Chem. 283 (2008) 8005–8013.

[13] A. Sarikas, T. Hartmann, Z.Q. Pan, The cullin protein family, Genome Biol. 12 (2011) 220.

[14] M.A. Read, J.E. Brownell, T.B. Gladysheva, M. Hottelet, L.A. Parent, M.B. Coggins, J.W. Pierce, V.N. Podust, R.S. Luo, V. Chau, V.J. Palombella, NEDD8 modification of cul-1 activates SCF(β-TrCP)-dependent ubiquitination of IκBα, Mol. Cell Biol. 20 (2000) 2326–2333.

[15] J.R. Lydeard, B.A. Schulman, J.W. Harper, Building and remodelling Cullin-RING E3 ubiquitin ligases, EMBO Rep. 14 (2003) 1050–1061.

[16] S. Lyapina, G. Cope, A. Shevchenko, G. Serino, T. Tsuge, C. Zhou, D.A. Wolf, N. Wei, R.J. Deshaies, Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome, Science 292 (2001) 1382–1385.

[17] G.A. Cope, G.S. Suh, L. Aravind, S.E. Schwarz, S.L. Zipursky, E.V. Koonin, R.J. Deshaies, Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of NEDD8 from Cul1, Science 298 (2002) 608–611.

[18] T. Kamura, M.N. Conrad, Q. Yan, R.C. Conaway, J.W. Conaway, The Rbx1 subunit of SCF and VHL E3 ubiquitin ligase activates Rub1 modification of cullins Cdc53 and CUL2, Genes Dev. 13 (1999) 2928–2933.

[19] D.T. Huang, O. Ayrault, H.W. Hunt, A.M. Taherbhoy, D.M. Duda, D.C. Scott, L.A. Borg, G. Neale, P.J. Murray, M.F. Roussel, B.A. Schulman, E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification, Mol. Cell 33 (2009) 483–495.

[20] D.M. Duda, L.A. Borg, D.C. Scott, H.W. Hunt, M. Hammel, B.A. Schulman, Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation, Cell 134 (2008) 995–1006.

[21] B.K. Boh, P.G. Smith, T. Hagen, Neddylation-induced conformational control regulates cullin RING ligase activity in vivo, J. Mol. Biol. 409 (2011) 136–145.

[22] N.W. Pierce, G. Kleiger, S.O. Shan, R.J. Deshaies, Detection of sequential polyubiquitylation on a millisecond timescale, Nature 462 (2009) 615–619.

[23] J.K. Monda, D.C. Scott, D.J. Miller, J. Lydeard, D. King, J.W. Harper, E.J. Bennett, B.A. Schulman, Structural conservation of distinctive N-terminal acetylation-dependent interactions across a family of mammalian NEDD8 ligation enzymes, Structure 21 (2013) 42–53.

[24] T. Kurz, Y.C. Chou, A.R. Willems, N. Meyer-Schaller, M.L. Hecht, M. Tyers, M. Peter, F. Sicheri, DCN1 functions as a scaffold-type E3 ligase for cullin neddylation, Mol. Cell 29 (2008) 23–35.

[25] D.C. Scott, J.K. Monda, E.J. Bennett, J.W. Harper, B.A. Schulman, N-terminal acetylation acts as an avidity enhancer within an interconnected multi-protein complex, Science 334 (2011) 674–678.

[26] D.C. Scott, V.O. Siderskiy, J.K. Monda, J.R. Lydeard, S.E. Cho, J.W. Harper, B.A. Schulman, Structure of a RING E3 trapped in action reveals ligation mechanism for the ubiquitin-like protein NEDD8, Cell 157 (2014) 1671–1684.

[27] L. Pintard, T. Kurz, S. Glaser, J.H. Willis, M. Peter, B. Bowerman, Neddylation and deneddylation of CUL3 is required to target MEI-1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans, Curr. Biol. 13 (2003) 911–921.

[28] M. Sharon, H. Mao, E. Boeri Erba, E. Stephens, N. Zheng, C.V. Robinson, Symmetrical modularity of the COP9 signalosome complex suggests its multifunctionality, Structure 17 (2009) 31–40.

[29] A. Echalier, Y. Pan, M. Birol, N. Tavernier, L. Pintard, F. Hoh, C. Ebel, N. Galophe, F.X. Claret, C. Dumas, Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 1273–1278.

[30] E.J. Bennett, J. Rush, S.P. Gygi, J.W. Harper, Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics, Cell 143 (2010) 951–965.

[31] L.K. Teixeira, S.I. Reed, Ubiquitin ligases and cell cycle control, Annu. Rev. Biochem. 82 (2013) 387–414.

[32] F.C. Guibal, C. Moog-Lutz, P. Smolewski, Y. Di Gioia, Z. Darzynkiewicz, P.G. Lutz, Y.E. Cayre, ASB2 inhibits growth and promotes commitment in myeloid leukemia cells, J. Biol. Chem. 277 (2002) 218–224.

[33] N.F. Bello, I. Lamsoul, M.L. Heuze, A. Metais, G. Moreaux, D.A. Calderwood, D. Duprez, C. Moog-Lutz, P.G. Lutz, The E3 ubiquitin ligase specificity subunit ASB2β is a novel regulator of muscle differentiation that targets filamin B to proteasomal degradation, Cell Death Differ. 16 (2009) 921–932.

[34] I. Lamsoul, A. Metais, E. Gouot, M.L. Heuze, A.M. Lennon-Dumenil, C. Moog-Lutz, P.G. Lutz, ASB2α regulates migration of inactive dendritic cells, Blood 122 (2013) 533–541.

[35] M.W. Schmidt, P.R. McQuary, S. Wee, K. Hofmann, D.A. Wolf, F-box-directed CRL complex assembly and regulation by the CSN and CAND1, Mol. Cell 35 (2009) 586–597.

[36] N.W. Pierce, J.E. Lee, X. Liu, M.J. Sweredoski, R.L. Graham, E.A. Larimore, M. Rome, N. Zheng, B.E. Curman, S.E. Hess, S.O. Shan, R.J. Deshaies, CAND1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins, Cell 153 (2013) 206–215.

[37] S. Wu, W. Zhu, T. Nhan, J.I. Toth, M.D. Petroski, D.A. Wolf, CAND1 controls in vivo dynamics of the cullin 1-RING ubiquitin ligase repertoire, Nat. Commun. 4 (2013) 1642.

[38] D.J. Hilton, R.T. Richardson, W.S. Alexander, E.M. Viney, T.A. Willson, N.S. Sprigg, R. Starr, S.E. Nicholson, D. Metcalf, N.A. Nicola, Twenty proteins containing a C-terminal SOCS box form five structural classes, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 114–119.

[39] X. Yu, Y. Yu, B. Liu, K. Luo, W. Kong, P. Mao, X.F. Yu, Induction of APOBEC3G ubiquitination and degradation by an HIV-1 Vif-CUL5-SCF complex, Science 302 (2003) 1056–1060.

[40] E. Querido, P. Blanchette, Q. Yan, T. Kamura, M. Morrison, D. Boivin, W.G. Kaelin, R.C. Conaway, J.W. Conaway, P.E. Branton, Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a cullin-containing complex, Genes Dev. 15 (2001) 3104–3117.

[41] Q.L. Cai, J.S. Knight, S.C. Verma, P. Zald, E.S. Robertson, EC55 ubiquitin complex is recruited by KSHV latent antigen LANA for degradation of the VHL and p53 tumor suppressors, PLoS Pathog. 2 (2006) e116.

[42] J.Jin, X.L. Ang, T. Shirogane, J. Wade Harper, Identification of substrates for F-box proteins, Methods Enzymol. 399 (2005) 287–309.

[43] F. Verdier, S. Chretien, O. Muller, P. Varlet, A. Yoshimura, S. Gisselbrecht, C. Lacombe, P. Mayeux, Proteasomes regulate erythropoietin receptor and signal transducer and activator of transcription 5 (STAT5) activation. Possible involvement of the ubiquitinated Cis protein, J. Biol. Chem. 273 (1998) 28185–28190.

[44] P. De Sepulveda, S. Ilangumaran, R. Rottapel, Suppressor of cytokine signaling-1 inhibits VAV function through protein degradation, J. Biol. Chem. 275 (2000) 14005–14008.

[45] A. Ryo, F. Suizu, Y. Yoshida, K. Perrem, Y.C. Liou, G. Wulf, R. Rottapel, S. Yamaoka, K.P. Lu, Regulation of NF-κB signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA, Mol. Cell 12 (2003) 1413–1426.

[46] E. Liu, J.F. Cote, K. Vuori, Negative regulation of FAK signaling by SOCS proteins, Embo J. 22 (2003) 5036–5046.

[47] A. Mansell, R. Smith, S.L. Doyle, P. Gray, J.E. Fenner, P.J. Crack, S.E. Nicholson, D.J. Hilton, L.A. O'Neill, P.J. Hertzog, Suppressor of cytokine signaling 1 negatively regulates Toll-like receptor signaling by mediating Mal degradation, Nat. Immunol. 7 (2006) 148–155.

[48] S. Kamizono, T. Hanada, H. Yasukawa, S. Minoguchi, R. Kato, M. Minoguchi, K. Hattori, S. Hatakeyama, M. Yada, S. Morita, T. Kitamura, H. Kato, K. Nakayama, A. Yoshimura, The SOCS box of SOCS1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2, J. Biol. Chem. 276 (2001) 12530–12538.

[49] J. Frantsve, J. Schwaller, D.W. Sternberg, J. Kutok, D.G. Gilliland, Socs-1 inhibits TEL-JAK2-mediated transformation of hematopoietic cells through inhibition of JAK2 kinase activity and induction of proteasome-mediated degradation, Mol. Cell Biol. 21 (2001) 3547–3557.

[50] D. Ungureanu, P. Saharinen, I. Juntilta, D.J. Hilton, O. Silvennoinen, Regulation of Jak2 through the ubiquitin-proteasome pathway involves phosphorylation of JAK2 on Y1007 and interaction with SOCS1, Mol. Cell Biol. 22 (2002) 3316–3326.

[51] L. Rui, M. Yuan, D. Frantz, S. Shoelson, M.F. White, SOCS1 and SOCS3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2, J. Biol. Chem. 277 (2002) 42394–42398.

[52] S.J. Orr, N.M. Morgan, J. Elliott, J.F. Burrows, C.J. Scott, D.W. McVicar, J.A. Johnston, CD33 responses are blocked by SOCS3 through accelerated proteasomal-mediated turnover, Blood 109 (2007) 1061–1068.

[53] F. Zadjali, A.C. Pike, M. Vesterlund, J. Sun, C. Wu, S.S. Li, L. Ronnstrand, S. Knapp, A.N. Bullock, A. Flores-Morales, Structural basis for c-KIT inhibition by the suppressor of cytokine signaling 6 (SOCS6) ubiquitin ligase, J. Biol. Chem. 286 (2011) 480–490.

[54] Y.B. Choi, M. Son, M. Park, J. Shin, Y. Yun, SOCS-6 negatively regulates T cell activation through targeting p56lck to proteasomal degradation, J. Biol. Chem. 285 (2010) 7271–7280.

[55] M.L. Heuze, F.C. Guibal, C.A. Banks, J.W. Conaway, R.C. Conaway, Y.E. Cayre, A. Benecke, P.G. Lutz, ASB2 is an elongin BC-interacting protein that can assemble with cullin 5 and RBX1 to reconstitute an E3 ubiquitin ligase complex, J. Biol. Chem. 280 (2005) 5468–5474.

[56] M.L. Heuze, I. Lamsoul, M. Baldassarre, Y. Lad, S. Leveque, Z. Razinia, C. Moog-Lutz, D.A. Calderwood, P.G. Lutz, ASB2 targets filamins A and B to proteasomal degradation, Blood 112 (2008) 5130–5140.

[57] C.F. Burande, M.L. Heuze, I. Lamsoul, B. Monsarrat, S. Uttenweiler-Joseph, P.G. Lutz, A label-free quantitative proteomics strategy to identify E3 ubiquitin ligase substrates targeted to proteasome degradation, Mol. Cell Proteomics 8 (2009) 1719–1727.

[58] M. Baldassarre, Z. Razinia, C.F. Burande, I. Lamsoul, P.G. Lutz, D.A. Calderwood, Filamins regulate cell spreading and initiation of cell migration, PLoS One 4 (2009) e7830.

[59] I. Lamsoul, C.F. Burande, Z. Razinia, T.C. Houles, D. Menoret, M. Baldassarre, M. Erard, C. Moog-Lutz, D.A. Calderwood, P.G. Lutz, Functional and structural insights into ASB2α, a novel regulator of integrin-dependent adhesion of hematopoietic cells, J. Biol. Chem. 286 (2011) 30571–30581.

[60] L. Nie, Y. Zhao, W. Wu, Y.Z. Yang, H.C. Wang, X.H. Sun, Notch-induced ASB2
expression promotes protein ubiquitination by forming non-canonical E3 ligase complexes, Cell Res. 21 (2011) 754–769.

[61] W. Wu, X.H. Sun, A mechanism underlying NOTCH-induced and ubiquitin-mediated JAK3 degradation, J. Biol. Chem. 286 (2011) 41153–41162.

[62] I. Lamsoul, M. Erard, P.F. van der Ven, P.G. Lutz, Filamins but not janus kinases are substrates of the ASB2α cullin-ring E3 ubiquitin ligase in hematopoietic cells, PLoS One 7 (2012) e43798.

[63] J.H. Martens, A.B. Brinkman, F. Simmer, K.J. Francois, A. Nebbioso, F. Ferrara, L. Altucci, H.G. Stunnenberg, PML-RARα/RXR alters the epigenetic landscape in acute promyelocytic leukemia, Cancer Cell 17 (2010) 173–185.

[64] J.H. Martens, A. Mandoli, F. Simmer, B.J. Wierenga, S. Saeed, A.A. Singh, L. Altucci, E. Vellenga, H.G. Stunnenberg, ERG and FLI1 binding sites demarcate targets for aberrant epigenetic regulation by AML1-ETO in acute myeloid leukemia, Blood 120 (2012) 4038–4048.

[65] S. Spicuglia, C. Vincent-Fabert, T. Benoukraf, G. Tiberi, A.J. Saurin, J. Zacarias-Cabeza, D. Grimwade, K. Mills, B. Calmels, F. Bertucci, M. Sieweke, P. Ferrier, E. Duprez, Characterisation of Genome-Wide PLZF/RARA Target Genes, PLoS One 6 (2011) e24176.

[66] A.S. Chung, Y.J. Guan, Z.L. Yuan, J.E. Albina, Y.E. Chin, Ankyrin repeat and SOCS box 3 (ASB3) mediates ubiquitination and degradation of tumor necrosis factor receptor II, Mol. Cell Biol. 25 (2005) 4716–4726.

[67] J.Y. Li, B. Chai, W. Zhang, X. Wu, C. Zhang, D. Fritze, Z. Xia, C. Patterson, M.W. Mulholland, Ankyrin repeat and SOCS box containing protein 4 (ASB4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation, BMC Neurosci. 12 (2011) 95.

[68] W.H. Townley-Tilson, Y. Wu, J.E. Ferguson 3rd, C. Patterson, The ubiquitin ligase ASB4 promotes trophoblast differentiation through the degradation of ID2, PLoS One 9 (2014) e89451.

[69] M.A. Debrincat, J.G. Zhang, T.A. Willson, J. Silke, L.M. Connolly, R.J. Simpson, W.S. Alexander, N.A. Nicola, B.T. Kile, D.J. Hilton, Ankyrin repeat and suppressors of cytokine signaling box protein ASB9 targets creatine kinase B for degradation, J. Biol. Chem. 282 (2007) 4728–4737.

[70] S.H. Diks, M.A. Sartori da Silva, J.L. Hillebrands, R.J. Bink, H.H. Versteeg, C. van Rooijen, A. Brouwers, A.B. Chitnis, M.P. Peppelenbosch, D. Zivkovic, d-ASB11 is an essential mediator of canonical Delta-Notch signalling, Nat. Cell Biol. 10 (2008) 1190–1198.

[71] M.A. Sartori da Silva, J.M. Tee, J. Paridaen, A. Brouwers, V. Runtuwene, D. Zivkovic, S.H. Diks, D. Guardavaccaro, M.P. Peppelenbosch, Essential role for the d-ASB11 CUL5 box domain for proper notch signaling and neural cell fate decisions *in vivo*, PLoS One 5 (2010) e14023.

[72] S. Liu, T. Nheu, R. Luwor, S.E. Nicholson, H.J. Zhu, SPSB1, a novel negative regulator of the TGF-β signaling pathway targeting the Type II receptor, J. Biol. Chem. 290 (2015) 17894–17908.

[73] Z. Kuang, R.S. Lewis, J.M. Curtis, Y. Zhan, B.M. Saunders, J.J. Babon, T.B. Kolesnik, A. Low, S.L. Masters, T.A. Willson, L. Kedzierski, S. Yao, E. Handman, R.S. Norton, S.E. Nicholson, The SPRY domain-containing SOCS box protein SPSB2 targets iNOS for proteasomal degradation, J. Cell Biol. 190 (2010) 129–141.

[74] T. Nishiya, K. Matsumoto, S. Maekawa, E. Kajita, T. Horinouchi, M. Fujimuro, K. Ogasawara, T. Uehara, S. Miwa, Regulation of inducible nitric-oxide synthesis by the SPRY domain- and SOCS box-containing proteins, J. Biol. Chem. 286 (2011) 9009–9019.

[75] Y. Feng, T.C. Pan, D.K. Pant, K.R. Chakrabarti, J.V. Alvarez, J.R. Ruth, L.A. Chodosh, SPSB1 promotes breast cancer recurrence by potentiating c-MET signaling, Cancer Discov. 4 (2014) 790–803.

[76] D.W. Choi, Y.M. Seo, E.A. Kim, K.S. Sung, J.W. Ahn, S.J. Park, S.R. Lee, C.Y. Choi, Ubiquitination and degradation of homeodomain-interacting protein kinase 2 by WD40 repeat/SOCS box protein WSB-1, J. Biol. Chem. 283 (2008) 4682–4689.

[77] M. Dentice, A. Bandyopadhyay, B. Gereben, I. Callebaut, M.A. Christoffolete, B.W. Kim, S. Nissim, J.P. Mornon, A.M. Zavacki, A. Zeold, L.P. Capelo, C. Curcio-Morelli, R. Ribeiro, J.W. Harney, C.J. Tabin, A.C. Bianco, The Hedgehog-inducible ubiquitin ligase subunit WSB-1 modulates thyroid hormone activation and PTHrP secretion in the developing growth plate, Nat. Cell Biol. 7 (2005) 698–705.

[78] T. Yasukawa, T. Kamura, S. Kitajima, R.C. Conaway, J.W. Conaway, T. Aso, Mammalian Elongin A complex mediates DNA-damage-induced ubiquitylation and degradation of Rpb1, EMBO J. 27 (2008) 3256–3266.

[79] Y. Yu, Z. Xiao, E.S. Ehrlich, X. Yu, X.F. Yu, Selective assembly of HIV-1 Vif-Cul5-ElonginB-ElonginC E3 ubiquitin ligase complex through a novel SOCS box and upstream cysteines, Genes Dev. 18 (2004) 2867–2872.

[80] B. Liu, P.T. Sarkis, K. Luo, Y. Yu, X.F. Yu, Regulation of Apobec3F and human immunodeficiency virus type 1 Vif by Vif-CUL5-ElonB/C E3 ubiquitin ligase, J. Virol. 79 (2005) 9579–9587.

[81] J.N. Harada, A. Shevchenko, D.C. Pallas, A.J. Berk, Analysis of the adenovirus E1B-55K-anchored proteome reveals its link to ubiquitination machinery, J. Virol. 76 (2002) 9194–9206.

[82] P. Blanchette, C.Y. Cheng, Q. Yan, G. Ketner, D.A. Ornelles, T. Dobner, R.C. Conaway, J.W. Conaway, P.E. Branton, Both BC-box motifs of adenovirus protein E4orf6 are required to efficiently assemble an E3 ligase complex that degrades p53, Mol. Cell Biol. 24 (2004) 9619–9629.

[83] P. Blanchette, C.Y. Cheng, Q. Yan, G. Ketner, D.A. Ornelles, T. Dobner, R.C. Conaway, J.W. Conaway, P.E. Branton, Both BC-box motifs of adenovirus protein E4orf6 are required to efficiently assemble an E3 ligase complex that degrades p53, Mol. Cell Biol. 24 (2004) 9619–9629.

[84] K. Luo, E. Ehrlich, Z. Xiao, W. Zhang, G. Ketner, X.F. Yu, Adenovirus E4orf6 assembles with Cullin5-ElonginB-ElonginC E3 ubiquitin ligase through an

HIV/SIV Vif-like BC-box to regulate p53, FASEB J. 21 (2007) 1742–1750.

[85] T.H. Stracker, C.T. Carson, M.D. Weitzman, Adenovirus oncoproteins inactivate the Mre11-Rad50-NBS1 DNA repair complex, Nature 418 (2002) 348–352.

[86] A. Baker, K.J. Rohleder, L.A. Hanakahi, G. Ketner, Adenovirus E4 34k and E1b 55k oncoproteins target host DNA ligase IV for proteasomal degradation, J. Virol. 81 (2007) 7034–7040.

[87] F. Dallaire, P. Blanchette, P. Groitl, T. Dobner, P.E. Branton, Identification of integrin α3 as a new substrate of the adenovirus E4orf6/E1B 55-kilodalton E3 ubiquitin ligase complex, J. Virol. 83 (2009) 5329–5338.

[88] R. Nayak, D.J. Pintel, Positive and negative effects of adenovirus type 5 helper functions on adeno-associated virus type 5 (AAV5) protein accumulation govern AAV5 virus production, J. Virol. 81 (2007) 2205–2212.

[89] R. Boggio, A. Passafaro, S. Chiocca, Targeting SUMO E1 to ubiquitin ligases: a viral strategy to counteract sumoylation, J. Biol. Chem. 282 (2007) 15376–15382.

[90] Y. Sato, T. Kamura, N. Shirata, T. Murata, A. Kudoh, S. Iwahori, S. Nakayama, H. Isomura, Y. Nishiyama, T. Tsurumi, Degradation of phosphorylated p53 by viral protein-EC5 E3 ligase complex, PLoS Pathog. 5 (2009) e1000530.

[91] Y. Sato, N. Shirata, A. Kudoh, S. Iwahori, S. Nakayama, T. Murata, H. Isomura, Y. Nishiyama, T. Tsurumi, Expression of Epstein-Barr virus BZLF1 immediate- early protein induces p53 degradation independent of MDM2, leading to repression of p53-mediated transcription, Virology 388 (2009) 204–211.

[92] L. Rodrigues, J. Filipe, M.P. Seldon, L. Fonseca, J. Anrather, M.P. Soares, J.P. Simas, Termination of NF-κB activity through a gammaherpesvirus protein that assembles an EC55 ubiquitin-ligase, EMBO J. 28 (2009) 1283–1295.

[93] L.R. Dick, P.E. Fleming, Building on bortezomib: second-generation proteasome inhibitors as anti-cancer therapy, Drug Discov. Today 15 (2010) 243–249.

[94] D.F. Ceccarelli, X. Tang, B. Pelletier, S. Orlicky, W. Xie, V. Plantevin, D. Neculai, Y.C. Chou, A. Ogunjimi, A. Al-Hakim, X. Varelas, J. Koszela, G.A. Wasney, M. Vedadi, S. Dhe-Paganon, S. Cox, S. Xu, A. Lopez-Girona, F. Mercurio, J. Wrana, D. Durocher, S. Meloche, D.R. Webb, M. Tyers, F. Sicheri, An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme, Cell 145 (2011) 1075–1087.

[95] H. Huang, D.F. Ceccarelli, S. Orlicky, D.J. St-Cyr, A. Ziemba, P. Garg, S. Plamondon, M. Auer, S. Sidhu, A. Marinier, G. Kleiger, M. Tyers, F. Sicheri, E2 enzyme inhibition by stabilization of a low-affinity interface with ubiquitin, Nat. Chem. Biol. 10 (2014) 156–163.

[96] J.E. Brownell, M.D. Sintchak, J.M. Gavin, H. Liao, F.J. Bruzzese, N.J. Bump, T.A. Soucy, M.A. Milhollen, X. Yang, A.L. Burkhardt, J. Ma, H.K. Loke, T. Lingaraj, D. Wu, K.B. Hamman, J.J. Spelman, C.A. Cullis, S.P. Langston, S. Vyskocil, T.B. Sells, W.D. Mallender, I. Visiers, P. Li, C.F. Claiborne, M. Rolfe, J.B. Lolen, R.D. Dick, Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8-AMP mimetic *in situ*, Mol. Cell 37 (2010) 102–111.

[97] M.A. Milhollen, T. Traore, J. Adams-Duffy, M.P. Thomas, A.J. Berger, L. Dang, L.R. Dick, J.J. Garnsey, E. Koenig, S.P. Langston, M. Manfredi, U. Narayanan, M. Rolfe, L.M. Staudt, T.A. Soucy, J. Yu, J. Zhang, J.B. Bolen, P.G. Smith, MLN4924, a NEDD8-activating enzyme inhibitor, is active in diffuse large B-cell lymphoma models: rationale for treatment of NF-(kappa)B-dependent lymphoma, Blood 116 (2010) 1515–1523.

[98] J.C. Godbisen, L.A. Humphries, O.V. Danilova, P.E. Kebbekus, J.R. Brown, A. Eastman, A.V. Danilov, The NEDD8-activating enzyme inhibitor MLN4924 thwarts microenvironment-driven NF-kappaB activation and induces apoptosis in chronic lymphocytic leukemia B cells, Clin. Cancer Res. 20 (2014) 1576–1589.

[99] R.T. Swords, K.R. Kelly, P.G. Smith, J.J. Garnsey, D. Mahalingam, E. Medina, K. Oberheus, S. Padmanabhan, M. O'Dwyer, S.T. Nawrocki, F.J. Giles, J.S. Carew, Inhibition of NEDD8-activating enzyme: a novel approach for the treatment of acute myeloid leukemia, Blood 115 (2010) 3796–3800.

[100] D.W. McMillin, H.M. Jacobs, J.E. Delmore, L. Buon, Z.R. Hunter, V. Monrose, J. Yu, P.G. Smith, P.G. Richardson, K.C. Anderson, S.P. Treon, A.L. Kung, C.S. Mitsiades, Molecular and cellular effects of NEDD8-activating enzyme inhibition in myeloma, Mol. Cancer Ther. 11 (2012) 942–951.

[101] C. Mackintosh, D.J. Garcia-Dominguez, J.L. Ordonez, A. Ginel-Picardo, P.G. Smith, M.P. Sacristan, E. de Alva, WEE1 accumulation and deregulation of S-phase proteins mediate MLN4924 potent inhibitory effect on Ewing sarcoma cells, Oncogene 32 (2013) 1441–1451.

[102] L. Jia, H. Li, Y. Sun, Induction of p21-dependent senescence by an NAE inhibitor, MLN4924, as a mechanism of growth suppression, Neoplasia 13 (2011) 561–569.

[103] D.J. Hughes, J.J. Wood, B.R. Jackson, B. Baquero-Perez, A. Whitehouse, NED-Dylation is essential for Kaposi's sarcoma-associated herpesvirus latency and lytic reactivation and represents a novel anti-KSHV target, PLoS Pathog. 11 (2015) e1004771.

[104] Z. Luo, G. Yu, H.W. Lee, L. Li, L. Wang, D. Yang, Y. Pan, C. Ding, J. Qian, L. Wu, Y. Chu, J. Yi, X. Wang, Y. Sun, L.S. Jeong, J. Liu, L. Jia, The NEDD8-activating enzyme inhibitor MLN4924 induces autophagy and apoptosis to suppress liver cancer cell growth, Cancer Res. 72 (2012) 3360–3371.

[105] Y. Zhao, X. Xiong, L. Jia, Y. Sun, Targeting Cullin-RING ligases by MLN4924 induces autophagy via modulating the HIF1-REDD1-TSC1-mTORC1-DEPTOR axis, Cell Death Dis. 3 (2012) e386.

[106] D. Yang, M. Tan, G. Wang, Y. Sun, The p21-dependent radiosensitization of human breast cancer cells by MLN4924, an investigational inhibitor of
NEDD8 activating enzyme, PLoS One 7 (2012) e34079.

[107] D. Wei, H. Li, J. Yu, J.T. Sebolt, L. Zhao, T.S. Lawrence, P.G. Smith, M.A. Morgan, Y. Sun, Radiosensitization of human pancreatic cancer cells by MLN4924, an investigational NEDD8-activating enzyme inhibitor, Cancer Res. 72 (2012) 282–293.

[108] A.A. Jazaeri, E. Shibata, J. Park, J.L. Bryant, M.R. Conaway, S.C. Modesitt, P.G. Smith, M.A. Milhollen, A.J. Berger, A. Dutta, Overcoming platinum resistance in preclinical models of ovarian cancer using the neddylation inhibitor MLN4924, Mol. Cancer Ther. 12 (2013) 1958–1967.

[109] S.T. Nawrocki, K.R. Kelly, P.G. Smith, C.M. Espitia, A. Possemato, S.A. Beausoleil, M. Milhollen, S. Blakemore, M. Thomas, A. Berger, J.S. Carew, Disrupting protein NEDDylation with MLN4924 is a novel strategy to target cisplatin resistance in ovarian cancer, Clin. Cancer Res. 19 (2013) 3577–3590.

[110] K. Garcia, J.L. Blank, D.C. Bouck, X.J. Liu, D.S. Sappal, G. Hather, K. Cosmopoulos, M.P. Thomas, M. Kuranda, M.D. Pickard, R. Liu, S. Bandi, P.G. Smith, E.S. Lightcap, NEDD8-activating enzyme inhibitor MLN4924 provides synergy with mitomycin C through interactions with ATR, BRCA1/ BRCA2, and chromatin dynamics pathways, Mol. Cancer Ther. 13 (2014) 1625–1635.

[111] S.T. Nawrocki, K.R. Kelly, P.G. Smith, M. Keaton, H. Carraway, M.A. Sekeres, J.P. Maciejewski, J.S. Carew, The NEDD8-activating enzyme inhibitor MLN4924 disrupts nucleotide metabolism and augments the efficacy of cytarabine, Clin. Cancer Res. 21 (2015) 439–447.

[112] M.A. Milhollen, M.P. Thomas, U. Narayanan, T. Traore, J. Riceberg, B.S. Amidon, N.F. Bence, J.B. Bolen, J. Brownell, L.R. Dick, H.K. Loke, A.A. McDonald, J. Ma, M.G. Manfredi, T.B. Sells, M.D. Sintchak, X. Yang, Q. Xu, E.M. Koenig, J.M. Gavin, P.G. Smith, Treatment-emergent mutations in NAEβ confer resistance to the NEDD8-activating enzyme inhibitor MLN4924, Cancer Cell 21 (2012) 388–401.

[113] G.W. Xu, J.J. Toth, S.R. da Silva, S.L. Paiva, J.L. Lukkarila, R. Hurren, N. Maclean, M.A. Sukhai, R.N. Bhattacharjee, C.A. Goard, B. Medeiros, P.T. Gunning, S. Dhe-Paganon, M.D. Petroski, A.D. Schimmer, Mutations in UBA3 confer resistance to the NEDD8-activating enzyme inhibitor MLN4924 in human leukemic cells, PLoS One 9 (2014) e93530.

[114] Z. Wang, P. Liu, H. Inuzuka, W. Wei, Roles of F-box proteins in cancer, Nat.

Rev. Cancer 14 (2014) 233–247.

[115] J.R. Skaar, J.K. Pagan, M. Pagano, SCF ubiquitin ligase-targeted therapies, Nat. Rev. Drug Discov. 13 (2014) 889–903.

[116] A.N. Bullock, J.E. Debreczeni, A.M. Edwards, M. Sundstrom, S. Knapp, Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 7637–7642.

[117] A.N. Bullock, M.C. Rodriguez, J.E. Debreczeni, Z. Songyang, S. Knapp, Structure of the SOCS4-ElonginB/C complex reveals a distinct SOCS box interface and the molecular basis for SOCS-dependent EGFR degradation, Structure 15 (2007) 1493–1504.

[118] Y.K. Kim, M.J. Kwak, B. Ku, H.Y. Suh, K. Joo, J. Lee, J.U. Jung, B.H. Oh, Structural basis of intersubunit recognition in elongin BC-cullin 5-SOCS box ubiquitin-protein ligase complexes, Acta Crystallogr. D Biol. Crystallogr. 69 (2013) 1587–1597.

[119] J.R. Muniz, K. Guo, N.J. Kershaw, V. Ayinampudi, F. von Delft, J.J. Babon, A.N. Bullock, Molecular architecture of the ankyrin SOCS box family of Cul5-dependent E3 ubiquitin ligases, J. Mol. Biol. 425 (2013) 3166–3177.

[120] J.C. Thomas, D. Matak-Vinkovic, I. Van Molle, A. Ciulli, Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases, Biochemistry 52 (2013) 5236–5246.

[121] Y. Guo, L. Dong, X. Qiu, Y. Wang, B. Zhang, H. Liu, Y. Yu, Y. Zang, M. Yang, Z. Huang, Structural basis for hijacking CBF-beta and CUL5 E3 ligase complex by HIV-1 Vif, Nature 505 (2014) 229–233.

[122] N. Zheng, B.A. Schulman, L. Song, J.J. Miller, P.D. Jeffrey, P. Wang, C. Chu, D.M. Koepp, S.J. Elledge, M. Pagano, R.C. Conaway, J.W. Conaway, J.W. Harper, N.P. Pavletich, Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex, Nature 416 (2002) 703–709.

[123] R. Nathans, H. Cao, N. Sharova, A. Ali, M. Sharkey, R. Stranska, M. Stevenson, T.M. Rana, Small-molecule inhibition of HIV-1 Vif, Nat. Biotechnol. 26 (2008) 1187–1192.

[124] T. Zuo, D. Liu, W. Lv, X. Wang, J. Wang, M. Lv, W. Huang, J. Wu, H. Zhang, H. Jin, L. Zhang, W. Kong, X. Yu, Small-molecule inhibition of human immunodeficiency virus type 1 replication by targeting the interaction between Vif and ElonginC, J. Virol. 86 (2012) 5497–5507.