E3 Ubiquitin Ligases in Signaling, Disease, and Therapeutics

Abstract

The ubiquitin–proteasome system (UPS) is a central regulator of protein turnover and signaling, with E3 ubiquitin ligases conferring substrate specificity and chain-type control. Recent advances have revealed new mechanistic classes of E3 ligases and expanded our understanding of their roles in disease, including cancer, neurodegeneration, and immune dysfunction. These insights have fueled the development of targeted protein degradation strategies that harness the UPS to eliminate disease-associated proteins. Approaches such as PROTACs, molecular glues, and antibody-based degraders are broadening the druggable proteome. Despite this progress, key challenges remain, including limited E3 ligase diversity, difficulties in degrader delivery, and resistance mechanisms. This review outlines recent advances in E3 ligase biology and therapeutic degradation, emphasizing opportunities to expand and refine UPS-targeted interventions.

Ubiquitination and the Central Role of E3 Ligases in Cellular Regulation

The ubiquitin (Ub; see Glossary) system is a fundamental regulator of protein homeostasis, orchestrating protein stability, localization, and degradation across nearly all cellular processes [1]. Central to this system is the modification of proteins with Ub and Ub-like proteins (UBLs), which modulate signaling, DNA repair, immunity, and proteostasis [2]. Dysregulation of this tightly controlled process contributes to numerous pathologies, including cancer, neurodegeneration, and immune disorders [3].

Ubiquitination proceeds through an enzymatic cascade involving Ub-activating (E1), Ub-conjugating (E2), and Ub ligating (E3) enzymes, with E3 ligases playing the most decisive role in determining substrate specificity (Box 1) [4]. The human genome encodes over 600 E3 ligases, comprising a vast and mechanistically diverse family that confers exquisite control over cellular signaling. Importantly, E3 ligases not only define substrate fate, but in many cases also dictate the topology of Ub chains, factors that collectively influence whether a protein is degraded, relocalized, or functionally modified [5].

Recent work has greatly expanded our understanding of E3 ligases, revealing novel classes, unconventional catalytic mechanisms, and cooperative architectures such as E3–E3 super-assemblies [6]. These discoveries have reframed our view of E3 ligases as dynamic signaling platforms rather than simple enzymatic relays. At the same time, targeted protein degradation (TPD) strategies, including proteolysis-targeting chimera (PROTACs) and molecular glues, have emerged as powerful tools for therapeutic intervention, many of which hijack or engage E3 ligases to selectively eliminate disease-driving proteins [7].

This review highlights recent advances in the classification, mechanism, and functional diversity of E3 ligases, their roles in cellular signaling and disease, and the growing landscape of therapeutic strategies that exploit these enzymes. We also examine how distinct Ub chain types and their regulation influence signaling outcomes, and how dysregulation of Ub networks contributes to disease. Finally, we explore current challenges and future directions, including efforts to expand the repertoire of druggable E3 ligases, elucidate E3-substrate relationships, and improve degrader pharmacology. By focusing on E3 ligases as both key biological regulators and emerging therapeutic targets, we aim to provide a cohesive synthesis of recent progress in Ub biology and to highlight opportunities for innovation in the development of next-generation degraders.

E3 Ligases: Diverse Mechanisms Underlying Substrate Specificity and Signaling Control

RING/U-box Ligases

RING (Really Interesting New Gene) and U-box E3 ligases act as scaffolds that align the E2~Ub conjugate with target substrates, catalyzing direct Ub transfer without forming an E3-Ub intermediate (Figure 1A) [8, 9]. This class includes the largest and most functionally diverse group of E3 ligases. RING ligases can function as monomers (e.g., c-CBL), homodimers (e.g., RNF4), heterodimers (e.g., BRCA1–BARD1), or as components of large multiprotein complexes such as Cullin-RING ligases (CRLs) [10].

Figure 1. Mechanistic Diversity of E3 Ubiquitin Ligases.

(A) RING E3 ligases act as scaffolds, bridging the E2~ubiquitin complex and the target substrate, facilitating ubiquitination directly by the E2. (B) HECT E3 ligases first transfer ubiquitin from E2 to an active site cysteine on the E3, followed by substrate recruitment and ubiquitin conjugation. (C) RBR E3 ligases transfer ubiquitin from the E2 to an active site cysteine on the RING2 domain, followed by ubiquitin conjugation to the target substrate. (D) Certain CRLs (Cullin-RING Ligases) recruit RBR E3 ligases, such as ARIH1, to transfer ubiquitin from the E2 to ARIH1, which then ARIH1 conjugates ubiquitin to the substrate. (E) RZ finger E3 ligase such as RNF213 uses E3 shell and CTD to bind to E2. RZ domain is used to recruit Ub. (F) RING-Cys-Relay E3 ligases transfer ubiquitin from E2 to an upstream cysteine on their TC domain before conjugating ubiquitin to the threonine residue of substrate.

CRLs are modular assemblies composed of a Cullin scaffold (CUL1–9), a RING-finger subunit (RBX1 or RBX2), and interchangeable adaptor and substrate receptor proteins [11]. Their catalytic activity is regulated by NEDD8 modification, which relieves autoinhibition and enhances E2~Ub recruitment [12]. The modular design of CRLs enables broad substrate specificity, and variation in complex architecture contributes to functional specialization. CRLs orchestrate the ubiquitination of numerous key regulators, including those involved in cell cycle control, hypoxic response, and innate immunity [13]. Owing to their broad expression and central roles in diverse cellular processes, RING E3 ligases have emerged as attractive targets for therapeutic intervention [14]. In particular, the substrate receptors of CRLs offer unique opportunities for targeted degradation strategies. For example, the conserved cysteine residue C1113 in the DCAF1 substrate receptor can be exploited for the development of electrophilic PROTACs, enabling covalent engagement and selective degradation of disease-relevant proteins [15].

Recent advances have shown that CRLs can also assemble into higher-order supercomplexes with RBR (RING-between-RING) ligases, such as ARIH1 and ARIH2 [16, 17]. In these cooperative assemblies, the CRL scaffold recruits the E2~Ub donor, while the RBR ligase performs the catalytic transfer of Ub to the substrate. This mechanism allows for precise modulation of chain topology and substrate selection, expanding the functional scope of each E3 class. These findings underscore the emergent regulatory complexity of E3–E3 crosstalk in shaping Ub signaling outcomes.

HECT Ligases

HECT (Homologous to E6AP C-Terminus) E3 ligases catalyze Ub transfer through a two-step mechanism that enables direct control over chain type and substrate modification (Figure 1B) [18]. Unlike RING ligases, which act as scaffolds, HECT ligases form a covalent E3~Ub intermediate via an active-site cysteine before transferring Ub to the substrate. This intermediate allows for greater regulatory precision, including chain editing and linkage specificity.

HECT E3 ubiquitin ligases share a conserved C-terminal HECT domain, which catalyzes ubiquitin transfer via a transthiolation reaction mediated by an E2-binding N-lobe and a catalytic C-lobe connected by a flexible hinge [18]. They are classified into three main families based on their N-terminal domain architecture, which dictates substrate recognition, localization, and regulatory inputs [19]. The NEDD4 family is defined by a C2 domain (for membrane association) and multiple WW domains that recognize PY motifs. Members include NEDD4–1, NEDD4–2, SMURF1, SMURF2, and ITCH, which regulate diverse pathways including receptor internalization, ion channel turnover, and TGF-β signaling [20, 21]. The HERC family, subdivided into large (HERC1, HERC2) and small (HERC3–6) members, contains one or more RCC1-like domains (RLDs) that mediate protein interactions involved in DNA repair, mitosis, and innate immunity [22]. The “Other” HECT ligases lack WW or RLD domains but contain alternative motifs such as armadillo repeats or IQ domains; members like HUWE1, E6AP, UBR5, and HECTD1 function in transcriptional silencing, apoptosis, and neurodevelopment [23].

HECT ligases are emerging as tissue-specific regulators and disease drivers. Loss-of-function mutations in neuronally imprinted UBE3A cause Angelman syndrome, a neurodevelopmental disorder marked by impaired synaptic plasticity and intellectual disability [24]. HUWE1 targets both tumor suppressors and oncogenes (e.g., p53, MYC, MCL1), and its dysregulation disrupts DNA repair and promotes tumor progression [25–27].

In addition to canonical K48-linked degradation, many HECT ligases assemble alternative chains that modulate signaling. For instance, SMURF2 mediates K63-linked ubiquitination of TGF-β components, regulating SMAD signaling and receptor trafficking without triggering degradation [28]. TRIP12 also catalyzes K29 chains that are associated with proteotoxic stress responses and K29/K48 branched chains that regulate substrates in oxidative, lipid, and pH stress responses and protein degradation [29]. UBR5 catalyzes K11/K48 chains important in cell cycle and quality control [30]

Despite their therapeutic relevance, HECT ligases have long been considered undruggable due to their conformational plasticity and transient intermediates [31]. With that said, recent advances have revealed dynamic conformational states and allosteric surfaces amenable to targeting including covalent allosteric inhibitors of SMURF1 that lock the αH10 helix in an inactive conformation [32]. Separately, reconstituted E3~Ub complexes using ubiquitin-propargylamine have enabled mapping of ligandable surfaces, supporting covalent inhibitor development that disrupts catalysis or substrate binding [33]. These strategies underscore the emerging therapeutic potential of selectively targeting HECT ligases in disease.

RBR Ligases

RBR (RING-between-RING) E3 ligases represent a mechanistic hybrid between RING and HECT-type enzymes, enabling finely tuned control over Ub transfer [34]. These ligases contain three conserved domains: a RING1 domain, which recruits E2~Ub conjugates; an IBR (In-Between-RING) domain, which provides structural scaffolding; and a RING2 domain, which contains an active-site cysteine that forms a transient E3~Ub thioester intermediate [34]. This two-step catalytic process allows RBR ligases to maintain both the recruitment flexibility of RING-type enzymes and the chain-editing control of HECT ligases (Figure 1C).

RBR ligases play essential roles in multiple signaling pathways. One of the most studied members, Parkin, is a key regulator of mitophagy, responsible for ubiquitinating damaged mitochondrial surface proteins and targeting them for autophagic clearance [35]. Mutations in Parkin (PARK2) are among the most common genetic causes of autosomal recessive Parkinson’s disease, and loss of Parkin activity results in mitochondrial dysfunction and oxidative stress [36]. Structural and biochemical studies of Parkin have revealed tightly regulated autoinhibition and activation mechanisms, including phosphorylation by PINK1 and conformational rearrangement of the RING domains to expose the catalytic site [37].

Other RBR ligases include ARIH1 and ARIH2, which participate in proteostasis and immune signaling [17]. ARIH1 and ARIH2 interact functionally with CRLs to form higher-order E3–E3 supercomplexes [17]. In this cooperative mechanism, the CRL complex positions the E2~Ub conjugate, while the RBR ligase performs the catalytic transfer of Ub to the substrate (Figure 1D). Recent structural and functional studies have shown that this CRL–RBR interaction broadens the potential for substrate recognition and ubiquitin chain diversification, offering expanded regulatory capabilities that may impact diverse cellular signaling processes [38]. These supercomplexes illustrate a growing appreciation for modular coordination between distinct E3 ligase families, expanding the topological and temporal control of Ub signaling.

Another notable RBR ligase is HOIP, which, along with HOIL-1 and SHARPIN, forms the Linear Ub Assembly Complex (LUBAC). LUBAC uniquely generates M1-linked linear Ub chains, a modification that activates NF-κB signaling and promotes pro-survival and immune responses [39, 40]. Disruption of LUBAC components has been linked to autoinflammatory syndromes, immune dysregulation, and increased susceptibility to infection [41]. HOIP’s catalytic specificity is achieved through coordination between its RING2 domain and the LDD (Linear chain Determining Domain), which orients the substrate Ub’s N-terminal methionine for chain initiation [42].

Recent structural studies have provided molecular insights into RBR ligase activation and chain-type specification, opening new avenues for drug development. Unlike RING ligases, which are typically regulated through scaffolding or substrate availability, RBR ligases often require conformational unlocking or posttranslational modification for activity [37]. This level of regulatory control, combined with their unique catalytic relay, makes RBR ligases attractive yet underexplored candidates for therapeutic targeting in neurodegeneration, immunity, and inflammation.

RZ and RCR Ligases

In addition to the well-characterized RING, HECT, and RBR families, recent work has identified mechanistically distinct classes of E3 ligases that operate through unconventional catalytic strategies [43]. These emerging ligases, RZ (derived from RNF213 and ZNFX1) finger [44] and RCR enzymes [45], expand the diversity of ubiquitination beyond classical lysine targeting and scaffold-based catalysis, offering new perspectives on E3 function in immune regulation, neurobiology, and stress adaptation.

RZ finger ligases contain a unique zinc-coordinating fold with an active-site cysteine that forms a covalent E3~Ub intermediate, similar to HECT and RBR ligases, but they lack canonical RING or HECT domains [43]. The best-characterized example is RNF213, a 586 kDa ligase mutated in Moyamoya disease, a cerebrovascular disorder marked by stenosis of the internal carotid artery [46]. Rather than relying on a RING domain, RNF213 uses its C-terminal domain (CtD) to mediate Ub transfer via a distinct “E3 shell” architecture that positions Ub near a catalytic cysteine within a noncanonical RZ fold (Figure 1E) [47]. RNF213 uniquely catalyzes ubiquitin transfer to serine and threonine residues via ester linkages and can also ubiquitinate bacterial lipopolysaccharides (LPS), suggesting a role in innate immune defense [43]. These findings identify RZ finger ligases as emerging players at the host–pathogen interface.

RCR ligases, exemplified by MYCBP2 (also known as PHR1), utilize a multi-step relay mechanism that transfers Ub between two conserved cysteine residues within the E3 before substrate modification (Figure 1F) [45]. In contrast to classical E3 ligases that ubiquitinate lysine residues, MYCBP2 modifies threonine side chains, introducing an entirely new layer of substrate diversity to the Ub code. RCR ligases are especially abundant in neuronal tissues and are implicated in axon guidance, synaptic remodeling, and neuronal degeneration [48]. These findings raise the possibility that RCR ligases may be particularly important in long-lived, post-mitotic cells that require noncanonical signaling modes to regulate homeostasis.

Although both RZ and RCR ligases remain underexplored compared to other E3 families, they reveal a remarkable degree of mechanistic and substrate diversity within the Ub system. Their ability to modify non-lysine residues, function outside of canonical domain architectures, and respond to unique cellular signals positions them as valuable new models for understanding Ub-dependent regulation and as potential targets for disease intervention, particularly in immune and neurological contexts.

Ubiquitin Chain Types and Their Role in Signaling

The type of Ub chain assembled by an E3 ligase is a critical determinant of substrate fate, guiding proteins toward degradation, trafficking, or non-proteolytic signaling [49]. Ub contains seven lysine residues and an N-terminal methionine (M1), each of which can serve as a linkage point for polyUb chain formation. These chain types encode distinct cellular messages that are interpreted by Ub-binding proteins and deubiquitinases (DUBs), thereby orchestrating diverse physiological outcomes (Figure 2A).

Figure 2. Structural diversity and linkage types in ubiquitination.

(A) Ubiquitin is a conserved 76-amino-acid polypeptide containing seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) and an N-terminal methionine (M1), each capable of forming distinct ubiquitin chain linkages.

(B) Ubiquitin modifications include monoubiquitination (single ubiquitin moiety attached to one or more substrate lysine residues), multi-monoubiquitination (multiple ubiquitin moieties attached individually to separate lysines on a substrate), polyubiquitination (chains of ubiquitin molecules attached to substrates), and branched ubiquitination (ubiquitin chains with multiple linkage points). Polyubiquitin chains composed exclusively of one type of linkage are termed homotypic, whereas chains incorporating multiple linkage types are termed heterotypic or mixed-linkage chains, providing diverse regulatory signals.

K48-linked ubiquitin chains are the canonical signal for proteasomal degradation [50]. UBR5, a HECT E3 ligase that helps assemble K48 linkages, maintains proteostasis in stem cells by targeting misfolded proteins such as mutant huntingtin [51]. These chains are recognized by proteasomal receptors like Rpn10 for efficient degradation [52]. K29-linked ubiquitination also contributes to protein quality control, especially in ER-associated degradation (ERAD). In this pathway, ER-localized E3 ligases such as Hrd1 and Doa10 tag misfolded proteins with K29, K48, or K11 chains to mark them for removal [53]. These tagged proteins are then extracted from the ER and targeted to the proteasome for degradation. In contrast, K63-linked ubiquitin chains form extended, non-degradative structures that act as scaffolds for signaling [54]. During DNA damage repair, for example, the E3 ligase TRAF6 assembles K63 chains that help recruit repair factors to sites of damage [55]. K63 chains also promote selective autophagy by linking ubiquitinated cargo to autophagosomes via adaptor proteins such as p62/SQSTM1 [56].

M1-linked chains, synthesized by LUBAC, are non-degradative and central to innate immunity and inflammation [40]. LUBAC-mediated linear ubiquitination of NEMO promotes its dimerization via UBAN domain binding, driving NF-κB phase separation and activation [39]. Other linkages have specialized roles: K6 and K27 in DNA damage and chromatin remodeling; K29 and K33 in ERAD, Wnt, and kinase signaling [57]. Though less well understood, these atypical linkages are increasingly recognized as modulators of spatially or temporally restricted signaling [57].

Beyond homotypic chains, E3 ligases can build branched and mixed-linkage chains that encode complex signaling instructions (Figure 2B) [58]. K11/K48 branches from APC/C enhance proteasomal targeting [59], while K63/K48 hybrids balance scaffolding with degradation [60]. These architectures are interpreted by linkage-specific DUBs and Ub-binding domains attuned to topology and accessibility, enabling the Ub system to “write,” “read,” and “erase” a multilayered signaling language.

Recent advances in Ub chain detection technologies have deepened our understanding of these complex modifications. Mass spectrometry-based proteomics approaches, such as Ub-clipping [61], diGly remnant profiling, and TUBE-based enrichment [62], now allow linkage-specific detection in vivo [63]. Chain-specific antibodies and engineered binding probes are also enabling more precise mapping of Ub architectures across signaling pathways [64].

Together, the topology of Ub chains provides an essential axis of cellular regulation, translating the enzymatic activity of E3 ligases into highly specific outcomes. Disruption of chain assembly, recognition, or editing underlies a wide spectrum of human diseases, from cancer and autoimmunity to neurodegeneration. In the next section, we explore how these regulatory principles are enforced, and how their breakdown contributes to pathology.

Regulation of Ubiquitin Signaling

The activity and specificity of the Ub system are tightly regulated by posttranslational modifications (PTMs), dynamic protein–protein interactions, and spatial compartmentalization [2]. These mechanisms control when, where, and how Ub is attached to substrates and are particularly critical at the level of E3 ligases, which act as gatekeepers of substrate fate.

One of the best-characterized regulatory modules involves CRLs, whose activity is governed by reversible neddylation of the Cullin scaffold [12]. NEDD8 conjugation relieves autoinhibition and enhances E2~Ub positioning [65], aided by DCN1 [66] and reversed by the COP9 signalosome (CSN) [67]. This cycle enables CRLs to respond dynamically to signals such as DNA damage and mitogens (Figure 3) [68].

Figure 3. Regulation CRL signaling.

(A) DCN1 enhances cullin neddylation by recruiting the NEDD8 E2 enzyme to cullin-RING ligases. Conjugation of Nedd8 to the C-terminus of cullin induces a conformational shift in the RING domain, enabling binding to E2~ubiquitin and activation of the CRL. (B) The neddylated CRL facilitates ubiquitination of the substrate. (C) DUBs can cleave the isopeptide bond between the substrate and ubiquitin. (D) CSN cleaves Nedd8 and shifts CRL to an inactive conformation. (E) CAND1 binds to the unneddylated cullin and displaces adaptor and receptor proteins. (F) New adaptor and receptor form a complex with cullin. (G) CAND1 dissociates from cullin complex.

Substrate selection by E3s is often controlled by phosphodegrons, short linear motifs exposed by phosphorylation, which are recognized by adaptors like β-TrCP in SCF (Skp, cullin, F-box containing) complexes [69]. This mechanism provides precise timing, as seen in the degradation of cell cycle regulators and IκBα during NF-κB activation [69]. Acetylation can also inhibit ubiquitination by masking lysine residues, stabilizing proteins like p53 and FOXO3a in stress responses [70].

Crosstalk with other PTMs further fine-tunes Ub signaling. For instance, SUMOylation can prime substrates for ubiquitination via SUMO-targeted Ub ligases (STUbLs) such as RNF4, which recognize polySUMO chains and add K48-linked Ub to drive proteasomal degradation [71]. This SUMO-to-Ub handoff is important in nuclear quality control and the DNA damage response [72]. Other modifications, such as as O-GlcNAcylation [73], ADP-ribosylation [74], and S-nitrosylation [75], can affect ubiquitination indirectly by altering substrate conformation or E3 activity.

Recent proteomic and structural studies have revealed how multiple PTMs converge to modulate E3 ligase function. High-throughput phosphoproteomics has uncovered novel degrons [76], while activity-based probes and structural analyses have mapped PTM-induced changes in E3–substrate interactions [77]. These findings highlight a broader principle: Ub signaling is embedded within a dense PTM network that filters cellular inputs into context-specific outcomes by shaping chain type, substrate localization, and signal duration. As discussed next, breakdowns in these regulatory layers can drive diverse pathologies.

Ubiquitin Dysregulation in Diseases

Dysfunction of the Ub system, particularly involving E3 ligase activity, Ub chain assembly, and posttranslational regulation, is increasingly recognized as a driver of human disease [48]. Genetic mutations, chronic stress, or pathogen interference can disrupt these processes, contributing to cancer, neurodegeneration, immune disorders, and infection [5]. Key mechanisms and examples are discussed below.

E3 ligases in tumorigenesis and cancer progression

E3 ligases critically shape tumor suppressor and oncogene stability. Indeed, loss-of-function mutations in F-box protein FBXW7 or VHL, both of which function as substrate recognition components of E3 ubiquitin ligase complexes, impair degradation of oncogenic drivers (e.g., cyclin E, c-Myc, hypoxia-inducible factors (HIFs)), promoting unchecked proliferation and angiogenesis [78, 79]. Conversely, amplification of oncogenic E3s such as MDM2 leads to excessive p53 degradation, suppressing apoptosis and enabling genomic instability [80]. These disruptions reflect how altered substrate recognition or chain topology (e.g., excessive K48-linkage) shifts the balance between cell survival and death.

E3 ligases and proteotoxic stress in neurodegeneration

Defective Ub-dependent clearance of misfolded proteins contributes to protein aggregation in neurodegenerative diseases [36]. Mutations in the Parkin E3 ligase hinder mitophagy in Parkinson’s disease, exacerbating oxidative stress [81]. In Alzheimer’s disease, failure to tag tau and amyloid-β for Ub–proteasome system (UPS) or autophagic degradation leads to toxic accumulation [82]. Emerging therapies aim to restore these clearance pathways via enhanced E3 function or degrader-based strategies.

Ub signaling control of immune responses and inflammation

E3 ligases modulate immune homeostasis by regulating key inflammatory pathways [83]. The LUBAC complex generates M1-linked chains that activate NF-κB via NEMO ubiquitination [39]. Mutations that impair LUBAC activity cause immunodeficiency [43], while gain-of-function variants drive chronic inflammation and autoimmunity [41]. The linkage-specific activity of E3 ligases like LUBAC exemplifies how chain topology encodes immune outcomes.

Viral hijacking of E3 ligases

Viruses exploit host E3 machinery to escape immune surveillance and enhance replication [84]. For example, human papillomavirus (HPV)’s E6 recruits E6AP/UBE3A to degrade p53, blocking apoptosis [85]. Human immunodeficiency virus (HIV) co-opts CRL complexes to degrade antiviral proteins such as APOBEC3G [86], while hepatitis B (HBV) and Hepatitis C virus (HCV) subvert host Ub signaling to persist in hepatocytes [87, 88]. These mechanisms illustrate how pathogens target E3–substrate interactions to manipulate host defenses. Collectively, these examples illustrate how dysregulation of the ubiquitin system, whether through genetic mutation, environmental stress, or pathogenic interference, can disrupt proteostasis and signaling across multiple physiological systems. Aberrant E3 activity, altered chain topology, and regulatory failure contribute to a wide spectrum of diseases, and as a result, targeting the ubiquitin system, particularly E3 ligases, offers promising therapeutic opportunities for restoring cellular balance.

Targeted Protein Degraders: Redirecting Ubiquitin for Therapeutic Benefit

Building on the mechanistic diversity of E3 ligases and their central role in protein fate, several innovative strategies have emerged to therapeutically redirect Ub signaling. Rather than inhibit proteins directly, these TPD approaches exploit the cell’s own UPS to eliminate pathogenic proteins (Table 1). This shift expands the druggable proteome and offers durable suppression of disease drivers.

Table 1.

Targeted protein degradation approaches.

Strategy	Mechanism	E3 Ligase Engagement	Advantages	Limitations
PROTACs	Bifunctional molecule links POI to E3 ligase	Androgen or estrogen receptors targeted by Cul3 VHL and Cul4 CRBN	Reversible, tunable design, catalytic mode of action	Large size affects cell permeability and pharmacokinetics
Molecular Glues	Small molecules stabilize interaction between POI and E3 ligase	IKZF1 targeted by Cul4 CRBN	Small, drug-like, can target undruggable proteins	Requires serendipitous or engineered compatibility
AbTACs	Bispecific antibodies engage POI and membrane-associated E3 ligase	RNF43	High specificity for membrane proteins	Limited to surface proteins; requires antibody delivery
IBG	Engages two adjacent domains of a POI in cis	BRD4 targeted by Cul4 DCAF16	May overcome weak affinities of monovalent glues	Design complexity; linker length critical
Degron Tails	Fusion of short degradation tags (“degrons”) to proteins	XIAP, NSD2 and FKBP12 targeted by Cul1 FBXO22	Can transform Non-degrading Ligands into Degraders	Can cause reduced solubility and may cause off-target reactivity

PROTACs

PROTACs are bifunctional small molecules that induce the selective degradation of intracellular proteins by co-opting the UPS [89]. Unlike conventional inhibitors, which merely block protein activity, PROTACs eliminate the entire target protein, preventing its function and reducing the likelihood of resistance caused by adaptive mutations.

Each PROTAC consists of three components: a ligand that binds the protein of interest (POI), a ligand that recruits an E3 Ub ligase, most commonly CRBN or VHL, and a chemical linker that bridges the two [89]. Upon ternary complex formation, the E3 ligase catalyzes the transfer of Ub moieties to the POI, marking it for recognition and degradation by the 26S proteasome (Figure 4A) [89]. This mechanism allows PROTACs to act catalytically, degrading multiple proteins per compound.

Figure 4. Therapeutic Strategies targeting Ub pathways.

(A) Proteolysis-targeting chimeras (PROTAC) induces the ubiquitination of protein of interest (POI). PROTACs are comprised of E3-recruiting ligand, a warhead that recruits protein of interest (POI), and a linker. (B) Molecular glue degraders are small molecules that induce the interaction between E3 ligase and POI. Rather than bridging two proteins, molecular glues stabilize novel interfaces at the E3–substrate binding site, triggering ubiquitination and degradation. (C) Intramolecular bivalent glues (IBGs) are a novel targeted protein degradation (TPD) strategy that induce conformational changes in a target protein by bridging adjacent domains, promoting its recognition and degradation by E3 ligases without directly binding the ligase. (D) Degradation tails are electrophilic or hydrophobic groups fused to non-degrading ligands that promote target protein ubiquitination by forming covalent bonds with E3 ligases, enabling degradation of POI. (E) AbTACs use bispecific antibodies to bridge membrane proteins with transmembrane E3 ligases like RNF43, promoting ubiquitination, endocytosis, and lysosomal degradation.

PROTACs have demonstrated broad therapeutic potential. Several CRBN- and VHL-based molecules targeting oncogenic drivers such as BRD4 [90], BCL-XL [91], and hormone receptors (AR, ER) [92, 93] have advanced into clinical trials. PROTACs are also explored for neurodegeneration [94], immune disorders [95], and infections [96]. Notably, they can target proteins traditionally considered “undruggable,” such as transcription factors and scaffold proteins, thereby expanding the pharmacologic landscape [7].

Molecular Glue Degraders

Molecular glues are monovalent small molecules that induce neomorphic interactions between an E3 ligase and a non-native substrate, stabilizing new interfaces that promote ubiquitination and degradation (Figure 4B) [97]. Unlike PROTACs, they do not require a linker, and their small size makes them more drug-like. Clinically validated examples include lenalidomide and pomalidomide, which reprogram CRBN to degrade IKZF1 and IKZF3 in multiple myeloma [97]. Molecular glues are particularly valuable for targeting proteins that lack ligand-binding pockets, such as transcription factors and regulatory proteins. More recently, glues targeting DCAF15 (e.g., indisulam) have broadened the E3 ligase landscape [98], and ongoing studies are identifying additional gluable ligases such as DCAF11 [99] and RNF126 [100].

Intramolecular Bivalent Glues (IBGs)

Intramolecular bivalent glues (IBGs) are a novel TPD strategy that bind two adjacent domains of a target protein in cis, inducing conformational changes that promote recognition by endogenous E3 ligases and subsequent ubiquitination (Figure 4C) [101]. For example, IBG1 was initially designed to target BRD4, a key regulator of transcription and the cell cycle, via its natural substrate receptor DCAF15, but was later found to promote degradation through a different E3 ligase, DCAF16 [102]. Structural analysis revealed that IBG1 bridges BRD4 bromodomains BD1 and BD2, inducing domain rearrangement that stabilizes interactions with the E3 ligase. Unlike conventional degraders that require high-affinity ligands to recruit E3s directly, IBGs reconfigure the target protein itself to enhance ligase binding. By simultaneously engaging two distinct domains, they create new interfacial contacts for E3 engagement and increase substrate specificity, offering a promising approach when direct E3 recruitment is limiting.

Degradation Tails

Degradation tails are electrophilic or hydrophobic moieties fused to non-degrading ligands to promote ubiquitination by recruiting E3 ligases (Figure 4D) [101]. For example, JQ1, a BET inhibitor, was modified with an electrophilic group that covalently binds Cys58 on DCAF16, enabling BRD4-dependent recruitment via template-assisted covalency [103]. Similarly, alkylamine tails can target FBXO22, a CUL1 substrate receptor, leading to degradation of XIAP, NSD2, and FKBP12 [104].

Degrading Extracellular and Membrane Proteins via Lysosomal Pathways Via AbTACs

While the UPS efficiently degrades intracellular proteins, extracellular and membrane-bound proteins require alternative degradation strategies, as they are inaccessible to the proteasome. Molecular degraders that harness lysosomal degradation pathways have emerged as promising therapeutic tools, allowing targeted removal of disease-associated proteins involved in cancer, neurodegenerative disorders, and immune dysregulation [105].

One key approach is antibody-based PROTACs (AbTACs), employs bispecific antibodies to simultaneously bind a target protein and a transmembrane E3 ligase, such as RNF43, inducing ubiquitination and lysosomal clearance of the target (Figure 4E) [105]. AbTACs recruit Ub machinery to membrane proteins, triggering their endocytosis and subsequent lysosomal degradation. This approach expands the scope of targeted protein degradation beyond intracellular proteins, offering new treatment avenues for conditions where extracellular signaling pathways drive disease progression.

Challenges in Targeted Protein Degradation

Despite remarkable progress, several challenges limit the full therapeutic potential of TPD. Chief among these is our incomplete understanding of E3 ligase substrate specificity, which constrains both mechanistic insight and rational degrader design. Although the human genome encodes over 600 E3 ligases, only a small subset has been functionally characterized, and even fewer adapted for therapeutic use. Substrate-ligase interactions are often transient, condition-dependent, and spatially compartmentalized, complicating efforts to predict or engineer degradative outcomes.

A related limitation is the restricted pool of recruitable E3s. Most current PROTACs rely on CRBN or VHL, which may not be expressed uniformly across tissues or tumor types. While promising new ligases such as DCAF15 [106], KEAP1 [107], and RNF114 [108] are emerging, many lack well-characterized, high-affinity ligands or structural data, a bottleneck that slows expansion into tissue-specific or resistance-resilient applications. A study leveraging RNA sequencing across human cancer cell lines identified 113 E3 ligases with tumor-specific expression patterns, offering a potential avenue for more targeted degradation strategies [109].

Beyond ligand development, technical barriers complicate the mapping of E3-substrate relationships. Ubiquitination is a rapid and reversible process, and many substrate-ligase interactions are low-affinity or short-lived. Substrates may exist at low abundance or be rapidly degraded, limiting their detection by conventional mass spectrometry. Proximity labeling tools (e.g., BioID, TurboID) [110], engineered E3 traps [111], and Ub-activated interaction profiling (UBAIT) [112] are helping overcome these limitations by capturing interactions in situ. A recent study introduced a powerful method called COMET (Combinatorial Mapping of E3 Ubiquitin Ligases to their Target Substrates), which uses genetic libraries to systematically test E3-substrate interactions [113]. In a complementary approach, Global Protein Stability (GPS)-peptidome screening functionally mapped over 15,000 degron candidates and their cognate E3 ligases, revealing diverse substrate recognition modes across CRLs and enabling motif clustering [114]. Both studies employed AlphaFold-Multimer to model E3–substrate complexes, helping to rationalize interaction specificity and degron recognition. Although originally developed to study native E3–substrate biology, these approaches are increasingly being leveraged to inform degrader design by revealing ligase specificity, degron architecture, and structural compatibility.

Even for well-characterized ligases, clinical application of degraders remains challenging. Tumors exposed to VHL- or CRBN-recruiting PROTACs can develop resistance through loss-of-function mutations or altered expression of the E3 ligase [115]. Expanding the pool of recruitable ligases may mitigate this vulnerability. In addition, PROTACs often have poor permeability, low bioavailability, and rapid clearance due to their large size (~1000 Da). Formulation strategies such as lipid nanoparticles are being explored to improve delivery and pharmacokinetics [115]. Molecular glues, while more compact and often orally bioavailable, rely on emergent and often unpredictable protein-protein interfaces, complicating rational design and sometimes leading to off-target toxicity [101]. Developing tunable molecular glues and optimizing degrader scaffolds for stability, selectivity, and delivery remain key to improving clinical efficacy.

To address the challenges of resistance, poor bioavailability, and limited E3 ligase diversity in degrader development, multidisciplinary integration is essential. Fragment-Based Ligand Discovery (FBLD) is a powerful strategy for identifying chemical fragments that bind to functional pockets on proteins, including E3 ligases [128]. The process begins with screening diverse chemical fragment library against the protein of interest. A combination of high-throughput biochemical techniques is used to identify the interacting chemical moieties with the protein of interest. Once the initial binding fragments are identified, they are further developed into high-affinity ligands [128]. For example, FBLD has been successfully applied to identify ligands for VHL, enabling the development of VHL-based PROTACs through structure-guided design [116]. Recent work has extended fragment-derived ligands to underexplored ligases such as RNF4 [117], where fragment screening has uncovered novel, ligandable pockets. These findings highlight how FBLD can reveal new ligandable E3 ligases, paving the way for more diverse strategies in targeted protein degradation.

Structural biology, AI-based modeling, and FBLD are revealing cryptic binding pockets on previously undruggable E3 ligases [135–138]. AI-driven substrate prediction, chemoproteomic profiling, and deep mutational scanning are also accelerating discovery of ligase-substrate pairs amenable to molecular glue recruitment [118]. For instance, chemoproteomic profiling recently enabled the identification of a covalent ligand for RNF114, enabling targeted degradation of BRD4 in the absence of known E3 recruiters [119]. Meanwhile, advances in delivery systems, such as nanoparticles and antibody–degrader conjugates, are improving the tissue distribution and cellular uptake of these therapeutics [139]. Together, these integrated approaches are critical to expanding the E3 toolkit, improving substrate mapping, and refining degrader pharmacology.

These efforts will be central to achieving tissue-specific, resistance-resilient, and disease-modifying degradation strategies across oncology, neurology, and immunology. In parallel, mechanistically distinct E3 ligases such as CRL-RBR super-assemblies, RZ finger ligases, and RCR enzymes offer intriguing opportunities for next-generation degrader strategies. The modularity of CRL-RBR complexes could, in principle, support programmable substrate handoff or chain-type control in engineered systems. RCR ligases such as MYCBP2, which catalyze threonine-directed ubiquitination via an internal relay, may enable noncanonical, cell-type-selective degradation, particularly in post-mitotic cells like neurons. While these ligases have not yet been co-opted in PROTAC or glue-based platforms, their unique catalytic logic broadens the conceptual toolkit for targeted degradation, especially as chemoproteomic mapping and structural modeling continue to advance.

Concluding remarks

Expanding the repertoire of druggable E3 ligases remains a central challenge and opportunity in TPD. Identifying high-quality small-molecule ligands for underexplored E3s will enable tissue- and disease-specific degrader strategies, reducing reliance on a narrow set of ligases such as CRBN, VHL, and MDM2. Advances in chemoproteomics, structure-guided ligand discovery, and AI-driven prediction are accelerating efforts to unlock new E3–substrate interactions and degrader-compatible ligase surfaces.

Equally important are innovations in delivery and control. Strategies such as nanoparticle conjugation, linker optimization, and prodrug design aim to improve tissue penetration and reduce off-target toxicity. Emerging approaches, including optogenetically activated and conditionally stabilized PROTACs, offer the promise of spatiotemporal precision, further enhancing therapeutic selectivity.

Looking forward, the integration of mechanistic insight, structural data, and high-throughput functional profiling will be essential to fully realize the potential of ubiquitin-directed therapies. As our understanding of E3 ligase biology deepens, rationally designed degraders, including PROTACs, glues, and next-generation modalities, will drive precision approaches to intractable diseases. Continued investment in Ub signaling and degradation technologies is critical for therapeutic innovation and understanding cellular regulation.

Box 1. The Ubiquitin Conjugation Cascade.

Ubiquitination is a tightly regulated PTM that involves the covalent attachment of Ub to target proteins, influencing their stability, localization, and function [120]. The process begins with the ATP-dependent activation of Ub by an E1 enzyme. ATP hydrolysis facilitates the adenylation of the C-terminal glycine residue of Ub, forming a high-energy Ub-adenylate intermediate [121]. The E1 enzyme then undergoes a nucleophilic attack via its active-site cysteine, forming a thioester-linked E1~Ub conjugate (Figure I) [122]. In humans, two E1 enzymes, UBA1 and UBA6, are responsible for Ub activation. While UBA1 is the primary E1 that loads Ub onto multiple E2s, UBA6 uniquely transfers Ub or the Ubl FAT10 to a distinct subset of E2s, thereby mediating specific downstream signaling pathways [122].

Activated Ub is then transferred from the E1 enzyme to an E2 conjugating enzyme via a transthioesterification reaction, preserving the thioester linkage (Figure I) [122]. The human genome encodes approximately 40 E2 enzymes, each varying in substrate specificity and Ub chain elongation properties [123]. Some E2s, such as UBE2T [124], mediate monoubiquitination by transferring a single Ub molecule, whereas others, like Cdc34, specialize in polyUb chain extension by adding additional Ub moieties to pre-existing Ub-modified proteins [125].

E3 Ub ligases are the final and most diverse components of the ubiquitination cascade, responsible for substrate recognition and Ub transfer from E2 to the target protein (Figure I). E3 ligases determine which proteins are ubiquitinated by selectively recognizing specific motifs or posttranslational modifications on substrates, while simultaneously interacting with the E2–Ub complex to facilitate Ub transfer. There are over 600 E3 ligases in humans, classified into three main families—RING, HECT, and RBR—based on their catalytic mechanisms. By controlling which proteins are ubiquitinated, E3 ligases govern key cellular processes such as protein degradation, signal transduction, and cell cycle progression [120].

Figure I. The Ubiquitin Activation and Transfer Pathway.

Ubiquitination is a stepwise process that tags proteins with ubiquitin. The cascade begins with ATP/Mg²+-dependent adenylation of ubiquitin by an E1 enzyme, forming a Ub-AMP intermediate. A thioester bond is then formed between the active-site cysteine of E1 and the Ub C-terminus. Ubiquitin is then transferred to an E2 conjugating enzyme through a thioester bond. Finally, an E3 ligase brings the E2~Ub complex and the target protein together, facilitating the transfer of ubiquitin onto specific lysine residues of the substrate.

Outstanding Questions Box.

• How can novel E3 ligases (e.g., RCR, RZ finger, CRL–RBR assemblies) be systematically characterized to identify substrates and reveal their biological roles?

• What approaches can effectively capture transient E3-substrate interactions and dynamic ubiquitination events under physiological conditions?

• How do branched and mixed-linkage ubiquitin chains precisely modulate cellular responses, and what molecular factors decode these signals?

• What strategies can expand the number of recruitable E3 ligases for PROTAC and molecular glue degraders, improving specificity and therapeutic efficacy?

• How can delivery systems (e.g., nanoparticles, prodrugs) optimize the pharmacokinetics and bioavailability of targeted protein degraders?

• What mechanisms drive resistance to targeted degraders, and how can future drug designs proactively mitigate such resistance?

• Can targeted degradation strategies be refined to selectively degrade pathogenic protein aggregates without perturbing normal proteostasis?

• How can ubiquitination crosstalk with other ubiquitin-like modifications (e.g., NEDDylation, SUMOylation) be leveraged for therapeutic intervention?

Highlights.

• Recent structural and biochemical studies have revealed new classes of E3 ligases, including RING-Cys-Relay, RZ finger, and CRL–RBR assemblies, expanding the mechanistic diversity of ubiquitin transfer.

• Branched and mixed-linkage ubiquitin chains serve as complex regulatory signals, integrating cellular stress, signaling, and degradation pathways.

• Advances in targeted protein degradation highlight the potential of E3-based strategies such as PROTACs and molecular glues to modulate previously undruggable proteins.

• Emerging structural, chemoproteomic, and AI-guided tools are accelerating discovery of E3–substrate interactions and enabling rational degrader design.

Glossary

AbTAC (antibody-based PROTAC)

Bispecific antibodies that recruit transmembrane E3 ligases to membrane-bound proteins, triggering lysosomal degradation. AbTACs enable targeted removal of extracellular or membrane proteins that are not accessible to the proteasome

CRL (Cullin-RING ligase)

A subclass of RING-type E3 ligases built around Cullin scaffold proteins. CRLs form modular complexes that ubiquitinate substrates involved in cell cycle, DNA repair, and signaling, and are regulated by NEDD8 conjugation

Degron

A short linear sequence or structural motif in a protein that is recognized by an E3 ligase, serving as a signal for ubiquitination and subsequent degradation

DUB (deubiquitinase)

Enzymes that remove Ub from proteins, opposing the activity of E3 ligases. DUBs regulate Ub signaling dynamics, protein stability, and cellular localization, and can edit or recycle Ub chains

E1 enzyme (ubiquitin-activating enzyme)

The initiating enzyme of the ubiquitination cascade. E1 uses ATP to activate ubiquitin, forming a high-energy thioester bond before transferring it to an E2 enzyme

E2 enzyme (ubiquitin-conjugating enzyme)

Enzymes that accept activated Ub from E1 enzymes and either transfer it directly to substrates or interact with E3 ligases to mediate ubiquitination. E2s influence Ub chain type and processivity

E3 ligase (Ub ligase)

Enzymes that confer substrate specificity in the Ub system. They catalyze or scaffold the transfer of Ub from E2 enzymes to target proteins, controlling chain topology and signaling outcomes. Families include RING, HECT, and RBR ligases

HECT (Homologous to E6AP C-Terminus) ligase

A class of E3 ligases that form a transient covalent intermediate with Ub through a catalytic cysteine before transferring it to a substrate. This two-step mechanism allows greater control over chain architecture

Molecular glue degrader

Small molecules that stabilize interactions between E3 ligases and non-native substrates by inducing a neomorphic binding interface, promoting selective ubiquitination and degradation without requiring a linker

PROTAC (proteolysis-targeting chimera)

Bifunctional small molecules that tether a target protein to an E3 ligase, inducing its ubiquitination and degradation by the proteasome. PROTACs expand the druggable proteome by removing rather than inhibiting proteins

RBR (RING-between-RING) ligase

A hybrid class of E3 ligases with two RING domains separated by an in-between RING (IBR). They function via a two-step mechanism involving a covalent E3~Ub intermediate, combining features of both RING and HECT ligases

RING (Really Interesting New Gene) ligase

The largest E3 ligase family. RING ligases function as scaffolds that bring E2~Ub and substrate into proximity, enabling direct Ub transfer without forming a covalent E3–Ub intermediate

TPD (targeted protein degradation)

Therapeutic strategies that exploit the cell’s own degradation systems (e.g., ubiquitin–proteasome system, autophagy) to eliminate disease-related proteins. Includes PROTACs, molecular glues, and AbTACs

Ub (ubiquitin)

A 76-amino acid protein modifier covalently attached to lysine residues on substrates or other Ub molecules. Ubiquitin regulates protein degradation, localization, and signaling through formation of diverse chain types

UPS (ubiquitin–proteasome system)

The principal cellular machinery for selective protein degradation. Substrates tagged with K48-linked Ub chains are recognized and degraded by the 26S proteasome