Publications by authors named "Eduard Sabido"

80 Publications

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

Int J Mol Sci 2021 May 3;22(9). Epub 2021 May 3.

Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), 48940 Leioa, Spain.

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 Ub 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.
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http://dx.doi.org/10.3390/ijms22094851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8124392PMC
May 2021

Quality standards in proteomics research facilities: Common standards and quality procedures are essential for proteomics facilities and their users.

EMBO Rep 2021 06 19;22(6):e52626. Epub 2021 May 19.

Centre de Regulació Genòmica, Barcelona Institute of Science and Technology (BIST), Barcelona, Spain.

Proteomics research infrastructures and core facilities within the Core for Life alliance advocate for community policies for quality control to ensure high standards in proteomics services.
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http://dx.doi.org/10.15252/embr.202152626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8183401PMC
June 2021

Proteomic Studies on the Management of High-Grade Serous Ovarian Cancer Patients: A Mini-Review.

Cancers (Basel) 2021 Apr 25;13(9). Epub 2021 Apr 25.

Centre de Regulació Genòmica, Barcelona Institute of Science and Technology (BIST), Dr Aiguader 88, 08003 Barcelona, Spain.

High-grade serous ovarian cancer (HGSC) remains the most common and deadly subtype of ovarian cancer. It is characterized by its late diagnosis and frequent relapse despite standardized treatment with cytoreductive surgery and platinum-based chemotherapy. The past decade has seen significant advances in the clinical management and molecular understanding of HGSC following the publication of the Cancer Genome Atlas (TCGA) researchers and the introduction of targeted therapies with anti-angiogenic drugs and poly(ADP-ribose) polymerase inhibitors in specific subgroups of patients. We provide a comprehensive review of HGSC, focusing on the most important molecular advances aimed at providing a better understanding of the disease and its response to treatment. We emphasize the role that proteomic technologies are now playing in these two aspects of the disease, through the identification of proteins and their post-translational modifications in ovarian cancer tumors. Finally, we highlight how the integration of proteomics with genomics, exemplified by the work performed by the Clinical Proteomic Tumor Analysis Consortium (CPTAC), can guide the development of new biomarkers and therapeutic targets.
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http://dx.doi.org/10.3390/cancers13092067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123279PMC
April 2021

Covalent Histone Modification by an Electrophilic Derivative of the Anti-HIV Drug Nevirapine.

Molecules 2021 Mar 3;26(5). Epub 2021 Mar 3.

Centro de Química Estrutural (CQE), Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisbon, Portugal.

Nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor widely used in combined antiretroviral therapy and to prevent mother-to-child transmission of the human immunodeficiency virus type 1, is associated with several adverse side effects. Using 12-mesyloxy-nevirapine, a model electrophile of the reactive metabolites derived from the NVP Phase I metabolite, 12-hydroxy-NVP, we demonstrate that the nucleophilic core and -terminal residues of histones are targets for covalent adduct formation. We identified multiple NVP-modification sites at lysine (e.g., H2BK47, H4K32), histidine (e.g., H2BH110, H4H76), and serine (e.g., H2BS33) residues of the four histones using a mass spectrometry-based bottom-up proteomic analysis. In particular, H2BK47, H2BH110, H2AH83, and H4H76 were found to be potential hot spots for NVP incorporation. Notably, a remarkable selectivity to the imidazole ring of histidine was observed, with modification by NVP detected in three out of the 11 histidine residues of histones. This suggests that NVP-modified histidine residues of histones are prospective markers of the drug's bioactivation and/or toxicity. Importantly, NVP-derived modifications were identified at sites known to determine chromatin structure (e.g., H4H76) or that can undergo multiple types of post-translational modifications (e.g., H2BK47, H4H76). These results open new insights into the molecular mechanisms of drug-induced adverse reactions.
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http://dx.doi.org/10.3390/molecules26051349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961589PMC
March 2021

In Utero Exposure to Mercury Is Associated With Increased Susceptibility to Liver Injury and Inflammation in Childhood.

Hepatology 2021 Mar 17. Epub 2021 Mar 17.

Mailman School of Public Health, Columbia University, New York, NY.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent cause of liver disease in children. Mercury (Hg), a ubiquitous toxic metal, has been proposed as an environmental factor contributing to toxicant-associated fatty liver disease. We investigated the effect of prenatal exposure to Hg on childhood liver injury by combining epidemiological results from a multicenter mother-child cohort with complementary in vitro experiments on monocyte cells that are known to play a key role in liver immune homeostasis and NAFLD. We used data from 872 mothers and their children (median age, 8.1 years; interquartile range [IQR], 6.5-8.7) from the European Human Early-Life Exposome (HELIX) cohort. We measured Hg concentration in maternal blood during pregnancy (median, 2.0 μg/L; IQR, 1.1-3.6). We also assessed serum levels of alanine aminotransferase (ALT), a common screening tool for pediatric NAFLD, and plasma concentrations of inflammation-related cytokines in children. We found that prenatal Hg exposure was associated with a phenotype in children that was characterized by elevated ALT (≥22.1 U/L for females and ≥25.8 U/L for males) and increased concentrations of circulating interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor α (TNF-α). Consistently, inflammatory monocytes exposed in vitro to a physiologically relevant dose of Hg demonstrated significant up-regulation of genes encoding these four cytokines and increased concentrations of IL-8 and TNF-α in the supernatants. CONCLUSION: These findings suggest that developmental exposure to Hg can contribute to inflammation and increased NAFLD risk in early life.
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http://dx.doi.org/10.1002/hep.31809DOI Listing
March 2021

QCloud2: An Improved Cloud-based Quality-Control System for Mass-Spectrometry-based Proteomics Laboratories.

J Proteome Res 2021 04 16;20(4):2010-2013. Epub 2021 Mar 16.

Centre de Regulació Genòmica (CRG), Barcelona Institute of Science and Technology (BIST), Dr. Aiguader 88, Barcelona 08003, Spain.

QCloud is a cloud-based system to support proteomics laboratories in daily quality assessment using a user-friendly interface, easy setup, and automated data processing. Since its release, QCloud has facilitated automated quality control for proteomics experiments in many laboratories. QCloud provides a quick and effortless evaluation of instrument performance that helps to overcome many analytical challenges derived from clinical and translational research. Here we present an improved version of the system, QCloud2. This new version includes enhancements in the scalability and reproducibility of the quality-control pipelines, and it features an improved front end for data visualization, user management, and chart annotation. The QCloud2 system also includes programmatic access and a standalone local version.
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http://dx.doi.org/10.1021/acs.jproteome.0c00853DOI Listing
April 2021

Ecto-GPR37: a potential biomarker for Parkinson's disease.

Transl Neurodegener 2021 Feb 26;10(1). Epub 2021 Feb 26.

Pharmacology Unit, Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, L'Hospitalet de Llobregat, Spain.

Objective: α-Synuclein has been studied as a potential biomarker for Parkinson's disease (PD) with no concluding results. Accordingly, there is an urgent need to find out reliable specific biomarkers for PD. GPR37 is an orphan G protein-coupled receptor that toxically accumulates in autosomal recessive juvenile parkinsonism. Here, we investigated whether GPR37 is upregulated in sporadic PD, and thus a suitable potential biomarker for PD.

Methods: GPR37 protein density and mRNA expression in postmortem substantia nigra (SN) from PD patients were analysed by immunoblot and RT-qPCR, respectively. The presence of peptides from the N-terminus-cleaved domain of GPR37 (i.e. ecto-GPR37) in human cerebrospinal fluid (CSF) was determined by liquid chromatography-mass spectrometric analysis. An engineered in-house nanoluciferase-based immunoassay was used to quantify ecto-GPR37 in CSF samples from neurological control (NC) subjects, PD patients and Alzheimer's disease (AD) patients.

Results: GPR37 protein density and mRNA expression were significantly augmented in sporadic PD. Increased amounts of ecto-GPR37 peptides in the CSF samples from PD patients were identified by mass spectrometry and quantified by the in-house ELISA method. However, the CSF total α-synuclein level in PD patients did not differ from that in NC subjects. Similarly, the cortical GPR37 mRNA expression and CSF ecto-GPR37 levels in AD patients were also unaltered.

Conclusion: GPR37 expression is increased in SN of sporadic PD patients. The ecto-GPR37 peptides are significantly increased in the CSF of PD patients, but not in AD patients. These results open perspectives and encourage further clinical studies to confirm the validity and utility of ecto-GPR37 as a potential PD biomarker.
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http://dx.doi.org/10.1186/s40035-021-00232-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908677PMC
February 2021

CSF SERPINA3 Levels Are Elevated in Patients With Progressive MS.

Neurol Neuroimmunol Neuroinflamm 2021 03 12;8(2). Epub 2021 Jan 12.

From the Servei de Neurologia-Neuroimmunologia (N.F., C.M.-B., C.C., R.P., V.B., X.M., M.C.L.), Centre d'Esclerosi Múltiple de Catalunya (Cemcat), Institut de Recerca Vall d'Hebron (VHIR), Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Spain; Biotech Research and Innovation Centre (BRIC) (M.O., S.I.-N.), University of Copenhagen, Denmark; Statistics and Bioinformatics Unit (B.M., A.S.), Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain; Genetics, Microbiology and Statistics Department (A.S.), Universitat de Barcelona, Spain; Department of Neurology (I.D.), University of Belgrade School of Medicine, Serbia; Department of Neurology (I.D.), University of North Carolina School of Medicine, Chapel Hill; Department of Neurology (M.V., M.K.), Medical University of Graz, Austria; Proteomics Unit (E.B., E.S.), Centre de Regulació Genòmica (CRG), Barcelona Institute of Science and Technology (BIST), Spain; Proteomics Unit (E.B., E.S.), Universitat Pompeu Fabra, Barcelona, Spain; and Center for Multiple Sclerosis (X.M.), St. Michael's Hospital, University of Toronto, ON, Canada.

Objective: To identify biomarkers associated with progressive phases of MS and with neuroprotective potential.

Methods: Combined analysis of the transcriptional and proteomic profiles obtained in CNS tissue during chronic progressive phases of experimental autoimmune encephalomyelitis (EAE) with the transcriptional profile obtained during the differentiation of murine neural stem cells into neurons. Candidate biomarkers were measured by ELISA in the CSF of 65 patients with MS (29 with relapsing-remitting MS [RRMS], 20 with secondary progressive MS, and 16 with primary progressive MS [PPMS]) and 30 noninflammatory neurologic controls (NINCs).

Results: Integrative analysis of gene and protein expression data identified 2 biomarkers, the serine protease inhibitor Serpina3n and the calcium-binding protein S100A4, which were upregulated in chronic progressive EAE and whose expression was induced during neuronal differentiation. Immunofluorescence studies revealed a primarily neuronal expression of S100A4 and Serpina3n during EAE. CSF levels of SERPINA3, the human ortholog of murine Serpina3n, and S100A4 were increased in patients with MS compared with NINCs (SERPINA3: 1,320 vs 838.6 ng/mL, = 0.0001; S100A4: 1.6 vs 0.8 ng/mL, = 0.02). Within the MS group, CSF SERPINA3 levels were significantly elevated in patients with progressive forms, mainly patients with PPMS compared with patients with RRMS (1,617 vs 1,129 ng/mL, = 0.02) and NINCs (1,617 vs 838.6 ng/mL, = 0.0001). Of interest, CSF SERPINA3 levels significantly correlated with CSF neurofilament light chain levels only in the PPMS group (r = 0.62, = 0.01).

Conclusion: These results point to a role of SERPINA3 as a biomarker associated with the progressive forms of MS, particularly PPMS.
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http://dx.doi.org/10.1212/NXI.0000000000000941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105904PMC
March 2021

Editorial: Proteomics as a Tool for Biomarker and Drug Target Discovery: Improving the Diagnosis and Treatment of Neurodegenerative Diseases.

Front Aging Neurosci 2020 26;12:232. Epub 2020 Nov 26.

IRMB, University of Montpellier, INSERM, CHU Montpellier, (LBPC-PPC), Montpellier, France.

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http://dx.doi.org/10.3389/fnagi.2020.00232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726015PMC
November 2020

Novel Protein Biomarkers of Monoamine Metabolism Defects Correlate with Disease Severity.

Mov Disord 2021 03 5;36(3):690-703. Epub 2020 Nov 5.

Synaptic Metabolism Laboratory, Sant Joan de Déu Foundation, Research Pediatric Institute (IPR), Sant Joan de Déu Hospital, Barcelona, Spain.

Background: Genetic defects of monoamine neurotransmitters are rare neurological diseases amenable to treatment with variable response. They are major causes of early parkinsonism and other spectrum of movement disorders including dopa-responsive dystonia.

Objectives: The objective of this study was to conduct proteomic studies in cerebrospinal fluid (CSF) samples of patients with monoamine defects to detect biomarkers involved in pathophysiology, clinical phenotypes, and treatment response.

Methods: A total of 90 patients from diverse centers of the International Working Group on Neurotransmitter Related Disorders were included in the study (37 untreated before CSF collection, 48 treated and 5 unknown at the collection time). Clinical and molecular metadata were related to the protein abundances in the CSF.

Results: Concentrations of 4 proteins were significantly altered, detected by mass spectrometry, and confirmed by immunoassays. First, decreased levels of apolipoprotein D were found in severe cases of aromatic L-amino acid decarboxylase deficiency. Second, low levels of apolipoprotein H were observed in patients with the severe phenotype of tyrosine hydroxylase deficiency, whereas increased concentrations of oligodendrocyte myelin glycoprotein were found in the same subset of patients with tyrosine hydroxylase deficiency. Third, decreased levels of collagen6A3 were observed in treated patients with tetrahydrobiopterin deficiency.

Conclusion: This study with the largest cohort of patients with monoamine defects studied so far reports the proteomic characterization of CSF and identifies 4 novel biomarkers that bring new insights into the consequences of early dopaminergic deprivation in the developing brain. They open new possibilities to understand their role in the pathophysiology of these disorders, and they may serve as potential predictors of disease severity and therapies. © 2020 International Parkinson and Movement Disorder Society.
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http://dx.doi.org/10.1002/mds.28362DOI Listing
March 2021

Chromatin-Bound Proteome Profiling by Genome Capture.

STAR Protoc 2020 Jun 3;1(1):100014. Epub 2020 Jun 3.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain.

identification of chromatin interactors can reveal unexpected pathways relevant to physiology and human disease. Inspired by the DNA mediated chromatin pull-down (Dm-ChP) technology (also known as iPOND [isolation of proteins on nascent DNA]) for the proteomic characterization of nascent DNA, we have recently reported a new experimental protocol that allows for the identification of proteins on total DNA (iPOTD) for bulk chromatome profiling and identification of chromatin-bound proteins. Here, we detail a step-by-step protocol to survey the cellular chromatin-bound proteome in a simple, robust, and unbiased manner. For complete details on the use and execution of this protocol, please refer to Aranda et al. (2019).
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http://dx.doi.org/10.1016/j.xpro.2020.100014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580076PMC
June 2020

Genome-wide postnatal changes in immunity following fetal inflammatory response.

FEBS J 2021 Apr 24;288(7):2311-2331. Epub 2020 Oct 24.

Department of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF), Barcelona, Spain.

The fetal inflammatory response (FIR) increases the risk of perinatal brain injury, particularly in extremely low gestational age newborns (ELGANs, < 28 weeks of gestation). One of the mechanisms contributing to such a risk is a postnatal intermittent or sustained systemic inflammation (ISSI) following FIR. The link between prenatal and postnatal systemic inflammation is supported by the presence of well-established inflammatory biomarkers in the umbilical cord and peripheral blood. However, the extent of molecular changes contributing to this association is unknown. Using RNA sequencing and mass spectrometry proteomics, we profiled the transcriptome and proteome of archived neonatal dried blood spot (DBS) specimens from 21 ELGANs. Comparing FIR-affected and unaffected ELGANs, we identified 782 gene and 27 protein expression changes of 50% magnitude or more, and an experiment-wide significance level below 5% false discovery rate. These expression changes confirm the robust postnatal activation of the innate immune system in FIR-affected ELGANs and reveal for the first time an impairment of their adaptive immunity. In turn, the altered pathways provide clues about the molecular mechanisms triggering ISSI after FIR, and the onset of perinatal brain injury. DATABASES: EGAS00001003635 (EGA); PXD011626 (PRIDE).
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http://dx.doi.org/10.1111/febs.15578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049052PMC
April 2021

MassIVE.quant: a community resource of quantitative mass spectrometry-based proteomics datasets.

Nat Methods 2020 10 14;17(10):981-984. Epub 2020 Sep 14.

Khoury College of Computer Sciences, Northeastern University, Boston, MA, USA.

MassIVE.quant is a repository infrastructure and data resource for reproducible quantitative mass spectrometry-based proteomics, which is compatible with all mass spectrometry data acquisition types and computational analysis tools. A branch structure enables MassIVE.quant to systematically store raw experimental data, metadata of the experimental design, scripts of the quantitative analysis workflow, intermediate input and output files, as well as alternative reanalyses of the same dataset.
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http://dx.doi.org/10.1038/s41592-020-0955-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541731PMC
October 2020

SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1.

Sci Adv 2020 Jul 24;6(30):eaaz2590. Epub 2020 Jul 24.

Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916 Badalona, Barcelona, Catalonia, Spain.

Sirtuins are key players of metabolic stress response. Originally described as deacetylases, some sirtuins also exhibit poorly understood mono-adenosine 5'-diphosphate (ADP)-ribosyltransferase (mADPRT) activity. We report that the deacetylase SirT7 is a dual sirtuin, as it also features auto-mADPRT activity. SirT7 mADPRT occurs at a previously undefined active site, and its abrogation alters SirT7 chromatin distribution. We identify an epigenetic pathway by which ADP-ribosyl-SirT7 is recognized by the ADP-ribose reader mH2A1.1 under glucose starvation, inducing SirT7 relocalization to intergenic regions. SirT7 promotes mH2A1 enrichment in a subset of nearby genes, many of them involved in second messenger signaling, resulting in their specific up- or down-regulation. The expression profile of these genes under calorie restriction is consistently abrogated in SirT7-deficient mice, resulting in impaired activation of autophagy. Our work provides a novel perspective on sirtuin duality and suggests a role for SirT7/mH2A1.1 axis in glucose homeostasis and aging.
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http://dx.doi.org/10.1126/sciadv.aaz2590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439345PMC
July 2020

In utero and childhood exposure to tobacco smoke and multi-layer molecular signatures in children.

BMC Med 2020 08 19;18(1):243. Epub 2020 Aug 19.

ISGlobal, Barcelona, Spain.

Background: The adverse health effects of early life exposure to tobacco smoking have been widely reported. In spite of this, the underlying molecular mechanisms of in utero and postnatal exposure to tobacco smoke are only partially understood. Here, we aimed to identify multi-layer molecular signatures associated with exposure to tobacco smoke in these two exposure windows.

Methods: We investigated the associations of maternal smoking during pregnancy and childhood secondhand smoke (SHS) exposure with molecular features measured in 1203 European children (mean age 8.1 years) from the Human Early Life Exposome (HELIX) project. Molecular features, covering 4 layers, included blood DNA methylation and gene and miRNA transcription, plasma proteins, and sera and urinary metabolites.

Results: Maternal smoking during pregnancy was associated with DNA methylation changes at 18 loci in child blood. DNA methylation at 5 of these loci was related to expression of the nearby genes. However, the expression of these genes themselves was only weakly associated with maternal smoking. Conversely, childhood SHS was not associated with blood DNA methylation or transcription patterns, but with reduced levels of several serum metabolites and with increased plasma PAI1 (plasminogen activator inhibitor-1), a protein that inhibits fibrinolysis. Some of the in utero and childhood smoking-related molecular marks showed dose-response trends, with stronger effects with higher dose or longer duration of the exposure.

Conclusion: In this first study covering multi-layer molecular features, pregnancy and childhood exposure to tobacco smoke were associated with distinct molecular phenotypes in children. The persistent and dose-dependent changes in the methylome make CpGs good candidates to develop biomarkers of past exposure. Moreover, compared to methylation, the weak association of maternal smoking in pregnancy with gene expression suggests different reversal rates and a methylation-based memory to past exposures. Finally, certain metabolites and protein markers evidenced potential early biological effects of postnatal SHS, such as fibrinolysis.
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http://dx.doi.org/10.1186/s12916-020-01686-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437049PMC
August 2020

Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence.

Nat Commun 2020 06 2;11(1):2761. Epub 2020 Jun 2.

ISGlobal, Hospital Clínic - Universitat de Barcelona, Barcelona, 08036, Spain.

Plasmodium vivax is the most widely distributed human malaria parasite. Previous studies have shown that circulating microparticles during P. vivax acute attacks are indirectly associated with severity. Extracellular vesicles (EVs) are therefore major components of circulating plasma holding insights into pathological processes. Here, we demonstrate that plasma-derived EVs from Plasmodium vivax patients (PvEVs) are preferentially uptaken by human spleen fibroblasts (hSFs) as compared to the uptake of EVs from healthy individuals. Moreover, this uptake induces specific upregulation of ICAM-1 associated with the translocation of NF-kB to the nucleus. After this uptake, P. vivax-infected reticulocytes obtained from patients show specific adhesion properties to hSFs, reversed by inhibiting NF-kB translocation to the nucleus. Together, these data provide physiological EV-based insights into the mechanisms of human malaria pathology and support the existence of P. vivax-adherent parasite subpopulations in the microvasculature of the human spleen.
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http://dx.doi.org/10.1038/s41467-020-16337-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265481PMC
June 2020

The dental proteome of Homo antecessor.

Nature 2020 04 1;580(7802):235-238. Epub 2020 Apr 1.

Evolutionary Genomics Section, Globe Institute, University of Copenhagen, Copenhagen, Denmark.

The phylogenetic relationships between hominins of the Early Pleistocene epoch in Eurasia, such as Homo antecessor, and hominins that appear later in the fossil record during the Middle Pleistocene epoch, such as Homo sapiens, are highly debated. For the oldest remains, the molecular study of these relationships is hindered by the degradation of ancient DNA. However, recent research has demonstrated that the analysis of ancient proteins can address this challenge. Here we present the dental enamel proteomes of H. antecessor from Atapuerca (Spain) and Homo erectus from Dmanisi (Georgia), two key fossil assemblages that have a central role in models of Pleistocene hominin morphology, dispersal and divergence. We provide evidence that H. antecessor is a close sister lineage to subsequent Middle and Late Pleistocene hominins, including modern humans, Neanderthals and Denisovans. This placement implies that the modern-like face of H. antecessor-that is, similar to that of modern humans-may have a considerably deep ancestry in the genus Homo, and that the cranial morphology of Neanderthals represents a derived form. By recovering AMELY-specific peptide sequences, we also conclude that the H. antecessor molar fragment from Atapuerca that we analysed belonged to a male individual. Finally, these H. antecessor and H. erectus fossils preserve evidence of enamel proteome phosphorylation and proteolytic digestion that occurred in vivo during tooth formation. Our results provide important insights into the evolutionary relationships between H. antecessor and other hominin groups, and pave the way for future studies using enamel proteomes to investigate hominin biology across the existence of the genus Homo.
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http://dx.doi.org/10.1038/s41586-020-2153-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582224PMC
April 2020

DYRK1A Overexpression Alters Cognition and Neural-Related Proteomic Pathways in the Hippocampus That Are Rescued by Green Tea Extract and/or Environmental Enrichment.

Front Mol Neurosci 2019 15;12:272. Epub 2019 Nov 15.

Systems Biology Program, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain.

Down syndrome (DS), caused by trisomy of chromosome 21, is the most common genetic cause of intellectual disability. We recently discovered that green tea extracts containing epigallocatechin-3-gallate (EGCG) improve cognition in mice transgenic for (TgDyrk1A) and in a trisomic DS mouse model (Ts65Dn). Interestingly, paired with cognitive stimulation, green tea has beneficial pro-cognitive effects in DS individuals. Dual Specificity Tyrosine-Phosphorylation-Regulated Kinase 1A () is a major candidate to explain the cognitive phenotypes of DS, and inhibiting its activity is a promising pro-cognitive therapy. DYRK1A kinase activity can be normalized in the hippocampus of transgenic DYRK1A mice administering green tea extracts, but also submitting the animals to environmental enrichment (EE). However, many other mechanisms could also explain the pro-cognitive effects of green tea extracts and EE. To underpin the overall alterations arising upon DYRK1A overexpression and the molecular processes underneath the pro-cognitive effects, we used quantitative proteomics. We investigated the hippocampal (phospho)proteome in basal conditions and after treatment with a green tea extract containing EGCG and/or EE in TgDyrk1A and control mice. We found that overexpression alters protein and phosphoprotein levels of key postsynaptic and plasticity-related pathways and that these alterations were rescued upon the cognitive enhancer treatments.
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http://dx.doi.org/10.3389/fnmol.2019.00272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873902PMC
November 2019

Molecular signature of cardiogenic shock.

Eur Heart J 2020 10;41(39):3839-3848

Department of Cardiology, Heart Institute, Hospital Universitari Germans Trias i Pujol, Carretera de Canyet s/n 08916, Barcelona, Spain.

The incidence of cardiogenic shock (CS) has increased remarkably over the past decade and remains a challenging condition with mortality rates of ∼50%. Cardiogenic shock encompasses cardiac contractile dysfunction; however, it is also a multiorgan dysfunction syndrome, often complicated by a systemic inflammatory response with severe cellular and metabolic dysregulations. Here, we review the evidence on the biochemical manifestations of CS, elaborating on current gold standard biomarkers and novel candidates from molecular signatures of CS. Glucose and lactate, both identified over a century ago, remain the only clinically used biomarkers in current predictive risk scores. Novel genomic, transcriptomic, and proteomic data are discussed, and a recently reported molecular score derived from unbiased proteomic discovery, the CS4P, which includes liver fatty acid-binding protein, beta-2-microglobulin, fructose-bisphosphate aldolase B, and SerpinG1 is comprehensively described. Recent advances in -omics technologies provide new insight into a more holistic molecular signature of CS. Thus, we need to open new diagnostic and therapeutic avenues if we aim to improve outcomes.
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http://dx.doi.org/10.1093/eurheartj/ehz783DOI Listing
October 2020

IKKα Kinase Regulates the DNA Damage Response and Drives Chemo-resistance in Cancer.

Mol Cell 2019 08 10;75(4):669-682.e5. Epub 2019 Jul 10.

Cancer Research Program, Institut Mar d'Investigacions Mèdiques, CIBERONC, Hospital del Mar, Doctor Aiguader 88, Barcelona 08003, Spain. Electronic address:

Phosphorylated IKKα(p45) is a nuclear active form of the IKKα kinase that is induced by the MAP kinases BRAF and TAK1 and promotes tumor growth independent of canonical NF-κB signaling. Insights into the sources of IKKα(p45) activation and its downstream substrates in the nucleus remain to be defined. Here, we discover that IKKα(p45) is rapidly activated by DNA damage independent of ATM-ATR, but dependent on BRAF-TAK1-p38-MAPK, and is required for robust ATM activation and efficient DNA repair. Abolishing BRAF or IKKα activity attenuates ATM, Chk1, MDC1, Kap1, and 53BP1 phosphorylation, compromises 53BP1 and RIF1 co-recruitment to sites of DNA lesions, and inhibits 53BP1-dependent fusion of dysfunctional telomeres. Furthermore, IKKα or BRAF inhibition synergistically enhances the therapeutic potential of 5-FU and irinotecan to eradicate chemotherapy-resistant metastatic human tumors in vivo. Our results implicate BRAF and IKKα kinases in the DDR and reveal a combination strategy for cancer treatment.
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http://dx.doi.org/10.1016/j.molcel.2019.05.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715775PMC
August 2019

Proteomic study of the membrane components of signalling cascades of Botrytis cinerea controlled by phosphorylation.

Sci Rep 2019 07 8;9(1):9860. Epub 2019 Jul 8.

Andalusian Center for Grape and Grapevine Research (IVAGRO), Microbiology Lab, University of Cadiz, Puerto Real, 11510, Spain.

Protein phosphorylation and membrane proteins play an important role in the infection of plants by phytopathogenic fungi, given their involvement in signal transduction cascades. Botrytis cinerea is a well-studied necrotrophic fungus taken as a model organism in fungal plant pathology, given its broad host range and adverse economic impact. To elucidate relevant events during infection, several proteomics analyses have been performed in B. cinerea, but they cover only 10% of the total proteins predicted in the genome database of this fungus. To increase coverage, we analysed by LC-MS/MS the first-reported overlapped proteome in phytopathogenic fungi, the "phosphomembranome" of B. cinerea, combining the two most important signal transduction subproteomes. Of the 1112 membrane-associated phosphoproteins identified, 64 and 243 were classified as exclusively identified or overexpressed under glucose and deproteinized tomato cell wall conditions, respectively. Seven proteins were found under both conditions, but these presented a specific phosphorylation pattern, so they were considered as exclusively identified or overexpressed proteins. From bioinformatics analysis, those differences in the membrane-associated phosphoproteins composition were associated with various processes, including pyruvate metabolism, unfolded protein response, oxidative stress response, autophagy and cell death. Our results suggest these proteins play a significant role in the B. cinerea pathogenic cycle.
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http://dx.doi.org/10.1038/s41598-019-46270-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614480PMC
July 2019

Protein-based cardiogenic shock patient classifier.

Eur Heart J 2019 08;40(32):2684-2694

Heart Institute, Hospital Universitari Germans Trias i Pujol, c/ Canyet SN, 08916 Badalona, Spain.

Aims: Cardiogenic shock (CS) is associated with high short-term mortality and a precise CS risk stratification could guide interventions to improve patient outcome. Here, we developed a circulating protein-based score to predict short-term mortality risk among patients with CS.

Methods And Results: Mass spectrometry analysis of 2654 proteins was used for screening in the Barcelona discovery cohort (n = 48). Targeted quantitative proteomics analyses (n = 51 proteins) were used in the independent CardShock cohort (n = 97) to derive and cross-validate the protein classifier. The combination of four circulating proteins (Cardiogenic Shock 4 proteins-CS4P), discriminated patients with low and high 90-day risk of mortality. CS4P comprises the abundances of liver-type fatty acid-binding protein, beta-2-microglobulin, fructose-bisphosphate aldolase B, and SerpinG1. Within the CardShock cohort used for internal validation, the C-statistic was 0.78 for the CardShock risk score, 0.83 for the CS4P model, and 0.84 (P = 0.033 vs. CardShock risk score) for the combination of CardShock risk score with the CS4P model. The CardShock risk score with the CS4P model showed a marked benefit in patient reclassification, with a net reclassification improvement (NRI) of 0.49 (P = 0.020) compared with CardShock risk score. Similar reclassification metrics were observed in the IABP-SHOCK II risk score combined with CS4P (NRI =0.57; P = 0.032). The CS4P patient classification power was confirmed by enzyme-linked immunosorbent assay (ELISA).

Conclusion: A new protein-based CS patient classifier, the CS4P, was developed for short-term mortality risk stratification. CS4P improved predictive metrics in combination with contemporary risk scores, which may guide clinicians in selecting patients for advanced therapies.
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http://dx.doi.org/10.1093/eurheartj/ehz294DOI Listing
August 2019

Molecular basis for the protective effects of low-density lipoprotein receptor-related protein 1 (LRP1)-derived peptides against LDL aggregation.

Biochim Biophys Acta Biomembr 2019 07 8;1861(7):1302-1316. Epub 2019 May 8.

Group of Lipids and Cardiovascular Pathology, Biomedical Research Institute Sant Pau (IIB Sant Pau), Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; Institute of Biomedical Research of Barcelona (IIBB), Spanish National Research Council (CSIC), Barcelona, Spain; CIBER Enfermedades Cardiovasculares (CIBERcv), Spain. Electronic address:

Aggregated LDL is the first ligand reported to interact with the cluster II CR9 domain of low-density lipoprotein receptor-related protein 1 (LRP1). In particular, the C-terminal half of domain CR9, comprising the region Gly-Cys exclusively recognizes aggregated LDL and it is crucial for aggregated LDL binding. Our aim was to study the effect of the sequence Gly-Cys (named peptide LP3 and its retro-enantio version, named peptide DP3) on the structural characteristics of sphingomyelinase- (SMase) and phospholipase 2 (PLA)-modified LDL particles. Turbidimetry, gel filtration chromatography (GFC) and transmission electronic microscopy (TEM) analysis showed that LP3 and DP3 peptides strongly inhibited SMase- and PLA-induced LDL aggregation. Nondenaturing polyacrylamide gradient gel electrophoresis (GGE), agarose gel electrophoresis and high-performance thin-layer chromatography (HPTLC) indicated that LP3 and DP3 prevented SMase-induced alterations in LDL particle size, electric charge and phospholipid content, respectively, but not those induced by PLA. Western blot analysis showed that LP3 and DP3 counteracted changes in ApoB-100 conformation induced by the two enzymes. LDL proteomics (LDL trypsin digestion followed by mass spectroscopy) and computational modeling methods evidenced that peptides preserve ApoB-100 conformation due to their electrostatic interactions with a basic region of ApoB-100. These results demonstrate that LRP1-derived peptides are protective against LDL aggregation, even in conditions of extreme lipolysis, through their capacity to bind to ApoB-100 regions critical for ApoB-100 conformational preservation. These results suggests that these LRP1(CR9) derived peptides could be promising tools to prevent LDL aggregation induced by the main proteolytic enzymes acting in the arterial intima.
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http://dx.doi.org/10.1016/j.bbamem.2019.05.003DOI Listing
July 2019

A comprehensive proteomics-based interaction screen that links DYRK1A to RNF169 and to the DNA damage response.

Sci Rep 2019 04 12;9(1):6014. Epub 2019 Apr 12.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), 08003, Barcelona, Spain.

Dysregulation of the DYRK1A protein kinase has been associated with human disease. On the one hand, its overexpression in trisomy 21 has been linked to certain pathological traits of Down syndrome, while on the other, inactivating mutations in just one allele are responsible for a distinct yet rare clinical syndrome, DYRK1A haploinsufficiency. Moreover, altered expression of this kinase may also provoke other human pathologies, including cancer and diabetes. Although a few DYRK1A substrates have been described, its upstream regulators and downstream targets are still poorly understood, an information that could shed light on the functions of DYRK1A in the cell. Here, we carried out a proteomic screen using antibody-based affinity purification coupled to mass spectrometry to identify proteins that directly or indirectly bind to endogenous DYRK1A. We show that the use of a cell line not expressing DYRK1A, generated by CRISPR/Cas9 technology, was needed in order to discriminate between true positives and non-specific interactions. Most of the proteins identified in the screen are novel candidate DYRK1A interactors linked to a variety of activities in the cell. The in-depth characterization of DYRK1A's functional interaction with one of them, the E3 ubiquitin ligase RNF169, revealed a role for this kinase in the DNA damage response. We found that RNF169 is a DYRK1A substrate and we identified several of its phosphorylation sites. In particular, one of these sites appears to modify the ability of RNF169 to displace 53BP1 from sites of DNA damage. Indeed, DYRK1A depletion increases cell sensitivity to ionizing irradiation. Therefore, our unbiased proteomic screen has revealed a novel activity of DYRK1A, expanding the complex role of this kinase in controlling cell homeostasis.
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http://dx.doi.org/10.1038/s41598-019-42445-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461666PMC
April 2019

The cochaperone CHIP marks Hsp70- and Hsp90-bound substrates for degradation through a very flexible mechanism.

Sci Rep 2019 03 25;9(1):5102. Epub 2019 Mar 25.

Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049, Madrid, Spain.

Some molecular chaperones are involved not only in assisting the folding of proteins but also, given appropriate conditions, in their degradation. This is the case for Hsp70 and Hsp90 which, in concert with the cochaperone CHIP, direct their bound substrate to degradation through ubiquitination. We generated complexes between the chaperones (Hsp70 or Hsp90), the cochaperone CHIP and, as substrate, a p53 variant containing the GST protein (p53-TMGST). Both ternary complexes (Hsp70:p53-TMGST:CHIP and Hsp90:p53-TMGST:CHIP) ubiquitinated the substrate at a higher efficiency than in the absence of the chaperones. The 3D structures of the two complexes, obtained using a combination of cryoelectron microscopy and crosslinking mass spectrometry, showed the substrate located between the chaperone and the cochaperone, suggesting a ubiquitination mechanism in which the chaperone-bound substrate is presented to CHIP. These complexes are inherently flexible, which is important for the ubiquitination process.
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http://dx.doi.org/10.1038/s41598-019-41060-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433865PMC
March 2019

Isotopologue Multipoint Calibration for Proteomics Biomarker Quantification in Clinical Practice.

Anal Chem 2019 04 25;91(8):4934-4938. Epub 2019 Mar 25.

Proteomics Unit, Center for Genomics Regulation , Barcelona Institute of Science and Technology (BIST) , 08003 , Barcelona , Spain.

Targeted proteomics has become the method of choice for biomarker validation in human biopsies due to its high sensitivity, reproducibility, accuracy, and precision. However, for targeted proteomics to be transferred to clinical routine there is the need to reduce its complexity, make its procedures simpler, increase its throughput, and improve its analytical performance. Here we present the Isotopologue Multipoint Calibration (ImCal) quantification strategy, which uses a mix of isotopologue peptides to generate internal multipoint calibration curves for each individual sample and to accurately quantify biomarker peptides in clinical applications without the need of expert supervision. ImCal relies on the use of five different isotopically-labelled peptides of different nominal mass mixed at different concentrations to be used as an internal calibration curve for each endogenous peptide. The use of internal multipoint calibration curves is well-suited for the generation of ready-to-use biomarker kits for clinical applications as it is compatible with both high- and low-resolution mass spectrometers and different levels of endogenous peptide, it eliminates the need for blank matrixes required in external curves, it allows the evaluation of matrix effects and the valid quantification range in each individual sample, and it does not require expert adjustment. We used the ImCal method to quantify HER2 in 35 breast cancer formalin-fixed paraffin-embedded patient samples, revealing a high degree of heterogeneity among patients, which contrasts with the homogeneous immunohistochemistry patient classification. Our work illustrates how an improvement of mass spectrometry methods for biomarker quantification can provide fine-grain patient stratification, and thus better disease diagnostic and prognosis.
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http://dx.doi.org/10.1021/acs.analchem.8b05802DOI Listing
April 2019

Chromatin capture links the metabolic enzyme AHCY to stem cell proliferation.

Sci Adv 2019 03 6;5(3):eaav2448. Epub 2019 Mar 6.

Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain.

Profiling the chromatin-bound proteome (chromatome) in a simple, direct, and reliable manner might be key to uncovering the role of yet uncharacterized chromatin factors in physiology and disease. Here, we have designed an experimental strategy to survey the chromatome of proliferating cells by using the DNA-mediated chromatin pull-down (Dm-ChP) technology. Our approach provides a global view of cellular chromatome under normal physiological conditions and enables the identification of chromatin-bound proteins de novo. Integrating Dm-ChP with genomic and functional data, we have discovered an unexpected chromatin function for adenosylhomocysteinase, a major one-carbon pathway metabolic enzyme, in gene activation. Our study reveals a new regulatory axis between the metabolic state of pluripotent cells, ribosomal protein production, and cell division during the early phase of embryo development, in which the metabolic flux of methylation reactions is favored in a local milieu.
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http://dx.doi.org/10.1126/sciadv.aav2448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402848PMC
March 2019

Changes in Synaptic Proteins Precede Neurodegeneration Markers in Preclinical Alzheimer's Disease Cerebrospinal Fluid.

Mol Cell Proteomics 2019 Mar 22;18(3):546-560. Epub 2020 Sep 22.

§Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031 Madrid, Spain;; ¶Biomedical Research Institute Sant Pau (IIB Sant Pau), 08025Barcelona, Spain.

A biomarker of synapse loss, an early event in Alzheimer's disease (AD) pathophysiology that precedes neuronal death and symptom onset, would be a much-needed prognostic biomarker. With direct access to the brain interstitial fluid, the cerebrospinal fluid (CSF) is a potential source of synapse-derived proteins. In this study, we aimed to identify and validate novel CSF biomarkers of synapse loss in AD. Discovery: Combining shotgun proteomics of the CSF with an exhaustive search of the literature and public databases, we identified 251 synaptic proteins, from which we selected 22 for further study. Verification: Twelve proteins were discarded because of poor detection by Selected Reaction Monitoring (SRM). We confirmed the specific expression of 9 of the remaining proteins (Calsyntenin-1, GluR2, GluR4, Neurexin-2A, Neurexin-3A, Neuroligin-2, Syntaxin-1B, Thy-1, Vamp-2) at the human synapse using Array Tomography microscopy and biochemical fractionation methods. Exploration: Using SRM, we monitored these 9 synaptic proteins (20 peptides) in a cohort of CSF from cognitively normal controls and subjects in the pre-clinical and clinical AD stages (n = 80). Compared with controls, peptides from 8 proteins were elevated 1.3 to 1.6-fold (p < 0.04) in prodromal AD patients. Validation: Elevated levels of a GluR4 peptide at the prodromal stage were replicated (1.3-fold, p = 0.04) in an independent cohort (n = 60). Moreover, 7 proteins were reduced at preclinical stage 1 (0.6 to 0.8-fold, p < 0.04), a finding that was replicated (0.7 to 0.8-fold, p < 0.05) for 6 proteins in a third cohort (n = 38). In a cross-cohort meta-analysis, 6 synaptic proteins (Calsyntenin-1, GluR4, Neurexin-2A, Neurexin-3A, Syntaxin-1B and Thy-1) were reduced 0.8-fold (p < 0.05) in preclinical AD, changes that precede clinical symptoms and CSF markers of neurodegeneration. Therefore, these proteins could have clinical value for assessing disease progression, especially in preclinical stages of AD.
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http://dx.doi.org/10.1074/mcp.RA118.001290DOI Listing
March 2019

Unraveling the hidden universe of small proteins in bacterial genomes.

Mol Syst Biol 2019 02 22;15(2):e8290. Epub 2019 Feb 22.

EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain

Identification of small open reading frames (smORFs) encoding small proteins (≤ 100 amino acids; SEPs) is a challenge in the fields of genome annotation and protein discovery. Here, by combining a novel bioinformatics tool (RanSEPs) with "-omics" approaches, we were able to describe 109 bacterial small ORFomes. Predictions were first validated by performing an exhaustive search of SEPs present in proteome via mass spectrometry, which illustrated the limitations of shotgun approaches. Then, RanSEPs predictions were validated and compared with other tools using proteomic datasets from different bacterial species and SEPs from the literature. We found that up to 16 ± 9% of proteins in an organism could be classified as SEPs. Integration of RanSEPs predictions with transcriptomics data showed that some annotated non-coding RNAs could in fact encode for SEPs. A functional study of SEPs highlighted an enrichment in the membrane, translation, metabolism, and nucleotide-binding categories. Additionally, 9.7% of the SEPs included a N-terminus predicted signal peptide. We envision RanSEPs as a tool to unmask the hidden universe of small bacterial proteins.
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http://dx.doi.org/10.15252/msb.20188290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385055PMC
February 2019