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.

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.

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.

Phosphoproteomic and Functional Analyses Reveal Sperm-specific Protein Changes Downstream of Kappa Opioid Receptor in Human Spermatozoa.

G-protein coupled receptors (GPCRs) belong to the seven transmembrane receptor superfamily that transduce signals via G proteins in response to external stimuli to initiate different intracellular signaling pathways which culminate in specific cellular responses. The expression of diverse GPCRs at the plasma membrane of human spermatozoa suggests their involvement in the regulation of sperm fertility. However, the signaling events downstream of many GPCRs in spermatozoa remain uncharacterized. Here, we selected the kappa-opioid receptor (KOR) as a study model and applied phosphoproteomic approach based on TMT labeling and LC-MS/MS analyses. Quantitative coverage of more than 5000 proteins with over 3500 phosphorylation sites revealed changes in the phosphorylation levels of sperm-specific proteins involved in the regulation of the sperm fertility in response to a specific agonist of KOR, U50488H. Further functional studies indicate that KOR could be involved in the regulation of sperm fertile capacity by modulation of calcium channels. Our findings suggest that human spermatozoa possess unique features in the molecular mechanisms downstream of GPCRs which could be key regulators of sperm fertility and improved knowledge of these specific processes may contribute to the development of useful biochemical tools for diagnosis and treatment of male infertility.

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.

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.

Cylindromatosis Tumor Suppressor Protein (CYLD) Deubiquitinase is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor.

Cylindromatosis tumor suppressor protein (CYLD) is a deubiquitinase, best known as an essential negative regulator of the NFkB pathway. Previous studies have suggested an involvement of CYLD in epidermal growth factor (EGF)-dependent signal transduction as well, as it was found enriched within the tyrosine-phosphorylated complexes in cells stimulated with the growth factor. EGF receptor (EGFR) signaling participates in central cellular processes and its tight regulation, partly through ubiquitination cascades, is decisive for a balanced cellular homeostasis. Here, using a combination of mass spectrometry-based quantitative proteomic approaches with biochemical and immunofluorescence strategies, we demonstrate the involvement of CYLD in the regulation of the ubiquitination events triggered by EGF. Our data show that CYLD regulates the magnitude of ubiquitination of several major effectors of the EGFR pathway by assisting the recruitment of the ubiquitin ligase Cbl-b to the activated EGFR complex. Notably, CYLD facilitates the interaction of EGFR with Cbl-b through its Tyr15 phosphorylation in response to EGF, which leads to fine-tuning of the receptor's ubiquitination and subsequent degradation. This represents a previously uncharacterized strategy exerted by this deubiquitinase and tumors suppressor for the negative regulation of a tumorigenic signaling pathway.

Differential proteomic analysis of endometrial fluid suggests increased inflammation and impaired glucose metabolism in non-implantative IVF cycles and pinpoints PYGB as a putative implantation marker.

STUDY QUESTION: Is there any difference in the protein composition of the endometrial fluid aspirate (EFA) obtained the day of embryo transfer in in vitro fertilization (IVF) cycles achieving and not achieving pregnancy? SUMMARY ANSWER: Comparative analysis identified a differential protein expression pattern in 'implantative' and 'non-implantative' IVF cycles. WHAT IS KNOWN ALREADY: EFA allows non-invasive characterization of the endometrium, and may contain important information on its receptivity when performing (IVF) cycles. Endometrial side of implantation has usually been studied with endometrial biopsy in an IVF cycle prior to embryo transfer, focusing on 'receptive/non-receptive' endometria and with low-throughput proteomic techniques. STUDY DESIGN, SIZE, DURATION: We have compared the protein expression patterns in EFA from a total of 110 women undergoing IVF, corresponding to 50 implantative and 60 non-implantative IVF cycles. Discovery (38 patients) and Validation (42 patients) sample cohorts were analyzed using a high-throughput differential proteomic approach. Then, the differential expression of glycogen phosphorylase B (PYGB) was validated by western blotting in an additional cohort (30 patients). The study period was 18 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: The population under study consisted of 110 women aged 18-40 years old, undergoing their first or second IVF/ intracytoplasmic sperm injection cycle, with normal uterus and endometrium, and 1-2 good quality embryos, and embryo transfer being performed on Day 3. Endometrial fluid aspiration was performed immediately before the embryo transfer. Samples (80) were initially distributed in two independent cohorts and analyzed by liquid chromatography-mass spectrometry. The first cohort was used for the discovery and the second for the validation of the results. Filter-aided sample preparation was used for the in-solution tryptic digestion of the proteins present in the samples, followed by label-free mass spectrometry analysis. In order to unravel the molecular features of receptivity, the lists of differential proteins were thoroughly analyzed using different bioinformatic tools, including GSEA, IPA and GO analysis. MAIN RESULTS AND THE ROLE OF CHANCE: A false discovery rate-based correction of the t-test P-values was carried out in order to strengthen the reliability of the results. Functional analyses denoted the deregulation of important processes governing receptivity, such as antimicrobial response, cell-cell interaction, immune response and inflammatory signaling, among others. Overall eight proteins were commonly deregulated in both studied datasets and brain form glycogen phosphorylase (PYGB) was selected for confirmatory analysis. LIMITATIONS, REASONS FOR CAUTION: Our results were obtained from patients with normal uterus and endometrium and with good quality embryos, who had fresh Day-3 embryo transfer, in stimulated cycles. Therefore, our observations may not be applicable to poor prognosis cases or non-stimulated cycles. WIDER IMPLICATIONS OF THE FINDINGS: This work provides insights into the molecular features of implantative IVF cycles using non-invasive methods. It reveals that EFA may reflect an increased inflammatory state in non-implantative endometrium. Additionally, it proposes PYGB as a potential biomarker for endometrial receptivity or implantation success. This knowledge opens a new avenue for developing embryo transfer strategies, through the improvement of embryo culture media or modifying endometrial fluid composition to increase pregnancy rates. STUDY FUNDING/COMPETING INTEREST(S): This study was partially funded by a Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). Authors declare no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes.

Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4(+) T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer immunotherapies.

Characterization of Receptor-Associated Protein Complex Assembly in Interleukin (IL)-2- and IL-15-Activated T-Cell Lines.

It remains a paradox that IL-2 and IL-15 can differentially modulate the immune response using the same signaling receptors. We have previously dissected the phosphotyrosine-driven signaling cascades triggered by both cytokines in Kit225 T-cells, unveiling subtle differences that may contribute to their functional dichotomy. In this study, we aimed to decipher the receptor complex assembly in IL-2- and IL-15-activated T-lymphocytes that is highly orchestrated by site-specific phosphorylation events. Comparing the cytokine-induced interactome of the interleukin receptor beta and gamma subunits shared by the two cytokines, we defined the components of the early IL-2 and IL-15 receptor-associated complex discovering novel constituents. Additionally, phosphopeptide-directed analysis allowed us to detect several cytokine-dependent and -independent phosphorylation events within the activated receptor complex including novel phosphorylated sites located in the cytoplasmic region of IL-2 receptor ß subunit (IL-2Rß). We proved that the distinct phosphorylations induced by the cytokines serve for recruiting different types of effectors to the initial receptor/ligand complex. Overall, our study sheds new light into the initial molecular events triggered by IL-2 and IL-15 and constitutes a further step toward a better understanding of the early signaling aspects of the two closely related cytokines in T-lymphocytes.

Guanine nucleotide exchange factor αPIX leads to activation of the Rac 1 GTPase/glycogen phosphorylase pathway in interleukin (IL)-2-stimulated T cells.

Recently, we have reported that the active form of Rac 1 GTPase binds to the glycogen phosphorylase muscle isoform (PYGM) and modulates its enzymatic activity leading to T cell proliferation. In the lymphoid system, Rac 1 and in general other small GTPases of the Rho family participate in the signaling cascades that are activated after engagement of the T cell antigen receptor. However, little is known about the IL-2-dependent Rac 1 activator molecules. For the first time, a signaling pathway leading to the activation of Rac 1/PYGM in response to IL-2-stimulated T cell proliferation is described. More specifically, αPIX, a known guanine nucleotide exchange factor for the small GTPases of the Rho family, preferentially Rac 1, mediates PYGM activation in Kit 225 T cells stimulated with IL-2. Using directed mutagenesis, phosphorylation of αPIX Rho-GEF serines 225 and 488 is required for activation of the Rac 1/PYGM pathway. IL-2-stimulated serine phosphorylation was corroborated in Kit 225 T cells cultures. A parallel pharmacological and genetic approach identified PKCθ as the serine/threonine kinase responsible for αPIX serine phosphorylation. The phosphorylated state of αPIX was required to activate first Rac 1 and subsequently PYGM. These results demonstrate that the IL-2 receptor activation, among other early events, leads to activation of PKCθ. To activate Rac 1 and consequently PYGM, PKCθ phosphorylates αPIX in T cells. The biological significance of this PKCθ/αPIX/Rac 1 GTPase/PYGM signaling pathway seems to be the control of different cellular responses such as migration and proliferation.

Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics.

Common γ-chain family of cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, where IL stands for interleukin) are key regulators of the immune homeostasis that exhibit pleiotropic biological activities and even sometimes redundant roles as a result of the utilization of the same receptor subunit. However, they also exert distinct functions that make each of them to be indispensable. For instance, all family members can act as T-cell growth factors; however, we found that IL-15 but not IL-7 can replace IL-2 to promote and sustain the proliferation of Kit225T cells. In addition to the γ-chain, IL-2 and IL-15 share the ß-chain, which creates the paradox of how they can trigger diverse phenotypes despite signaling through the same receptors. To investigate this paradigm, we combined SILAC with enrichment of tyrosine-phosphorylated proteins and peptides followed by mass spectrometric analysis to quantitatively assess the signaling networks triggered downstream IL-2/IL-2R and IL-15/IL-15R. This study confirmed that the transduction pathways initiated by both cytokines are highly similar and revealed that the main signaling branches, JAK/STAT, RAS/MAPK and PI3K/AKT, were nearly equivalently activated in response to both ILs. Despite that, our study revealed that receptor internalization rates differ in IL-2- and IL-15-treated cells indicating a discrete modulation of cytokine signaling. All MS data have been deposited in the ProteomeXchange with identifier PXD001129 (http://proteomecentral.proteomexchange.org/dataset/PXD001129).

The nuclear protein ALY binds to and modulates the activity of transcription factor E2F2.

E2F transcription factors control the expression of genes involved in a variety of essential cellular processes and consequently their activity needs to be tightly regulated. Protein-protein interactions are thought to be key modulators of E2F activity. To gain insight into the mechanisms that regulate the activity of E2F2, we searched for novel proteins that associate with this transcription factor. We show that the nuclear protein ALY (THO complex 4), originally described as a transcriptional co-activator, associates with DNA-bound E2F2 and represses its transcriptional activity. The capacity of ALY to modulate gene expression was analyzed with expression microarrays by characterizing the transcriptome of E2F2 expressing HEK293T cells in which ALY was either overexpressed or silenced. We show that ALY influences the expression of more than 400 genes, including 98 genes bearing consensus E2F motifs. Thus, ALY emerges as a novel E2F2-interacting protein and a relevant modulator of E2F-responsive gene expression.

E2F2 and CREB cooperatively regulate transcriptional activity of cell cycle genes.

E2F2 is essential for the maintenance of T lymphocyte quiescence. To identify the full set of E2F2 target genes, and to gain further understanding of the role of E2F2 in transcriptional regulation, we have performed ChIP-chip analyses across the genome of lymph node-derived T lymphocytes. Here we show that during quiescence, E2F2 binds the promoters of a large number of genes involved in DNA metabolism and cell cycle regulation, concomitant with their transcriptional silencing. A comparison of ChIP-chip data with expression profiling data on resting E2f2(-)(/)(-) T lymphocytes identified a subset of 51 E2F2-specific target genes, most of which are upregulated on E2F2 loss. Luciferase reporter assays showed a retinoblastoma-independent role for E2F2 in the negative regulation of these target genes. Importantly, we show that the DNA binding activity of the transcription factor CREB contributes to E2F2-mediated repression of Mcm5 and Chk1 promoters. siRNA-mediated CREB knockdown, expression of a dominant negative KCREB mutant or disruption of CREB binding by mutating a CRE motif on Mcm5 promoter, relieved E2F2-mediated transcriptional repression. Taken together, our data uncover a new regulatory mechanism for E2F-mediated transcriptional control, whereby E2F2 and CREB cooperate in the transcriptional repression of a subset of E2F2 target genes.

Improving the mechanical and biological functions of cell sheet constructs: The interplay of human-derived periodontal ligament stem cells, endothelial cells and plasma rich in growth factors.

OBJECTIVE: The aim of this study was to produce and characterize triple-layered cell sheet constructs with varying cell compositions combined or not with the fibrin membrane scaffold obtained by the technology of Plasma Rich in Growth Factors (mPRGF). MATERIALS AND METHODS: Human primary cultures of periodontal ligament stem cells (hPDLSCs) were isolated, and their stemness nature was evaluated. Three types of triple-layered composite constructs were generated, composed solely of hPDLSCs or combined with human umbilical vein endothelial cells (HUVECs), either as a sandwiched endothelial layer or as coculture sheets of both cell phenotypes. These three triple-layered constructs were also manufactured using mPRGF as cell sheets' support. Necrosis, glucose consumption, secretion of extracellular matrix proteins and synthesis of proangiogenic factors were determined. Histological evaluations and proteomic analyses were also performed. RESULTS: The inclusion of HUVECs did not clearly improve the properties of the multilayered constructs and yet hindered their optimal conformation. The presence of mPRGF prevented the shrinkage of cell sheets, stimulated the metabolic activity and increased the matrix synthesis. At the proteome level, mPRGF conferred a dramatic advantage to the hPDLSC constructs in their ability to provide a suitable environment for tissue regeneration by inducing the expression of proteins necessary for bone morphogenesis and cellular proliferation. CONCLUSIONS: hPDLSCs' triple-layer construct onto mPRGF emerges as the optimal structure for its use in regenerative therapeutics. CLINICAL RELEVANCE: These results suggest the suitability of mPRGF as a promising tool to support cell sheet formation by improving their handling and biological functions.

PAnalyzer: a software tool for protein inference in shotgun proteomics.

BACKGROUND: Protein inference from peptide identifications in shotgun proteomics must deal with ambiguities that arise due to the presence of peptides shared between different proteins, which is common in higher eukaryotes. Recently data independent acquisition (DIA) approaches have emerged as an alternative to the traditional data dependent acquisition (DDA) in shotgun proteomics experiments. MSE is the term used to name one of the DIA approaches used in QTOF instruments. MSE data require specialized software to process acquired spectra and to perform peptide and protein identifications. However the software available at the moment does not group the identified proteins in a transparent way by taking into account peptide evidence categories. Furthermore the inspection, comparison and report of the obtained results require tedious manual intervention. Here we report a software tool to address these limitations for MSE data. RESULTS: In this paper we present PAnalyzer, a software tool focused on the protein inference process of shotgun proteomics. Our approach considers all the identified proteins and groups them when necessary indicating their confidence using different evidence categories. PAnalyzer can read protein identification files in the XML output format of the ProteinLynx Global Server (PLGS) software provided by Waters Corporation for their MSE data, and also in the mzIdentML format recently standardized by HUPO-PSI. Multiple files can also be read simultaneously and are considered as technical replicates. Results are saved to CSV, HTML and mzIdentML (in the case of a single mzIdentML input file) files. An MSE analysis of a real sample is presented to compare the results of PAnalyzer and ProteinLynx Global Server. CONCLUSIONS: We present a software tool to deal with the ambiguities that arise in the protein inference process. Key contributions are support for MSE data analysis by ProteinLynx Global Server and technical replicates integration. PAnalyzer is an easy to use multiplatform and free software tool.

The multifunctional role of SPANX-A/D protein subfamily in the promotion of pro-tumoural processes in human melanoma.

Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes encode a protein family (SPANX-A, -B, -C and -D), whose expression is limited to the testis and spermatozoa in normal tissues and various tumour cells. SPANX-A/D proteins have been detected in metastatic melanoma cells, but their contribution to cancer development and the underlying molecular mechanisms of skin tumourigenesis remain unknown. Combining functional and proteomic approaches, the present work describes the presence of SPANX-A/D in primary and metastatic human melanoma cells and how it promotes pro-tumoural processes such as cell proliferation, motility and migration. We provide insights into the molecular features of skin tumourigenesis, describing for the first time a multifunctional role of the SPANX-A/D protein family in nuclear function, energy metabolism and cell survival, considered key hallmarks of cancer. A better comprehension of the SPANX-A/D protein subfamily and its molecular mechanisms will help to describe new aspects of tumour cell biology and develop new therapeutic targets and tumour-directed pharmacological drugs for skin tumours.

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.

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.

Kappa- opioid receptor regulates human sperm functions via SPANX-A/D protein family.

The kappa-opioid receptor (KOR) is involved in the regulation of the fertilizing capacity of human sperm. Recently, a testicular-specific protein family, SPANX-A/D, has also been found to be involved in regulating this process. In order to determine if KOR has a role in the regulation of sperm fertility through the SPANX-A/D protein family, we activated the kappa opioid receptor adding its selective agonist, U50488H to normozoospermic human spermatozoa. Then, we performed immunofluorescence assays and immunoprecipitation experiments followed by LC-MS/MS. According to our results, KOR activation may cause the translocation of SPANX-A/D into the nucleus of human spermatozoa. Phosphoproteomic studies show that KOR does not cause phosphorylation changes in SPANX-A/D residues. However, interactome assays demonstrate that KOR activation provokes changes in SPANX-A/D potential interactors involved in sperm motility, energy metabolism and nuclear processes. Taking these results into account, KOR may regulate human sperm fertility through SPANX-A/D protein family, modifying its subcellular location and interactions. Although further studies are needed, this finding could help us describing the molecular mechanisms underlying sperm fertility as well as developing new strategies for treating infertility.