Ubiquitin proteasome system in immune regulation and therapeutics

Abstract

The ubiquitin proteasome system (UPS) is a proteolytic machinery for the degradation of protein substrates that are post-translationally conjugated with ubiquitin polymers through the enzymatic action of ubiquitin ligases, in a process termed ubiquitylation. Ubiquitylation of substrates precedes their proteolysis via proteasomes, a hierarchical feature of UPS. E3-ubiquitin ligases recruit protein substrates providing specificity for ubiquitylation. Innate and adaptive immune system networks are regulated by ubiquitylation and substrate degradation via E3-ligases/UPS. Deregulation of E3-ligases/UPS components in immune cells is involved in the development of lymphomas, neurodevelopmental abnormalities, and cancers. Targeting E3-ligases for therapeutic intervention provides opportunities to mitigate the unintended broad effects of 26S proteasome inhibition. Recently, bifunctional moieties such as PROTACs and molecular glues have been developed to re-purpose E3-ligases for targeted degradation of unwanted aberrant proteins, with a potential for clinical use. Here, we summarize the involvement of E3-ligases/UPS components in immune-related diseases with perspectives.

Introduction

The ubiquitin proteasome system (UPS)-mediated degradation of short-lived, misfolded, oxidized, or otherwise damaged protein substrates maintains cellular protein homeostasis. Substrate degradation via UPS regulates a wide range of cellular processes including immune response, apoptosis, cell cycle, cell differentiation, and signaling [1–6]. Ubiquitin, a tiny protein (~8 kDa) highly conserved throughout living species when conjugated to substrates in specific polymer forms, marks them for proteolysis via proteasomes. Ubiquitylation of substrates precedes proteolysis via proteasomes, a hierarchical feature of UPS. In a typical ubiquitylation reaction monomeric ubiquitin is activated by covalent attachment to E1 (ubiquitin-activating enzyme) followed by its transfer to E2 (ubiquitin-conjugating enzyme) for the conjugation reaction to a protein substrate. E3 (ubiquitin-ligase) acts as a substrate recruitment enzyme by binding protein substrates in the ubiquitylation cascade. Depending on the E3-substrate pair, activated ubiquitin on E2 is conjugated, typically to lysine residues, on the protein substrate recruited by E3. In monoubiquitylation reaction a single ubiquitin is conjugated, whereas in polyubiquitylation reaction, cycles of ubiquitin conjugation on the internal lysine residue(s) of previously added ubiquitin (primary sequence of ubiquitin protein contains seven acceptor lysines at positions K6, K11, K27, K29, K33, K48, and K63) forms ubiquitin-polymers of many topologies. It is generally believed that protein substrates conjugated with K48/K11 polyubiquitin polymers are targeted to proteasomes for complete degradation. Deubiquitinating enzymes (DUBs) are involved in the removal of ubiquitin polymers in a deubiquitylation reaction of the ubiquitin-modified proteins to prevent proteolysis or modulate signaling [7,8] (Figure 1).

Figure 1. Ubiquitin conjugations and protein degradation via UPS.

Ubiquitylation of proteins takes place through the sequential action of three enzymes, E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin-protein ligase). Ubiquitin is activated by E1 in an ATP-dependent reaction followed by transfer to E2, which finally conjugates the activated ubiquitin to an internal lysine of the substrate bound to E3 or to the lysine of the growing polyubiquitin chain. E3s provide specificity in the ubiquitylation cascade by directly binding and recruiting substrates. Ubiquitin-conjugated substrate may be degraded by the proteasome (K48/K11 ubiquitin linkages) or deubiquitinated by the DUBs. BioRender software was used for making the figure.

E3-ubiquitin ligase-mediated protein turnover in immune cells via UPS has functional implications for both innate and adaptive immunity [1,9]. Uncovering mechanisms for the maintenance of immunological homeostasis via E3-ligases is crucial because immune responses can be both detrimental (autoimmunity) or beneficial (antitumor immunity) [10]. Thus, understanding mechanisms of E3-ligase-mediated substrate degradation in immune cells to leverage targeting various UPS components is of great interest in both academic and pharmaceutical research. Ongoing and previous studies have provided proof-of-principal that targeting the UPS and associated components can lead to the development of effective cancer therapies [11,12].

Post-translational ubiquitin polymer conjugation

In eukaryotes, the UPS and autophagy regulate cellular protein homeostasis [13]. Autophagy degrades proteins and cellular components primarily with lysosomal hydrolysis. However, during UPS-mediated degradation, proteins are tagged with ubiquitin polymers of specific topology (e.g., K48/K11) for recognition by proteasomal adaptors for processive proteolysis by the proteasome. Thus, the UPS represents a selective proteolytic system, while autophagy is a bulk degradative system, although both utilize ubiquitin as a common modifier on their targets [13]. Proteasomal degradation by the UPS uses the K48 linkage as its primary ubiquitin chain type (Figure 2) which modulates the half-lives of thousands of short-lived proteins. Atypical ubiquitin chains of K11 and K29 topologies also are processed by proteasomes. K11-linked substrates may also include misfolded proteins in the endoplasmic reticulum (ER) destined for ERAD (ER-associated degradation) and STING (stimulator of interferon genes) during viral infection triggered innate immune response [13,14]. Ubiquitin chains on substrates may also be recognized and bound by ubiquitin-based degrons (UBDs) to transfer substrate information to downstream pathways. By processing the UBD-bound cargo, the UPS can also facilitate the removal of damaged or surplus subcellular organelles such as mitochondria or invading pathogens [15]. E3-ligases such as UBR1, UBR2, Parkin, CHIP, San1, E6-AP, and Hul5 ubiquitinate misfolded proteins with ubiquitin chains [15]. These chains are bound by UBDs of proteasome-associated adaptors, resulting in proteasomal targeting. Autophagy also utilizes ubiquitylation in protein quality control. Autophagic adaptors such as p62 or NBRI bind ubiquitin chains on target substrates for degradation via lysosomal hydrolases [13]. Autophagic adaptors preferentially bind K63 linkages on substrates promoting clearance of protein aggregates [16,17]. Moreover, K63 linkages often target damaged or surplus cellular components such as mitochondria, ER, ribosomes, and liposomes for autophagic turnover [15]. E3 ligases like Parkin, TRAF6, CHIP, and ITCH are involved in K63-linked ubiquitylation [16]. Interestingly, atypical ubiquitin chains such as K6 and K11 may also act as autophagic signals along with K48 in specific conditions [18].

Figure 2. Fate of various ubiquitin linkages.

K48- and K11-linked targets are the major substrates of proteasome-mediated degradation whereas autophagy substrates have mainly K63 linkage.

Ubiquitin proteasome system in immune system regulation

The UPS plays an important role in cell immunity specifically in cells of myeloid origin which regulate critical features of immunity, both adaptive and innate. Myeloid cells are crucial components of the immune system and include macrophages, dendritic cells (DC), monocytes, and granulocytes which monitor the body for possible intruders. Microbial products are identified by myeloid cells through pathogen-associated molecular patterns (PAMPs) which attach to their pattern recognition receptors (PRRs) [19,20] and trigger the innate immune response. PRR senses PAMP and launches a sequence of signaling pathways which induces the activation of vital transcription factors, specifically the nuclear factor- κ B (NF- κ B) and the interferon regulatory factors (IRF) 3 and 7, that manage inflammation.Once NF- κ B and IRF3/IRF7 reach the nucleus they promote the transcription of interferons (IFNs) and proinflammatory cytokines, which neutralizes invading pathogens [1]. That UPS is involved in activation and sustained PRRs signaling in response to microbial assaults suggests the importance of ubiquitin-mediated alterations of key substrate proteins in immune cell signaling [21–23]. Moreover, E3-ligases such as Inhibitors of apoptosis proteins (IAPs) are also known to act downstream of TLR, TNFR, and NOD2 to regulate NF- κ B and MAPK signaling via substrate ubiquitylation [24]. Ubiquitylation acts as a vital regulatory mechanism for IFN production to counter viruses that involves the NEMO-TBK1/IKK ε complex which plays a role in the nuclear translocation of the transcription factors IRF3 and IRF7 by driving their phosphorylation. For NEMO-TBK1/IKK ε to be assembled, the production of multiple non-proteolytic ubiquitin chains is essential. These chains include K63-linked polyubiquitin chains whose formation is catalyzed by the E3 ubiquitin ligase, TRAF3, once viral products are sensed by intracellular TLRs, such as TLR3, TLR7, TLR8, TLR9, and RIG-I-like receptors (RLRs). Suppression of RLR and TLR signaling in the presence of viral products requires the production of K48-linked polyubiquitin chains that direct vital components of the cascade for proteasomal degradation. These alterations by the E3 ubiquitin ligases RAUL, DTX4, TRIM27, or TRIP affect IRF3, IRF7, and TBK1 [25–28]. Moreover, ubiquitin hydrolases, including CYLD, USP21, OTUD5, and USP3, are target genes of IRF3 and IRF7 that trim non-proteolytic chains, and by doing so they deactivate vital signaling junctions and prevent type I IFN formation [29–31]. In the presence of a viral infection, the E3 ubiquitin ligase TRIM27 is recruited via the protein tyrosine phosphatase SHP2. Once TRIM27 is recruited, it leads to the degradation of TBK1 and negative regulation of type I IFN induction [1,32].

UPS also regulates cells of the adaptive immune system. Activation of T cells involves binding of the T cell receptor (TCR) to the antigen presented by the antigen-presenting cell (APC) through the major histocompatibility complex (MHC). Costimulation provided by binding CD28 to its ligands is required for the complete activation of T cells. In the absence of costimulation, T cells enter a long, unresponsive state termed as anergy. E3 ubiquitin ligases, Casitas B-cell lymphoma-b (Cbl-b) [33], Itch, and gene related to anergy in lymphocytes (GRAIL), block TCR signaling by targeted protein degradation in the anergic T cells. Cbl-b is a RING E3 ubiquitin ligase, which is best described as the main gatekeeper of T cell activation. Activation of T cells leads to the recruitment of Cbl-b to the TCR at the immune synapse, which implements multiple inhibitory mechanisms downstream of it [9,34]. CD4⁺ T cells can differentiate into T helper (Th) cells or regulatory T cells (Tregs) upon antigen binding. Th cells activate cytotoxic T cells to kill infected cells and B cells to secrete antibodies. In contrast, Tregs suppress the immune response to maintain homeostasis. Recently, E3 ubiquitin ligase, Cul5, was shown to promote CD4⁺ T cell fate to Treg by targeting pJak1 (Phospho Janus Kinase 1) for degradation [35]. Damage-specific DNA binding protein 1 (DDB1) E3 ligase is required for CD4⁺ Th cell expansion and antibody response to acute viral infection [36]. CRL4-DCAF12 ubiquitin ligase targets MOVO10 RNA helicase during T cell activation [37]. E3 ligase F-box/WD40 repeat-containing protein 7 (FBW7) is crucial for B cell antibody response [38]. UPS thus plays important roles in regulating the adaptive immune system.

Role of E3-ligases in inflammatory diseases and cancer

E3-ligases are deregulated in various inflammatory diseases like asthma, fibrosis, and inflammation-associated carcinogenesis (Table 1). The role of TRIM family E3-ligases in fibrosis has been extensively studied [39].ER-associated E3 ligase TRIM13 restrains inflammatory response provoked by pathogenic DNA. TRIM13 catalyzes Lys⁶-linked polyubiquitination of STING, delaying its exit from the ER and enhancing its degradation. STING is a key player in the pathogenic DNA-associated innate immune response. Trim13^−/−mice develop age-associated autoinflammation [40]. Several E3-ligases are deregulated in Inflammatory bowel disease (IBD) [41,42]. E3 ubiquitin ligase Speckle-type BTB–POZ protein (SPOP) restricts inflammation by ubiquitylation and degradation of myeloid differentiation primary response protein 88 (MYD88), an adaptor protein involved in IL1R and TLR signaling [43].

Table 1.

List of E3-ligases deregulated in inflammatory diseases and cancer. Disease associated substrates are highlighted.

Disease	E3-ligase/Component	Substrate(s)	References
Asthma Autocrine motility factor receptor (AMFR), an ER resident E3-ligase, targets cytokine inducible SH2-containing protein (CIS) for degradation and blocks its inhibitory effect on asthmatic inflammation. AMFR is upregulated in alveolar macrophages in asthma.	AMFR	CIS	[74]
Hypertensive renal disease (HRD) TGF-β1 pathway is involved in hypertensive renal fibrosis. E3-ligase TRIM31 inhibits TGF-β1 signaling by promoting degradation of the downstream player MAP3K7. TRIM31 is downregulated in HRD.	TRIM31	MAP3K7	[75]
Idiopathic pulmonary fibrosis (IPF) TGF- β is a well-characterized profibrotic factor. Fbxw7 downregulates the expression of TGF- β in profibrotic macrophages by targeting transcriptional factor Jun for Ubiquitylation and degradation. Fbxw7 expression is decreased in IPF.	Fbxw7	Jun	[76]
Inflammation-associated carcinogenesis (IAC) IL-6-STAT3 pathway plays an important role in IAC and the plasma membrane-associated E3 ubiquitin ligase MARCH3 negatively regulates the axis. MARCH3 promotes the polyUbiquitylation of the IL-6 receptor α-chain (IL-6Rα) and its coreceptor gp130, leading to their lysosomal degradation. MARCH3 deficiency promotes IAC and it has been shown to be downregulated in human colorectal cancer.	MARCH3	IL-6Rα, gp130	[45]
Colorectal carcinoma (CRC) and Hepatocellular carcinoma (HCC) Fbxo38 knockout promotes faster tumor progression by increasing the levels of PD-1 in tumor infiltrating T lymphocytes. In CRC and HCC, FBXO38 transcriptional levels have been shown to be downregulated in tumor infiltrating T cells. The most common symptoms of CRC are vague abdominal pain, blood in the stools or rectal bleeding, tenesmus, pain when defecating, and anemia. The symptoms of HCC include abdominal pain, palpable masses, loss of appetite, weight loss, lower extremity edema, jaundice, and fever.	FBXO38	PD-1	[49,77–79]
Non-Hodgkin’s lymphoma MDM2 plays a role in the progression of non-Hodgkin’s lymphoma and the relapse of the disease. The mechanism of how MDM2 promotes this disease is not yet known. The swelling of a lymph node in a location such as the neck or armpits is usually the first sign that leads to a diagnosis. Chest pain, fever, and ulcers are some of the other symptoms.	MDM2	Specific substrate(s) unknown	[51,52,80,81]

Inflammation is also associated with cancer [44]. E3 ligase MARCH3 deficiency promotes inflammation-associated carcinogenesis [45]. Many E3-ligases and DUBs have been identified in various immune and cancer cell types as key regulators of antitumor immunity and tumor-mediated immunosuppression. E3 dysregulation is common in cancer cells and contributes to immune evasion [10,46]. Anticancer immunotherapy tends to focus on programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) signaling, as it is required for immune evasion. PD-1 is a cell surface receptor found on activated T-cells, B-cells, monocytes, dendritic cells, natural killer T cells, and regulatory T-cells. Some types of tumor cells and APCs express PD-L1 at a high level [47,48]. FBXO38 is an E3-ligase specific to the PD-1 protein. It polyubiquitinates the Lys233 residue in the cytoplasmic domain of PD-1 protein and then degrades it, reducing surface PD-1 expression and blocking PD-1/PD-L1-mediated tumor immune escape [47,49]. In humans, FBXO38 transcriptional levels are downregulated in tumor-infiltrating T cells in colorectal carcinoma and hepato-cellular carcinoma [49]. A recent study shows that PD-L1 is targeted for degradation by ubiquitin ligase NEDD4, which itself is activated by fibroblast growth factor receptor 3 (FGFR3) [50]. MDM2, a RING-domain-containing E3-ligase, suppresses p53 expression in cancer cells [51]. MDM2 inhibits T-cell activation in a p53-independent manner [52]. MDM2 has been shown to be upregulated in a proportion of non-Hodgkin’s lymphoma patients [53].

E3-ligases and neurodevelopmental diseases

Recent studies explore the possibility of autoinflammation in the pathogenesis of neurodevelopmental diseases (NDDs). It is known that the accumulation of ubiquitin-tagged substrates contributes to neurodegeneration; however, the impact of the UPS on neurodevelopmental diseases and pathways associated with immune response is not as well understood. Monogenic forms of NDD have been reported to be caused by lesions in genes coding for specific components of the UPS [54]. Many of these proteins have a function in the negative regulation of innate immune response, suggesting autoinflammation as part of NDD pathogenesis. Parallels between immune dysregulation and neurodevelopmental diseases offer a greater understanding of the biological processes of NDDs and the design of therapeutic strategies [54].

In patients with neurological phenotypes, we see an increased number of genomic alterations in genes that code for components of the UPS (Table 2). This points to the involvement of the UPS in the pathogenesis of psychiatric disorders, further supported by an accumulation of ubiquitin aggregates found in children suffering from various NDDs. The UPS, via E1, E2 and E3 enzymes, results in the ubiquitylation of protein substrates on lysine, cysteine, serine, or threonine residues of proteins, determining their eventual outcome [55,56]. Several forms of NDDs display genomic alterations that affect one or several of the genes encoding the UPS, damaging its functionality. Studies so far show that any gene encoding for the UPS is vulnerable to impairment in an NDD. These observations suggest a causal relationship between the damaged UPS function and the pathogenesis of NDD. Ubiquitin ligases constitute the largest group of genes identified as causing NDDs [54]. For example, loss of function in the UBE3A gene encoding the E3 ubiquitin ligase E6-AP HECT has been shown to cause Angelman syndrome [57]. Since then, 45 further genes encoding E3 ubiquitin ligases or CRL, Cullin-RING-type ligases (multi-subunit E3 ligases with cullin scaffold bridging E2 enzyme to the substrate), have been identified as causing 48 types of NDDs. The phenotypic spectrum of these forms of NDDs has great variation in severity due to the large variety of functions performed by E3 ubiquitin ligases [54]. Studying the downstream effects of mutations in the genes encoding UPS constituents reveals that NDDs may occur due to the inability of cells to remove certain mature proteins; however, the multiple substrate targets of E3 ubiquitin ligases make it difficult to identify specific molecular pathogeneses of NDDs. More than two-thirds of NDD-causing E3 ubiquitin ligases have important functions in the innate and adaptive immune systems. Loss of function in any one of these genes results in a prolonged production of proinflammatory cytokines [58]. Since most genes causing NDDs are involved in type 1 IFN negative feedback loops, dysfunction would result in uncontrolled type 1 IFN responses and inflammation; however, a very small number of NDDs display symptoms of autoinflammation. The lack of peripheral immune manifestation in NDDs is initially surprising but it does not confirm a lack of autoinflammation/autoimmunity in pathogenesis. Immune-inflammatory parameters have been detected in NDD subjects devoid of clinical inflammatory symptoms [59]. Alterations in genes encoding deubiquitinating enzymes (DUBs) have also been reported as NDD-causing, four-fifths of which have specific roles within the immune system. Most recently, a group of UPS genes encoding proteasome subunits have also been associated with NDDs [60]. These loss-of-function mutations cause two clinically distinct phenotypes: the failure to develop systemic autoinflammation and the increased amounts of transcripts encoding IFN-stimulated genes in the immune system [54]. Possible explanations for the mutations in immune-related genes causing NDDs include tissue-specific effects of the impaired UPS components as most NDD patients do not display typical symptoms of inflammation and immune dysregulation [54]. Further study of the pro-inflammatory mediators in the UPS offers a new therapeutic strategy for the treatment of NDDs.

Table 2.

List of E3/E4-ligases deregulated in neurodevelopmental diseases. Predicted potential substrates are highlighted.

Disease	E3/E4-ligase/Component	Potential Substrate(s)	References
Autism spectrum disorder Impairment of synaptic plasticity and memory formation caused by proteostasis imbalance at (glutamatergic) synapses. Symptoms include delayed cognitive and learning skills, unusual social, eating and sleeping habits, hyperactivity, and impulsivity.	CUL 3	BTB/POZ- domain proteins	[82–85]
	CUL 5	IRS-1
	CUL 7	Cyclin D1, IRS-1, HPK1, GRASP65, TBC1D31
Intellectual disability X-linked syndromic or autosomal recessive mental retardation is caused by mutations in one or more E3-ligase components regulating the inflammasome and NF-kB signaling. Symptoms include delayed cognitive and learning abilities.	KLHL24	Receptor for keratin 14	[86–90]
	UBE4A	K48- and K63-linked substrates.
	CUL4B	p21(Cip1/WAF)
	HERC2	XPA, BRCA1
	HUWE1	AIM2, NLRP3 and NLRC4
Angelman Syndrome Loss of function mutations in UBE3A, exclusively expressed from the maternal allele, impair synaptic function and activity-dependent synaptic plasticity. Symptoms include developmental delays in motor and cognitive skills, frequent smiling, and laughter.	HERC2	XPA, BRCA1	[54,91–93]
	E6-AP HECT	MAPK1, CDK1, CDK4, PRMT5, β-catenin
Restless legs syndrome & Tourette syndrome Variations in BTBD9 cause predisposition to restless legs syndrome and periodic limb movements during sleep associated with Tourette syndrome. Symptoms include motor and/or vocal tics of varying severity and frequency along with obsessive-compulsiveness in some cases.	BTBD9	IRP2	[94,95]

Targeted protein degradation and pharmacological UPS inhibition for drug development

Theoretically, potential druggable candidates of UPS include two E1 enzymes, about 40 E2 enzymes, around 100 DUBs, nearly 600 E3-ligases, and approximately 32 proteasome subunits. Drugs like bortezomib, which target the UPS at the terminal end by inhibiting the proteasome, have very broad substrate inhibitory impact with unintended side effects. Similarly, inhibition at the E1 and E2 levels may be less specific. Targeting individual E3s, and to a lesser extent DUBs, represents a more precise druggable node in UPS, due to their ability to directly bind substrates, with minimum non-specific side effects [8] (Figure 3a). At the E3 level, FBXO3 and βTrCP have been previously targeted in inflammation using the small molecule inhibitors BC-1215 and GS143 respectively [8,42,61,62]. E3s have also been inhibited for cancer immunotherapy such as IAPs [63]. A combinatorial approach to IAP inhibition with birinapant and PD-1 blockade potentiates cancer cell killing by cytotoxic lymphocytes [64]. Pharmacological inhibition of MDM2 by AMG-232 in high MDM2 cancer cells has also been shown to increase T cell killing [65].

Figure 3. Targeting UPS components for therapeutics.

(a) Potential targets in the UPS for small molecule-mediated inhibition. Due to the direct binding of substrates to E3s, amongst all the potential UPS targets, E3 inhibition could be highly specific with minimum side effects (b) PROTAC-mediated targeted protein degradation. The PROTAC consists of an E3 binding region, a linker, and a substrate recruiting region and induces target protein degradation by drawing the substrate in proximity to the ligase. (c) Molecular glue-mediated target protein degradation. Molecular glue is a small molecule that interacts with both the ligase and the substrate and brings them in proximity to induce degradation. BioRender software was used for making the figure.

The UPS has been utilized for targeted protein degradation, a new platform to re-purpose E3s to target neo-substrates. Protein-targeting chimeric molecule (PROTAC) is an excellent example of targeted protein degradation. PROTACs consist of three parts, an E3 binding region, a linker, and the substrate binding moiety. PROTACs target the substrate for ubiquitin-mediated proteasomal degradation by bringing the substrate in proximity to the E3-ligase (Figure 3b). Sakamoto et al. designed Protac 1 consisting of IκBα phosphopeptide (IPP) and ovalicin (OVA) where IPP ligates SCF^β−TRCP and OVA binds MetAp-2 bringing it close to the E3-ligase for ubiquitylation and degradation [66].P300/CBP-associated factor (PCAF) and general control nonderepressible 5 (GCN5) are epigenetic proteins with roles in immune function. Bassi et al. designed a PROTAC to promote ubiquitin-mediated degradation of PCAF/GCN5 resulting in a decrease in the inflammatory response in LPS-stimulated macrophages and dendritic cells [67]. Similarly, Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) PROTAC promotes cytokine inhibition in peripheral blood mononuclear cells [68]. PROTACs-targeting IRAK4 and BTK (Bruton’s tyrosine kinase) are in Phase I clinical trials for the treatment of B cell malignancies and autoimmune diseases [12]. These studies show that PROTACs may be useful in the treatment of immunological diseases. However, there are disadvantages in using PROTACs as the design is complex and diffusion across the cell membrane can be challenging because of their larger size.

An alternative strategy to targeted protein degradation which can overcome the disadvantages of PROTACs is using molecular glues. A molecular glue is a small molecule that promotes protein—protein interaction and can be utilized for proximity-induced protein degradation [69]. A molecular glue for targeted protein degradation binds both the substrate and an E3-ligase promoting ubiquitin-mediated degradation of the former by the latter (Figure 3c). An example of the molecular glue for targeted protein degradation is Auxin, which stabilizes the interaction between the FBOX protein Arabidopsis auxin receptor TIR1 and its substrate [70]. Molecular glues can be utilized in the treatment of various diseases including those involving the deregulation of the immune system. Lenalidomide is a drug used for the treatment of multiple myeloma. Its mechanism of action involves ubiquitin-mediated degradation of B cell transcription factors IKZF1 and IKZF3 by the ubiquitin ligase CRBN-CRL4 [71,72]. Lenalidomide seems to act as a molecular glue between the ubiquitin ligase and the substrates [73].

Future perspectives

Due to substrate specificity provided by E3s during UPS-mediated substrate degradation, approximately 600 E3-ligases coded by the human genome are attractive targets for therapeutic intervention. However, the substrate repertoire, hence our perspective, is limited to only a handful of well-studied E3s at this stage. Thus, future work should focus on identifying the complete repertoire of E3-ligase/substrate pairs and relevant cellular pathways in which substrate degradation has functional consequences. Studies to uncover the molecular mechanism of substrate degradation will be crucial to target aberrant E3/substrate pairs with the potential to identify new drug targets in various diseases like cancer, neurological abnormalities, and immune disorders. Targeted protein degradation via PROTACs and/or molecular glues are emerging platforms for drug discovery where studies of E3-ligase/substrate pairs will be impactful. Furthermore, E3-ligase-mediated compartmentalized protein degradation is poorly understood and hence warrants further investigations. Innovative procedures such as gene editing tools (e.g., Crispr-Cas9) to introduce E3-ligase recognizable degrons in aberrantly expressed genes warrant further investigations for potential therapeutic adoption in disease settings.