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The nuclear receptor PPAR-β/δ (PPARD) has essential roles in fatty acid catabolism and energy homeostasis as well as cell differentiation, inflammation, and metabolism. However, its contributions to tumorigenesis are uncertain and have been disputed. Here, we provide evidence of tumor suppressive activity of PPARD in prostate cancer through a noncanonical and ligand-independent pathway. PPARD was downregulated in prostate cancer specimens. In murine prostate epithelium, PPARD gene deletion resulted in increased cellularity. Genetic modulation of PPARD in human prostate cancer cell lines validated the tumor suppressive activity of this gene in vitro and in vivo. Mechanistically, PPARD exerted its activity in a DNA binding-dependent and ligand-independent manner. We identified a novel set of genes repressed by PPARD that failed to respond to ligand-mediated activation. Among these genes, we observed robust regulation of the secretory trefoil factor family (TFF) members, including a causal and correlative association of TFF1 with prostate cancer biology in vitro and in patient specimens. Overall, our results illuminate the oncosuppressive function of PPARD and understanding of the pathogenic molecular pathways elicited by this nuclear receptor.Significance: These findings challenge the presumption that the function of the nuclear receptor PPARβ/δ in cancer is dictated by ligand-mediated activation. Cancer Res; 78(2); 399–409. ©2017 AACR.

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

The Ocular Comfort Index (OCI) assesses ocular surface irritation and grades the severity of dry eye disease. This study sought to adapt the OCI questionnaire into Spanish, and then to assess the psychometric performance and validity of the new adapted version (OCI-versión española, OCIVE). The questionnaire was translated, back translated, and then cross-culturally adapted for use with Spanish-speaking individuals. The OCIVE was completed by 450 participants, including 53 subjects that were diagnosed with dry eye disease. Through a Rasch analysis, the psychometric properties of item fit, targeting, person separation, reliability, and differential item functioning (DIF) were assessed. To test the convergent validity, we examined the correlation between the OCIVE and the Computer Vision Symptom Scale (CVSS17). Validity was tested in a subgroup of participants with and without dry eye, and test-retest repeatability was determined in a subset of 151 individuals. We also compared, via DIF, the performance of the OCIVE with that of the original OCI. Our Rasch analysis revealed a good model fit, high accuracy, good targeting, unidimensionality, and no DIF according to gender. The validity and repeatability were good. The OCIVE shows comparable psychometric properties to the original English version, making it a valid tool for measuring dry eye symptoms in Spanish adults.

The Spanish Society for Developmental Biology organized its 17th meeting in November 2020. The meeting, organized by CIC bioGUNE, the University of the Basque Country and the University of Cantabria, gathered about 280 registrants and received 132 scientific abstracts. Participants ranged from undergraduate to senior researchers, with a broad participation of Ph.D. students. The meeting was organized in 8 sessions: Growth and Scaling, Self-organization, Neurodevelopment, Genomes, Cell Biology, Development and Disease, Evo-Devo and Regeneration (Araújo et al., 2021). These sessions focused on the new tendencies in Developmental Biology research and, based on the science presented there, we organized this special issue on The 17th Edition of the Spanish Society for Developmental Biology Meeting: New Trends in Developmental Biology. This collection of articles gathers several scientific contributions in this area, featuring collaborative and interdisciplinary approaches among developmental biologists. With the focus on organogenesis and gene regulation, our selected content embraces novel discoveries on muscle development, regeneration and the transcriptional control and role of miRNAs in development, while highlighting advances in organogenesis and gonadal development.

Townes–Brocks syndrome (TBS) is a rare syndrome characterized by triad of anal, ear, and thumb anomalies. Further malformations/anomalies include congenital heart diseases, foot malformations, sensorineural and/or conductive hearing impairment, genitourinary malformations, and anomalies of eye and nervous system. Definitive diagnosis for TBS is confirmed by molecular analysis for mutations in the SALL1 gene. Only one known case of TBS with absent pulmonary valve syndrome (APVS) has been previously described to our knowledge. Here, we report a newborn diagnosed with TBS with APVS and tetralogy of Fallot (TOF) who was found to carry the most common pathogenic SALL1 gene mutation c.826C > T (p.R276X), with its surgical repair and postoperative follow-up. To our knowledge, this is the first genotyped case of TBS from Turkey to date. TBS should be suspected in the presence of ear, anal, and thumb malformations in a neonate. If a patient with TBS and TOF-APVS needs preoperative ventilation within the first months of life, this implies prolonged postoperative intubation and increased risk of mortality.

Townes–Brocks syndrome (TBS) is a rare syndrome characterized by triad of anal, ear, and thumb anomalies. Further malformations/anomalies include congenital heart diseases, foot malformations, sensorineural and/or conductive hearing impairment, genitourinary malformations, and anomalies of eye and nervous system. Definitive diagnosis for TBS is confirmed by molecular analysis for mutations in the SALL1 gene. Only one known case of TBS with absent pulmonary valve syndrome (APVS) has been previously described to our knowledge. Here, we report a newborn diagnosed with TBS with APVS and tetralogy of Fallot (TOF) who was found to carry the most common pathogenic SALL1 gene mutation c.826C > T (p.R276X), with its surgical repair and postoperative follow-up. To our knowledge, this is the first genotyped case of TBS from Turkey to date. TBS should be suspected in the presence of ear, anal, and thumb malformations in a neonate. If a patient with TBS and TOF-APVS needs preoperative ventilation within the first months of life, this implies prolonged postoperative intubation and increased risk of mortality. https://www.thieme-connect.com/products/ejournals/abstract/10.1055/s-0041-1740371

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

The Spanish Society for Developmental Biology (SEBD) organized its 17th meeting in November 2020 (herein referred to as SEBD2020). This meeting, originally programmed to take place in the city of Bilbao, was forced onto an online format due to the SARS-CoV2, COVID-19 pandemic. Although, we missed the live personal interactions and missed out on the Bilbao social scene, we were able to meet online to present our work and discuss our latest results. An overview of the activities that took place around the meeting, the different scientific sessions and the speakers involved are presented here. The pros and cons of virtual meetings are discussed.

Objective Neddylation is a druggable and reversible ubiquitin-like post-translational modification upregulated in many diseases, including liver fibrosis, hepatocellular carcinoma, and more recently, non-alcoholic fatty liver disease (NAFLD). Herein, we propose to address the effects of neddylation inhibition and the underlying mechanisms in pre-clinical models of NAFLD. Methods Hepatic neddylation measured by immunohistochemical analysis and NEDD8 serum levels measured by ELISA assay were evaluated in NAFLD clinical and pre-clinical samples. The effects of neddylation inhibition by using a pharmacological small inhibitor, MLN4924, or molecular approaches were assessed in isolated mouse hepatocytes and pre-clinical mouse models of diet-induced NAFLD, male adult C57BL/6 mice, and the AlfpCre transgenic mice infected with AAV-DIO-shNedd8. Results Neddylation inhibition reduced lipid accumulation in oleic acid-stimulated mouse primary hepatocytes and ameliorated liver steatosis, preventing lipid peroxidation and inflammation in the mouse models of diet-induced NAFLD. Under these conditions, increased Deptor levels and the concomitant repression of mTOR signaling were associated with augmented fatty acid oxidation and reduced lipid content. Moreover, Deptor silencing in isolated mouse hepatocytes abolished the anti-steatotic effects mediated by neddylation inhibition. Finally, serum NEDD8 levels correlated with hepatic neddylation during the disease progression in the clinical and pre-clinical models Conclusions Overall, the upregulation of Deptor, driven by neddylation inhibition, is proposed as a novel effective target and therapeutic approach to tackle NAFLD., Graphical abstract Image 1, Highlights • Hepatic neddylation is induced in clinical and pre-clinical NAFLD. • Neddylation inhibition reduces hepatic steatosis in pre-clinical models of NAFLD. • Neddylation inhibition boosts fatty acid oxidation through a DEPTOR/mTOR axis. • Neddylation inhibition reduces hepatic inflammation and lipid peroxidation in NAFLD. • Serum NEDD8 levels correlate positively with liver disease in patients and pre-clinical models of NAFLD.

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 &ldquo, protein polyubiquitination&rdquo, 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.

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. Keywords: liver fibrosis; ubiquitination; metabolomics; proliferating cell nuclear antigen (PCNA); DNA damage response (DDR)

Primary cilia are sensory organelles crucial for cell signaling during development and organ homeostasis. Cilia arise from centrosomes and their formation and function is governed by numerous factors. Through our studies on Townes-Brocks Syndrome (TBS), a rare disease linked to abnormal cilia formation in human fibroblasts, we uncovered the leucine-zipper protein LUZP1 as an interactor of truncated SALL1, a dominantly-acting protein causing the disease. Using TurboID proximity labeling and pulldowns, we show that LUZP1 associates with factors linked to centrosome and actin filaments. Here, we show that LUZP1 is a cilia regulator. It localizes around the centrioles and to actin cytoskeleton. Loss of LUZP1 reduces F-actin levels, facilitates ciliogenesis and alters Sonic Hedgehog signaling, pointing to a key role in cytoskeleton-cilia interdependency. Truncated SALL1 increases the ubiquitin proteasome-mediated degradation of LUZP1. Together with other factors, alterations in LUZP1 may be contributing to TBS etiology., eLife digest Primary cilia are the ‘antennae’ of animal cells: these small, flexible protrusions emerge from the surface of cells, where they help to sense and relay external signals. Cilia are assembled with the help of the cytoskeleton, a dynamic network of mesh-like filaments that spans the interior of the cell and controls many different biological processes. If cilia do not work properly, human diseases called ciliopathies can emerge. Townes-Brocks Syndrome (TBS) is an incurable disease that presents a range of symptoms such as malformations of the toes or fingers, hearing impairment, and kidney or heart problems. It is caused by a change in the gene that codes for a protein called SALL1, and recent work has also showed that the cells of TBS patients have defective cilia. In addition, this prior research identified a second protein that interacted with the mutant version of SALL1; called LUZP1, this protein is already known to help maintain the cytoskeleton. In this study, Bozal-Basterra et al. wanted to find out if LUZP1 caused the cilia defects in TBS. First, the protein was removed from mouse cells grown in the laboratory, which dramatically weakened the cytoskeleton. In keeping with this observation, both the number of cilia per cell and the length of the cilia were abnormal. Cells lacking LUZP1 also had defects in a signalling process that transmits signals received by cilia to different parts of the cell. All these defects were previously observed in cells isolated from TBS patients. In addition, LUZP1-deficient mouse cells showed the same problems with their cilia and cytoskeleton as the cells from individuals with TBS. Crucially, the cells from human TBS patients also had much lower levels of LUZP1 than normal, suggesting that the protein may contribute to the cilia defects present in this disease. The work by Bozal-Basterra et al. sheds light on how primary cilia depend on the cytoskeleton, while also providing new insight into TBS. In the future, this knowledge could help researchers to develop therapies for this rare and currently untreatable disease.

During the development of multicellular organisms, transcriptional regulation plays an important role in the control of cell growth, differentiation and morphogenesis. SUMOylation is a reversible post-translational process involved in transcriptional regulation through the modification of transcription factors and through chromatin remodelling (either modifying chromatin remodelers or acting as a `molecular glue' by promoting recruitment of chromatin regulators). SUMO modification results in changes in the activity, stability, interactions or localization of its substrates, which affects cellular processes such as cell cycle progression, DNA maintenance and repair or nucleocytoplasmic transport. This review focuses on the role of SUMO machinery and the modification of target proteins during embryonic development and organogenesis of animals, from invertebrates to mammals. We apologize to those whose related publication could not be cited due to space limitations. We are grateful to all members of Barrio's Lab for comments and suggestions. R.B. acknowledges grants BFU2017-84653-P (MINECO/AEI/FEDER/EU), SEV-2016-0644 (Severo Ochoa Excellence Program, MINECO/AEI), 765445-EU (UbiCODE Program, EU), SAF2017-90900-REDT (UBIRed Program, MINECO/AEI).

This study unravels an unprecedented murine model of lethal metastatic prostate cancer, based on the combined deletion of Pten and Lkb1. Importantly, minimal activity of LKB1 is sufficient to hamper prostate cancer cell aggressiveness, thus redefining the relationship between gene dosage and tumor suppression., Gene dosage is a key defining factor to understand cancer pathogenesis and progression, which requires the development of experimental models that aid better deconstruction of the disease. Here, we model an aggressive form of prostate cancer and show the unconventional association of LKB1 dosage to prostate tumorigenesis. Whereas loss of Lkb1 alone in the murine prostate epithelium was inconsequential for tumorigenesis, its combination with an oncogenic insult, illustrated by Pten heterozygosity, elicited lethal metastatic prostate cancer. Despite the low frequency of LKB1 deletion in patients, this event was significantly enriched in lung metastasis. Modeling the role of LKB1 in cellular systems revealed that the residual activity retained in a reported kinase-dead form, LKB1K78I, was sufficient to hamper tumor aggressiveness and metastatic dissemination. Our data suggest that prostate cells can function normally with low activity of LKB1, whereas its complete absence influences prostate cancer pathogenesis and dissemination.

Oncogene addiction postulates that the survival and growth of certain tumor cells is dependent upon the activity of one oncogene, despite their multiple genetic and epigenetic abnormalities. This phenomenon provides a foundation for molecular targeted therapy and a rationale for oncogene-based stratification. We have previously reported that the Promyelocytic Leukemia protein (PML) is upregulated in triple negative breast cancer (TNBC) and it regulates cancer-initiating cell function, thus suggesting that this protein can be therapeutically targeted in combination with PML-based stratification. However, the effects of PML perturbation on the bulk of tumor cells remained poorly understood. Here we demonstrate that TNBC cells are addicted to the expression of this nuclear protein. PML inhibition led to a remarkable growth arrest combined with features of senescence in vitro and in vivo. Mechanistically, the growth arrest and senescence were associated to a decrease in MYC and PIM1 kinase levels, with the subsequent accumulation of CDKN1B (p27), a trigger of senescence. In line with this notion, we found that PML is associated to the promoter regions of MYC and PIM1, consistent with their direct correlation in breast cancer specimens. Altogether, our results provide a feasible explanation for the functional similarities of MYC, PIM1, and PML in TNBC and encourage further study of PML targeting strategies for the treatment of this breast cancer subtype. Aologies to those whose related publications were not cited due to space limitations. LA, AS, MPu, AA-A, and LV-J were supported by the Basque Government of education. IH was supported by the Program "Juan de la Cierva" from MINECO. FC acknowledges the Proteomics Unit at VHIO is a member ProteoRed, PRB3 (Grant IPT17/0019-ISCIII-SGEFI/ERDF. RB acknowledges projects BFU2017-84653-P, Consolider BFU2014-57703-REDC, and SAF2017-90900-REDT (MINECO/FEDER, EU). The work of VT is founded by Fundacion Vasca de Innovacion e Investigacion Sanitarias, BIOEF (BIO15/CA/052), the AECC J.P. Bizkaia, the Basque Department of Health (2016111109) and the MINECO (RTI2018-097267-B-I00 (MCIU/AEI/FEDER, UE)). AA was supported by ERC consolidator (683136) and Swiss Cancer League (KFS4267-08-2017) grant, Dr. Josef Steiner Foundation, Swiss CardOnco-Grant of Alfred and Annemarie von Sick grant, Helmut Horten Foundation, SNSF (310030_176045) and PCUK (RIA15-ST2-018). NM-M was supported by the Spanish Association Against Cancer (AECC), AECC JP Vizcaya and CIBERONC. The work of A. Carracedo is supported by the Basque Department of Industry, Tourism and Trade (Elkartek) and the department of education (IKERTALDE IT1106-16), the BBVA foundation, the MINECO (SAF2016-79381R (FEDER/EU); Severo Ochoa Excellence Accreditation SEV2016-0644; Excellence Networks SAF2016-81975-REDT), European Training Networks Project (H2020-MSCA-ITN-308 2016 721532), the AECC (IDEAS175CARR, GCTRA18006CARR), La Caixa Foundation (HR17-00094), FERO foundation, the AstraZeneca Oncology prize and the European Research Council (Starting Grant 336343, PoC 754627). CIBERONC was co-funded with FEDER funds and funded by ISCIII.

This book, written by members of the European network PROTEOSTASIS, provides an up-to-date review of the research regarding protein homeostasis in health and disease. With new discoveries contributing to the increasing complexity of this topic, the book offers a detailed overview of the pathways regulating protein homeostasis, including autophagy and the ubiquitin protein family. Following a basic introduction, it explains how defects in protein homeostasis contribute to numerous pathologies, including cancer, neurodegeneration, inflammation and a number of rare diseases. In addition, it discusses, the role of protein homeostasis in cellular development and physiology. Highlighting the latest research in the field of protein homeostasis and its implications for various clinically relevant diseases, the book appeals to researchers and clinicians, while also offering a reference guide for scholars who are new to the field.

Cancer cells can develop a strong addiction to discrete molecular regulators, which control the aberrant gene expression programs that drive and maintain the cancer phenotype. Here, we report the identification of the RNA-binding protein HuR/ELAVL1 as a central oncogenic driver for malignant peripheral nerve sheath tumors (MPNSTs), which are highly aggressive sarcomas that originate from cells of the Schwann cell lineage. HuR was found to be highly elevated and bound to a multitude of cancer-associated transcripts in human MPNST samples. Accordingly, genetic and pharmacological inhibition of HuR had potent cytostatic and cytotoxic effects on tumor growth, and strongly suppressed metastatic capacity in vivo. Importantly, we linked the profound tumorigenic function of HuR to its ability to simultaneously regulate multiple essential oncogenic pathways in MPNST cells, including the Wnt/beta-catenin, YAP/TAZ, RB/E2F, and BET pathways, which converge on key transcriptional networks. Given the exceptional dependency of MPNST cells on HuR for survival, proliferation, and dissemination, we propose that HuR represents a promising therapeutic target for MPNST treatment. This work was funded by grants to AW from the Spanish Association Against Cancer (AECC; JP Vizcaya); Spanish Ministry of Science, Innovation and Universities (MCIU)/Agencia Estatal de Investigacion (AEI)/European Regional Development Fund (FEDER), European Union (EU) (Subprograma Ramon y Cajal RYC2010-06901; Proyectos Retos Investigacion RTI2018-097503-B-I00, SAF2015-65360-R; Proyectos Explora Ciencia SAF2015-72416-EXP; Proyectos Europa Excelencia SAF2015-62588-ERC); the BBVA Foundation; Basque Department of Industry, Tourism and Trade (Elkartek) and Education (PI2013-46); Fundacion Vasca de Innovacion e Investigacion Sanitarias EiTB Maratoia (BIO13/CI/015); the Neurofibromatosis Therapeutic Acceleration Program; and the European Research Council (consolidator grant under the EU's Horizon 2020 research and innovation programme; grant agreement 865157). MVR is grateful for the support of a 2017 Leonardo Grant for Researchers and Cultural Creators (BBVA Foundation), Accion Estrategica Ciber Emergentes 2018 (Ciberehd-ISCIII), and Gilead Sciences International Research Scholars Program in Liver Disease. The work of AC is supported by the Basque Department of Industry, Tourism and Trade (Elkartek) and Education (IKERTALDE IT1106-16); the BBVA Foundation; the MCIU/AEI (SAF2016-79381-R [FEDER/EU]; Severo Ochoa Excellence Accreditation SEV-2016-0644; Excellence Networks SAF20168-1975-REDT); the European Training Networks Project (H2020-MSCA-ITN-308 2016 721532); the AECC (IDEAS175CARR, GCTRA18006CARR); La Caixa Foundation (HR17-00094); and the European Research Council (Starting Grant 336343, PoC 754627). MG was supported by the Intramural Research Program of the National Institute on Aging, NIH. MPI is grateful for the support of the Basque Government of Education fellowship. MTC is grateful for the support of "Ayudas para contratos predoctorales para la formacion de doctores" (MCIU/AEI/FEDER, EU). COH acknowledges funding from Brain Tumour Research. RB acknowledges MCIU/AEI/FEDER, EU (BFU2014-52282-P and BFU2017-84653-P). ES and CL are supported by the Carlos III National Health Institute funded by FEDER funds - a way to build Europe (CIBERONC) and the Government of Catalonia (2017SGR496). MLM-C acknowledges support from MCIU/AEI/FEDER, EU (SAF2017-87301-R); Asociacion Espanola Contra El Cancer (AECC); AECC Canceres Raros; BBVA Biomedicina (UMBRELLA); and La CAIXA Foundation (LCF/PR/HP17/52190004). AP was supported by Associazione Italiana Ricerca sul Cancro (AIRC; IG 2018 Id.21548). CIBERehd is funded by the Instituto de Salud Carlos III. We thank MCIU for the Severo Ochoa Excellence Accreditation (SEV-2016-0644).

Some viruses take advantage of conjugation of ubiquitin or ubiquitin-like proteins to enhance their own replication. One example is Ebola virus, which has evolved strategies to utilize these modification pathways to regulate the viral proteins VP40 and VP35 and to counteract the host defenses. Here, we show a novel mechanism by which Ebola virus exploits the ubiquitin and SUMO pathways. Our data reveal that minor matrix protein VP24 of Ebola virus is a bona fide SUMO target. Analysis of a SUMOylation-defective VP24 mutant revealed a reduced ability to block the type I interferon (IFN) pathway and to inhibit IFN-mediated STAT1 nuclear translocation, exhibiting a weaker interaction with karyopherin 5 and significantly diminished stability. Using glutathione S-transferase (GST) pulldown assay, we found that VP24 also interacts with SUMO in a noncovalent manner through a SIM domain. Mutation of the SIM domain in VP24 resulted in a complete inability of the protein to downmodulate the IFN pathway and in the monoubiquitination of the protein. We identified SUMO deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7) as an interactor and a negative modulator of VP24 ubiquitination. Finally, we show that mutation of one ubiquitination site in VP24 potentiates the IFN modulatory activity of the viral protein and its ability to block IFN-mediated STAT1 nuclear translocation, pointing to the ubiquitination of VP24 as a negative modulator of the VP24 activity. Altogether, these results indicate that SUMO interacts with VP24 and promotes its USP7-mediated deubiquitination, playing a key role in the interference with the innate immune response mediated by the viral protein.IMPORTANCE The Ebola virus VP24 protein plays a critical role in escape of the virus from the host innate immune response. Therefore, deciphering the molecular mechanisms modulating VP24 activity may be useful to identify potential targets amenable to therapeutics. Here, we identify the cellular proteins USP7, SUMO, and ubiquitin as novel interactors and regulators of VP24. These interactions may represent novel potential targets to design new antivirals with the ability to modulate Ebola virus replication., Funding at the laboratory of C.R. is provided by Ministry of Science, Innovation and Universities and FEDER (BFU-2017-88880-P). C.R. also acknowledges grants GRC GI-2119 (Xunta de Galicia) and SAF2017-90900-REDT (UBIRed Program). S.V. is a predoctoral fellow funded by Xunta de Galicia. M.B.-M. is a postdoctoral fellow funded by Xunta de Galicia (Consellería de Cultura, Educación e Ordenación Universitaria). A.E.M. is the recipient of a fellowship of the Spanish FPI program. This work was partially funded by the German Center for Infection Research (DZIF TTU 01.702_00 to C.M.-F.). R.B. and J.D.S. acknowledge grants BFU2017-84653-P (MINECO/FEDER, EU), SEV-2016-0644 (Severo Ochoa Excellence Program), 765445-EU (UbiCODE Program), and SAF2017- 90900-REDT (UBIRed Program). C.S.M. acknowledges grant BFU2016-74868-P.

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.

Salp15, a salivary protein of Ixodes ticks, inhibits the activation of naïve CD4 T cells. Treatment with Salp15 results in the inhibition of early signaling events and the production of the autocrine growth factor, interleukin-2. The fate of the CD4 T cells activated in the presence of Salp15 or its long-term effects are, however, unknown. We now show that Salp15 binding to CD4 is persistent and induces a long-lasting immunomodulatory effect. The activity of Salp15 results in sustained diminished cross-antigenic antibody production even after interruption of the treatment with the protein. Transcriptionally, the salivary protein provokes an acute effect that includes known activation markers, such as Il2 or Cd44, and that fades over time. The long-term effects exerted by Salp15 do not involve the induction of either anergy traits nor increased populations of regulatory T cells. Similarly, the treatment with Salp15 does not result in B cell anergy or the generation of myeloid suppressor cells. However, Salp15 induces the increased expression of the ectoenzyme, CD73, in regulatory T cells and increased production of adenosine. Our study provides a profound characterization of the immunomodulatory activity of Salp15 and suggests that its long-term effects are due to the specific regulation of CD73. Introduction, Supported by grants from the Department of Education of the Basque Government (PI2013-49 to JA and PI2012-42 to RB). JA is funded by the European Union (Grant Agreement number 602272). AMA and JLL’s work was supported by the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek Programs), the Innovation Technology Department of Bizkaia and the CIBERehd Network. The work of AC is supported by a Ramón y Cajal award, the Basque Department of Industry, Tourism and Trade (Etortek), ISCIII (PI13/00031), FERO VIII Fellowship, the BBVA foundation, MINECO (SAF2016-79381-R) and the European Research Council Starting Grant (336343). CIBERonc was co-funded with FEDER funds. AC-M was funded by a Juan de la Cierva program award and the European Union MSCA program (CIG 660191). RB was funded by MINECO grants BFU2011-25986 and BFU2014-52282-P and the Consolider Program (BFU2014-57703-REDC). FJB was funded by a MINECO grant (CTQ2014-56966-R). D.B. is funded by a MINECO FPI fellowship. We thank the MINECO for the Severo Ochoa Excellence accreditation (SEV-2016-0644).

Primary cilia are sensory organelles that are crucial for cell signaling during development and organ homeostasis. Cilia arise from the centrosome and their formation is governed by numerous regulatory factors. We show that the leucine-zipper protein LUZP1 localizes to the pericentriolar material and actin cytoskeleton. Using TurboID proximity labeling and pulldowns, LUZP1 associates with factors linked to centrosome and actin filaments. Loss of LUZP1 reduces F-actin levels, facilitating ciliogenesis and altering Sonic Hedgehog signaling, pointing to a key role in the cytoskeleton-cilia interdependency. Moreover, we show that LUZP1 interacts with a truncated form of the transcription factor SALL1 that causes Townes-Brocks Syndrome. TBS is characterized by digit, heart and kidney malformations and is linked in part to defective cilia. Truncated SALL1 increases the ubiquitin proteasome-mediated degradation of LUZP1. Alteration of LUZP1 levels may be a contributing factor to TBS, suggesting possible therapies using modulators of cilia and cytoskeletal function.

Post-translational modifications (PTMs) by ubiquitin (Ub) are versatile, highly dynamic, and involved in nearly all aspects of eukaryote biological function. The reversibility and heterogeneity of Ub chains attached to protein substrates have complicated their isolation, quantification, and characterization. Strategies have emerged to isolate endogenous ubiquitylated targets, including technologies based on the use of Ub-binding peptides, such as tandem-repeated Ub-binding entities (TUBEs). TUBEs allow the identification and characterization of Ub chains, and novel substrates for deubiquitylases (DUBs) and Ub ligases (E3s). Here we review their impact on purification, analysis of pan or chain-selective polyubiquitylated proteins and underline the biological relevance of this information. Together with peptide aptamers and other Ub affinity-based approaches, TUBEs will contribute to unraveling the secrets of the Ub code.

Since its discovery, the high-affinity interaction between biotin and avidin has formed the basis of numerous tools and techniques in molecular biology and biochemistry. The post-translational modifications of cellular proteins via conjugation of ubiquitin (Ub) and ubiquitin-like (UbL) proteins contribute to crucial cellular processes, such as protein homeostasis and the DNA damage response, yet they are challenging to study due to their dynamic nature, scarcity, and sensitivity to removal by proteases. Understanding how the Ub/UbL-modified proteome changes during development or in response to environmental insults or pathological conditions may yield new biomarkers or identify new drug targets. The use of biotin-based technologies for Ub/UbL studies is relatively new but has already contributed to the identification of new substrates and promises much more. In this review, we focus on two separate approaches: biotin-tagged Ub/UbLs to modify and capture target proteins in vivo, and BioID, a tool to facilitate the labeling and identification of proximal interactors of Ub/UbL enzymes. Coupled with ongoing advances in proteomics to increase sensitivity in peptide identification, and gene-editing techniques to avoid overexpression artifacts, biotin-based systems promise to reveal new information about the role of Ub/UbL modifications in development and disease.