Esperanza diseñada en el laboratorio

Si imaginem l’ésser humà en un entorn salvatge i primitiu, entendrem que la vista és un sentit essencial per a la supervivència: cal evitar els depredadors, cal detectar les preses... Ho deixa de ser en un entorn social i civilitzat? Seguint l’últim informe de la OMS, publicat l’octubre del 2011 (WHO, Fact sheet 282), el 90% de les persones amb deficiències visuals viuen en països desenvolupats. Globalment, la xifra és de 285 milions d’afectats, dels quals 39 milions són cecs –respecte la població mundial, 7 mil milions de persones. Estudiar-ne les bases moleculars, és a dir, la diana –la molécula– que es veu directament afectada en aquestes malalties, és el primer pas per trobar-ne una curaSi imaginamos el ser humano en un entorno salvaje y primitivo, entenderemos que la vista es un sentido esencial para la supervivencia: hay que evitar los depredadores, hay que detectar las presas ... ¿Lo deja de ser en un entorno social y civilizado? Según el último informe de la OMS, publicado en octubre de 2011 (WHO, Fact sheet 282), el 90% de las personas con deficiencias visuales viven en países desarrollados. Globalmente, la cifra es de 285 millones de afectados, de los cuales 39 millones son ciegos- respecto a la población mundial, 7 mil millones de personas. Estudiar las bases moleculares, es decir, la diana-la molécula-que se ve directamente afectada en estas enfermedades, es el primer paso para encontrar una cura..

Ubiquitin-Specific Protease 25 Functions in Endoplasmic Reticulum-Associated Degradation

Endoplasmic Reticulum (ER)-associated degradation (ERAD) discards abnormal proteins synthesized in the ER. Through coordinated actions of ERAD components, misfolded/anomalous proteins are recognized, ubiquitinated, extracted from the ER and ultimately delivered to the proteasome for degradation. It is not well understood how ubiquitination of ERAD substrates is regulated. Here, we present evidence that the deubiquitinating enzyme Ubiquitin-Specific Protease 25 (USP25) is involved in ERAD. Our data support a model where USP25 counteracts ubiquitination of ERAD substrates by the ubiquitin ligase HRD1, rescuing them from degradation by the proteasome

Blocking GM-CSF receptor α with mavrilimumab reduces infiltrating cells, pro-inflammatory markers and neoangiogenesis in ex vivo cultured arteries from patients with giant cell arteritis

Effective and safe therapies are needed for the treatment of patients with giant cell arteritis (GCA). Emerging as a key cytokine in inflammation, granulocyte-macrophage colony stimulating factor (GM-CSF) may play a role in promoting inflammation in GCA.To investigate expression of GM-CSF and its receptor in arterial lesions from patients with GCA. To analyse activation of GM-CSF receptor-associated signalling pathways and expression of target genes. To evaluate the effects of blocking GM-CSF receptor α with mavrilimumab in ex vivo cultured arteries from patients with GCA.Quantitative real time PCR, in situ RNA hybridisation, immunohistochemistry, immunofluorescence and confocal microscopy, immunoassay, western blot and ex vivo temporal artery culture.GM-CSF and GM-CSF receptor α mRNA and protein were increased in GCA lesions; enhanced JAK2/STAT5A expression/phosphorylation as well as increased expression of target genes CD83 and Spi1/PU.1 were observed. Treatment of ex vivo cultured GCA arteries with mavrilimumab resulted in decreased transcripts of CD3ε, CD20, CD14 and CD16 cell markers, and reduction of infiltrating CD16 and CD3ε cells was observed by immunofluorescence. Mavrilimumab reduced expression of molecules relevant to T cell activation (human leukocyte antigen-DR [HLA-DR]) and Th1 differentiation (interferon-γ), the pro-inflammatory cytokines: interleukin 6 (IL-6), tumour necrosis factor α (TNFα) and IL-1β, as well as molecules related to vascular injury (matrix metalloprotease 9, lipid peroxidation products and inducible nitric oxide synthase [iNOS]). Mavrilimumab reduced CD34 + cells and neoangiogenesis in GCA lesions.The inhibitory effects of mavrilimumab on multiple steps in the GCA pathogenesis cascade in vitro are consistent with the clinical observation of reduced GCA flares in a phase 2 trial and support its development as a therapeutic option for patients with GCA.© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ

New protein-protein interactions of mitochondrial connexin 43 in mouse heart

Connexin 43 (Cx43), the gap junction protein involved in cell-to-cell coupling in the heart, is also present in the subsarcolemmal fraction of cardiomyocyte mitochondria. It has been described to regulate mitochondrial potassium influx and respiration and to be important for ischaemic preconditioning protection, although the molecular effectors involved are not fully characterized. In this study, we looked for potential partners of mitochondrial Cx43 in an attempt to identify new molecular pathways for cardioprotection. Mass spectrometry analysis of native immunoprecipitated mitochondrial extracts showed that Cx43 interacts with several proteins related with mitochondrial function and metabolism. Among them, we selected for further analysis only those present in the subsarcolemmal mitochondrial fraction and known to be related with the respiratory chain. Apoptosis-inducing factor () and the beta-subunit of the electron-transfer protein (), two proteins unrelated to date with Cx43, fulfilled these conditions, and their interaction with Cx43 was proven by direct and reverse co-immunoprecipitation. Furthermore, a previously unknown molecular interaction between and was established, and protein content and sub-cellular localization appeared to be independent from the presence of Cx43. Our results identify new protein-protein interactions between -Cx43, -Cx43 and - as possible players in the regulation of the mitochondrial redox state

The UBA-UIM Domains of the USP25 Regulate the Enzyme Ubiquitination State and Modulate Substrate Recognition

USP25m is the muscle isoform of the deubiquitinating (DUB) enzyme USP25. Similarly to most DUBs, data on USP25 regulation and substrate recognition is scarce. In silico analysis predicted three ubiquitin binding domains (UBDs) at the N-terminus: one ubiquitin-associated domain (UBA) and two ubiquitin-interacting motifs (UIMs), whereas no clear structural homology at the extended C-terminal region outside the catalytic domains were detected. In order to asses the contribution of the UBDs and the C-terminus to the regulation of USP25m catalytic activity, ubiquitination state and substrate interaction, serial and combinatorial deletions were generated. Our results showed that USP25m catalytic activity did not strictly depend on the UBDs, but required a coiled-coil stretch between amino acids 679 to 769. USP25 oligomerized but this interaction did not require either the UBDs or the C-terminus. Besides, USP25 was monoubiquitinated and able to autodeubiquitinate in a possible loop of autoregulation. UBDs favored the monoubiquitination of USP25m at the preferential site lysine 99 (K99). This residue had been previously shown to be a target for SUMO and this modification inhibited USP25 activity. We showed that mutation of K99 clearly diminished USP25-dependent rescue of the specific substrate MyBPC1 from proteasome degradation, thereby supporting a new mechanistic model, in which USP25m is regulated through alternative conjugation of ubiquitin (activating) or SUMO (inhibiting) to the same lysine residue (K99), which may promote the interaction with distinct intramolecular regulatory domains

Esperanza diseñada en el laboratorio

Si imaginem l’ésser humà en un entorn salvatge i primitiu, entendrem que la vista és un sentit essencial per a la supervivència: cal evitar els depredadors, cal detectar les preses... Ho deixa de ser en un entorn social i civilitzat? Seguint l’últim informe de la OMS, publicat l’octubre del 2011 (WHO, Fact sheet 282), el 90% de les persones amb deficiències visuals viuen en països desenvolupats. Globalment, la xifra és de 285 milions d’afectats, dels quals 39 milions són cecs –respecte la població mundial, 7 mil milions de persones. Estudiar-ne les bases moleculars, és a dir, la diana –la molécula– que es veu directament afectada en aquestes malalties, és el primer pas per trobar-ne una curaSi imaginamos el ser humano en un entorno salvaje y primitivo, entenderemos que la vista es un sentido esencial para la supervivencia: hay que evitar los depredadores, hay que detectar las presas ... ¿Lo deja de ser en un entorno social y civilizado? Según el último informe de la OMS, publicado en octubre de 2011 (WHO, Fact sheet 282), el 90% de las personas con deficiencias visuales viven en países desarrollados. Globalmente, la cifra es de 285 millones de afectados, de los cuales 39 millones son ciegos- respecto a la población mundial, 7 mil millones de personas. Estudiar las bases moleculares, es decir, la diana-la molécula-que se ve directamente afectada en estas enfermedades, es el primer paso para encontrar una cura..

Dominis estructurals i noves interaccions proteiques de l'enzim deubiquitinant USP25

La present Tesi Doctoral es presenta com una agrupació de quatre publicacions que resumeixen el treball realitzat al Departament de Genètica de la Facultat de Biologia de la Universitat de Barcelona. El principal objectiu és la caracterització funcional de les regions estructurals de la isoforma muscular de l’enzim deubiquitinat USP25, inicialment definides amb eines bioinformàtiques. A més a més, es pretén fer un estudi de noves interaccions moleculars tipus proteïna-proteïna per la cerca de nous substrats o reguladors enzimàtics. La cèl•lula eucariota posseeix, entre altres, un sistema senyalitzador intracel•lular basat en una família de pèptids, el representant dels quals és la ubiquitina. Aquest sistema presenta diferents categories funcionals, entre elles, els enzims deubiquitinants, un centenar a l’espècie humana. Aquests enzims hidrolitzen l’enllaç que uneix la ubiquitina als seus precursors o substrats, mantenint així l’homeostasi d’aquest pèptid dins la cèl•lula. Una alteració de la seva funció pot portar diferents conseqüències depenent de la via metabòlica que es vegi afectada, doncs suposa una desregulació estequiomètrica dels substrats ubiquitinants respecte els no ubiquitinats. L’enzim deubiquitinant USP25 es va descriure en el grup d’investigació d’aquesta tesi durant la cerca de nous gens relacionats amb la síndrome de Down. Un cop caracteritzat funcionalment com una proteasa específica d’ubiquitina, els estudis d’expressió van mostrar l’existència de tres isoformes proteiques, una d’elles, USP25m, restringida al teixit muscular i cardíac. Tenint en compte que en el fenotip dels pacients amb síndrome de Down, entre altres trets, hi ha deficiència cardiovascular i atonia muscular, els esforços del grup es van centrar en la descripció i anàlisi d’aquesta isoforma. Els primers estudis van mostrar la seva situació citosòlica, l’expressió correlativa amb la diferenciació de cèl•lules musculars i la relació específica amb diverses proteines del sarcòmer. En el treball realitzat per la present Tesi Doctoral, s’han caracteritzat funcionalment diferents regions reguladores descrites a nivell bioinformàtic, així com també s’ha analitzat la seva implicació fisiológica a la funció d’USP25m. A més a més, mitjançant un estudi de cerca de nous interactors proteics, s’ha trobat una nova molècula que pertany a la mateixa via senyalitzadora i que es relaciona de manera específica amb USP25m, la lligasa d’ubiquititna MKRN1 (makorin 1) Mitjançant l’ús de diferents construccions amb delecions i mutacions puntuals de la proteïna que afecten a les regions d’interès, s’ha arribat a diferents conclusions, entre elles, que USP25m és monoubiquitinat i té la capacitat d’autodeubiquitinar-se. La monoubiquitinació en regula la seva activitat enzimàtica i es proposa un mecanisme de regulació basat en la conjugació alternativa de SUMO (una altra molècula de la família de la ubiquitina) i ubiquitina, en el mateix residu aminoacídic, la lisina 99 (Lys99). Els dominis d’unió a ubiquitina regulen el reconeixement de substrat i afavoreixen la monoubiquitinació. USP25m oligomeritza dins la cèl•lula i es troba present en diferents formacions proteiques d’elevat pes molecular. S’ha comprovat la relació específica amb la nova lligasa MKRN1 i es suggereixen diferents escenaris moleculars on poden trobar-se inclosos els dos pèptidsThe main aim of the present PhD work, titled “Structural domains and new protein interactions of the deubiquitinating enzyme USP25”, is the functional characterisation of the structural domains of the muscle isoform of USP25, USP25m, as well as the analysis of new protein‐protein interactions. The ubiquitin‐proteasome pathway is widely known as the preferential system to get ride of old or non‐functional proteins. Recently, it has become more apparent that this is not the only function. Ubiquitin (Ub) and all ubiquitin–like (UbLs) molecules acted as regulatory tags involved in different cellular events as subcellular localization, enzyme activation, DNA repair, etc. The intricate Ub‐signalling networks require a tight regulation of both conjugation and deconjugationprocesses, which are controlled by ubiquitin ligases and deubiquitinating enzymes (DUBs), respectively. USP25 is a DUB described while looking for novel genes involved in Down syndrome phenotype. First studies showed that it encoded three alternative protein isoforms, one of them, muscle specific. This muscle isoform, USP25m, is a cytosolic protein, upregulated during myogenesis that interacts in a specific manner with different sarcomeric proteins. Using an “in silico” approach, we were able to identify different structural domains, among them three ubiquitin binding domains (UBDs), and we aimed to characterise its role on USP25m function. By generating a collection of deletion and punctual mutants of the regions of interest, we conclude that USP25m is monoubiquitinated and that the UBDs modulate this modification. The preferential site for monoubiquitination is lysine 99 (K99), a residue that has been reported to undergo sumoylation (SUMO conjugation, being SUMO an UbL). According to our results, mutation of the K99 residue diminishes the deubiquitinating function, proposing a mechanistic model for USP25m regulation based on alternative conjugation of Ub and SUMO on the same residue, K99. Futhermore, while seeking new protein interactions of USP25m we identified Makorin Ring finger protein 1 (MKRN1), which belongs to an ubiquitin ligase family, as a putative interactor. We were capable of characterise its interaction and propose different cellular scenarios were they could interact.The main aim of the present PhD work, titled “Structural domains and new protein interactions of the deubiquitinating enzyme USP25”, is the functional characterisation of the structural domains of the muscle isoform of USP25, USP25m, as well as the analysis of new protein‐protein interactions. The ubiquitin‐proteasome pathway is widely known as the preferential system to get ride of old or non‐functional proteins. Recently, it has become more apparent that this is not the only function. Ubiquitin (Ub) and all ubiquitin–like (UbLs) molecules acted as regulatory tags involved in different cellular events as subcellular localization, enzyme activation, DNA repair, etc. The intricate Ub‐signalling networks require a tight regulation of both conjugation and deconjugationprocesses, which are controlled by ubiquitin ligases and deubiquitinating enzymes (DUBs), respectively. USP25 is a DUB described while looking for novel genes involved in Down syndrome phenotype. First studies showed that it encoded three alternative protein isoforms, one of them, muscle specific. This muscle isoform, USP25m, is a cytosolic protein, upregulated during myogenesis that interacts in a specific manner with different sarcomeric proteins. Using an “in silico” approach, we were able to identify different structural domains, among them three ubiquitin binding domains (UBDs), and we aimed to characterise its role on USP25m function. By generating a collection of deletion and punctual mutants of the regions of interest, we conclude that USP25m is monoubiquitinated and that the UBDs modulate this modification. The preferential site for monoubiquitination is lysine 99 (K99), a residue that has been reported to undergo sumoylation (SUMO conjugation, being SUMO an UbL). According to our results, mutation of the K99 residue diminishes the deubiquitinating function, proposing a mechanistic model for USP25m regulation based on alternative conjugation of Ub and SUMO on the same residue, K99. Futhermore, while seeking new protein interactions of USP25m we identified Makorin Ring finger protein 1 (MKRN1), which belongs to an ubiquitin ligase family, as a putative interactor. We were capable of characterise its interaction and propose different cellular scenarios were they could interact

Ubiquitin-specific protease USP25 functions in endoplasmic reticulum-associated degradation

Endoplasmic Reticulum (ER)-associated degradation (ERAD) discards abnormal proteins synthesized in the ER. Through coordinated actions of ERAD components, misfolded/anomalous proteins are recognized, ubiquitinated, extracted from the ER and ultimately delivered to the proteasome for degradation. It is not well understood how ubiquitination of ERAD substrates is regulated. Here, we present evidence that the deubiquitinating enzyme Ubiquitin-Specific Protease 25 (USP25) is involved in ERAD. Our data support a model where USP25 counteracts ubiquitination of ERAD substrates by the ubiquitin ligase HRD1, rescuing them from degradation by the proteasome

USP25 interacts with ERAD components.

<p>A) Schematics depict known domains of common (USP25(WT)) and muscle-specific (USP25(m)) isoforms of USP25 that are expressed in mammals <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Denuc1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Meulmeester1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Valero1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Valero2" target="_blank">[42]</a>. B) HEK-293 cells were transfected with HA-USP25. 48 hours later cells were fixed, probed as indicated and imaged with laser confocal microscopy. Panels IA-IC are single optical plane images (1 µM) of a cell immunolabeled for ER (KDEL, endogenous marker), HA-USP25 and nucleus (DAPI). Panel IC is the merged view of panels IA (green channel), IB (red channel) and DAPI (blue channel; not shown as a separate channel). Panels II and III are merged views of other cells stained similarly to panel I. Scale bars: 10 µM. C–G) HEK-293 cells were transfected as shown. Indicated constructs were immunopurified with bead-bound antibodies. Similar results were obtained from COS-7 cells for panels B–E (not shown). All USP25 constructs used in this figure were the common isoform (USP25(WT)).</p

USP25 and HRD1 have opposing effects on CD3

<p>δ <b>protein levels and ubiquitination.</b> A) HEK-293 cells were transfected as indicated and harvested 48 hours later. Western blots are from whole cell lysates. HRD1(WT): normal HRD1; HRD1(CA): catalytically inactive HRD1, in which the catalytic cysteine is substituted by an alanine residue <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Kikkert1" target="_blank">[7]</a>. Histograms on the right: semi-quantification of data from the left and other independent experiments. Shown are means +/− standard deviations. CD3δ levels were normalized to loading control. P values from Student T tests are shown below histograms. B and C) HEK-293 cells were transfected with the indicated constructs. 48 hours post transfection, cells were treated for 6 hours with MG132 (15 µM) and HA-CD3δ was immunopurified using bead-bound anti-HA antibody after a stringent denature/renature step (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#s4" target="_blank">Materials and Methods</a> for details). Histograms: semi-quantification of bracketed ubiquitin smears from the experiment on the left and other similar, independent experiments. Shown are means +/− standard deviations. P values for panel C are from Student T-tests. </p