Abstract
Ubiquitylation is one of the most common post-translational modifications (PTMs) of proteins that frequently targets substrates for proteasomal degradation. However it can also result in non-proteolytic events which play important functions in cellular processes such as intracellular signaling, membrane trafficking, DNA repair and cell cycle. Emerging evidence demonstrates that dysfunction of non-proteolytic ubiquitylation is associated with the development of multiple human diseases. In this review, we summarize the current knowledge and the latest concepts on how non-proteolytic ubiquitylation pathways are involved in cellular signaling and in disease-mediating processes. Our review, may advance our understanding of the non-degradative ubiquitylation process.
Similar content being viewed by others
Introduction
Ubiquitin (Ub) was first discovered in 1975 by Gideon Goldstein1. It is a small and highly conserved protein that is expressed in all eukaryotic cells2,3. There are four human genes encoding Ub precursors: RPS27A, UBA52, UBB, and UBC4. Deubiquitylases (DUBs) play a key role in generating free Ub from these precursors5. Ub can be attached covalently to lysine (K) residue/s of substrate proteins to determine their fate in a process called ubiquitylation. Alternatively, K residues can also be covalently attached by Ub-like modifiers such as small ubiquitin-like modifier (SUMO)6 or neuronal precursor cell-expressed, developmentally down-regulated protein 8 (NEDD8)92. Interestingly, Lunapark ubiquitylation is not required for its ER localization and does not regulate ER architecture or dynamics, but is indispensable for the translocation and activation of mTORC1 (mechanistic target of rapamycin complex-1) at the lysosome. Non-ubiquitylated Lunapark fails to interact with mTOR at ER-lysosome contact sites, leading to hyperactivation of downstream growth signaling pathways in response to nutrients. Moreover, zebrafish embryos defective for CUL3/KLHL12- mediated Lunapark ubiquitylation displayed increased locomotion activity and neurodevelopmental defects, suggesting that future investigation on how Lunapark ubiquitylation might be implicated in neurodegenerative diseases associated with mTOR hyperactivation should be warranted. Another screen identified DUB USP32 as a critical modulator of endosomal membrane dynamics and architecture by directly deubiquitylating the small GTPase Rab7 at K191 and promoting its functions in transport and recycling from the multi-vesicular body via two distinct mechanisms93. Deubiquitylation of Rab7 by USP32 promotes its release from the membrane to the cytosol and its binding to the late endosome (LE) transport effector Rab-interacting lysosomal protein (RILP), thereby favoring LE transport toward the nucleus. Moreover, in the presence of USP32, Rab7 association with the retromer (a complex of proteins important in recycling transmembrane receptors from endosomes to the trans-Golgi network) is destabilized and leads to enhanced membrane recycling from the LE. According to another study related to endosomal organization and function, Rab7 is also subjected to non-proteolytic ubiquitylation by the E3 ligase Parkin94. Parkin ubiquitylates Rab7 at K38 and increases its activity, its binding to the RILP effector and its membrane association, and is suggested to at least partially be responsible for reduced exosome secretion. Considering that all above-mentioned components (USP32, Parkin, Rab7), as well as deregulation of endosomal membrane dynamics have been implicated in the pathogenesis of Parkinson’s disease (PD), we could speculate that non-proteolytic ubiquitylation signaling within the endolysosomal system might provide new perspectives into the development of therapeutic strategies against PD.
Non-proteolytic ubiquitylation and development
Components of the ubiquitin machinery have been shown to control different aspects of embryonic development by modulating a variety of developmental signaling pathways. Here, we outline some recent examples of how non-proteolytic ubiquitylation directs developmental cell-fate decisions.
Neural patterning is the biological process by which cells in the develo** nervous system acquire distinct identities according to their specific spatial positions95. One of its major regulators is the Sonic Hedgehog signaling which fine-tunes the balance of Gli activators (GliA) and repressors (GliR) in the ventral neural tube to govern the local expression of a group of cell-fate-determining transcription factors in responding neural progenitors96. A recent study addressed the role of non-proteolytic ubiquitylation in regulating the Gli gradient and identified the RING E3 ligase RNF220 as a novel important modulator of neural patterning in mice embryos97 (Fig. 3a). RNF220 interacts and ubiquitylates both activators and repressors of the Gli family and controls their nucleocytoplasmic shuttling by unmasking a nuclear export signal that is then recognized by the exportin CRM1. Disruption of this ubiquitylation leads to enhanced Gli activation in the nucleus and subsequently interferes with the GliA and GliR gradient equilibrium, thereby resulting in irregular neural cell differentiation patterns.
Examples of reported E2/E3/DUB enzymes (left boxes) and different non-proteolytic Ub signals on the substrate proteins (right boxes) regulating a development and b NF-κB signaling pathways.
Studies from the Hu lab have identified the RING-type E3 ligase MKRN3 (Makorin 3) as a critical repressor of premature mammalian puberty initiation through the non-proteolytic ubiquitylation of several substrates within the gonadotropin-releasing hormone (GnRH) signaling pathway at both translational and epigenetic level98,99 (Fig. 3a). MKRN3 ubiquitylates methyl-DNA binding protein 3 (MBD3) in order to inhibit both its binding to the promoter of GNRH1 and its interaction with the demethylase TET2 (ten-eleven translocation methylcytosine dioxygenase 2), hence leading to the epigenetic silencing of GNRH1 transcription98. Moreover, MKRN3 ubiquitylates (Poly (A)-binding protein 1(PABC1)) in order to decrease its binding to the poly (A) tails of GNRH1 mRNA, which results in GNRH1 mRNA destabilization and disruption of the translation initiation complex formation99. Both studies have provided important genetic evidence for the role of MKRN3-dependent non-proteolytic ubiquitylation in the inhibition of the early activation of hypothalamic axis upstream of puberty initiation and development, suggesting new targets for the treatment of clinical conditions like central precocious puberty.
Bone remodeling is a critical process of the adult skeleton homeostasis that depends on the balanced actions of osteoblasts and osteoclasts. Among the master regulators that are involved in the commitment of mesenchymal progenitors to differentiate into osteoblasts, are the bone morphogenetic proteins (BMPs), the RUNX Family Transcription Factor 2 (RUNX2), and the proteolytic signaling mediated by the NEDD4 family of HECT type E3 ligases100. A recent study provides evidence for the involvement of the WWP2 E3 ligase in osteogenic differentiation by catalyzing the non-proteolytic mono-ubiquitylation of RUNX2 in order to stimulate its transcriptional and osteoblastic actitivity, a modification that is shown to be further enhanced upon BMP signaling activation101 (Fig. 3a). Considering that missense mutations linked to severe skeletal disorders have been identified for the WWP2 targeted lysine residues of RUNX2, it would be important to further dissect the role of non-proteolytic ubiquitylation events in osteogenesis and skeleton development.
Yes-associated protein (YAP) is a transcription factor with established roles in development and in oncogenic processes. YAP continuously shuttles between the nucleus and the cytoplasm and the regulation of its activity has mostly been studied downstream of the Hippo pathway102. A Hippo independent mechanism based on the non-proteolytic ubiquitylation of YAP was recently described to finetune its localization and function through the counteracting actions of the SCFSKP2 E3 ligase complex and the DUB OTUD1103 (Fig. 3a). SKP2 ubiquitylation enhances the interaction of YAP with its nuclear binding partner TEAD and induces YAP nuclear localization, transcriptional activity, and growth-promoting function, while OTUD1-mediated deubiquitylation promotes the retention of YAP in the cytoplasm and blocks its activity.
Dynamic remodeling of the actin cytoskeleton is a key process driving intracellular communication and fusion during tissue formation that needs to be precisely coordinated to prevent developmental defects104. Non-proteolytic monoubiquitylation mediated by the CUL3/KCTD10 complex was recently identified as a molecular rheostat that finetunes actin bundling in a model of myogenic cell fusion by exerting dual counteracting activities on the EPS8–IRSp53 complex105 (Fig. 3a). The authors demonstrate that CUL3/KCTD10-dependent ubiquitylation of EPS8-IRSp53 is regulating both the recruitment as well as the displacement of the complex to the plasma membrane in order to timely orchestrate the actin-bundling at cell–cell contact sites to promote myoblast fusion. This mechanism was further shown to not only be required for myoblast-myotube fusion, but rather to be responsible for generally rearranging the actin network and restricting actin-bundling when it is no longer required, opening research avenues that exploit this signaling in pathological conditions characterized by defective cell fusion.
Non-proteolytic ubiquitylation in NF-κB signaling
Nuclear factor kappa light chain enhancer of activated B cells (NF-κB) is a widely studied family of transcription factors, highly involved in various processes such as inflammation, immune response, and cell survival. The multiple biological mechanisms that mediate NF-κB signaling, as well as the genetic diseases that are associated with deregulated NF-κB pathways have been extensively reviewed106. Mounting evidence demonstrates that non-proteolytic ubiquitylation is one of the major PTMs that control the NF-κB cellular response, with the M1-linked ubiquitylation mediated by the RBR-type E3 ligase linear ubiquitin chain assembly complex (LUBAC) remaining in the spotlight107. Furthermore, the deubiquitylase OTULIN (OTU domain-containing deubiquitinase with linear linkage specificity) has emerged as the DUB responsible for deactivating the NF-κB signaling downstream of LUBAC through multiple mechanisms discussed in the referred review108. Recently, a study revealed a novel regulatory mechanism upstream of OTULIN-mediated NF-κB inactivation that involves non-proteolytic ubiquitylation of OTULIN by the RING-type E3 ligase3 tripartite motif-containing protein 32 (TRIM32)109 (Fig. 3b). The authors show that TRIM32 conjugates K63-linked polyubiquitin onto OTULIN which perturbs the interaction between OTULIN and the LUBAC E3 ligase component HOIP (E3 ubiquitin-protein ligase RNF31). As a result, OTULIN can no longer cleave the linear ubiquitin chains generated by LUBAC and NF-κB signaling is maintained.
One of the major regulators of NF-κB pathway activation in response to pro-inflammatory cytokines stimulation is the transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1). Many studies have investigated so far the different PTMs of TAK1, including ubiquitylation and deubiquitylation events that are critical for the regulation of TAK1-mediated NF-kB activation and which are discussed in the suggested review110. A more recent study identified the ubiquitin-specific protease 19 (USP19) as a novel DUB that targets TAK1 and acts as a negative regulator of the TNFα and IL1β-triggered NF-kB activation111 (Fig. 3b). More specifically, USP19-mediated TAK1 deubiquitylation abrogated TAK1 auto-phosphorylation as well as the formation of the TAK1–TAB2/3 complex, both of which are steps required for efficient NF-kB signaling and thus resulted in attenuated inflammatory response. NF-κB pathway activation is among the major oncogenic signatures in activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) primary tumors. A new molecular mechanism of non-proteolytic ubiquitylation was discovered to play a key role in NF-κB constitutive activation, where the RING E3 ligases cellular inhibitors of apoptosis 1 and 2 polyubiquitylate the B-cell lymphoma/leukemia 10 (BCL10) factor within the CARD11-MALT1-BCL10 (CBM) adapter complex and thus facilitate the recruitment and activation of the established LUBAC/IKK/NEMO signaling axis upstream of NF-κΒ activation112 (Fig. 3b). The authors show that second mitochondria-derived activator of caspase (SMAC) mimetics target cIAP1/2 for destruction, thereby suppressing NF-κB and selectively killing ABC DLBCL cells depending on this signaling cascade, providing the perspective of future therapeutic strategies against this lymphoma subtype.
Non-proteolytic ubiquitylation in cancer signaling
The role of the ubiquitin-proteasome system in cancer is well recognized and is highlighted by the development of proteasome inhibitors as the front line of anticancer treatments currently tested in clinical trials113. Nevertheless, the notion that non-K48-linked ubiquitylation can drive oncogenic pathways, has led to a growing demand for the identification of components that could exploit the therapeutic potential of the non-degradative functions of ubiquitylation114. Here, we describe some recent examples illustrating new roles of non-proteolytic ubiquitylation in regulating oncogenic and tumor-suppressive signaling pathways in cancer.
Recent findings demonstrate that the autophagy regulator UVRAG (UV radiation resistance-associated) is subjected to non-proteolytic ubiquitylation by the HECT E3 ligase SMURF1 (SMAD specific E3 ubiquitin-protein ligase 1), which promotes autophagosome maturation and inhibits HCC tumor growth in vivo115 (Fig. 4). Mechanistically, the SMURF1/UVRAG-dependent autophagic flux is perturbed by counterbalancing modifications on UVRAG including direct deubiquitylation by zinc finger RANBP2-type containing 1 (ZRANB1) and phosphorylation by Casein Kinase 1 alpha 1 (CSNK1A1), thereby providing novel candidates for combined therapeutic intervention against HCC. Suppressor of Fused (SuFu) is a tumor suppressor protein that acts as a critical regulator of the Hedgehog signaling, a conserved pathway with an important role in cerebellar development and tumorigenesis. SuFu is ubiquitylated in a non-proteolytic manner by the HECT E3 ligase ITCH (ubiquitin-protein ligase Itchy homolog) in complex with the adapter protein β-arrestin2, which increases the binding of SuFu to Gli transcription factors and eventually switches off the Hedgehog signaling116 (Fig. 4). Considering that medulloblastoma patients often bear SuFu mutations which are insensitive to ITCH ligase activity, it would be worth investigating whether ITCH-mediated ubiquitylation could act as protective mechanism toward the tumor suppressor function of SuFu in cancer types with deregulated Hedgehog signaling. Another example of non-proteolytic ubiquitylation by the ITCH E3 ligase that also acts in concert with phosphorylation events, is proposed to have a pivotal role in melanoma tumorigenesis through the coordination of MER/ERK signaling activation in response to proinflammatory cytokines117. The authors demonstrate that cytokine stimulation triggers the activation of ITCH by the C-Jun N-terminal kinase (JNK), which in turn catalyzes the K27-linked polyubiquitylation of the BRAF oncoprotein in order to sustain BRAF activity in transducing mitogenic signals (Fig. 4). Mechanistically, ITCH-mediated BRAF ubiquitylation disrupts its interaction with the 14-3-3 protein, a complex required for maintaining the inhibitory state of BRAF kinase based on structural studies118, thus leading to aberrant ERK signaling activation that further promotes melanoma tumor growth.
Examples of reported E2/E3/DUB enzymes (left boxes) and different non-proteolytic Ub signals on the substrate proteins (right boxes) regulating cancer cell proliferation, tumor growth, and metastasis.
A recent study provided for the first time evidence that SMAD4 (mothers against decapentaplegic homolog 4), the common signal transducer within the TGF-β and BMP signaling pathways, is subjected to non-proteolytic ubiquitylation by the CRL based on CUL4 (CRL4) and its substrate receptor AMBRA1 (activating molecule in BECN1-regulated autophagy protein 1)119 (Fig. 4). The interaction of the CUL4/AMBRA1 complex with SMAD4 enhances its association with the transcriptional co-activator CBP (CREB-binding protein) and subsequently the transcriptional activity of SMAD4, suggesting that this ubiquitylation might create a platform that facilitates the recruitment of essential transcription components to mediate the TGF-β response. AMBRA1 depletion potentiated the metastatic potential of breast cancer cells toward the bones and the lungs in vivo, indicating that the CUL4/AMBRA1-mediated SMAD4 ubiquitylation should be therapeutically investigated in TGF-β-dependent metastatic cancers. In triple-negative breast cancer (TNBC) cellular models, the E3 ligase RING finger protein 181 (RNF181) ubiquitylates and stabilizes YAP, the key effector of the Hippo signaling pathway, potentially protecting it from K48 linked proteolytic ubiquitylation events mediated by the E3 ligases FBW7 and SCF120 (Fig. 4). Aberrant activation of the Hippo/YAP signaling downstream of RNF181 promotes migration and invasion phenotypes, suggesting that this modification could serve as a new marker for TNBC patient prognosis.
β-Catenin is the core component of the Wnt/β-Catenin pathway which has established roles in embryonic development and differentiation and its deregulation is strongly linked to colorectal tumorigenesis. Recently, the ubiquitin-conjugating E2S (UBE2S) was found to form a complex with the Cdc27 subunit of the APC/C E3 ligase in order to ubiquitylate β-Catenin and increase it protein stability121 (Fig. 4). This modification is non-proteolytic, but seems to antagonize known phosphorylation-induced degradation signals, hence protecting β-Catenin from being recognized and targeted for degradation by the E3 complex Skp1/Cul1/F-boxβ-TrCP. The signaling mediated by UBE2S is proposed to be required for both physiological and pathological processes downstream of β-Catenin, since its ubiquitylation enhances the differentiation of embryonic stem cells into mesoendoderm lineages but also increases the metastatic potential of colorectal cancer cells in vitro and in vivo.
TNF receptor-associated factor 4 (TRAF4) represents another RING E3 ligase which has been suggested to promote cancer cell invasiveness and metastasis through non-proteolytic ubiquitylation in cellular models of prostate cancer122. TRAF4 binds to and ubiquitylates the tyrosine receptor kinase A specifically at the cell membrane before its internalization, leading to hyperactivation of its downstream pathways which stimulate the expression of genes associated to invasive phenotypes (Fig. 4). Although the exact mechanism on how TRAF4-mediated ubiquitylation regulates TRKA’s kinase activity remains unclear, the authors propose that ubiquitin attachment might induce conformational changes within the activation loop of TRKA. It would be interesting to investigate if this regulatory mode acts as a more general mechanism in cancers characterized by aberrant tyrosine kinase signaling. Within the context of targeting non-proteolytic ubiquitylation of TRKs in cancer, a recent study on the epidermal growth factor receptor (EGFR) revealed the involvement of the RING E3 ligase CBLC in sustaining an hyperactive EGFR signaling in lung adenocarcinoma both in vitro and in vivo39 (Fig. 4). Interestingly, CBLC leads to increased stability and prolonged activation of EGFR and further promotes its trafficking to the nucleus and recycling to the plasma membrane, through competition with another E3 ligase of the same family, known to regulate EGFR turnover123. According to the study, CBLC depletion increased the sensitivity of EGFR-mutant lung cancer cells to tyrosine kinase inhibitors (TKIs), which could suggest an alternative strategy to enhance the efficacy of therapeutics in lung cancer patients by the use of combinatory treatments with TKIs and CBLC targeting molecules.
Non-proteolytic ubiquitylation and metabolism-related diseases
Although the role of ubiquitin signaling has not been firmly associated with the development of metabolic disorders, a considerable number of recent findings suggest that non-proteolytic ubiquitylation events are part of the molecular mechanisms underlying metabolic syndromes. Nonalcoholic fatty liver disease (NAFLD) is a chronic liver condition that can severely damage the liver and ultimately lead to cancer124. The HECT E3 ligase SMURF1 was found to target the peroxisome proliferator-activated receptor γ (PPARγ), a lipid-sensing nuclear receptor with established role in NAFLD pathogenesis, in order to suppress its transcriptional activity in hepatocytes and balance the PPARγ-mediated fatty acid uptake and lipid synthesis, thereby inhibiting steatosis125 (Fig. 5a). Moreover, the fact that the PPARγ antagonist, GW9662, was shown to completely reverse the liver lipid accumulation in mice deficient for SMURF1, suggests that activation of SMURF1-dependent non-proteolytic ubiquitylation could be exploited as a new strategy for NAFLD treatment.
Examples of reported E2/E3/DUB enzymes (left boxes) and different non-proteolytic Ub signals on the substrate proteins (right boxes) regulating a metabolic and b innate immunity-related diseases.
Diabetes is a metabolic disorder characterized by hyperglycemia126. There are two main types of diabetes: Type 1 resulting from the autoimmune destruction of insulin-producing pancreatic β cells and type 2 resulting from compromised cellular response to insulin127. Several PTMs including ubiquitylation can critically contribute to pancreatic β-cell function and insulin-mediated signal transduction. While the role of proteolytic ubiquitylation in the regulation of diabetes is firmly established, emerging evidence also suggest critical involvement of non-proteolytic ubiquitylation in the development of this disease. Insulin signaling plays a vital role in the regulation of energy homeostasis and its dysregulation is causally linked to diabetes128. Protein kinase B (PKB or AKT) is an essential intermediate signaling node, downstream of phosphatidylinositol 3-kinase, in the insulin signaling pathway129,130, also implicated in diabetes131. It has been reported that the activation of AKT is regulated through K63-linked ubiquitylation upon stimulation with growth factors132, suggesting that K63-linked polyubiquitylation plays a critical role in insulin signaling.
Ubiquitin-conjugating enzyme E2 N (UBE2N or Ubc13) is a key E2 enzyme that promotes the formation of non-proteolytic K63-linked polyUb chains128. Interestingly, G-protein pathway suppressor 2 (GPS2) is a small multifunctional protein that can directly inhibit UBE2N mediated ubiquitylation133 (Fig. 5a). Deletion of UBE2N decreases AKT ubiquitylation and significantly inhibits AKT phosphorylation upon insulin stimulation. In contrast, deletion of GPS2 not only enhances ubiquitylation and activation of AKT but also phosphorylation of the AKT substrate, glycogen synthase kinase 3 beta. Consequently, fat-specific deletion of GPS2 is associated with increased body weight in mice, as a result of lipid accumulation and shift from lipid utilization towards their storage in the adipose tissue128. It has been reported that enhanced lipid storing capacity in the adipose tissue can lead to improved systemic insulin sensitivity134,135. Hence, it is not surprising that fat-specific deletion of GPS2 results in a positive effect on systemic insulin sensitivity128. In conclusion, non-proteolytic ubiquitylation of AKT represents a vital regulatory node in the insulin signaling transduction pathway. In addition, ubiquitin-conjugating E2 enzyme variant 1 (UBE2v1) is a cofactor of Ubc13, able to catalyze Lys63-linked ubiquitylation. Both levels of UBE2v1 and K63-linked ubiquitylated proteins are increased in diabetic patients136, suggesting that the UBE2v1-mediated synthesis of K63-linked polyubiquitylation is crucially involved in the development of diabetes.
Metabolic reprogramming is a hallmark of tumorigenesis and it refers to the well-described phenomenon of the Warburg effect in which cancer cells switch to glycolysis even in the presence of oxygen in order to sustain their hyper proliferation rate within a nutrient-deprived tumor microenvironment137. An interesting study unraveled a novel signaling axis that regulates cancer metabolism through the non-proteolytic ubiquitylation of hexokinase 2 (HK2), a critical enzyme upstream of many glycolytic pathways, by the HECT-type E3 ligase HECTH9 (or HUWE1)138 (Fig. 5a). The authors demonstrated in several models of metabolism-addicted tumors that HECTH9-mediated ubiquitylation orchestrates the trafficking of HK2 to mitochondria and its association with the voltage-dependent anion-selective channel protein at the outer mitochondrial membrane. This ubiquitylation confers to HK2-dependent apoptosis resistance, enhanced glycolysis, and reduced reactive oxygen species generation, ultimately leading to cancer stem cell (CSC) self-renewal and tumor progression. Targeting the HECTH9-HK2 signaling could emerge as an effective strategy to limit CSC expansion and cancer progression in metabolism-addicted tumors. The importance of non-proteolytic ubiquitylation in the metabolic reprogramming of tumor cells has also been confirmed by another recent study which reported a role for the DUB OTUB2 in promoting colorectal cancer progression by elevating the Pyruvate kinase M2 (PKM2) enzymatic activity, subsequently upregulating glycolysis139 Upon conditions of nutrient deficiency, OTUB2 co-localizes and interacts with PKM2 in the cytoplasm and blocks its ubiquitylation. Interestingly, this interaction occurs independently of OTUB2 catalytic activity but is instead mediated via competing with the E3 ligase Parkin140 for binding to PKM2, highlighting the significance of monitoring the balance between ubiquitylation/deubiquitylation events on key metabolic enzymes as a means to understand how cancer cells respond to metabolic stress (Fig. 5a).
Non-proteolytic ubiquitylation and innate immunity-related diseases
The role of ubiquitin chains inducing degradation-dependent ubiquitin signaling within the context of the antiviral innate immune response (IIR) has been recently discussed in the suggested review141. Here we focus on some recent advances in the regulation of innate immunity during viral infection, governed by non-proteolytic ubiquitylation events. Innate immunity generates a fast non-specific inflammatory response against all types of pathogens by activating the production of interferons (IFNs) and proinflammatory cytokines. Interferon regulatory factor 3 (IRF3) is a transcriptional factor with key functions in the IIR against DNA and RNA viruses and its interplay with the DUB enzyme ovarian tumor domain-containing deubiquitylase (OTUD1) in a non-proteolytic context has gained significant attention. IRF3 is subjected to viral infection-induced K6-linked ubiquitylation which is recognized and cleaved by OTUD1 in a way that impairs the capacity of IRF3 to bind to the promoter region of its target genes.142 (Fig. 5b). As a result, the IRF3-dependent production of interferons and pro-inflammatory cytokines is attenuated, leading to increased viral infection sensitivity. Moreover, under inflammatory conditions, IRF3 is deubiquitylated by OTUD1 at K63 ubiquitin chains which inhibits its nuclear translocation and transcriptional activity143. The authors also provide evidence that OTUD1 mutations which impair its ability to interact with IRF3, lead to sustained immune response and excessive interferon production that is strongly associated with the development of autoimmune disorders. The above studies highlight the importance of exploiting possible therapeutics targeting the OTUD1-IRF3 signaling axis as a means to ensure immune homeostasis.
One potentially fatal autoinflammatory condition linked to the ubiquitylation pathway is OTULIN-related autoinflammatory syndrome (ORAS), which is caused by a homozygous hypomorphic mutation in the human OTULIN gene108. Detailed analysis demonstrated that loss-of-function mutations in OTULIN and accumulation of M1-polyUb levels in myeloid cells in a TNF-dependent manner recapitulate the severe phenotypes of inflammation and autoimmunity observed in ORAS patients, which however can be ameliorated by treatment with the anti-TNF neutralizing antibody Infliximab144. The important role of OTULIN in restraining inflammation was also suggested by another study in patients suffering from otulipenia due to OTULIN missense mutations that were associated with excessive linear ubiquiylation of target proteins such as NEMO, RIPK1, and TNFR1, resulting in severe inflammation and further confirming OTULIN as a gatekeeper of innate immunity145.
IL-17-producing T helper (Th17) cells are a distinct subset of CD4 + T cells whose differentiation and function are controlled by several cytokines and transcription factors and which are considered key determinants in the pathogenesis of autoimmune diseases146. Recent evidence in experimental autoimmune encephalomyelitis suggests that HECTD3-mediated non-proteolytic ubiquitylation promotes pathogenic Th17 cell differentiation and function downstream of NF-kB activation147. HECTD3 E3 ligase targets two distinct components upstream of T17 cell differentiation, namely MALT1 (mucosa-associated lymphoid tissue 1) and STAT3 (signal transducer and activator of transcription 3) in a non-degradative manner, leading to robust NF-kB signaling activation (Fig. 5b). Moreover, ubiquitylation of both substrates is required for enhancing the expression of RAR-related orphan receptor gamma (RORγt), the master transcription factor controlling Th17 cell identity148, further supporting a role for HECTD3 in the development of T17-related autoimmune disorders.
A non-proteolytic role has been suggested for the F-box only protein Fbxo21 in viral-triggered IIR, which until now was a functionally unknown component of the SCF (Skp1–Cul1–F-box protein) complex. Upon viral infection, Fbxo21 ubiquitylates and activates apoptosis signal-regulating kinase 1 (ASK1), a protein with pivotal roles in stress and immune responses, thereby leading to the activation of c-Jun N-terminal kinases (JNK1/2) and p38 MAPKs downstream signaling149 (Fig. 5b). Disruption of this modification reduces the production of proinflammatory cytokines and type I interferons and results in attenuated antiviral innate response and enhanced virus replication, suggesting that Fbxo21 might play an indispensable role in regulating innate antiviral response. The first example of K29-linked, non-proteolytic ubiquitylation in the regulation of viral infection was recently described for the PB2 replication protein, an essential component of the viral transcription/replication machinery and a key determinant of host range during influenza A virus (IAV) infection150. The authors demonstrate that PB2 undergoes non-degradative ubiquitylation by two distinct RING E3 CRL4 ligase-based multi complexes containing the DDB1 adapter and either DCAF12L1 (D12L1) or DCAF11 (D11) as substrate recognition factors, which however do not complement each other functionally (Fig. 5b). Loss of PB2 ubiquitylation leads to attenuated viral load, indicating that this modification is essential for optimal IAV infection. Further investigation on how CRL4-mediated PB2 ubiquitylation can affect the viral cycle progression would be of great interest and could lead to design of future antiviral therapeutic strategies. The RING-type E3 ligase tripartite motif-containing protein 21 (TRIM21) was shown to display antiviral activity and to facilitate the IIR by exerting non-proteolytic ubiquitylation on a critical component of the innate immune system that is responsible for the downstream transmission of the pathogen recognition signal151. Upon conditions of RNA viral infection, TRIM21 is upregulated through the IFN/JAK/STAT pathway, targets the mitochondrial adapter molecule and promotes its interaction with the TBK1, thereby initiating the activation of IRF3 and NF-κB signaling which can then stimulate the production of IFNs and proinflammatory cytokines (Fig. 5b).
Outlook and conclusions
Deciphering the multifaceted ubiquitin code has proved fundamental for cell biology and for understanding the etiology of several detrimental human diseases. During the last decade, we have witnessed breakthroughs in the development of biochemical tools and cell-imaging technologies that allow us to visualize and monitor the dynamic complexity of the ubiquitin network with unprecedented detail152. Based on our existing knowledge, it appears well documented that non-proteolytic ubiquitylation pathways play many important roles in cellular signaling and in the development of various diseases. Unlike ubiquitylation signals that target proteins for their proteasomal degradation, non-proteolytic ubiquitylation mainly regulates proteins’ signaling and trafficking, localization, interplay with other PTMs and interactions. Although the underlying molecular and cellular mechanisms of human pathologies are relatively well understood, the direct links of non-proteolytic ubiquitylation pathways to their etiology are poorly characterized. The use of genetic, and genome-editing approaches in animal disease models are needed to understand the role of non-proteolytic ubiquitylation substrates and their modifying enzymes. Finally, the development of inhibitors targeting the key E3 ligases and DUBs could open interesting experimental possibilities and help in the long-term future to the identification of new therapies against human pathologies.
References
Goldstein, G. et al. Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells. Proc. Natl Acad. Sci. 72, 11–15 (1975).
Bonifacino, J. S. & Weissman, A. M. Ubiquitin and the control of protein fate in the secretory and endocytic pathways. Annu. Rev. Cell Dev. Biol. 14, 19–57 (1998).
Leestemaker, Y. & Ovaa, H. Tools to investigate the ubiquitin proteasome system. Drug Discov. Today.: Technol. 26, 25–31 (2017).
Kimura, Y. & Tanaka, K. Regulatory mechanisms involved in the control of ubiquitin homeostasis. J. Biochem. 147, 793–798 (2010).
Komander, D., Clague, M. J. & Urbé, S. Breaking the chains: structure and function of the deubiquitinases. Nat. Rev. Mol. Cell Biol. 10, 550–563 (2009).
Flotho, A. & Melchior, F. Sumoylation: a regulatory protein modification in health and disease. Annu Rev. Biochem 82, 357–385 (2013).
Abidi, N. & **rodimas, D. P. Regulation of cancer-related pathways by protein NEDDylation and strategies for the use of NEDD8 inhibitors in the clinic. Endocr.-Relat. Cancer 22, T55–T70 (2015).
Gâtel, P., Piechaczyk, M. & Bossis, G. Ubiquitin, SUMO, and Nedd8 as therapeutic targets in cancer. In Proteostasis and Disease (eds. Barrio, R., Sutherland, J. D. & Rodriguez, M. S.) vol. 1233, pp 29–54 (Springer International Publishing, 2020).
Sumara, I., Maerki, S. & Peter, M. E3 ubiquitin ligases and mitosis: embracing the complexity. Trends Cell Biol. 18, 84–94 (2008).
Tokunaga, F. Linear ubiquitination-mediated NF- B regulation and its related disorders. J. Biochem. 154, 313–323 (2013).
Olsen, S. K. & Lima, C. D. Structure of a ubiquitin E1-E2 complex: insights to E1-E2 thioester transfer. Mol. Cell 49, 884–896 (2013).
David, Y. et al. E3 ligases determine ubiquitination site and conjugate type by enforcing specificity on E2 enzymes. J. Biol. Chem. 286, 44104–44115 (2011).
Chen, Z. J. & Sun, L. J. Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286 (2009).
Morreale, F. E. & Walden, H. Types of ubiquitin ligases. Cell 165, 248–248.e1 (2016).
George, A. J., Hoffiz, Y. C., Charles, A. J., Zhu, Y. & Mabb, A. M. A comprehensive atlas of E3 ubiquitin ligase mutations in neurological disorders. Front. Genet. 9, 29 (2018).
Morreale, F. E. & Walden, H. Types of ubiquitin ligases. Cell 165, 248–248.e1 (2016).
Ebner, P., Versteeg, G. A. & Ikeda, F. Ubiquitin enzymes in the regulation of immune responses. Crit. Rev. Biochem. Mol. Biol. 52, 425–460 (2017).
Cardozo, T. & Pagano, M. The SCF ubiquitin ligase: insights into a molecular machine. Nat. Rev. Mol. Cell Biol. 5, 739–751 (2004).
Hare A.E. & Parvin D.J. Processes that regulate the ubiquitination of chromatin and chromatin-associated proteins. In Ubiquitin proteasome system—current insights into mechanism cellular regulation and disease (ed. Summers, M.) (IntechOpen, 2019). https://doi.org/10.5772/intechopen.82567.
Ohta, T. & Fukuda, M. Ubiquitin and breast cancer. Oncogene 23, 2079–2088 (2004).
Li, W. & Ye, Y. Polyubiquitin chains: functions, structures, and mechanisms. Cell. Mol. Life Sci. 65, 2397–2406 (2008).
Mulder, M. P. C., Witting, K. F. & Ovaa, H. Cracking the ubiquitin code: the ubiquitin toolbox. Curr. Issues Mol. Biol. 1–20 https://doi.org/10.21775/cimb.037.001 (2020).
Kwon, Y. T. & Ciechanover, A. The ubiquitin code in the ubiquitin-proteasome system and autophagy. Trends Biochem. Sci. 42, 873–886 (2017).
Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).
Chau, V. et al. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243, 1576–1583 (1989).
Finley, D. Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513 (2009).
Hicke, L. Protein regulation by monoubiquitin. Nat. Rev. Mol. Cell Biol. 2, 195–201 (2001).
Xu, P. et al. Quantitative proteomics reveals the function of VCP/p97 l ubiquitin chains in proteasomal degradation. Cell 137, 133–145 (2009).
Dittmar, G. & Winklhofer, K. F. Linear ubiquitin chains: cellular functions and strategies for detection and quantification. Front. Chem. 7, 915 (2020).
Spit, M., Rieser, E. & Walczak, H. Linear ubiquitination at a glance. J. Cell. Sci. 132, jcs208512 https://pubmed.ncbi.nlm.nih.gov/30659056/ (2019).
Erpapazoglou, Z., Walker, O. & Haguenauer-Tsapis, R. Versatile roles of K63-linked ubiquitin chains in trafficking. Cells 3, 1027–1088 (2014).
Pontrelli, P., Conserva, F. & Gesualdo, L. The role of lysine 63-linked ubiquitylation in health and disease. In Ubiquitin Proteasome System—Current Insights into Mechanism Cellular Regulation and Disease (ed. Summers, M.) (IntechOpen, 2019). https://doi.org/10.5772/intechopen.83659.
Wang, G. et al. K63-linked ubiquitination in kinase activation and cancer. Front. Oncol. 2, 5 https://doi.org/10.3389/fonc.2012.00005 (2012).
Tracz, M. & Bialek, W. Beyond K48 and K63: non-canonical protein ubiquitination. Cell Mol. Biol. Lett. 26, 1 (2021).
Cunningham, C. N. et al. USP30 and parkin homeostatically regulate atypical ubiquitin chains on mitochondria. Nat. Cell Biol. 17, 160–169 (2015).
Durcan, T. M. et al. USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin. EMBO J. 33, 2473–2491 (2014).
Srivastava, D. & Chakrabarti, O. Mahogunin-mediated α-tubulin ubiquitination via noncanonical K6 linkage regulates microtubule stability and mitotic spindle orientation. Cell Death Dis. 5, e1064 (2014).
Mukherjee, R., Majumder, P. & Chakrabarti, O. MGRN1-mediated ubiquitination of α-tubulin regulates microtubule dynamics and intracellular transport. Traffic 18, 791–807 (2017).
Hong, S.-Y., Kao, Y.-R., Lee, T.-C. & Wu, C.-W. Upregulation of E3 ubiquitin ligase CBLC enhances EGFR dysregulation and signaling in lung adenocarcinoma. Cancer Res 78, 4984–4996 (2018).
Paul, A. & Wang, B. RNF8- and Ube2S-dependent ubiquitin lysine 11-linkage modification in response to DNA damage. Mol. Cell 66, 458–472.e5 (2017).
Xue, B. et al. TRIM21 promotes innate immune response to RNA viral infection through Lys27-linked polyubiquitination of MAVS. J. Virol. 92, e00321–18 (2018). /jvi/92/14/e00321-18.atom.
Nucifora, F. C. et al. Ubiqutination via K27 and K29 chains signals aggregation and neuronal protection of LRRK2 by WSB1. Nat. Commun. 7, 11792 (2016).
Fei, C. et al. Smurf1-mediated Lys29-linked nonproteolytic polyubiquitination of axin negatively regulates Wnt/ -catenin signaling. Mol. Cell. Biol. 33, 4095–4105 (2013).
Yuan, W.-C. et al. K33-linked polyubiquitination of coronin 7 by Cul3-KLHL20 ubiquitin E3 ligase regulates protein trafficking. Mol. Cell 54, 586–600 (2014).
McClellan, A. J., Laugesen, S. H. & Ellgaard, L. Cellular functions and molecular mechanisms of non-lysine ubiquitination. Open Biol. 9, 190147 (2019).
Cadwell, K. & Coscoy, L. Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase. Science 309, 127–130 (2005).
Grou, C. P. et al. Members of the E2D (UbcH5) family mediate the ubiquitination of the conserved cysteine of Pex5p, the peroxisomal import receptor. J. Biol. Chem. 283, 14190–14197 (2008).
Carvalho, A. F. et al. Ubiquitination of mammalian Pex5p, the peroxisomal import receptor. J. Biol. Chem. 282, 31267–31272 (2007).
Shimizu, Y., Okuda-Shimizu, Y. & Hendershot, L. M. Ubiquitylation of an ERAD substrate occurs on multiple types of amino acids. Mol. Cell 40, 917–926 (2010).
Needham, P. G., Guerriero, C. J. & Brodsky, J. L. Chaperoning endoplasmic reticulum–associated degradation (ERAD) and protein conformational diseases. Cold Spring Harb. Perspect. Biol. 11, a033928 (2019).
Bhogaraju, S. et al. Phosphoribosylation of ubiquitin promotes serine ubiquitination and impairs conventional ubiquitination. Cell 167, 1636–1649.e13 (2016).
Shin, D. et al. Regulation of phosphoribosyl-linked serine ubiquitination by deubiquitinases DupA and DupB. Mol. Cell 77, 164–179.e6 (2020).
Kalousi, A. & Soutoglou, E. Nuclear compartmentalization of DNA repair. Curr. Opin. Genet. Dev. 37, 148–157 (2016).
Uckelmann, M. & Sixma, T. K. Histone ubiquitination in the DNA damage response. DNA Repair 56, 92–101 (2017).
Mattiroli, F. & Penengo, L. Histone ubiquitination: an integrative signaling platform in genome stability. Trends Genet. 37, 566–581 (2021).
Gatti, M. et al. RNF168 promotes noncanonical K27 ubiquitination to signal DNA damage. Cell Rep. 10, 226–238 (2015).
Thorslund, T. et al. Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage. Nature 527, 389–393 (2015).
Xu, Y. et al. RNF8-mediated regulation of Akt promotes lung cancer cell survival and resistance to DNA damage. Cell Rep. 37, 109854 (2021).
Maekawa, M. & Higashiyama, S. The roles of SPOP in DNA damage response and DNA replication. IJMS 21, 7293 (2020).
Ma, J. et al. SPOP mutation induces replication over-firing by impairing Geminin ubiquitination and triggers replication catastrophe upon ATR inhibition. Nat. Commun. 12, 5779 (2021).
Wang, D. et al. ATM-phosphorylated SPOP contributes to 53BP1 exclusion from chromatin during DNA replication. Sci. Adv. 7, eabd9208 (2021).
Luo, K. et al. A phosphorylation–deubiquitination cascade regulates the BRCA2–RAD51 axis in homologous recombination. Genes Dev. 30, 2581–2595 (2016).
Peters, J. M. The anaphase-promoting complex/cyclosome: a machine designed to destroy. Nat. Rev. Mol. Cell Biol. 7, 644–656 (2006).
Fournane, S., Krupina, K., Kleiss, C. & Sumara, I. Decoding ubiquitin for mitosis. Genes Cancer 3, 697–711 (2012).
Jerabkova, K. & Sumara, I. Cullin 3, a cellular scripter of the non-proteolytic ubiquitin code. Semin. Cell Dev. Biol. 93, 100–110 (2019).
Beck, J. et al. Ubiquitylation-dependent localization of PLK1 in mitosis. Nat. Cell Biol. 15, 430–439 (2013).
Metzger, T., Kleiss, C. & Sumara, I. CUL3 and protein kinases: Insights from PLK1/KLHL22 interaction. Cell Cycle 12, 2291–2296 https://pubmed.ncbi.nlm.nih.gov/24067371/ (2013).
Beck, J. & Peter, M. Regulating PLK1 dynamics by Cullin3/KLHL22-mediated ubiquitylation. Cell Cycle 12, 2528–2529 (2013).
de Cárcer, G. The mitotic cancer target Polo-like kinase 1: oncogene or tumor suppressor? Genes 10, 208 https://doi.org/10.3390/genes10030208 (2019).
Sumara, I. et al. A Cul3-based E3 ligase removes Aurora B from mitotic chromosomes, regulating mitotic progression and completion of cytokinesis in human cells. Dev. Cell 12, 887–900 (2007).
Sumara, I. & Peter, M. A Cul3-based E3 ligase regulates mitosis and is required to maintain the spindle assembly checkpoint in human cells. Cell Cycle 6, 3004–3010 https://pubmed.ncbi.nlm.nih.gov/18075312/ (2007).
Maerki, S. et al. The Cul3–KLHL21 E3 ubiquitin ligase targets Aurora B to midzone microtubules in anaphase and is required for cytokinesis. J. Cell Biol. 187, 791–800 (2009).
Krupina, K. et al. Ubiquitin receptor protein UBASH3B drives aurora B recruitment to mitotic microtubules. Dev. Cell 36, 63–78 (2016).
Goyama, S. et al. UBASH3B/Sts-1-CBL axis regulates myeloid proliferation in human preleukemia induced by AML1-ETO. Leukemia 30, 728–739 (2016).
Lee, S. T. et al. Protein tyrosine phosphatase UBASH3B is overexpressed in triple-negative breast cancer and promotes invasion and metastasis. Proc. Natl Acad. Sci. USA 110, 11121–11126 (2013).
Krupina, K. et al. UBASH3B-mediated silencing of the mitotic checkpoint: therapeutic perspectives in cancer. Mol. Cell. Oncol. 5, e1271494 (2018).
Cheng, J. et al. Functional analysis of Cullin 3 E3 ligases in tumorigenesis. Biochim. Biophys. Acta Rev. Cancer 1869, 11–28 (2018).
Jerabkova, K. et al. Deubiquitylase UCHL3 regulates bi-orientation and segregation of chromosomes during mitosis. FASEB J. 34, 12751–12767 (2020).
Niikura, Y. et al. CENP-A K124 ubiquitylation is required for CENP-A deposition at the centromere. Dev. Cell 32, 589–603 (2015).
Niikura, Y., Kitagawa, R. & Kitagawa, K. CENP-A ubiquitylation is inherited through dimerization between cell divisions. Cell Rep. 15, 61–76 (2016).
Niikura, Y., Kitagawa, R., Fang, L. & Kitagawa, K. CENP-A ubiquitylation is indispensable to cell viability. Developmental Cell 50, 683–689.e6 (2019).
Niikura, Y., Kitagawa, R. & Kitagawa, K. CENP-A ubiquitylation contributes to maintaining the chromosomal location of the centromere. Molecules 24, 402 (2019).
Fachinetti, D. et al. CENP-A modifications on Ser68 and Lys124 are dispensable for establishment, maintenance, and long-term function of human centromeres. Dev. Cell 40, 104–113 (2017).
Barra, V. & Fachinetti, D. The dark side of centromeres: types, causes and consequences of structural abnormalities implicating centromeric DNA. Nat. Commun. 9, 4340 (2018).
Ferreira, L. T., Figueiredo, A. C., Orr, B., Lopes, D. & Maiato, H. Dissecting the role of the tubulin code in mitosis. in Methods Cell Biol. 144 33–74 (Elsevier, 2018).
Lopes, D. & Maiato, H. The tubulin code in mitosis and cancer. Cells 9, 2356 (2020).
Meitinger, F. et al. TRIM37 controls cancer-specific vulnerability to PLK4 inhibition. Nature 585, 440–446 (2020).
Meitinger, F. et al. TRIM37 prevents formation of condensate-organized ectopic spindle poles to ensure mitotic fidelity. J. Cell Biol. 220, e202010180 (2021).
Yu, Y. et al. K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle. Nat. Chem. Biol. 17, 896–905 (2021).
Jongsma, M. L. M. et al. An ER-associated pathway defines endosomal architecture for controlled cargo transport. Cell 166, 152–166 (2016).
Cremer, T. et al. The ER-embedded UBE2J1/RNF26 ubiquitylation complex exerts spatiotemporal control over the endolysosomal pathway. Cell Rep. 34, 108659 (2021).
Yuniati, L. et al. Ubiquitylation of the ER-sha** protein Lunapark via the CRL3KLHL12 ubiquitin ligase complex. Cell Rep. 31, 107664 (2020).
Sapmaz, A. et al. USP32 regulates late endosomal transport and recycling through deubiquitylation of Rab7. Nat. Commun. 10, 1454 (2019).
Song, P., Trajkovic, K., Tsunemi, T. & Krainc, D. Parkin modulates endosomal organization and function of the endo-lysosomal pathway. J. Neurosci. 36, 2425–2437 (2016).
Sagner, A. & Briscoe, J. Establishing neuronal diversity in the spinal cord: a time and a place. Development 146, dev182154 (2019).
Stamataki, D. A gradient of Gli activity mediates graded Sonic Hedgehog signaling in the neural tube. Genes Dev. 19, 626–641 (2005).
Ma, P. et al. Fine-tuning of Shh/Gli signaling gradient by non-proteolytic ubiquitination during neural patterning. Cell Rep. 28, 541–553.e4 (2019).
Li, C. et al. MKRN3 regulates the epigenetic switch of mammalian puberty via ubiquitination of MBD3. Natl Sci. Rev. 7, 671–685 (2020).
Li, C. et al. MKRN3-mediated ubiquitination of Poly(A)-binding proteins modulates the stability and translation of GNRH1 mRNA in mammalian puberty. Nucleic Acids Res. 49, 3796–3813 (2021).
Halloran, D., Durbano, H. W. & Nohe, A. Bone morphogenetic protein-2 in development and bone homeostasis. JDB 8, 19 (2020).
Zhu, W. et al. The E3 ubiquitin ligase WWP2 facilitates RUNX2 protein transactivation in a mono-ubiquitination manner during osteogenic differentiation. J. Biol. Chem. 292, 11178–11188 (2017).
Morciano, G. et al. An updated understanding of the role of YAP in driving oncogenic responses. Cancers 13, 3100 (2021).
Yao, F. et al. SKP2- and OTUD1-regulated non-proteolytic ubiquitination of YAP promotes YAP nuclear localization and activity. Nat. Commun. 9, 2269 (2018).
Martin, S. G. Role and organization of the actin cytoskeleton during cell-cell fusion. Semin. Cell Dev. Biol. 60, 121–126 (2016).
Rodríguez-Pérez, F. et al. Ubiquitin-dependent remodeling of the actin cytoskeleton drives cell fusion. Dev. Cell 56, 588–601.e9 (2021).
Zhang, Q., Lenardo, M. J. & Baltimore, D. 30 years of NF-κB: a blossoming of relevance to human pathobiology. Cell 168, 37–57 (2017).
Ikeda, F. Linear ubiquitination signals in adaptive immune responses. Immunol. Rev. 266, 222–236 (2015).
Verboom, L., Hoste, E. & van Loo, G. OTULIN in NF-κB signaling, cell death, and disease. Trends Immunol. 42, 590–603 (2021).
Zhao, M. et al. Non-proteolytic ubiquitination of OTULIN regulates NF-κB signaling pathway. J. Mol. Cell Biol. 12, 163–175 (2020).
Hirata, Y., Takahashi, M., Morishita, T., Noguchi, T. & Matsuzawa, A. Post-translational modifications of the TAK1-TAB complex. IJMS 18, 205 (2017).
Lei, C.-Q. et al. USP19 inhibits TNF-α- and IL-1β-triggered NF-κB activation by deubiquitinating TAK1. J. Immunol. 203, 259–268 (2019).
Yang, Y. et al. Essential role of the linear ubiquitin chain assembly complex in lymphoma revealed by rare germline polymorphisms. Cancer Discov. 4, 480–493 (2014).
Manasanch, E. E. & Orlowski, R. Z. Proteasome inhibitors in cancer therapy. Nat. Rev. Clin. Oncol. 14, 417–433 (2017).
Dwane, L., Gallagher, W. M., Ní Chonghaile, T. & O’Connor, D. P. The emerging role of non-traditional ubiquitination in oncogenic pathways. J. Biol. Chem. 292, 3543–3551 (2017).
Feng, X. et al. Ubiquitination of UVRAG by SMURF1 promotes autophagosome maturation and inhibits hepatocellular carcinoma growth. Autophagy 15, 1130–1149 (2019).
Infante, P. et al. Itch/β-arrestin2-dependent non-proteolytic ubiquitylation of SuFu controls Hedgehog signalling and medulloblastoma tumorigenesis. Nat. Commun. 9, 976 (2018).
Yin, Q. et al. K27-linked ubiquitination of BRAF by ITCH engages cytokine response to maintain MEK-ERK signaling. Nat. Commun. 10, 1870 (2019).
Park, E. et al. Architecture of autoinhibited and active BRAF–MEK1–14-3-3 complexes. Nature 575, 545–550 (2019).
Liu, J. et al. AMBRA1 promotes TGF-β signaling via non-proteolytic polyubiquitylation of Smad4. Cancer Res. canres.0431.2021 https://doi.org/10.1158/0008-5472.CAN-21-0431 (2021).
Zhou, R., Ding, Y., Xue, M., **ong, B. & Zhuang, T. RNF181 modulates Hippo signaling and triple negative breast cancer progression. Cancer Cell Int. 20, 291 (2020).
Li, Z. et al. Ube2s stabilizes β-Catenin through K11-linked polyubiquitination to promote mesendoderm specification and colorectal cancer development. Cell Death Dis. 9, 456 (2018).
Singh, R. et al. TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis. J. Clin. Invest. 128, 3129–3143 (2018).
Schmidt, M. H. H. & Dikic, I. The Cbl interactome and its functions. Nat. Rev. Mol. Cell Biol. 6, 907–919 (2005).
Charrez, B., Qiao, L. & Hebbard, L. Hepatocellular carcinoma and non-alcoholic steatohepatitis: the state of play. World J. Gastroenterol. 22, 2494–2502 (2016).
Zhu, K. et al. Non-proteolytic ubiquitin modification of PPARγ by Smurf1 protects the liver from steatosis. PLoS Biol. 16, e3000091 (2018).
Huang, X., Liu, G., Guo, J. & Su, Z. The PI3K/AKT pathway in obesity and type 2 diabetes. Int. J. Biol. Sci. 14, 1483–1496 (2018).
Atkinson, M. A., Eisenbarth, G. S. & Michels, A. W. Type 1 diabetes. Lancet 383, 69–82 (2014).
Cederquist, C. T. et al. Systemic insulin sensitivity is regulated by GPS2 inhibition of AKT ubiquitination and activation in adipose tissue. Mol. Metab. 6, 125–137 (2017).
Summers, S., Whiteman, E. & Birnbaum, M. Insulin signaling in the adipocyte. Int. J. Obes. 24, S67–S70 (2000).
Jiang, Z. Y. et al. Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing. Proc. Natl Acad. Sci. 100, 7569–7574 (2003).
Cantley, L. C. The phosphoinositide 3-Kinase pathway. Science 296, 1655–1657 (2002).
Yang, W.-L. et al. The E3 ligase TRAF6 regulates Akt ubiquitination and activation. Science 325, 1134–1138 (2009).
Lentucci, C. et al. Inhibition of Ubc13-mediated ubiquitination by GPS2 regulates multiple stages of B cell development. J. Biol. Chem. 292, 2754–2772 (2017).
Chen, H. C., Stone, S. J., Zhou, P., Buhman, K. K. & Farese, R. V. Dissociation of obesity and impaired glucose disposal in mice overexpressing acyl coenzyme A:diacylglycerol acyltransferase 1 in white adipose tissue. Diabetes 51, 3189–3195 (2002).
Morley, T. S., **a, J. Y. & Scherer, P. E. Selective enhancement of insulin sensitivity in the mature adipocyte is sufficient for systemic metabolic improvements. Nat. Commun. 6, 7906 (2015).
Pontrelli, P. et al. Lysine 63 ubiquitination is involved in the progression of tubular damage in diabetic nephropathy. FASEB J. 31, 308–319 (2017).
Martínez-Reyes, I. & Chandel, N. S. Cancer metabolism: looking forward. Nat. Rev. Cancer 21, 669–680 (2021).
Lee, H.-J. et al. Non-proteolytic ubiquitination of Hexokinase 2 by HectH9 controls tumor metabolism and cancer stem cell expansion. Nat. Commun. 10, 2625 (2019).
Yu, S., Zang, W., Qiu, Y., Liao, L. & Zheng, X. Deubiquitinase OTUB2 exacerbates the progression of colorectal cancer by promoting PKM2 activity and glycolysis. Oncogene https://doi.org/10.1038/s41388-021-02071-2 (2021).
Liu, K. et al. Parkin regulates the activity of pyruvate kinase M2. J. Biol. Chem. 291, 10307–10317 (2016).
van Huizen, M. & Kikkert, M. The role of atypical ubiquitin chains in the regulation of the antiviral innate immune response. Front Cell Dev. Biol. 7, 392 (2019).
Zhang, Z., Wang, D., Wang, P., Zhao, Y. & You, F. OTUD1 negatively regulates type I IFN induction by disrupting noncanonical ubiquitination of IRF3. J. I. 204, 1904–1918 (2020).
Lu, D. et al. Mutations of deubiquitinase OTUD1 are associated with autoimmune disorders. J. Autoimmun. 94, 156–165 (2018).
Damgaard, R. B. et al. The deubiquitinase OTULIN is an essential negative regulator of inflammation and autoimmunity. Cell 166, 1215–1230.e20 (2016).
Zhou, Q. et al. Biallelic hypomorphic mutations in a linear deubiquitinase define otulipenia, an early-onset autoinflammatory disease. Proc. Natl Acad. Sci. USA 113, 10127–10132 (2016).
Yasuda, K., Takeuchi, Y. & Hirota, K. Correction to: The pathogenicity of Th17 cells in autoimmune diseases. Semin Immunopathol. 41, 299–299 (2019).
Cho, J. J. et al. Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation. Nat. Commun. 10, 701 (2019).
Capone, A. & Volpe, E. Transcriptional regulators of T helper 17 cell differentiation in health and autoimmune diseases. Front. Immunol. 11, 348 (2020).
Yu, Z. et al. Lys29-linkage of ASK1 by Skp1-Cullin 1-Fbxo21 ubiquitin ligase complex is required for antiviral innate response. Elife 5, e14087 (2016).
Karim, M. et al. Nonproteolytic K29-linked ubiquitination of the PB2 replication protein of influenza A viruses by proviral cullin 4-based E3 ligases. mBio 11, e00305–e00320 (2020). /mbio/11/2/mBio.00305-20.atom.
Xue, B. et al. TRIM21 promotes innate immune response to RNA viral infection through Lys27-linked polyubiquitination of MAVS. J. Virol. 92, e00321-18 https://pubmed.ncbi.nlm.nih.gov/29743353/ (2018).
van Wijk, S. J., Fulda, S., Dikic, I. & Heilemann, M. Visualizing ubiquitination in mammalian cells. EMBO Rep. 20, e46520 (2019).
Acknowledgements
We would like to thank Lucile Guerber and all members of Sumara group, for their helpful discussions and corrections on the manuscript. Figures were created with BioRender.com. Y. Liao is supported by a Ph.D. fellowship from the China Scholarship Council (CSC). E.P. was supported by postdoctoral fellowships from the Foundation pour la recherché Médicale (FRM) and ANR-10-LABX-0030-INRT. Research in I.S. laboratory was supported by the grant ANR-10-LABX-0030-INRT, a French State fund managed by the Agence Nationale de la Recherche under the frame program Investissements d’Avenir ANR-10-IDEX-0002-02, IGBMC, CNRS, Fondation ARC pour la recherche sur le cancer, Institut National du Cancer (INCa), Agence Nationale de la Recherche (ANR), Ligue Nationale contre le Cancer, USIAS, Sanofi iAward Europe and Programme Fédérateur Aviesan, Plan Cancer, National collaborative project: NANOTUMOR.
Author information
Authors and Affiliations
Contributions
Conceptualization and writing: Y.L., I.S., and E.P.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Liao, Y., Sumara, I. & Pangou, E. Non-proteolytic ubiquitylation in cellular signaling and human disease. Commun Biol 5, 114 (2022). https://doi.org/10.1038/s42003-022-03060-1
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s42003-022-03060-1
- Springer Nature Limited