Data on interleukin (IL)-2- and IL-15-dependent changes in IL-2Rβ and IL-2Rγ complexes

We provide detailed datasets from our analysis of the proteins that associate with IL-2Rbeta and IL-2Rgamma in T-cells stimulated with IL-2 or IL-15 compared with resting T-cells, as identified by SILAC-based quantitative proteomics. We also include quantitative data regarding site-specific phosphorylation events observed both in IL-2Rbeta and IL-2Rgamma. Moreover, we provide results demonstrating the specific protein recruitment capacity of four of those site-specific phosphorylations. The proteomics and phosphoproteomics data described in this article is associated with a research article entitled "Characterization of receptor-associated protein complex assembly in Interleukin (IL)-2- and IL-15-activated T-lymphocytes" (Osinalde et al., 2016 [1]). The mass spectrometry data have been deposited to the ProteomeEXchange Constorium with the identifier PXD002386

How to Inactivate Human Ubiquitin E3 Ligases by Mutation

E3 ubiquitin ligases are the ultimate enzymes involved in the transfer of ubiquitin to substrate proteins, a process that determines the fate of the modified protein. Numerous diseases are caused by defects in the ubiquitin-proteasome machinery, including when the activity of a given E3 ligase is hampered. Thus, inactivation of E3 ligases and the resulting effects at molecular or cellular level have been the focus of many studies during the last few years. For this purpose, site-specific mutation of key residues involved in either protein interaction, substrate recognition or ubiquitin transfer have been reported to successfully inactivate E3 ligases. Nevertheless, it is not always trivial to predict which mutation(s) will block the catalytic activity of a ligase. Here we review over 250 site-specific inactivating mutations that have been carried out in 120 human E3 ubiquitin ligases. We foresee that the information gathered here will be helpful for the design of future experimental strategies.This work was supported by Spanish MINECO (grant SAF2016-76898-P) cofinanced with FEDER funds. JR was funded with a postdoctoral fellowship from the University of the Basque Country (UPV/EHU)

Neuronal Proteomic Analysis Of The Ubiquitinated Substrates Of The Disease-Linked E3 Ligases Parkin And Ube3a

Both Parkin and UBE3A are E3 ubiquitin ligases whose mutations result in severe brain dysfunction. Several of their substrates have been identified using cell culture models in combination with proteasome inhibitors, but not in more physiological settings. We recently developed the (bio)Ub strategy to isolate ubiquitinated proteins in flies and have now identified by mass spectrometry analysis the neuronal proteins differentially ubiquitinated by those ligases. This is an example of how flies can be used to provide biological material in order to reveal steady state substrates of disease causing genes. Collectively our results provide new leads to the possible physiological functions of the activity of those two disease causing E3 ligases. Particularly, in the case of Parkin the novelty of our data originates from the experimental setup, which is not overtly biased by acute mitochondrial depolarisation. In the case of UBE3A, it is the first time that a nonbiased screen for its neuronal substrates has been reported.The authors thank Michael Clague for insightful comments on an early version of the manuscript. Ugo Mayor, Nerea Osinalde, and Jesus M. Arizmendi are supported by the Spanish MINECO (Grant no. SAF2016-76898-P)

The Ubiquitin Ligase Ariadne-1 Regulates Neurotransmitter Release Via Ubiquitination of NSF

Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, we used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. We identified sixteen candidates that met the established criteria: a significant change of at least two-fold increase on ubiquitination, with at least two unique peptides identified. Amongst those candidates, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release. Using a pulldown approach that relies on the overexpression and stringent isolation of a GFP-fused construct, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. We tested the possible functional relevance of this modification using Ari-1 loss of function mutants, which displayed a lower rate of spontaneous neurotransmitter release due to failures at the pre-synaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared to controls in a Ca2+ dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous versus evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. Our results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitinationThis research was funded by grants BFU2015-65685 and PGC2018-094630-B-100 from the Spanish Ministry of Economy to A. F. and grant SAF2016-76898-P from the Spanish Ministry of Economy cofinanced with FEDER funds to U. M. J. R. was supported with a postdoctoral research fellowship from the University of the Basque Country (UPV/EHU

Identification of substrates for human deubiquitinating enzymes (DUBs): An up-to-date review and a case study for neurodevelopmental disorders

Similar to the reversal of kinase-mediated protein phosphorylation by phosphatases, deubiquitinating enzymes (DUBs) oppose the action of E3 ubiquitin ligases and reverse the ubiquitination of proteins. A total of 99 human DUBs, classified in 7 families, allow in this way for a precise control of cellular function and homeostasis. Ubiquitination regulates a myriad of cellular processes, and is altered in many pathological conditions. Thus, ubiquitination-regulating enzymes are increasingly regarded as potential candidates for therapeutic intervention. In this context, given the predicted easier pharmacological control of DUBs relative to E3 ligases, a significant effort is now being directed to better understand the processes and substrates regulated by each DUB. Classical studies have identified specific DUB substrate candidates by traditional molecular biology techniques in a case-by-case manner. Lately, single experiments can identify thousands of ubiquitinated proteins at a specific cellular context and narrow down which of those are regulated by a given DUB, thanks to the development of new strategies to isolate and enrich ubiquitinated material and to improvements in mass spectrometry detection capabilities. Here we present an overview of both types of studies, discussing the criteria that, in our view, need to be fulfilled for a protein to be considered as a high-confidence substrate of a given DUB. Applying these criteria, we have manually reviewed the relevant literature currently available in a systematic manner, and identified 650 high-confidence substrates of human DUBs. We make this information easily accessible to the research community through an updated version of the DUBase website (https://ehubio.ehu.eus/dubase/). Finally, in order to illustrate how this information can contribute to a better understanding of the physiopathological role of DUBs, we place a special emphasis on a subset of these enzymes that have been associated with neurodevelopmental disorders.This work was supported by Spanish MINECO grant SAF2016-76898-P to UM and by the University of the Basque Country (UPV/EHU), grant numbers US19/05 and COLAB19/18

SPANX-A/D protein subfamily plays a key role in nuclear organisation, metabolism and flagellar motility of human spermatozoa

Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes encode a protein family (SPANX-A, -B, -C and -D), whose expression is limited to the testis and spermatozoa in normal tissues and to a wide variety of tumour cells. Present only in hominids, SPANX-A/D is exclusively expressed in post-meiotic spermatids and mature spermatozoa. However, the biological role of the protein family in human spermatozoa is largely unknown. Combining proteomics and molecular approaches, the present work describes the presence of all isoforms of SPANX-A/D in human spermatozoa and novel phosphorylation sites of this protein family. In addition, we identify 307 potential SPANX-A/D interactors related to nuclear envelop, chromatin organisation, metabolism and cilia movement. Specifically, SPANX-A/D interacts with fumarate hydratase and colocalises with both fumarate hydratase and Tektin 1 proteins, involved in meeting energy demands for sperm motility, and with nuclear pore complex nucleoporins. We provide insights into the molecular features of sperm physiology describing for the first time a multifunctional role of SPANX-A/D protein family in nuclear envelope, sperm movement and metabolism, considered key functions for human spermatozoa. SPANX-A/D family members, therefore, might be promising targets for sperm fertility management.Spanish Health Department ISCIII-DT, Basque Government and Danish Medical Research Council. IU-A was supported by a fellowship from the University of the Basque Country (UPV/EHU). IM-H was supported by a fellowship from the Basque Government. I.K. was supported by a grant from the Danish Medical Research Counci

The Multifunctional Role of SPANX-A/D Protein Subfamily in the Promotion of Pro-Tumoural Processes in Human Melanoma

Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes encode a protein family (SPANX-A, -B, -C and -D), whose expression is limited to the testis and spermatozoa in normal tissues and various tumour cells. SPANX-A/D proteins have been detected in metastatic melanoma cells, but their contribution to cancer development and the underlying molecular mechanisms of skin tumourigenesis remain unknown. Combining functional and proteomic approaches, the present work describes the presence of SPANX-A/D in primary and metastatic human melanoma cells and how it promotes pro-tumoural processes such as cell proliferation, motility and migration. We provide insights into the molecular features of skin tumourigenesis, describing for the first time a multifunctional role of the SPANX-A/D protein family in nuclear function, energy metabolism and cell survival, considered key hallmarks of cancer. A better comprehension of the SPANX-A/D protein subfamily and its molecular mechanisms will help to describe new aspects of tumour cell biology and develop new therapeutic targets and tumour-directed pharmacological drugs for skin tumoursUniversity of the Basque Country (UPV/EHU) (GIU19/018). IU-A is supported by a fellowship from the University of the Basque Country (UPV/EHU). IM-H is supported by a fellowship from the Basque Government

Nola eragiten du 2-Interleukinak T-zelulen nukleoko proteinen fosforilazio mailan?

Immunoterapiaren helburua da gaixoen immunitate-sistema indartuz minbizia bezalako gaitz larriak menderatzea. Horretarako, gaixoak maiz 2-interleukina (IL-2) deitzen den konposatu batekin tratatzen dira, izan ere molekula honek berebi-ziko ahalmena baitauka T-zelulen edo T linfozitoen hazkuntza sustatzeko. Zoritxarrez, IL-2ren erabilerak albo kalte desatseginak eragiten ditu gaixoetan, eta hauek ekiditeko ezinbestekoa da IL-2k T-zeluletan eragiten dituen molekula mailako aldaketak sakon aztertu eta ulertzea. Zelulen gainazalera heltzen diren seinaleak zelulan barrena ga-rraiatzen dira nagusiki proteinen behin behineko fosforilazioen bidez. Hori horrela, au-rreko lan batean, IL-2k T linfozitoetan pizten dituen seinalizazio-bidezidorrak aztertu genituen zelulen zitoplasman fosforilatzen diren proteinak masa espektrometriaren bi-dez identifikatuz. Oraingo lan honetan berriz, masa espektrometriaren laguntzaz aztertu da IL-2k nola erregulatzen duen T-zelulen nukleoko proteinen fosforilazio maila, izan ere horrek zuzenean eragingo baitu geneen erregulazioan eta horrenbestez baita zelu-laren patuan ere. Esperimentu bera 3 aldiz errepikatu ostean, gutxienez behin identifi-katu eta kuantifikatu dira lan honetan T-zelulen nukleoko proteinetan fosforilatutako 8.521 aminoazido, horietako haietako asko aurrez ezagutugabeak. Bestalde, ikusi da orkorrean berdinantzera mantentzen dela IL-2rekin tratatutako eta tratatu gabeko T-ze-luletako fosforilazio maila. Halere, azpimarragarria da IL-2ren ondorioz 391 aminoa-zidoren fosforilazio maila nabarmen aldatzen dela T-zelulen nukleoetan. Etorkizunean egingo diren ikerkuntzek argituko dute zein den IL-2ren menpeko fosforilazio hauen esanahi biologikoa.; Immunotherapy aims to fight against diseases such as cancer by boosting the immune system of the patients. For that purpose, patients are usually treated with a molecule called interleukin-2 (IL-2), which plays a pivotal role in modulating the im-mune system and specially in promoting the proliferation of T-cells. Unfortunately, ad-ministration of IL-2 is usually accompanied by severe side effects that must be over-come. To achieve this, it is mandatory to understand in detail the molecular effects triggered in T-cells upon IL-2 stimulation. It is known that signaling events initiated at the cell surface are mainly transduced inside the cell through transient phosphoryla-tions occurring in distinct proteins. For that reason our previous study focused on dis-secting the signaling pathways triggered in IL-2-treated T lymphocytes by studying the proteins that become tyrosine phosphorylation upon the stimuli using mass spectrome-try. In the present study, we aimed to unveil how IL-2 modulates the phosphorylation status of the nuclear proteins in T-cells, which ultimately will affect gene expression and the fate of the cell. Following a quantitative mass spectrometry-based approach a total of 8,521 phosphorylated aminoacids were quantified at least in one of the 3 repli-cas of the same experiment that were performed. Whereas the phosphorylation levels of most phosphosites remained unaffected in IL-2-treated and untreated T-cells, the phosphorylation status of 391 phosphosites was found to be dramatically modulated upon IL-2 stimulation. Further investigation will unveil the biological significance of such finding

UBE3Aren substratuak identifikatzen Angelman sindromearen ingurukoak argitzeko.

Angelman syndrome (AS) is a rare neurodevelopmental disease that results from loss of function of the maternal UBE3A gene. UBE3A codes for an E3 ubiquitin ligase, that coordinately with E1 and E2 enzymes, attaches ubiquitin to proteins. Ubiquitination plays a key role in the fate of proteins. For instance, it can target a protein for degradation, relocate the protein within the cell or determine protein-protein interactions. Therefore, considering the relevance of ubiquitination, and the function of UBE3A, it could be anticipated that in the absence of UBE3A, the ubiquitination pattern of its substrates will be inadequate, thus affecting not only proteins, but also cell physiology. Consequently, to understand the aetiology of the neuronal defects in AS patients, it is mandatory to know which the substrates of UBE3A are. With that aim, we compared the proteins that are ubiquitinated in Drosophila flies overexpressing and not overexpressing UBE3A. Briefly, we combined the BioUb strategy developed in our lab with mass spectrometry, to isolate and detect ubiquitinated proteins, respectively. From the hundreds of proteins detected, 79 fulfilled the criteria to be considered as putative UBE3A substrate. One of those proteins was Rpn10, previously reported to be a UBE3A substrate. Additionally, we confirmed MS results indicating that Rngo might be a UBE3A substrate. Moreover, using human cells we proved that human Rngo homolog DDI1 is also a UBE3A substrate. Now we plan to (i) validate more UBE3A substrates, (ii) check whether those proteins are also substrates in human, and (iii) characterize their biological role.; Angelman sindromea neurogarapenezko gaixotasun bakana da, eta, arrazoiak arrazoi, neuronetan amarengandik jasotako UBE3A genearen kopia adierazten ez duten gizabanakoek pairatzen dute. UBE3Ak E3 ubikuitina ligasa entzima bat kodetzen du, zeina, E1 eta E2 entzimekin elkarlanean, itu-proteinei ubikuitina deritzon molekula txiki bat itsasteaz arduratzen den. Proteina bati ubikuitina gehitzeak berebiziko eragina izan dezake beraren patuan; izan ere, besteak beste, proteinen biziraupena, zelula barruko kokapena edota proteinen arteko elkarrekintzak baldintzatu ditzake. Beraz, ubikuitinazio-prozesuaren garrantzia ikusirik, eta UBE3Aren funtzioa zein den aintzat hartuta, pentsatzekoa da UBE3Aren faltan beraren itu-proteinen ubikuitinazio-maila gutxitua egongo dela, eta horrek eragin zuzena izango duela ez soilik proteinetan, baita zelularen funtzionamenduan ere. Angelman sindromea pairatzen dutenen zelula mailako kalteen jatorria ulertzeko, ezinbestekoa da UBE3Ak kodetzen duen E3 ubikuitina ligasaren itu-proteinak edo substratuak zeintzuk diren jakitea. Hori argitze aldera, UBE3A gainadierazten duten eta gainadierazten ez duten Drosophila eulietan ubikuitinatuta dauden proteinak aztertu eta konparatu ditugu. Ubikuitinatutako proteinak arrantzatzeko, gure laborategian garatutako BioUb estrategia erabili dugu, eta, ondoren, horiek identifikatzeko eta kuantifikatzeko, masa-espektrometria (MS) erabili dugu. Identifikatutako ehunka proteinetatik, 79k betetzen dituzte UBE3Aren substratu izateko irizpideak. Horien artean dago Rnp10, aurretiaz UBE3Aren itu-proteina gisa deskribatu den proteina. Horrez gain, Rngo deituriko proteinak piztu zuen gure arreta, eta, biologia molekularreko teknika osagarriak erabiliz, MS bidezko emaitzak berretsi ditugu. Gainera, giza zelulak erabiliz, Rngo-ren homologoa den DDI1 ere UBE3Aren substratua dela frogatu dugu. Etorkizunean, gure helburu nagusiak hiru dira: (i) MS bidez identifikatutako UBE3Aren substratu-izangai gehiago egiaztatzea, (ii) proteina horiek gizakietan ere UBE3Aren substratuak direnentz frogatzea, eta (iii) UBE3Aren substratuek zelula mailan betetzen dituzten funtzioak aztertzea

2-Interleukina (IL-2) eta IL-15 seinalizazio bidezidorrak T linfozitoetan: antzekoak baina aldiberean ezberdinak

2-interleukina (IL-2) eta IL-15 zitokinek berebiziko garrantzia daukate sistema immunologikoaren erregulazioan, besteak beste T linfozitoen proliferazioa sustatzen dutelako. Zitokina biek bi hartzaile-azpiunitate partekatzen dituztenez (IL-2Rβ eta IL-2Rγ) ez da harritzekoa teilakatutako hainbat funtzio izatea, baina bai da harrigarria, ordea, bakoitzak erantzun immunologiko espezikoak eta maiz kontrajarriak eragitea. Nahiz eta mekanismo asko proposatu diren IL-2 eta IL-15 arteko desberdintasunak azaltzeko, ez dago argi nola den posible biek, hartzaile berdinak erabiltzen dituztelarik, zeluletan erantzun desberdinak sustatzea. Horren gakoa aurkitzeko asmoz, IL-2 eta IL-15ez kitzikatutako T-zeluletan piztutako seinalizazio-bidezidorrak aztertu eta elkarren artean konparatu ditugu, masa-espektrometria erabilita. Gure lanak erakutsi du bi zitokinek kitzikatutako T linfozitoen seinalizazio-sareak oso parekoak izan arren, badirela ñabardura batzuk bi zitokinen arteko alde funtzionala azaltzen lagun dezaketenak.; L-2 and IL-15 are key cytokines regulating the immune system i n part by promoting T-cell proliferation. Both cytokines signal through the same receptor subunits (IL-2Rβ and IL-2Rγ) and not surprisingly they exert some overlapping functions. However they also have specific and even sometimes opposing roles raising the paradigm of how they mediate distinct immune reponses by using the same receptors. Although several options have been suggested, it remains controversial the molecular mechanism underlying the functional dichotomy of IL-2 and IL-15. Aiming to find the key to answering such enigma, we assessed and compared by mass spectrometry the signaling networks activated by both cytokines. Our study revealed that the transduction pathways initiated by IL-2 and IL-15 are highly similar but not identical since we detected faint differences that may account for the functional discrepancy observed between both cytokines