Cullin-RING Ubiquitin Ligase Regulatory Circuits: a Quarter Century\nBeyond the F-box Hypothesis
Cullin-RING ubiquitin ligases (CRLs) are dynamic modular platforms that regulate myriad biological processes through target-specific ubiquitylation. Our knowledge of this system emerged from the F-box hypothesis, posited a quarter century ago: numerous interchangeable F-box proteins confer specific substrate recognition for a core CUL1-based RING E3 ubiquitin ligase. This paradigm has been expanded through the evolution of a superfamily of analogous modular CRLs, with five major families and over 200 different substrate-binding receptors in humans. Regulation is achieved by numerous factors organized in circuits that dynamically control CRL activation and substrate ubiquitylation. CRLs also serve as a vast landscape for developing small molecules that reshape interactions and promote targeted ubiquitylation-dependent turnover of proteins of interest. Here, we review molecular principles underlying CRL function, the role of allosteric and conformational mechanisms in controlling substrate timing and ubiquitylation, and how the dynamics of substrate receptor interchange drives the turnover of selected target proteins to promote cellular decision making.
INTRODUCTION<!><!>CULLIN\xe2\x80\x93RBX PROTEIN\xe2\x80\x93PROTEIN INTERACTIONS FORM INTEGRATED REGULATORY\nCIRCUITS<!>THE ELONGATED CULLIN-RING CORE IS COMPRISED OF MULTIPLE SUBDOMAINS THAT SERVE AS\nHUBS FOR DYNAMIC MULTISITE INTERACTIONS<!>CULLIN-SPECIFIC SUBSTRATE RECEPTOR MODULES BINDING CULLIN-REPEAT 1 DOMAINS\nESTABLISH A FOUNDATION FOR REGULATORY CIRCUITRY<!>STIMULUS-REGULATED DYNAMIC SUBSTRATE RECRUITMENT TO CULLIN-RING UBIQUITIN\nLIGASES<!>Posttranslational Modifications Positively and Negatively Regulate\nSubstrate\xe2\x80\x93Substrate Receptor Interactions<!>A Cullin-RING Ligase 1 Substrate Receptor Mediating Dimerization Quality\nControl of Cullin-RING Ligase 3 Substrate Receptors<!>Metabolic Cofactors Driving or Blocking Substrate\xe2\x80\x93Substrate Receptor\nInteractions<!>Viral Hijacking<!>Small Molecule\xe2\x80\x93Driven Substrate Binding: Lessons from Plant Hormone\nMolecular Glues and Allosteric Regulators<!>Development of Therapeutics: from Degraders to Inhibitors<!>Regulation by Substrate Receptor Localization: from Phase-Separated\nCondensates to Membrane Association<!>CULLIN-RING TRANSDUCER AND EFFECTOR RESPONSES DEPEND ON TRANSIENT PARTNERING WITH\nNEDD8- AND UBIQUITIN-CARRYING ENZYMES<!>Neddylation<!>UBE2D-Family E2-Catalyzed Priming of Neddylated Cullin-RING Ubiquitin Ligase\nSubstrates<!>Substrate Priming by ARIH-family RING-between-RING E3s<!>Polyubiquitylation by UBE2R-Family and UBE2G1 E2s<!>FEEDBACK THROUGH DYNAMIC CULLIN-RING UBIQUITIN LIGASE ASSEMBLY AND NETWORK\nCONTROL BY THE COP9 SIGNALOSOME (CSN) AND CULLIN-ASSOCIATED NEDD8-DISSOCIATED (CAND)\nREGULATORS<!>COP9 Signalosome Deneddylase<!>Cullin-Associated NEDD8-Dissociated 1: a Substrate Receptor/Cullin Exchange\nFactor<!>Regulatory Circuit Controls Cullin-RING Ubiquitin Ligase Assembly and\nActivation On Demand from a Stimulus<!>ROLES FOR CULLIN-RING UBIQUITIN LIGASE DYNAMICS IN THE NEXT QUARTER CENTURY OF\nDISCOVERY
<p>Cullin-RING ligase (CRL) E3-mediated ubiquitylation regulates virtually every
facet of eukaryotic biology, including cell division, signal transduction,
transcription, metabolism, hormone perception, circadian rhythms, differentiation,
development, and more. Numerous viruses and bacteria usurp CRL activities to disarm
host antimicrobial defenses and to promote their own propagation. Moreover, CRLs
serve as the platform for drug discovery efforts aimed at redirecting ubiquitylation
to target disease-causing proteins for degradation. This vast system of regulation,
dysregulation, and therapeutic potential depends on an accordingly vast system of
modular and exchangeable parts that share homologous structural features and
mechanistic principles across five major CRL families.</p><p>The CRL field was born a quarter century ago, with the discovery of the
F-box. Named based on its presence in Cyclin F, this ~40-residue motif was
defined both as a conserved eukaryotic domain that binds SKP1 and as a connection
between its bearers and the ubiquitin-mediated proteolysis machinery (1). Prescient early observations in the budding yeast
Saccharomyces cerevisiae included that (a)
different F-box proteins were essential for the degradation of distinct proteins,
(b) these pathways also depended on Cdc53 (yeast CUL1) and the
E2 ubiquitin conjugating enzyme Cdc34, and (c) beyond the F-box
domain, F-box proteins display various protein–protein interaction domains,
e.g., WD40 and LRR, which were proposed to recruit specific substrates for
ubiquitylation (1–4). By 1997, with biochemically reconstituted
ubiquitylation of the budding yeast cyclin-dependent kinase inhibitor Sic1, F-box
proteins were demonstrated to be substrate receptors (SRs), recruiting specific
proteins to Skp1–Cdc53 complexes for Cdc34-dependent polyubiquitylation via
an assembly referred to as SCF (Skp1–Cdc53–F-box protein)—the
founding CRL system (note: here we use sentence case for names of yeast proteins,
and upper case for names of proteins from higher eukaryotes) (5–8).
Although the CRL field was established to a large degree on the mechanisms
underlying S-phase entry through degradation of Sic1, it rapidly became clear that
the SCF system was involved in a number of distinct processes in yeast, and that a
given F-box protein may have multiple substrates. SCFs were recognized to regulate
degradation of G1 (gap phase 1) cyclins (specifically Cln2 via F-box protein Grr1),
and transcriptional responses to nutrient availability (specifically Gic2 via F-box
protein Grr1 and Met4 via F-box protein Met30) (1, 2, 8, 9). These roles
reflect the universal activities of the SCF core subunits Cdc53 and Skp1 in target
turnover but with distinct functions and regulatory networks that varied with the
target and could be traced genetically or biochemically to specific F-box proteins.
Over the next several years, pieces of a larger, more complex system of modular
cullin-based E3 ligases in multicellular eukaryotes emerged, seeded by the
identification of multiple cullin proteins homologous to Cdc53 in higher organisms
(10). Indeed, by the end of the twentieth
century, a flurry of biochemical and domain-mapping experiments established many of
the fundamental molecular principles underlying the CRL superfamily and the
mechanisms underlying ubiquitylation, linking CRLs to a vast array of substrates and
pathways (Figure 1a):</p><!><p>A suite of homologous CRLs are nucleated by a core consisting of a
cullin protein and its dedicated RING domain–containing RBX partner.
This review describes canonical CRLs, which in humans involve one of six
cullin proteins (CUL1, CUL2, CUL3, CUL4A, CUL4B, and CUL5) partnering with a
dedicated RBX protein, RBX1 (CUL1–4) or RBX2 (CUL5).</p><p>The ~100 kDa cullin-RING core has multiple key regions
mediating protein ubiquitylation. Chief among these are a domain in the
cullin that binds interchangeable substrate receptor (SR) modules, and the
RBX RING domain for catalysis.</p><p>The CRL system depends on vast numbers of SRs that each recruit
specific substrates. In humans, hundreds of CRL SRs bind to thousands of
substrates, while some organisms, including plants, rely on even more.
Ubiquitylation depends on SRs partnering with a particular cullin-RING core.
The N-terminal region of a cullin binds interchangeably with a suite of
numerous cullin-specific SRs, either directly or via adaptors (11). For simplicity, the modular
subcomplexes combining SR proteins and their cognate adaptors are referred
to as SRs. It is now known that substrate-binding F-box proteins are
complexed with SKP1, which is an adaptor that binds CUL1–RBX1 (1); substrate binding BC-box proteins
are complexed with Elongin B and Elongin C (EloBC), a multiprotein adaptor
for CUL2–RBX1 and CUL5–RBX2 (12); BTB-3-box domain–containing proteins encompass
homology to SKP1 and its bound F-box in a single polypeptide, dimerize, and
act as SRs for CUL3–RBX1 (13–17); and
substrate-binding DCAFs form complexes with DDB1, which is an adaptor for
CUL4A and CUL4B (collectively referred to hereafter as CUL4) to form the
DCAF–DDB1 SR module (18–21). A uniform
nomenclature has been adopted to designate the numerous distinct E3
complexes as CRL#SR, where # is the cullin family member, and SR
is the name of the substrate receptor. The presence of the family-specific
SR- and cullin-binding adaptor (i.e., SKP1 for a CRL1, EloBC for a CRL2 or
CRL5, and DDB1 for a CRL4) is implicit.</p><p>SR binding to substrate proteins is tightly regulated in coordination
with stimuli triggering specific cellular signaling pathways.</p><p>At the opposite end of the assembly, the hallmark E3 ligase RING
domain does not directly catalyze ubiquitylation but is an adaptor that
recruits and activates a partner ubiquitin-carrying enzyme (22–28). RING E3s typically promote the transfer of
ubiquitin's C terminus from a thioester linkage with the catalytic
cysteine of a ubiquitin-carrying enzyme to a remotely bound substrate. The
first ubiquitin-carrying enzyme identified as mediating CRL ubiquitylation
was the E2 Cdc34, in yeast (5–8). However, it
is now known that, analogous to a cullin's pairing with different SRs
to recruit various substrates, CRLs collaborate with a broad repertoire of
ubiquitin-carrying enzymes including ARIH-family RING-between-RING (RBR)
E3s, as well as E2s (29). Therefore,
we use the term ubiquitin-carrying enzyme to collectively refer to the E2
and ARIH E3 enzymes that carry and deliver ubiquitin to CRL substrates.</p><!><p>Each end of the CUL–RBX core is capable of associating interchangeably
with many protein partners—substrate-bound SRs and ubiquitin-carrying enzymes
with distinct specificities. Extensive communication across an elongated
CUL–RBX complex is achieved by three key regulators (Figure 1a): (a) neddylation, via an
E1-E2-E3 cascade analogous to ubiquitylation cascades but instead links a specific
cullin lysine to the C terminus of the ubiquitin-like protein NEDD8, which activates
CRL ubiquitin ligase activity, (b) NEDD8 deconjugation by the
multiprotein COP9 signalosome (CSN), and (c) CUL–SR exchange
catalyzed by cullin-associated NEDD8-dissociated (CAND) proteins. Moreover, in the
context of a cell, these three general CRL regulators establish regulatory circuits
that determine which CRLs are assembled and activated as needed.</p><p>A regulatory circuit comprises a stimulus, sensor, transducer, effector
response, and feedback relay (Figure 1b). Here,
the stimulus is a signal that triggers a posttranslational modification (PTM),
production of a crucial metabolite, or another form of regulation that drives
substrate binding to a SR. The transducer is cullin neddylation, made possible upon
SR-dependent displacement of CAND1 (30–39). Whether or not a
cullin remains neddylated depends on the CSN, which serves as a sensor to
distinguish substrate-bound versus substrate-free CRLs (40–42).
Where tested, CRL binding to a substrate is mutually-exclusive with deneddylation by
the CSN. By virtue of maintaining NEDD8, substrate-binding feeds forward to promote
the effector response, i.e., NEDD8-activated substrate ubiquitylation (40, 43–46) (Figure 1b). After a ubiquitylated substrate dissociates
(or is degraded, ultimately reducing its concentration), then CSN catalyzes
deneddylation. CAND-catalyzed SR exchange provides feedback that the CRL is no
longer needed.</p><p>In the next sections, we describe each of the components of the regulatory
circuit and how they synergistically specify target degradation in space and
time.</p><!><p>At the heart of CRLs—and their regulation at individual and
system-wide levels—is the cullin–RBX core. CUL1–5 and their
bound RBX1 (or RBX2) adopt homologous elongated structures comprised of multiple
subdomains (Figure 1a,c) (47).</p><p>The N-terminal region of a cullin is composed of four subdomains arranged in
a linear manner from the N terminus: three tandem cullin-repeat domains (CR1, CR2,
and CR3) and a 4-helix bundle (4HB) (47). As
described in the section titled Cullin-Specific Substrate Receptor Modules Binding
Cullin-Repeat 1 Domains Establish Foundation Of Regulatory Circuitry, the CR1
subdomain engages SRs, while all four cullin N-terminal subdomains engage the SR
exchange factor CAND1 (Figure 1c) (32, 47).</p><p>The 4HB subdomain makes extensive hydrophobic interactions with the
subsequent domain, which intertwines the cullin and RBX proteins and is thus
referred to as the intermolecular C/R domain (Figure
1c). Briefly, the C/R domain is nucleated by a β-sheet that is
largely from the cullin but completed by a central strand from RBX1's N
terminus (Figure 1c) (47). In addition to affixing the cullin and RBX proteins,
the C/R domain engages the CSN for cullin deneddylation (40–42).</p><p>On the other side of the C/R domain, the C-terminal domains of the cullin
and the RBX protein are dynamically tethered. The C-terminal cullin winged-helix B
(WHB) domain contains the conserved lysine that is reversibly modified by NEDD8.
Meanwhile the RBX strand embedded in the C/R domain continues into a linker, which
tethers the C-terminal RING domain (Figure
1a,c). Biochemical and structural
data suggest that both the cullin WHB and the RBX RING domains are mobile relative
to the intermolecular C/R domain (36, 48, 49).</p><p>The overall elongated shape of a cullin-RING complex has been likened to a
banana. However, the differing dynamic properties at the two ends suggest a more
appropriate analogy would be a partially peeled, partially eaten banana (49). The cullin's N-terminal domain
corresponds to the relatively rigid unpeeled portion, the C/R domain corresponds to
the junction between the banana and the flexibly bound peel, and the RBX C-terminal
RING domain and cullin C-terminal WHB domain correspond to the two halves of the
peel flexibly tethered to the remainder of the complex.</p><p>The CRL regulatory circuit depends on dynamic regulation mediated by the
cullin WHB and the RBX RING domains. In the absence of other factors, these domains
preferentially pack against each other and favor inactive assemblies. Interestingly,
deleting the WHB domain increases ubiquitylation in conjunction with some
ubiquitin-carrying enzymes (48, 50). Neddylation alters the conformational
equilibrium and provides an additional binding site for CRL partner proteins (48–52). The cullin and RBX C-terminal WHB and RING domains rearrange for
the neddylation reaction and to corecruit ubiquitin-carrying enzymes, CAND1, or CSN,
depending on whether or not the cullin WHB domain is neddylated, which is
coordinated by the association of substrate-bound SR with the cullin's
N-terminal domain (Figure 1a,c).</p><!><p>CUL1, CUL2, CUL3, and CUL5 form structurally homologous complexes with their
cognate SRs, either through direct interaction or via one or more dedicated SR
adaptors (Figure 2). This was first shown for
CUL1, whose CR1 domain binds a composite surface between SKP1 and its associated
F-box (47). These SR–CUL interactions
involve a portion of SKP1 that superimposes with EloC and the BTB domain fold (17, 53,
54). In addition, a helix-turn-helix
motif in an F-box, BC-box, or the 3-box sequence immediately following a BTB domain,
binds cullins and contributes to SR–CUL interaction specificity (17, 47,
55). Most importantly, F-box, BC-box, and
BTB-3-box proteins typically display another adjacent but distinct
protein–protein interaction domain (e.g., WD40, LRR, SH2, or Kelch) that
recruits substrates for ubiquitylation.</p><p>The surface of CUL4's CR1 domain recruits DDB1, the adaptor for DCAF
(DDB1- and cullin 4–associated factors)-family SRs (18). DDB1 adopts a unique fold comprising three
β-propellers (BPs): BPA, BPB, and BPC. BPB binds CUL4's CR1 domain,
distinctively engaging the surface that other cullins use to bind the
SKP1–F-box fold (18). CUL4 also
contains an extended N-terminal peptide-like sequence that wraps around the side of
the DDB1 BPB propeller (18) (Figure 2). Meanwhile, the DDB1 BPA and BPC together
generate a V-shaped groove that engages helical motifs from DCAF SRs (18). The DDB1 BPB–BPA/BPC interface is
flexible, allowing DDB1–DCAF complexes to adopt varying positions relative to
CUL4 (45). The DDB1 BPA can also engage a
partner protein, DDA1, via a distal groove (56), although the reason why only a subset of CUL4s seem to bind DDA1
and the ways in which DDA1 modulates their regulation remain unclear.</p><!><p>The fundamental feature of the CRL regulatory circuitry is that substrate
binding to a CRL is tightly controlled in response to cellular stimuli, to ensure
the proper timing of ubiquitylation. This concept was first established by the
discovery of F-box proteins as receptors for phosphorylated substrates. For some of
the first discovered CRL substrates, their ubiquitylation depended on
phosphorylation that was regulated by the cell cycle, responses to nutrient
availability, or proinflammatory stimuli. Subsequently, substrate–SR
interactions have been found to depend on an extraordinary array of diverse
regulatory stimuli.</p><!><p>At the time F-box proteins were discovered, connections between
phosphorylation and protein degradation were amassing. So-called PEST (Pro, Glu,
Ser, Thr) sequences—in which the Ser and Thr were
phosphorylated—were increasingly found to confer protein instability,
although the biochemical basis for this property was far from clear.
Furthermore, studies in budding yeast revealed many genetic interactions between
regulators of phosphorylation and protein stability. Among those pathways
involving an F-box protein, the stability of the budding yeast cell cycle
inhibitor, Sic1, was found to depend on cyclin-dependent kinase activity (4, 7).
Ultimately, biochemical reconstitution demonstrated that this phosphorylation
mediates Sic1 binding to the WD40 propeller domain of the F-box protein Cdc4,
which established the notion that some F-box proteins are receptors for
phosphorylated substrates (5, 6) (Figure
3a).</p><p>A flurry of subsequent studies identified many SRs that recognize
substrate phosphothreonine-, phosphoserine-, and/or phosphotyrosine-centered
peptide-like motifs called phosphodegrons. Crystal structures revealed how
various SRs recruit phosphodegrons. As was shown crystallographically for yeast
Cdc4, its human counterpart FBXW7, β-TRCP (FBXW1), and SOCS3, SRs display
constellations of basic residues to recruit phosphorylated residues (57–60). Sequence specificity is determined by adjacent pockets in the
SR that complement features surrounding the substrate's phosphorylation
site.</p><p>An additional twist on this mechanism is seen in the context of the
human CDK inhibitor, phosphorylated p27, which is recognized by the F-box
protein SKP2 (FBXL1) while in a complex with CKSHS1. In this case, the p27
phosphodegron is bound to the phosphate-binding site on CKSHS1 while the
surrounding composite CKSHS1–SKP2 interface binds adjacent residues in a
sequence-specific manner (61, 62) (Figure
1a). In this case, CKSHS1 can be viewed as a glue between the degron
and the SR. Notably, affinity for p27 is further potentiated through its other
binding partners (Cyclin E– or Cyclin A–bound CDK2), which
simultaneously bind elsewhere on CKSHS1 and on SKP2.</p><p>The phosphorylation–E3 ligase code is remarkably complex and
depends not only on the substrate binding to its cognate SR but also on the
upstream regulation of the kinases that drive these interactions (Figure 3a). In some cases, a single kinase is not
sufficient to transmit the signal from the stimulus. Two sequential
phosphorylations typically determine substrate binding to F-box proteins in the
β-TRCP family. The C-terminal serine in its DSGXXXS phosphodegron is
phosphorylated first by one of various priming kinases (63). The priming phosphorylation serves as a docking
site for a different kinase, typically GSK3β, that mediates the second
phosphorylation (64). The two
phosphorylations are recognized by distinct basic patches on
β-TRCP's substrate-binding WD40 propeller domain (58).</p><p>Phosphorylation can also negatively regulate substrate–SR
interactions (Figure 3a). For example,
substrates of CRL3SPOP are recruited via SPOP-binding consensus (SBC)
sequences that are rich in unphosphorylated serines and threonines (17). Phosphorylation of a
substrate's SBC prevents binding to and ubiquitylation by SPOP (17, 65).</p><p>Notably, some F-box proteins and the BTB domains of CRL3 SRs dimerize,
such that each protomer binds to a cullin (13–17, 66). Dimerization of β-TRCP and FBXW7 in
principle allows the simultaneous engagement of two separate phosphodegrons. For
example, Cyclin E displays two phosphodegrons, one N-terminal and the other
C-terminal of the cyclin domain. It is thought that a dimer simultaneously
recognizing two distinct degrons in its substrate enables avid engagement of
both (60, 66, 67). This principle has
clearly been established for recognition of a nonphosphorylated substrate of
CRL3KEAP1: Avid association depends on two distinct degrons in
the transcription factor NRF2, one binding to each KEAP1 protomer in the BTB
domain–mediated dimer (68). For
FBXW7, it seems that dimerization also influences its stability in a manner
involving CRL1-mediated autoubiquitylation (67).</p><p>In addition to phosphorylation, a plethora of other PTMs direct
substrates to many SRs (Figure 3b). Other
PTMs driving CRL substrate interactions include prolyl hydroxylation, which is
stimulated by normal oxygen tension (69–71); glycans such
as those displayed on misassembled or misfolded proteins emerging from the
endoplasmic reticulum or exposed on damaged lysosomes (72); and acetylation (73). Often perceived as a more drastic modification, proteolytic
cleavage can also direct a substrate to its SR. This concept emerged from a
series of recent systems biology studies that together identified eight BC-box
and two DCAF SRs binding to specific sequences, termed N- or C-degrons, at
proteins' N or C termini (74–76). One of these,
KLHDC2, recognizes a C-terminal Gly-Gly sequence, including that found in the
USP1 N-terminal fragment produced during DNA damage–induced
autoproteolysis (77). Other N- or
C-degron-recognizing CRLs perform quality control functions, for example,
recognizing C-degrons from truncated selenoproteins that are generated upon
failure to translate UGA as selenocysteine (75, 76). Interestingly, the
N-degron-recognizing SRs ZYG11B and ZER1 promote degradation of proteins with
exposed N-terminal glycines that are formed upon N-myristoylation failure. Here,
the N-myristoyl PTM prevents SR binding and thus protects properly processed
proteins from degradation (76).</p><p>Interactions can also be regulated by PTM of SRs (Figure 3c). A classic example involves regulation of
the cysteine-rich CRL3 SR, KEAP1. Under homeostatic conditions, KEAP1 forms an
active E3 ligase complex that mediates continuous turnover of the NRF2
transcription factor. However, during oxidative stress conditions, oxidizing and
electrophilic agents react with KEAP1 cysteines. KEAP1 modification prevents
ubiquitin-mediated proteolysis of NRF2 (78–80). KEAP1's
reactive cysteines do not directly map to known functional protein-interacting
residues, so the precise basis for this inhibition remains to be determined.</p><!><p>Improper protein complex formation is a particularly fascinating signal
for CRL1-dependent ubiquitylation, because the substrates themselves are CRL3
SRs. Misassembled BTB dimers (i.e., heterodimers) are recognized by the F-box
protein FBXL17 (81, 82). FBXL17 promotes dissociation of BTB dimers into
monomers and binds a single monomeric BTB domain via residues that are normally
buried in the dimer interface (Figure 3d).
When would such residues be exposed? An answer comes from FBXL17 preferentially
binding to mismatched BTB proteins that do not normally heterodimerize: It seems
that a weak dimer interface, or aberrant exposure of normal BTB interface
residues, triggers CRL1FBXL17-dependent ubiquitin-mediated
proteolysis and dimerization quality control.</p><!><p>A variety of SRs and substrates are regulated by binding to metabolic
cofactors. Substrate binding to the F-box protein FBXL5 is positively regulated
by iron in a multifaceted activation mechanism (83, 84). First, FBXL5, which
is otherwise subjected to rapid turnover, is stabilized by iron binding to its
N-terminal hemerythrin-like domain. Second, oxidation-dependent formation of an
iron-sulfur cluster coordinates the folding of FBXL5's substrate-binding
domain, which recruits the IRP2 translational inhibitor for ubiquitylation.
Overall, the folding of FBXL5 serves as a sensor for high iron and high oxygen,
ultimately triggering the production of iron-responsive proteins (85).</p><p>Meanwhile, an emerging theme in CRL-mediated regulation of metabolic
enzymes is that occupation of their ligand binding sites blocks SR binding. This
was first described for recognition of mammalian cryptochrome CRY2, where an
empty cofactor binding pocket directly engages the C-terminal tail of the F-box
protein FBXL3. Here, the cofactor FAD or artificial ligands block the FBXL3
binding site of CRY2 and inhibit its ubiquitin-mediated degradation (86).</p><!><p>As predicted upon the discovery of F-box proteins (1), many viruses express proteins that hijack CRLs to
ubiquitylate host restriction factors and subvert immunity. Some viral proteins
mimic SRs. For example, HIV-1 Vif displays a BC-box, and hepatitis B virus HBx
binds DDB1 via a DCAF-like helix (87,
88). Others (e.g., HIV-1 Vpu and Vpr)
latch onto SRs (the F-box proteins FBXW1 or FBXW11 and DCAF1, respectively) via
degron-like sequences (89, 90). These viral hijackers display
additional protein–protein interaction domains that either alone, or in
partnership with other cellular proteins, recruit host antiviral proteins for
ubiquitylation.</p><!><p>To rapidly alter growth, development, and metabolism, plants often
circulate small-molecule phytohormones that bind to existing proteins and switch
their activities. Some plant hormones are perceived by CRLs to regulate
ubiquitylation. Notably, plants have an enormous capacity for CRL regulation, as
reflected by the nearly 700 F-box proteins in Arabidopsis
thaliana (91). Some plant
phytohormones, such as auxins and jasmonate, act as molecular glues by filling a
hole at the interface between an SR and its substrate (Figure 3b). Crystal structures showed hormone glues
simultaneously engage pockets in F-box proteins (TIR1 and COI1) and in their
substrates (hormone-inactivated transcription factors) in a manner that allows
for efficient ubiquitylation (92–94).</p><p>Interestingly, other plant hormones play more nuanced roles in
regulating substrate binding to SRs. Although further structural details remain
to be elucidated, it seems that both gibberellin- and strigolactone-type
phytohormones induce conformational changes in their partner proteins GID1 and
D14, respectively. Gibberellin-bound GID1 acts as a protein glue to bridge
substrates with specific F-box proteins (95, 96). It seems that
strigolactone mediates the assembly of a substrate-targeting CRL1 complex in a
conceptually similar manner but with the added twist that after substrate
degradation, strigolactone hydrolysis within this complex leads to the
ubiquitin-mediated proteolysis of D14 to reset the pathway (97–99).</p><!><p>There is much pharmaceutical interest in small molecule–mediated
modulation of human SRs, particularly in degraders that artificially promote SR
binding to neo-substrates that are not their endogenous targets. Degrader-driven
proteolysis offers a route to extinguishing proteins that have been
traditionally viewed as undruggable, including many disease-causing proteins.
Interestingly, degraders may offer opportunities for substoichiometric
therapeutic targeting, because after a neo-substrate is eliminated, the degrader
and E3 ligase are both liberated to ubiquitylate another molecule of the
targeted protein.</p><p>The initial proof of principle for degrader technology came from a
bifunctional molecule: an IÎºBÎ±-derived phosphodegron peptide that
binds the F-box protein β-TRCP connected to ovalicin (100). Ovalicin is a small molecule angiogenesis
inhibitor that binds methionine amino peptidase 2 (METAP2). By mediating
CRL1β-TRCP-dependent ubiquitin-mediated proteolysis of
METAP2, the designed molecule proved to be a protein-targeting chimeric molecule
(PROTAC) (Figure 3e).</p><p>In the subsequent two decades, small molecule degraders have advanced
from concept to reality. Entirely synthetic PROTACs—with two distinct
small molecule binders connected by a linker (Figure 3e)—were systematically developed to target the CRL2
SR VHL and show in vivo efficacy in rodents (101). Moreover, the mechanism of action of FDA-approved
immunomodulatory imide drugs (IMiDs), which include thalidomide and less toxic
analogs including lenalidomide and pomalidomide, proved to act as degraders.
IMiDs approved as chemotherapeutics for refractory multiple myeloma function
analogously to plant hormone molecular glues. Rather than having two distinct
handles for E3 and the target, IMiDs function as singular chemical entities that
complete nonnatural interfaces between the CRL4 SR CRBN and neo-substrates
including Ikaros-family transcription factors and casein kinase 1α (102–104). IMiDs simultaneously bind CRBN and a glycine
residue in a surface turn conserved among neo-substrates (105) (Figure
3e). Structure-based elaboration of the thalidomide chemical has yielded
novel analogs with antitumor efficacy that connect CRBN with yet other
neo-substrates (106).</p><p>The discovery of bona fide drugs acting as degraders has propelled
targeted protein degradation into position as a major therapeutic discovery
platform. An explosion in the number of known PROTAC and molecular-glue
degraders, and their mechanisms of action, has expanded our understanding of key
functional characteristics. First, many SRs and diverse substrate features are
amenable to adjoining by degraders. Second, the lines between PROTACs and
molecular glues are blurred, because linkers connecting the two binding moieties
in PROTACs can actually function in a glue-like manner by further buttressing
nonnative but substantial interfaces between SRs and neo-substrates (107). Remarkably, the nonnative interfaces
between a given SR and a targeted neo-substrate can vary based simply on how
they are glued by different linkers (108). Also, the prospective discovery and development of a molecular
glue–type small molecule that can restore β-TRCP interactions with
its mutant substrate raises hope that future high-throughput screening and
subsequent medicinal chemistry efforts can identify degraders that compensate
for disease-causing mutations at SR–substrate interfaces (109).</p><p>Small molecules can also allosterically inhibit SR–substrate
interactions. Cdc4–I2—the top hit in a screen for inhibitors of
degron peptide binding to the F-box protein Cdc4—binds 25 Å away
from the degron-binding groove. A crystal structure showed that Cdc4–I2
elicits long-range structural rearrangements that blocks the degron-binding site
(110). Interestingly, the
Cdc4–I2 binding pocket was not observed in crystal structures without the
small molecule, highlighting the conformational plasticity of CRL SRs that may
prove useful for therapeutic targeting.</p><!><p>It is important that substrates and SRs interact not only at the right
time but also at the right place in a cell. The CRL3 receptor SPOP, which is a
tumor suppressor protein originally identified in cellular speckles, colocalizes
with substrates in phase-separated condensates (111). Many SPOP substrates, including the death
domain–associated protein DAXX, display numerous SBC motifs. Meanwhile,
SPOP contains two self-associating domains: the BTB domain and another
homodimerizing domain called BACK. Coexpression of SPOP with DAXX results in
their colocalization along with CUL3 in nuclear SPOP/DAXX bodies thought to be
phase-separated membraneless organelles that serve as ubiquitylation hubs (112) (Figure
3f). Notably, SPOP/DAXX body formation depends on features required
for their liquid-liquid phase separation (i.e., multiple of DAXX's SBCs
and SPOP's homodimerization domains) in vitro, and these features are
disrupted by cancer-associated mutations in SPOP.</p><p>CRLs that ubiquitylate membrane proteins must localize to particular
lipid-encased organelles. For the F-box protein FBXL2, this is specified by its
dedicated geranylgeranyltransferase (GGTase3). A recent crystal structure showed
that the concave interior surface of FBXL2's LRR domain complements a
dome-like structure of the GGTase3 regulatory subunit (113). This drives FBXL2 prenylation, which in turn
localizes FBXL2 for ubiquitylation of its membrane-associated substrates.</p><p>Membrane localization is regulated in a different way for the dimeric
BTB CRL3 SR KHLH12. KLHL12 binding to its substrate SEC31A is enhanced by two
calcium-binding regulatory proteins, PEF1 and ALG2. Transient increases in
calcium concentration induce the localization of KLHL12 bound to SEC31A, PEF1,
and ALG2 at vesicular structures (114).
Monoubiquitylation of SEC31A at the membrane in turn drives COPII coat
formation, ultimately enabling the encapsulation and secretion of collagen
(114).</p><!><p>The ultimate function of CRLs is to decorate recruited substrates with
ubiquitin modifications, as required for stimulus-induced regulation. CRLs were
first discovered as mediators of polyubiquitylation-dependent degradation (5, 6).
However, it is now recognized that substrates are regulated by distinct forms of
ubiquitin, with another extreme being monoubiquitylation regulating assembly of the
substrate protein into multiprotein complexes (114).</p><p>E3 ligase activity depends on multiple, tunable functions of RBX RING
domains. RBX RING domains serve as E3 ligases for both NEDD8 and ubiquitin,
promoting their transfer from specific partner E2s and E3s (Figure 4) (22–26, 115). Biochemical and cellular data suggest that
many—potentially most or all—active human CRLs are neddylated (116). Neddylation, in turn, activates a
variety of forms of ubiquitylation determined by CRL partner ubiquitin-carrying
enzymes (Figure 4).</p><p>UBE2D-family E2s directly mark SR-bound substrates with ubiquitin on one or
more sites in substrate-priming reactions and generate polyubiquitin chains,
including branched versions in which multiple lysines in one ubiquitin are linked to
other ubiquitins (117, 118). UBE2G1 and UBE2R-family E2s are specialized to
extend Lys48-linked polyubiquitin chains (119). Some CRLs may have alternative E2 partners or activities; for
example, CRL4s have been associated with UBE2E-family E2s, and a specific CRL3 was
reported to modify its substrate with distinctive K33-linked polyubiquitin chains
(18, 120). In addition to E2 enzymes, many CRLs can also use ARIH-type RBR E3
enzymes to prime their SR-bound substrates (29).</p><p>Reactions ranging from neddylation of the cullin's WHB domain to
polyubiquitylation of an SR-bound substrate depend on the capacity of the terminal
cullin-RING domains to dynamically rearrange and to bind, activate, and orient their
wide range of cognate NEDD8- or ubiquitin-linked carrying enzymes (48).</p><!><p>Much like that of ubiquitin, NEDD8's C terminus is ligated to
cullin targets via specific E1-E2-E3 cascades (121–124). NEDD8 is
activated by its own dedicated E1, NEDD8 activating enzyme (NAE), which
catalyzes the formation of a transient thioester bond between NEDD8's C
terminus and the catalytic cysteine of a NEDD8-specific E2 enzyme, either UBE2M
or UBE2F (125). UBE2M is specific for
RBX1-bound cullins. UBE2F can neddylate RBX1- or RBX2-bound cullins and is
therefore the requisite neddylating E2 for CUL5–RBX2. In addition to
their canonical E2 catalytic domains, UBE2M and UBE2F feature unique N-terminal
disordered regions that are N-terminally acetylated and mediate interactions
with upstream and downstream enzymes in the neddylation cascade. These
N-terminal extensions adopt distinct conformations when bound to NAE to promote
NEDD8 conjugation to the E2 active site or to proteins in the DCN (defective in
cullin neddylation) family that support NEDD8 transfer to a specific cullin
lysine (K720 in CUL1) (36, 125, 126).</p><p>A crystal structure representing CUL1 neddylation showed specialized
orientations of RBX1's RING domain and CUL1's WHB domain that are
required to juxtapose the RING-bound UBE2M~NEDD8 intermediate and target
lysine in the cullin WHB domain (~ represents a thioester bond between a
carrying-enzyme cysteine and the NEDD8 C terminus; similar bonds can also form
between the carrying enzyme and ubiquitin) (36). RBX1 activates the UBE2M~NEDD8 intermediate through the
canonical RING mechanism, binding both NEDD8 and its linked E2 in a
well-characterized activated conformation also formed by
RING–E2~ubiquitin intermediates (36). Numerous interactions—mediated by DCN1, RBX1, NEDD8 and
its linked E2, and the cullin—direct the flexibly tethered
RING–UBE2M~NEDD8 subassembly to the WHB-domain lysine (36).</p><p>Neddylated and unneddylated cullin–RBX complexes preferentially
interact with different protein partners. Neddylation ultimately transduces the
signal that an SR is substrate-bound to the effector machinery (38) by activating the effector response—i.e.,
CRL-mediated substrate ubiquitylation—through multiple mechanisms,
including by promoting conformational changes, increasing affinities for
ubiquitin-loaded ubiquitin-carrying enzymes, and stimulating catalysis of
ubiquitin transfer (48–52).</p><!><p>UBE2D E2s (also known as UBCH4 or UBCH5) were identified through
fractionation of mammalian cell lysates as mediating
CRL1β-TRCP-dependent ubiquitylation of IκBα
(127). Indeed, knockdown of UBE2D
E2s stabilizes IÎºBÎ±, as well as a neo-substrate of
CRL4CRBN (117, 128).</p><p>Neddylation activates numerous facets of UBE2D-catalyzed ubiquitylation
of CRL1β-TRCP substrates (48–52, 129, 130). The mechanism was recently revealed by a structure
representing ubiquitin transfer from UBE2D to an IÎºBÎ±-derived
peptide substrate recruited to neddylated CRL1β-TRCP (49). RBX1's RING-bound
UBE2D~ubiquitin adopts the canonical activated conformation, and this
portion of the ubiquitylation assembly superimposes with the isolated RBX1
RING–UBE2M~NEDD8 subcomplex. However, the flexibly tethered RBX1
RING—and its associated UBE2D~ubiquitin—and CUL1's
WHB domain linked to NEDD8 are strikingly reoriented for ubiquitylation.
RBX1's RING is swiveled around to juxtapose the UBE2D~ubiquitin
active site with the β-TRCP-bound substrate. NEDD8 binds directly to
UBE2D's back side surface opposite the active site, and to two domains
from CUL1. The data suggest that NEDD8 (a) activates the
intrinsic reactivity of the RBX1 RING–UBE2D~ubiquitin
intermediate, (b) stabilizes interactions with the
UBE2D~ubiquitin intermediate, and (c) positions the
ubiquitylation active site proximal to the substrate recruited to β-TRCP
(49).</p><p>Recent studies have shown that some RING E3s elicit UBE2D-mediated
ubiquitin chain branching, where a ubiquitin is itself modified on many of its
lysines (Figure 4) (118). If and how this occurs with CRLs remains
unknown. One question for the future is whether, after NEDD8 activates an
initial substrate modification, a substrate-linked ubiquitin could make some of
the structurally observed interactions, for example, binding UBE2D's back
side, to then stimulate polyubiquitylation, including with ubiquitin-chain
branching.</p><!><p>Proteomic and in vitro reconstitution studies collectively showed that
many neddylated human CRLs employ the mechanistically distinct thioester-forming
Ariadne family RBR-type E3s, ARIH1 or ARIH2, to ubiquitylate their SR-bound
substrates (Figure 4) (29, 131).
ARIH1 binds many RBX1-bound CRLs, while ARIH2 is specific for those with
neddylated CUL5-RBX2. The biochemical requirements for ARIH RBR E3-mediated
ubiquitylation match those for CRL substrate regulation, including dependence on
substrate recruitment to cullin-bound SRs and on neddylation. Neddylated cullins
bind ARIH E3s, relieve ARIH autoinhibition, and activate ubiquitin transfer from
an ARIH-specific E2 to the ARIH RBR E3 catalytic cysteine (29, 131). The
ARIH-linked ubiquitin is then transferred to a CRL's SR-bound substrate
(29). The underlying mechanism was
recently revealed by a suite of cryo EM structures of chemically-stabillized
complexes representing the transition states in ARIH1 ubiquitylation of CRL
substrates (131a).</p><p>When and which neddylated CRLs employ UBE2D E2s or ARIH E3s to prime
their substrates remains an open question, although in vitro ARIH1 can mediate
ubiquitylation of some substrates that UBE2D E2s cannot (29). A recent CRISPR screen revealed a CRL5 pathway
that depends on ARIH2: HIV-1 Vif-dependent degradation of APOBEC host
restriction factors, and HIV infectivity, depend on ARIH2 (132). Notably, the Caenorhabditis
elegans ortholog of ARIH1 is associated with CRL1-dependent
regulation in vivo (133).</p><!><p>Early studies identified Cdc34 as the E2 mediating ubiquitylation of
substrates recruited to S. cerevisiae F-box proteins (5, 6).
However, the human orthologs in the UBE2R family preferentially extend
K48-linked polyubiquitin chains on substrates already modified by a priming
ubiquitin (117). Comparative enzyme
kinetics showed that UBE2R2 modification of a substrate with the second
ubiquitin is 200-fold faster than adding the first ubiquitin (119). When measurements were made with physiological
concentrations of ubiquitin-carrying enzymes, priming of substrates for two
different neddylated CRL1s was negligible by UBE2R2, whereas they were robustly
ubiquitylated by UBE2D3 or ARIH1 (119,
131a).</p><p>The polyubiquitylation reaction involves transfer of the C terminus of
the donor ubiquitin from the thioester linkage connecting it to the E2 catalytic
cysteine onto a lysine on the acceptor ubiquitin. Beyond binding to
RBX1's RING domain, several unique elements of UBE2R-family E2s were
found to influence polyubiquitylation, including an acidic loop and an
intrinsically disordered acidic tail located C-terminal of a UBE2R catalytic
domain (134–136). A crystal structure showed part of the acidic
tail buttressing the interface between the UBE2R catalytic domain and its linked
donor ubiquitin in the closed conformation of the UBE2R~ubiquitin
intermediate (137). The end of the
acidic tail also drives an uncommonly high association rate with CUL1,
established by electrostatic interactions with a basic canyon between the C/R
and 4HB domains that is conserved among cullins (138). Rapid association of UBE2R~ubiquitin
with CRLs underlies efficient polyubiquitylation.</p><p>Despite the essential nature of S. cerevisiae Cdc34,
UBE2R1/UBE2R2 double knockout human cells are viable (119). A series of studies using CRISPR technology
identified UBE2G1 as another CRL partner E2. In vitro, UBE2G1 showed no
detectable substrate-priming activity, but it efficiently generates Lys48-linked
polyubiquitin chains on a priming ubiquitin that is already linked to CRL
substrates (119, 139). UBE2G1 mediates IMiD-induced
CRL4CRBN-dependent degradation of neo-substrates (128, 140). Also, knocking down UBE2G1 confers synthetic lethality to
UBE2R1/UBE2R2 double knockout cells and results in increased levels of several
CRL1 and CRL2 substrates (119). For
these latter substrates, it is thought that the large collection of potential
ubiquitin-carrying enzymes provides buffering capacity such that any of them
could mediate degradative polyubiquitylation if one becomes limiting.</p><p>Altogether, the available data suggest that many CRLs employ UBE2D E2s
and/or ARIH-family E3s to directly modify their substrates in priming reactions,
while UBE2R and/or UBE2G1 E2s are used for polyubiquitylation (Figure 4).</p><p>The dynamic features of ubiquitin-carrying enzymes—the loops,
extensions, and domains that are reconfigured for catalysis—are
attractive targets for allosteric modulation by small molecules. Indeed, this
concept was validated by an inhibitor of UBE2R1, CC0651, which lodges in a
crevice between UBE2R1 and the donor ubiquitin to stabilize the
UBE2R1~ubiquitin intermediate and prevent ubiquitin transfer (141).</p><!><p>CRL-mediated ubiquitylation is intrinsically a dynamic process, involving
transient interactions with a substrate and cycles involving binding of a
ubiquitin-loaded carrying enzyme, ubiquitin transfer to the substrate, and the
exchange of ubiquitin-free and ubiquitin-charged carrying enzymes to add another
ubiquitin to the substrate or build a polyubiquitin chain. It is now clear that
feedback between the signal from the stimulus and the effector outcome is regulated
at the level of cullin–SR assembly and disassembly. CRL assembly and
activation are regulated by CSN, together with CAND-family proteins, integrating
information across both ends of a CRL (Figures
1 and 5). Although CSN and CAND1
biochemically block an individual CRL's ubiquitylation activity, in the
context of cellular regulation of CRL assembly, they are required for substrate
ubiquitylation. CSN and CAND1 collaborate with neddylation enzymes to establish the
system-wide regulatory circuit that responds to cellular cues through rapid CRL
assembly and activation on demand (Figure
1b).</p><!><p>CSN is a multiprotein deneddylating enzyme comprised of eight core
subunits (CSN1–8), with a JAMM metalloprotease active site in CSN5 (142, 143). CSN is remarkably selective, binding CRLs and cleaving the
isopeptide bond between a cullin and NEDD8's C terminus. Mechanistic
understanding of CSN's cullin deneddylase activity comes from
high-resolution crystal structures of an eight-protein CSN complex and of CSN5
alone, together with lower resolution electron microscopy structures of
catalytically inactive CSN complexes with several neddylated CRL1–4s
(40–42, 144–146). Structures
of CSN bound to different CRLs are similar, so fundamentally common mechanisms
are thought to apply generally across CRLs.</p><p>CSN's deneddylase active site is housed in the CSN5 subunit,
which heterodimerizes with CSN6 (145).
All other CSN subunits contain PCI domains, arranged in a semicircular PCI ring
(145). The PCI ring, together with a
bundle comprising C-terminal helices from all subunits, forms a central scaffold
from which elongated domains from CSN1, CSN2, and CSN4 protrude outward (145).</p><p>The CSN2 and CSN4 protrusions clamp around the C/R domain and
encapsulate RBX1's RING between them. In so doing, they trigger a series
of conformational changes that ultimately activate CSN5's deneddylase
activity (40–42, 145).
Quantitative enzymology data and mutational analyses suggest that unique CSN5
loops direct an additional conformational cycle for binding neddylated cullins,
severing the bond to NEDD8 and releasing deneddylated cullins.</p><p>Meanwhile, structural and biochemical data suggest that the CSN1
protrusion essentially scans for substrate-free neddylated CRLs, and such
dynamic scanning underlies CRL remodeling through the neddylation-deneddylation
cycle (Figure 5) (40–46).
In structures of CSN–CRL1 complexes, CSN1's protrusion contacts
the F-box protein (40, 41). Substrate binding would sterically occlude CSN1.
CSN–CRL2 and CSN–CRL4 complexes showed more nuanced roles for the
CSN1 protrusion, contacting the adaptors EloBC or DDB1, respectively (42, 146). Overall, whether or not a substrate is bound influences
whether or not a CRL binds CSN and is deneddylated.</p><!><p>Although CSN inhibits CRL ubiquitylation activity, genetic experiments
showed that CSN can be required for F-box protein pathways in cells (147, 148). Similarly, paradoxical differences were observed for the roles
of CAND1 in vitro and in vivo. At steady state, CAND1 binds nonneddylated,
SR-free cullin–RBX complexes in vitro and in cells (30, 31).
Indeed, a crystal structure showed that CAND1 adopts a two-pronged, clamp-shaped
superhelical structure that binds both ends of CUL1–RBX1 (Figure 5) (32).
CAND1's N terminus forms a three-way junction with RBX1's RING and
CUL1's WHB and blocks the neddylation site, explaining why neddylation
blocks CAND1 binding and vice versa. CAND1's C terminus encases
CUL1's N-terminal domain. A crucial CAND1 β-hairpin binds the same
CR1 subdomain surface that would otherwise recruit an SR module. Thus, it was
surprising that, similar to the effects of depleting CSN, decreased cellular
CAND1 levels led to increases in many substrates (31, 149,
150). These CSN and CAND1 paradoxes
were ultimately resolved by the understanding that neddylation, deneddylation,
and CAND-dependent SR exchange underlie CRL regulation.</p><p>Elegant biochemical, genetic, and proteomics studies showed that CAND1
is an SR/cullin–RBX exchange factor that allows one CUL1-RBX1 to switch
which SKP1-F-box protein module it is bound to (33–35). This activity
is apparently shared by the paralog CAND2, whose deletion in some cells is
required to expose CAND1 function (38).
In the current model (Figure 5), either an
F-box protein–SKP1 complex or CAND1 individually forms a tight, slowly
dissociating complex with CUL1–RBX1. However, if an F-box
protein–SKP1–CUL1–RBX1 complex is confronted by CAND1, or
CAND1–CUL1–RBX1 encounters an F-box protein–SKP1 complex, a
fleeting intermediate with both the F-box protein–SKP1 and CAND1 bound to
CUL1–RBX1 increases the rate of dissociation of the original partner
(33, 37). Moreover, adding CAND1 to an inert mixture between a preformed
F-box protein–SKP1–CUL1–RBX1 complex and a different F-box
protein–SKP1 establishes a dynamic equilibrium that grants the free F-box
protein access to the CUL1–RBX1 complex. CRL4s are regulated analogously
(33).</p><!><p>Although each mechanism—substrate–SR association,
SR–cullin binding, cullin neddylation, ubiquitylation, CSN-mediated NEDD8
deconjugation, and CAND1-catalyzed SR exchange—was deciphered
independently, the model that emerged shows that they coordinately form a
regulatory circuit controlling CRL assembly and activation on demand (Figures 1b and 5) (33–35, 150). As
described in the COP9 Signalosome Deneddylase section above, the
CRL system is dynamic and responds to a stimulus for ubiquitylation: the
stimulus induces substrate binding to an SR, which is sensed by CSN and inhibits
deneddylation. As such, the substrate indirectly preserves NEDD8 on the cullin.
NEDD8 alters the dynamics of the cullin WHB domain and RBX RING to favor
catalytic conformations. NEDD8 stimulates substrate ubiquitylation by indirectly
stabilizing the assembly - by preventing CAND1-mediated SR exchange, and by
directly promoting interactions between the neddylated CRL and a
ubiquitin-carrying enzyme. The manifold roles of neddylation and deneddylation
presumably underlie the effectiveness of the NAE inhibitor, the investigational
anticancer drug Pevonedistat, at arresting CRL activities (116).</p><p>Quantifying the extent and properties of cellular CRL assembly and
activation on demand in response to a stimulus required overcoming two major
challenges: (a) depleting all cellular CAND1 and CAND2 to
demonstrate their importance and (b) examining regulation in
their presence, while preventing CAND–CUL–SR exchange from skewing
the apparent repertoire of cullin-bound SRs (11). These challenges were solved by using CRISPR-Cas9 technology to
delete the CAND1 and CAND2 genes and by adding recombinant, untagged
cullin–RBX to lysates prior to purifying a cullin that was endogenously
epitope tagged (38). Recombinant
cullin–RBX acts as a molecular sponge that soaks up unassembled CAND
proteins and SR modules. This prevents postlysis exchange and enables the
capture of quantitative proteomics snapshots of proteins bound to tagged
cullin–RBX across varying cellular conditions. Consistent with the model,
the repertoire of assembled CRL complexes responds to stimuli (33–35).
CAND1-, CSN-, and neddylation-dependent, stimulus-mediated CRL assembly on
demand has been quantified for a variety of signals from TNFÎ± to
anticancer therapeutic degraders (38,
39). Amazingly, a series of kinetic
measurements indicated that, in cells, each CUL1 molecule goes through the
entire neddylation-deneddylation-SR exchange cycle every few minutes (37).</p><!><p>Our understanding of CRLs has been transformed by the discovery of molecular
components and their cellular functions and structures. We now recognize that
regulation depends on the ongoing interconversion between cullin–RBX
assemblies with factors mediating ubiquitylation and those scanning for SRs bound to
substrates. It is also appreciated that the elaborate system of varying CRL
regulators allows substrates to essentially demand their own ubiquitylation. The
regulatory circuit depends on the multidomain and dynamic nature of the
cullin–RBX core, which enables varying multisite interactions. The circuit is
robust because of the avid nature of the multisite interactions. Transient complexes
disassemble when certain contacts cannot engage; for example, after ubiquitin
transfer to a substrate, interactions with the carrying enzyme–linked
ubiquitin are lost. The circuit also relies on the dynamic nature of neddylated RBX
RING and cullin WHB portions of the cullin–RBX complex, which bind
functionally distinct partners bind in particular conformations that specify each
activity. In coming years, we expect structural studies to provide insight into the
fleeting regulatory and catalytic reaction trajectories, as well as the ways in
which these dynamics are integrated with cellular and developmental signals.</p><p>The CRL system is a vast landscape on which to generate new chemical matter
to act as degraders. It remains to be seen the extent to which the discovery of new
molecular glues will be serendipitous, the primary route to date. We anticipate that
in the future, it will be commonplace for systematic screens to prospectively
identify small molecules that bind both an SR and an important cellular target for
productive ubiquitylation. New technologies are needed to rapidly scan for such very
special molecules and to truly reveal the promise of molecular glues for
therapeutics.</p><p>In addition, fundamental discoveries related to the
neddylation-deneddylation cycle and SR exchange illuminate how dynamic the system
is. CRL assembly thus provides a roadmap for understanding the properties of
cellular stimuli. Those generating sufficiently long-lived
SR–substrate–cullin–RBX interactions have the potential to
convert the associated CRL into an active neddylated form, thereby stabilizing the
complex and enabling efficient substrate ubiquitylation. Understanding how the
dynamics of the CRL system are intimately linked with novel degraders and other
stimuli will unveil their underlying system-wide biological and therapeutic
regulatory circuits.</p>