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.

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.

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.

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.

Opposite feedback from mTORC1 to H-ras and K-ras4B downstream of SREBP1.

As a major growth factor transducer, Ras is an upstream activator of mTORC1, which further integrates nutrient and energy inputs. To ensure a contextual coupling of cell division via Ras/MAPK-signalling and growth via mTORC1-signalling, feedback loops from one pathway back to the other are required. Here we describe a novel feedback from mTORC1, which oppositely affects oncogenic H-ras- and K-ras-signalling output, and as a consequence stemness properties of tumourigenic cells. Amino acid stimulation of mTORC1 increases the processed form of SREBP1, a major lipidome regulator. We show that modulation of the SREBP1 levels downstream of S6K1 has opposite effects on oncogenic H-ras and K-ras nanoscale membrane organisation, ensuing signalling output and promotion of mammospheres expressing these oncogenes. Our data suggest that modulation of phosphatidic acid, a major target of SREBP1 controlled lipid metabolism, is sufficient to affect H-ras and K-ras oppositely in the membrane. Thus mTORC1 activation increases H-ras-, but decreases K-ras-signalling output in cells transformed with the respective oncogene. Given the different impact of these two Ras isoforms on stemness, our results could have implications for stem cell biology and inhibition of cancer stem cells.

Rapalogs can promote cancer cell stemness in vitro in a Galectin-1 and H-ras-dependent manner.

Currently several combination treatments of mTor- and Ras-pathway inhibitors are being tested in cancer therapy. While multiple feedback loops render these central signaling pathways robust, they complicate drug targeting.Here, we describe a novel H-ras specific feedback, which leads to an inadvertent rapalog induced activation of tumorigenicity in Ras transformed cells. We find that rapalogs specifically increase nanoscale clustering (nanoclustering) of oncogenic H-ras but not K-ras on the plasma membrane. This increases H-ras signaling output, promotes mammosphere numbers in a H-ras-dependent manner and tumor growth in ovo. Surprisingly, also other FKBP12 binders, but not mTor-inhibitors, robustly decrease FKBP12 levels after prolonged (>2 days) exposure. This leads to an upregulation of the nanocluster scaffold galectin-1 (Gal-1), which is responsible for the rapamycin-induced increase in H-ras nanoclustering and signaling output. We provide evidence that Gal-1 promotes stemness features in tumorigenic cells. Therefore, it may be necessary to block inadvertent induction of stemness traits in H-ras transformed cells by specific Gal-1 inhibitors that abrogate its effect on H-ras nanocluster. On a more general level, our findings may add an important mechanistic explanation to the pleiotropic physiological effects that are observed with rapalogs.

Quantitative proteomic analysis of Parkin substrates in Drosophila neurons.

BACKGROUND: Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson's Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings. METHODS: Here we utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression we analysed by mass spectrometry and stringent bioinformatics analysis those proteins differentially ubiquitinated to provide the first survey of steady state Parkin substrates using an in vivo model. We further used an in vivo ubiquitination assay to validate one of those substrates in SH-SY5Y cells. RESULTS: We identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, ALiX/PDCD6IP and Vps4. We also identified the retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin. Importantly, we validated Parkin-dependent ubiquitination of VPS35 in human neuroblastoma cells. CONCLUSIONS: Collectively our results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation.

ASPP2 Is a Novel Pan-Ras Nanocluster Scaffold.

Ras-induced senescence mediated through ASPP2 represents a barrier to tumour formation. It is initiated by ASPP2's interaction with Ras at the plasma membrane, which stimulates the Raf/MEK/ERK signaling cascade. Ras to Raf signalling requires Ras to be organized in nanoscale signalling complexes, called nanocluster. We therefore wanted to investigate whether ASPP2 affects Ras nanoclustering. Here we show that ASPP2 increases the nanoscale clustering of all oncogenic Ras isoforms, H-ras, K-ras and N-ras. Structure-function analysis with ASPP2 truncation mutants suggests that the nanocluster scaffolding activity of ASPP2 converges on its α-helical domain. While ASPP2 increased effector recruitment and stimulated ERK and AKT phosphorylation, it did not increase colony formation of RasG12V transformed NIH/3T3 cells. By contrast, ASPP2 was able to suppress the transformation enhancing ability of the nanocluster scaffold Gal-1, by competing with the specific effect of Gal-1 on H-rasG12V- and K-rasG12V-nanoclustering, thus imposing ASPP2's ERK and AKT signalling signature. Similarly, ASPP2 robustly induced senescence and strongly abrogated mammosphere formation irrespective of whether it was expressed alone or together with Gal-1, which by itself showed the opposite effect in Ras wt or H-ras mutant breast cancer cells. Our results suggest that Gal-1 and ASPP2 functionally compete in nanocluster for active Ras on the plasma membrane. ASPP2 dominates the biological outcome, thus switching from a Gal-1 supported growth-promoting setting to a senescence inducing and stemness suppressive program in cancer cells. Our results support Ras nanocluster as major integrators of tumour fate decision events.

Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types.

Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells.

BACKGROUND: Ubiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Several ubiquitin proteomic approaches have been developed during the last decade but, as they have been mostly applied to non-neuronal cell culture, very little is yet known about neuronal ubiquitination pathways in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Using an in vivo biotinylation strategy we have isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of Drosophila melanogaster. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, we report the proof-of-principle validation of two lysine residues required for nSyb ubiquitination. CONCLUSIONS/SIGNIFICANCE: These data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The in vivo biotinylation methodology described here complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system.

Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation.

Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras. Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms. Mutants are biochemically inconspicuous, while their clustering into nanoscale signaling complexes on the plasma membrane, termed nanocluster, is augmented. Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation. Our results describe an unprecedented mechanism of signaling protein activation in cancer.

Ubiquitin profiling in liver using a transgenic mouse with biotinylated ubiquitin.

Ubiquitination is behind most cellular processes, with ubiquitin substrates being regulated variously according to the number of covalently conjugated ubiquitin molecules and type of chain formed. Here we report the first mammalian system for ubiquitin proteomics allowing direct validation of the MS-identified proteins. We created a transgenic mouse expressing biotinylated ubiquitin and demonstrate its use for the isolation of ubiquitinated proteins from liver and other tissues. The specificity and strength of the biotin-avidin interaction allow very stringent washes, so only proteins conjugated to ubiquitin are isolated. In contrast with recently available antibody-based approaches, our strategy allows direct validation by immunoblotting, therefore revealing the type of ubiquitin chains (mono or poly) formed in vivo. We also identify the conjugating E2 enzymes that are ubiquitin-loaded in the mouse tissue. Furthermore, our strategy allows the identification of candidate cysteine-ubiquitinated proteins, providing a strategy to identify those on a proteomic scale. The novel in vivo system described here allows broad access to tissue-specific ubiquitomes and can be combined with established mouse disease models to investigate ubiquitin-dependent therapeutical approaches.

Ube3a, the E3 ubiquitin ligase causing Angelman syndrome and linked to autism, regulates protein homeostasis through the proteasomal shuttle Rpn10.

Ubiquitination, the covalent attachment of ubiquitin to a target protein, regulates most cellular processes and is involved in several neurological disorders. In particular, Angelman syndrome and one of the most common genomic forms of autism, dup15q, are caused respectively by lack of or excess of UBE3A, a ubiquitin E3 ligase. Its Drosophila orthologue, Ube3a, is also active during brain development. We have now devised a protocol to screen for substrates of this particular ubiquitin ligase. In a neuronal cell system, we find direct ubiquitination by Ube3a of three proteasome-related proteins Rpn10, Uch-L5, and CG8209, as well as of the ribosomal protein Rps10b. Only one of these, Rpn10, is targeted for degradation upon ubiquitination by Ube3a, indicating that degradation might not be the only effect of Ube3a on its substrates. Furthermore, we report the genetic interaction in vivo between Ube3a and the C-terminal part of Rpn10. Overexpression of these proteins leads to an enhanced accumulation of ubiquitinated proteins, further supporting the biochemical evidence of interaction obtained in neuronal cells.

Recognition of intermolecular G-quadruplexes by full length nucleophosmin. Effect of a leukaemia-associated mutation.

Nucleophosmin (NPM) is a nucleolar protein involved in ribosome biogenesis. NPM1 gene is frequently mutated in acute myeloid leukaemia (AML), correlating with aberrant cytoplasmic localization of the protein. NPM attachment to the nucleolus in physiological conditions probably depends on binding to nucleic acids, and this recognition could be altered in AML. NPM associates to guanine-rich DNA sequences, able to fold as "G-quadruplexes". We have analyzed the interaction of pentameric, full length NPM with G-rich oligonucleotides, finding that the protein binds preferentially high-order G-quadruplexes. AML-associated mutation significantly hampers DNA binding, pointing to a possible mechanism contributing to pathological mislocalization of NPM.

The orexigenic activity of the hypothalamic neuropeptide 26RFa is mediated by the neuropeptide Y and proopiomelanocortin neurons of the arcuate nucleus.

26RFa is a hypothalamic RFamide neuropeptide that was identified as the endogenous ligand of the orphan G protein-coupled receptor, GPR103, and that stimulates appetite in mice. Up until now, the mechanism of action of 26RFa in the hypothalamic control of food intake remains unknown. The high density of GPR103 in the arcuate nucleus (Arc) prompted us to investigate, in the present study, the effects of 26RFa on the rat neuropeptide Y (NPY)/proopiomelanocortin (POMC) system. Intracerebroventricular injection of 26RFa stimulated NPY expression and release in the basal hypothalamus, whereas it decreased POMC expression and alpha-MSH release, and these effects were associated with an increase in food intake. A double in situ hybridization procedure indicated that the 26RFa receptor is present in NPY neurons of the Arc, but not in POMC neurons. Central administration of NPY Y1 and Y5 receptor antagonists abolished the inhibitory effects of 26RFa on POMC expression and alpha-MSH release, and reversed 26RFa-induced food consumption. Finally, 26RFa antagonized the effects of leptin on NPY expression and release, POMC expression and alpha-MSH release, and food intake. Altogether, the present data demonstrate for the first time that 26RFa exerts its orexigenic activity by stimulating the release of NPY in the Arc, which in turn inhibits POMC neurons by activating the Y1 and Y5 receptors. It is also suggested that the balance 26RFa/leptin is an important parameter in the maintenance of energy homeostasis.

Characterization of urotensin II, distribution of urotensin II, urotensin II-related peptide and UT receptor mRNAs in mouse: evidence of urotensin II at the neuromuscular junction.

Urotensin II (UII) and UII-related peptide (URP) are paralog neuropeptides whose existence and distribution in mouse have not yet been investigated. In this study, we showed by HPLC/RIA analysis that the UII-immunoreactive molecule in the mouse brain corresponds to a new UII(17) isoform. Moreover, calcium mobilization assays indicated that UII(17) and URP were equally potent in stimulating UII receptor (UT receptor). Quantitative RT-PCR and in situ hybridization analysis revealed that in the CNS UII and URP mRNAs were predominantly expressed in brainstem and spinal motoneurons. Besides, they were differentially expressed in the medial vestibular nucleus, locus coeruleus and the ventral medulla. In periphery, both mRNAs were expressed in skeletal muscle, testis, vagina, stomach, and gall bladder, whereas only URP mRNA could be detected in the seminal vesicle, heart, colon, and thymus. By contrast, the UT receptor mRNA was widely expressed, and notably, very high amounts of transcript occurred in skeletal muscle and prostate. In the biceps femoris muscle, UII-like immunoreactivity was shown to coexist with synaptophysin in muscle motor end plate regions. Altogether these results suggest that (i) UII and URP may have many redundant biological effects, especially at the neuromuscular junction; (ii) URP may more specifically participate to autonomic, cardiovascular and reproductive functions.

Distribution of 26RFa binding sites and GPR103 mRNA in the central nervous system of the rat.

The novel RFamide peptide 26RFa, the endogenous ligand of the orphan receptor GPR103, affects food intake, locomotion, and activity of the gonadotropic axis. However, little is known regarding the localization of 26RFa receptors. The present report provides the first detailed mapping of 26RFa binding sites and GPR103 mRNA in the rat central nervous system (CNS). 26RFa binding sites were widely distributed in the brain and spinal cord, whereas the expression of GPR103 mRNA was more discrete, notably in the midbrain, the pons, and the medulla oblongata, suggesting that 26RFa can bind to a receptor(s) other than GPR103. Competition experiments confirmed that 26RFa interacts with an RFamide peptide receptor distinct from GPR103 that may be NPFF2. High densities of 26RFa binding sites were observed in olfactory, hypothalamic, and brainstem nuclei involved in the control of feeding behavior, including the piriform cortex, the ventromedial and dorsomedial hypothalamic nuclei, the paraventricular nucleus, the arcuate nucleus, the lateral hypothalamic area, and the nucleus of the solitary tract. The preoptic and anterior hypothalamic areas were also enriched with 26RFa recognition sites, supporting a physiological role of the neuropeptide in the regulation of the gonadotropic axis. A high density of 26RFa binding sites was detected in regions of the CNS involved in the processing of pain, such as the dorsal horn of the spinal cord and the parafascicular thalamic nucleus. The wide distribution of 26RFa binding sites suggests that 26RFa has multiple functions in the CNS that are mediated by at least two distinct receptors.

Anatomical distribution and biochemical characterization of the novel RFamide peptide 26RFa in the human hypothalamus and spinal cord.

26RFa is a novel RFamide peptide originally isolated in the amphibian brain. The 26RFa precursor has been subsequently characterized in various mammalian species but, until now, the anatomical distribution and the molecular forms of 26RFa produced in the CNS of mammals, in particular in human, are unknown. In the present study, we have investigated the localization and the biochemical characteristics of 26RFa-like immunoreactivity (LI) in two regions of the human CNS--the hypothalamus and the spinal cord. Immunohistochemical labeling using specific antibodies against human 26RFa and in situ hybridization histochemistry revealed that in the human hypothalamus 26RFa-expressing neurons are located in the paraventricular and ventromedial nuclei. In the spinal cord, 26RFa-expressing neurons were observed in the dorsal and lateral horns. Characterization of 26RFa-related peptides showed that two distinct molecular forms of 26RFa are present in the human hypothalamus and spinal cord, i.e. 26RFa and an N-terminally elongated form of 43 amino acids designated 43RFa. These data provide the first evidence that 26RFa and 43RFa are actually produced in the human CNS. The distribution of 26RF-LI suggests that 26RFa and/or 43RFa may modulate feeding, sexual behavior and transmission of nociceptive stimuli.