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.

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.

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.

The intrinsically disordered distal face of nucleoplasmin recognizes distinct oligomerization states of histones.

The role of Nucleoplasmin (NP) as a H2A-H2B histone chaperone has been extensively characterized. To understand its putative interaction with other histone ligands, we have characterized its ability to bind H3-H4 and histone octamers. We find that the chaperone forms distinct complexes with histones, which differ in the number of molecules that build the assembly and in their spatial distribution. When complexed with H3-H4 tetramers or histone octamers, two NP pentamers form an ellipsoidal particle with the histones located at the center of the assembly, in stark contrast with the NP/H2A-H2B complex that contains up to five histone dimers bound to one chaperone pentamer. This particular assembly relies on the ability of H3-H4 to form tetramers either in solution or as part of the octamer, and it is not observed when a variant of H3 (H3C110E), unable to form stable tetramers, is used instead of the wild-type protein. Our data also suggest that the distal face of the chaperone is involved in the interaction with distinct types of histones, as supported by electron microscopy analysis of the different NP/histone complexes. The use of the same structural region to accommodate all type of histones could favor histone exchange and nucleosome dynamics.

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.

Surfing transcriptomic landscapes. A step beyond the annotation of chromosome 16 proteome.

The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC-MS/MS and gel/LC-MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study.

Application of label-free shotgun nUPLC-MS(E) and 2-DE approaches in the study of Botrytis cinerea mycelium.

The phytopathogenic fungus Botrytis cinerea infects more than different 200 plant species and causes substantial losses in numerous crops. The B05.10 and T4 wild-type strain genomes have been recently sequenced, becoming a model system for necrotrophic pathogens, as well as opening up new alternatives in functional genomics, such as proteomics. We analyzed B. cinerea mycelium from these two wild-type strains, introducing label-free shotgun nUPLC-MS(E) methodology to complement the 2-DE-MS-based approach. We assessed the label-free nUPLC-MS(E) methodology for protein identification and quantification using five mycelium protein dilutions. A total of 225 and 170 protein species were identified by nUPLC-MS(E) in the B05.10 and T4 strains, respectively. Moreover, 129 protein species were quantified in both strains. Significant differences in protein abundance were found in 15 more abundant and 16 less abundant protein species in the B05.10 strain compared to the T4 strain. Twenty-nine qualitative and 15 significant quantitative differences were found using 2-DE. The label-free nUPLC-MS(E) was a reliable, reproducible and sensitive method for protein identification and quantification to study the B. cinerea mycelial proteome. Results obtained by gel-based and gel-free complementary approaches allow a deeper characterization of this fungus, as well as the identification of potential virulence factors.

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.