Biotin-Based Strategies to Explore the World of Ubiquitin and Ubiquitin-Like Modifiers.

A complex code of cellular signals is mediated by ubiquitin and ubiquitin-like (Ub/UbL) modifications on substrate proteins. The so-called Ubiquitin Code specifies protein fates, such as stability, subcellular localization, functional activation or suppression, and interactions. Hundreds of enzymes are involved in placing and removing Ub/UbL on thousands of substrates, while the consequences of modifications and the mechanisms of specificity are still poorly defined. Challenges include rapid and transient engagement of enzymes and Ub/UbL interactors, low stoichiometry of modified versus non-modified cellular substrates, and protease-mediated loss of Ub/UbL in lysates. To decipher this complexity and confront the challenges, many tools have been created to trap and identify substrates and interactors linked to Ub/UbL modification. This review focuses on an assortment of biotin-based tools developed for this purpose (for example BioUbLs, UbL-ID, BioE3, BioID), taking advantage of the strong affinity of biotin-streptavidin and the stringent lysis/washing approach allowed by it, paired with sensitive mass-spectrometry-based proteomic methods. Knowing how substrates change during development and disease, the consequences of substrate modification, and matching substrates to particular UbL-ligating enzymes will contribute new insights into how Ub/UbL signaling works and how it can be exploited for therapies.

BioE3 identifies specific substrates of ubiquitin E3 ligases.

Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network.

Linoleic acid improves PIEZO2 dysfunction in a mouse model of Angelman Syndrome.

Angelman syndrome (AS) is a neurogenetic disorder characterized by intellectual disability and atypical behaviors. AS results from loss of expression of the E3 ubiquitin-protein ligase UBE3A from the maternal allele in neurons. Individuals with AS display impaired coordination, poor balance, and gait ataxia. PIEZO2 is a mechanosensitive ion channel essential for coordination and balance. Here, we report that PIEZO2 activity is reduced in Ube3a deficient male and female mouse sensory neurons, a human Merkel cell carcinoma cell line and female human iPSC-derived sensory neurons with UBE3A knock-down, and de-identified stem cell-derived neurons from individuals with AS. We find that loss of UBE3A decreases actin filaments and reduces PIEZO2 expression and function. A linoleic acid (LA)-enriched diet increases PIEZO2 activity, mechano-excitability, and improves gait in male AS mice. Finally, LA supplementation increases PIEZO2 function in stem cell-derived neurons from individuals with AS. We propose a mechanism whereby loss of UBE3A expression reduces PIEZO2 function and identified a fatty acid that enhances channel activity and ameliorates AS-associated mechano-sensory deficits.

RNAi-Based Screening for the Identification of Specific Substrate-Deubiquitinase Pairs.

Ubiquitination signals are regulated in time and space due to the coordinated action of E3s and DUBs, which enables the precise control of cellular function and homeostasis. Mutations in all types of ubiquitin-proteasome system (UPS) components are related to pathological conditions. The identification of E3/DUBs' ubiquitinated substrates can provide a clearer view of the molecular mechanisms underlying those diseases. However, the analysis of ubiquitinated proteins is not trivial. Here, we propose a protocol to identify DUB/substrate pairs, by combining DUB silencing, specific pull-down of the substrate, and image analysis of its ubiquitinated fraction.

SUMO-ID: A Strategy for the Identification of SUMO-Dependent Proximal Interactors.

Posttranslational modifications by the ubiquitin-like family (UbL) of proteins determine the biological fate of a substrate, including new interaction partners. In the case of the small ubiquitin-like modifier (SUMO), this is achieved in part through its non-covalent interaction with SUMO-interacting motifs (SIMs) found in some proteins. Investigating such partner-complex formation is particularly challenging due to the fast dynamics and reversibility of SUMO modifications and the low affinity of SUMO-SIM interactions. Here, we present a detailed protocol of SUMO-ID, a technology that merges promiscuous proximity biotinylation by TurboID enzyme and protein-fragment complementation strategy to specifically biotinylate SUMO-dependent interactors of particular substrates. When coupled to streptavidin-affinity purification and mass spectrometry, SUMO-ID efficiently identifies SUMO-dependent interactors of a given protein. The methodology describes all the steps from SUMO-ID cell line generation to LC-MS sample preparation to study SUMO-dependent interactors of a particular protein. The protocol is generic and therefore adaptable to study other UbL-dependent interactors, such as ubiquitin.

HERC2 deficiency activates C-RAF/MKK3/p38 signalling pathway altering the cellular response to oxidative stress.

HERC2 gene encodes an E3 ubiquitin ligase involved in several cellular processes by regulating the ubiquitylation of different protein substrates. Biallelic pathogenic sequence variants in the HERC2 gene are associated with HERC2 Angelman-like syndrome. In pathogenic HERC2 variants, complete absence or marked reduction in HERC2 protein levels are observed. The most common pathological variant, c.1781C > T (p.Pro594Leu), encodes an unstable HERC2 protein. A better understanding of how pathologic HERC2 variants affect intracellular signalling may aid definition of potential new therapies for these disorders. For this purpose, we studied patient-derived cells with the HERC2 Pro594Leu variant. We observed alteration of mitogen-activated protein kinase signalling pathways, reflected by increased levels of C-RAF protein and p38 phosphorylation. HERC2 knockdown experiments reproduced the same effects in other human and mouse cells. Moreover, we demonstrated that HERC2 and RAF proteins form molecular complexes, pull-down and proteomic experiments showed that HERC2 regulates C-RAF ubiquitylation and we found out that the p38 activation due to HERC2 depletion occurs in a RAF/MKK3-dependent manner. The displayed cellular response was that patient-derived and other human cells with HERC2 deficiency showed higher resistance to oxidative stress with an increase in the master regulator of the antioxidant response NRF2 and its target genes. This resistance was independent of p53 and abolished by RAF or p38 inhibitors. Altogether, these findings identify the activation of C-RAF/MKK3/p38 signalling pathway in HERC2 Angelman-like syndrome and highlight the inhibition of RAF activity as a potential therapeutic option for individuals affected with these rare diseases.

Identification of substrates for human deubiquitinating enzymes (DUBs): An up-to-date review and a case study for neurodevelopmental disorders.

Similar to the reversal of kinase-mediated protein phosphorylation by phosphatases, deubiquitinating enzymes (DUBs) oppose the action of E3 ubiquitin ligases and reverse the ubiquitination of proteins. A total of 99 human DUBs, classified in 7 families, allow in this way for a precise control of cellular function and homeostasis. Ubiquitination regulates a myriad of cellular processes, and is altered in many pathological conditions. Thus, ubiquitination-regulating enzymes are increasingly regarded as potential candidates for therapeutic intervention. In this context, given the predicted easier pharmacological control of DUBs relative to E3 ligases, a significant effort is now being directed to better understand the processes and substrates regulated by each DUB. Classical studies have identified specific DUB substrate candidates by traditional molecular biology techniques in a case-by-case manner. Lately, single experiments can identify thousands of ubiquitinated proteins at a specific cellular context and narrow down which of those are regulated by a given DUB, thanks to the development of new strategies to isolate and enrich ubiquitinated material and to improvements in mass spectrometry detection capabilities. Here we present an overview of both types of studies, discussing the criteria that, in our view, need to be fulfilled for a protein to be considered as a high-confidence substrate of a given DUB. Applying these criteria, we have manually reviewed the relevant literature currently available in a systematic manner, and identified 650 high-confidence substrates of human DUBs. We make this information easily accessible to the research community through an updated version of the DUBase website (https://ehubio.ehu.eus/dubase/). Finally, in order to illustrate how this information can contribute to a better understanding of the physiopathological role of DUBs, we place a special emphasis on a subset of these enzymes that have been associated with neurodevelopmental disorders.

Identification of proximal SUMO-dependent interactors using SUMO-ID.

The fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We develop an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allow us to identify interactors of SUMOylated SALL1, a less characterized SUMO substrate. Furthermore, using TP53 as a substrate, we identify SUMO1, SUMO2 and Ubiquitin preferential interactors. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code.

A Proteomic Approach for Systematic Mapping of Substrates of Human Deubiquitinating Enzymes.

The human genome contains nearly 100 deubiquitinating enzymes (DUBs) responsible for removing ubiquitin moieties from a large variety of substrates. Which DUBs are responsible for targeting which substrates remain mostly unknown. Here we implement the bioUb approach to identify DUB substrates in a systematic manner, combining gene silencing and proteomics analyses. Silencing of individual DUB enzymes is used to reduce their ubiquitin deconjugating activity, leading to an increase of the ubiquitination of their substrates, which can then be isolated and identified. We report here quantitative proteomic data of the putative substrates of 5 human DUBs. Furthermore, we have built a novel interactive database of DUB substrates to provide easy access to our data and collect DUB proteome data from other groups as a reference resource in the DUB substrates research field.

Neddylation inhibition ameliorates steatosis in NAFLD by boosting hepatic fatty acid oxidation via the DEPTOR-mTOR axis.

OBJECTIVE: Neddylation is a druggable and reversible ubiquitin-like post-translational modification upregulated in many diseases, including liver fibrosis, hepatocellular carcinoma, and more recently, non-alcoholic fatty liver disease (NAFLD). Herein, we propose to address the effects of neddylation inhibition and the underlying mechanisms in pre-clinical models of NAFLD. METHODS: Hepatic neddylation measured by immunohistochemical analysis and NEDD8 serum levels measured by ELISA assay were evaluated in NAFLD clinical and pre-clinical samples. The effects of neddylation inhibition by using a pharmacological small inhibitor, MLN4924, or molecular approaches were assessed in isolated mouse hepatocytes and pre-clinical mouse models of diet-induced NAFLD, male adult C57BL/6 mice, and the AlfpCre transgenic mice infected with AAV-DIO-shNedd8. RESULTS: Neddylation inhibition reduced lipid accumulation in oleic acid-stimulated mouse primary hepatocytes and ameliorated liver steatosis, preventing lipid peroxidation and inflammation in the mouse models of diet-induced NAFLD. Under these conditions, increased Deptor levels and the concomitant repression of mTOR signaling were associated with augmented fatty acid oxidation and reduced lipid content. Moreover, Deptor silencing in isolated mouse hepatocytes abolished the anti-steatotic effects mediated by neddylation inhibition. Finally, serum NEDD8 levels correlated with hepatic neddylation during the disease progression in the clinical and pre-clinical models CONCLUSIONS: Overall, the upregulation of Deptor, driven by neddylation inhibition, is proposed as a novel effective target and therapeutic approach to tackle NAFLD.

The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF.

Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, we used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. We identified 16 candidates that met the established criteria: a significant change of at least twofold increase on ubiquitination, with at least two unique peptides identified. Among those candidates, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release. Using a pull-down approach that relies on the overexpression and stringent isolation of a GFP-fused construct, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. We tested the possible functional relevance of this modification using Ari-1 loss-of-function mutants, which displayed a lower rate of spontaneous neurotransmitter release due to failures at the presynaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared with controls in a Ca2+-dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous and evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. Our results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination.

Multi-Omics Integration Highlights the Role of Ubiquitination in CCl4-Induced Liver Fibrosis.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in chronic liver disease. Ubiquitination is a post-translational modification that is crucial for a plethora of physiological processes. Even though the ubiquitin system has been implicated in several human diseases, the role of ubiquitination in liver fibrosis remains poorly understood. Here, multi-omics approaches were used to address this. Untargeted metabolomics showed that carbon tetrachloride (CCl4)-induced liver fibrosis promotes changes in the hepatic metabolome, specifically in glycerophospholipids and sphingolipids. Gene ontology analysis of public deposited gene array-based data and validation in our mouse model showed that the biological process "protein polyubiquitination" is enriched after CCl4-induced liver fibrosis. Finally, by using transgenic mice expressing biotinylated ubiquitin (bioUb mice), the ubiquitinated proteome was isolated and characterized by mass spectrometry in order to unravel the hepatic ubiquitinated proteome fingerprint in CCl4-induced liver fibrosis. Under these conditions, ubiquitination appears to be involved in the regulation of cell death and survival, cell function, lipid metabolism, and DNA repair. Finally, ubiquitination of proliferating cell nuclear antigen (PCNA) is induced during CCl4-induced liver fibrosis and associated with the DNA damage response (DDR). Overall, hepatic ubiquitome profiling can highlight new therapeutic targets for the clinical management of liver fibrosis.

SUMOylation in the control of cholesterol homeostasis.

SUMOylation-protein modification by the small ubiquitin-related modifier (SUMO)-affects several cellular processes by modulating the activity, stability, interactions or subcellular localization of a variety of substrates. SUMO modification is involved in most cellular processes required for the maintenance of metabolic homeostasis. Cholesterol is one of the main lipids required to preserve the correct cellular function, contributing to the composition of the plasma membrane and participating in transmembrane receptor signalling. Besides these functions, cholesterol is required for the synthesis of steroid hormones, bile acids, oxysterols and vitamin D. Cholesterol levels need to be tightly regulated: in excess, it is toxic to the cell, and the disruption of its homeostasis is associated with various disorders like atherosclerosis and cardiovascular diseases. This review focuses on the role of SUMO in the regulation of proteins involved in the metabolism of cholesterol.

The role of SUMOylation during development.

During the development of multicellular organisms, transcriptional regulation plays an important role in the control of cell growth, differentiation and morphogenesis. SUMOylation is a reversible post-translational process involved in transcriptional regulation through the modification of transcription factors and through chromatin remodelling (either modifying chromatin remodelers or acting as a 'molecular glue' by promoting recruitment of chromatin regulators). SUMO modification results in changes in the activity, stability, interactions or localization of its substrates, which affects cellular processes such as cell cycle progression, DNA maintenance and repair or nucleocytoplasmic transport. This review focuses on the role of SUMO machinery and the modification of target proteins during embryonic development and organogenesis of animals, from invertebrates to mammals.

How to Inactivate Human Ubiquitin E3 Ligases by Mutation.

E3 ubiquitin ligases are the ultimate enzymes involved in the transfer of ubiquitin to substrate proteins, a process that determines the fate of the modified protein. Numerous diseases are caused by defects in the ubiquitin-proteasome machinery, including when the activity of a given E3 ligase is hampered. Thus, inactivation of E3 ligases and the resulting effects at molecular or cellular level have been the focus of many studies during the last few years. For this purpose, site-specific mutation of key residues involved in either protein interaction, substrate recognition or ubiquitin transfer have been reported to successfully inactivate E3 ligases. Nevertheless, it is not always trivial to predict which mutation(s) will block the catalytic activity of a ligase. Here we review over 250 site-specific inactivating mutations that have been carried out in 120 human E3 ubiquitin ligases. We foresee that the information gathered here will be helpful for the design of future experimental strategies.

Mass Spectrometry-Based Characterization of Ub- and UbL-Modified Proteins.

Regulation by ubiquitin (Ub) and ubiquitin-like (UbL) modifiers can confer their substrate proteins a myriad of assignments, such as inducing protein-protein interactions, the internalization of membrane proteins, or their degradation via the proteasome. The underlying code regulating those diverse endpoints appears to be based on the topology of the ubiquitin chains formed.Experimental characterization of the specific regulation mediated by Ub and UbLs is not trivial. The substoichiometric levels of Ub- and UbL-modified proteins greatly limit their analytical detection in a background of more abundant proteins. Therefore, modified proteins or peptides must be enriched prior to any downstream detection analysis. For that purpose, we recently developed a GFP-tag based isolation strategy. Here we illustrate the usefulness of combining GFP-tag isolation strategy with mass spectrometry (MS) to identify Ub- and UbL-modified residues within the GFP-tagged protein, as well as to uncover the types of Ub and UbL chains formed.

Detailed Dissection of UBE3A-Mediated DDI1 Ubiquitination.

The ubiquitin E3 ligase UBE3A has been widely reported to interact with the proteasome, but it is still unclear how this enzyme regulates by ubiquitination the different proteasomal subunits. The proteasome receptor DDI1 has been identified both in Drosophila photoreceptor neurons and in human neuroblastoma cells in culture as a direct substrate of UBE3A. Here, we further characterize this regulation, by identifying the UBE3A-dependent ubiquitination sites and ubiquitin chains formed on DDI1. Additionally, we found one deubiquitinating enzyme that is capable of reversing the action of UBE3A on DDI1. The complete characterization of the ubiquitination pathway of an UBE3A substrate is important due to the role of this E3 ligase in rare neurological disorders as Angelman syndrome.

Impaired proteostasis in rare neurological diseases.

Rare diseases are classified as such when their prevalence is 1:2000 or lower, but even if each of them is so infrequent, altogether more than 300 million people in the world suffer one of the ∼7000 diseases considered as rare. Over 1200 of these disorders are known to affect the brain or other parts of our nervous system, and their symptoms can affect cognition, motor function and/or social interaction of the patients; we refer collectively to them as rare neurological disorders or RNDs. We have focused this review on RNDs known to have compromised protein homeostasis pathways. Proteostasis can be regulated and/or altered by a chain of cellular mechanisms, from protein synthesis and folding, to aggregation and degradation. Overall, we provide a list comprised of above 215 genes responsible for causing more than 170 distinct RNDs, deepening on some representative diseases, including as well a clinical view of how those diseases are diagnosed and dealt with. Additionally, we review existing methodologies for diagnosis and treatment, discussing the potential of specific deubiquitinating enzyme inhibition as a future therapeutic avenue for RNDs.

Ubiquitylation Dynamics of the Clock Cell Proteome and TIMELESS during a Circadian Cycle.

Circadian clocks have evolved as time-measuring molecular devices to help organisms adapt their physiology to daily changes in light and temperature. Transcriptional oscillations account for a large fraction of rhythmic protein abundance. However, cycling of various posttranslational modifications, such as ubiquitylation, also contributes to shape the rhythmic protein landscape. In this study, we used an in vivo ubiquitin labeling assay to investigate the circadian ubiquitylated proteome of Drosophila melanogaster. We find that cyclic ubiquitylation affects MEGATOR (MTOR), a chromatin-associated nucleoporin that, in turn, feeds back to regulate the core molecular oscillator. Furthermore, we show that the ubiquitin ligase subunits CULLIN-3 (CUL-3) and SUPERNUMERARY LIMBS (SLMB) cooperate for ubiquitylating the TIMELESS protein. These findings stress the importance of ubiquitylation pathways in the Drosophila circadian clock and reveal a key component of this system.

Quantitative proteomics reveals neuronal ubiquitination of Rngo/Ddi1 and several proteasomal subunits by Ube3a, accounting for the complexity of Angelman syndrome.

Angelman syndrome is a complex neurodevelopmental disorder caused by the lack of function in the brain of a single gene, UBE3A. The E3 ligase coded by this gene is known to build K48-linked ubiquitin chains, a modification historically considered to target substrates for degradation by the proteasome. However, a change in protein abundance is not proof that a candidate UBE3A substrate is indeed ubiquitinated by UBE3A. We have here used an unbiased ubiquitin proteomics approach, the bioUb strategy, to identify 79 proteins that appear more ubiquitinated in the Drosophila photoreceptor cells when Ube3a is over-expressed. We found a significantly high number of those proteins to be proteasomal subunits or proteasome-interacting proteins, suggesting a wide proteasomal perturbation in the brain of Angelman patients. We focused on validating the ubiquitination by Ube3a of Rngo, a proteasomal component conserved from yeast (Ddi1) to humans (DDI1 and DDI2), but yet scarcely characterized. Ube3a-mediated Rngo ubiquitination in fly neurons was confirmed by immunoblotting. Using human neuroblastoma SH-SY5Y cells in culture, we also observed that human DDI1 is ubiquitinated by UBE3A, without being targeted for degradation. The novel observation that DDI1 is expressed in the developing mice brain, with a significant peak at E16.5, strongly suggests that DDI1 has biological functions not yet described that could be of relevance for Angelman syndrome clinical research.