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

La edición genómica aplicada a seres humanos: aspectos éticos, jurídicos y sociales

El artículo analiza las implicaciones éticas jurídicas y sociales del empleo de las técnicas de edición genómica en seres humanos y su capacidad para alterar el genoma de la descendencia.Examina su impacto en investigación básica,asi como su potencial en el uso terapéutico y en el llamado mejoramiento humano. El trabajo parte de la evidencia científica y técnica para profundizar en los valores implicados en las diferentes opciones y cursos de acción, y - previo estudio de los argumentos que la doctrina esgrime en la actualidad - proponer recomendaciones para encauzar el debate y orientar las actuaciones en este campo

Advancing in Schaaf-Yang syndrome pathophysiology: from bedside to subcellular analyses of truncated MAGEL2

Background Schaaf-Yang syndrome (SYS) is caused by truncating mutations in MAGEL2, mapping to the Prader-Willi region (15q11-q13), with an observed phenotype partially overlapping that of Prader-Willi syndrome. MAGEL2 plays a role in retrograde transport and protein recycling regulation. Our aim is to contribute to the characterisation of SYS pathophysiology at clinical, genetic and molecular levels. Methods We performed an extensive phenotypic and mutational revision of previously reported patients with SYS. We analysed the secretion levels of amyloid-β 1-40 peptide (Aβ1-40) and performed targeted metabolomic and transcriptomic profiles in fibroblasts of patients with SYS (n=7) compared with controls (n=11). We also transfected cell lines with vectors encoding wild- type (WT) or mutated MAGEL2 to assess stability and subcellular localisation of the truncated protein. Results Functional studies show significantly decreased levels of secreted Aβ1-40 and intracellular glutamine in SYS fibroblasts compared with WT. We also identified 132 differentially expressed genes, including non-coding RNAs (ncRNAs) such as HOTAIR, and many of them related to developmental processes and mitotic mechanisms. The truncated form of MAGEL2 displayed a stability similar to the WT but it was significantly switched to the nucleus, compared with a mainly cytoplasmic distribution of the WT MAGEL2. Based on the updated knowledge, we offer guidelines for the clinical management of patients with SYS. Conclusion A truncated MAGEL2 protein is stable and localises mainly in the nucleus, where it might exert a pathogenic neomorphic effect. Aβ1-40 secretion levels and HOTAIR mRNA levels might be promising biomarkers for SYS. Our findings may improve SYS understanding and clinical management

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

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

mRNA expression analysis of the SUMO pathway genes in the adult mouse retina

Contains fulltext : 154561.pdf (publisher's version ) (Open Access

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 inhibits degradation of the ERAD substrate CD3δ

<p>. A) Western blots of whole cell lysates. Top: HEK-293 cells were transfected as indicated and treated with the proteasome inhibitor MG132 where noted (15 µM, 6 hours) before harvesting. Bottom: semi-quantification of bands from western blots shown above and other similar, independent experiments. CD3δ protein levels were normalized to loading control. Shown are means +/− standard deviations. USP25(WT): common isoform of USP25; USP25(m): muscle-specific isoform of USP25. P values from Student T-tests are shown below histograms. B) Top: HEK-293 cells were transfected with the indicated constructs and harvested 48 hours later. Shown are western blots of whole cell lysates probed with the indicated antibodies. WT: wild type USP25, C178S: the catalytic cysteine of USP25 was replaced by a serine residue <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Denuc1" target="_blank">[18]</a>, ΔUBA: UBA deleted, ΔUIM: both UIMs deleted. Bottom: semi-quantification of data from the top and two other independent experiments. CD3δ protein levels were normalized to loading control. Shown are means +/− standard deviations. P values from Student T-tests are shown below histograms. C) Top: HEK-293 cells were transfected as indicated. 48 hours post-transfection cells were treated for the indicated periods of time with 75 µg/ml cycloheximide to inhibit synthesis of new protein. Bottom: semi-quantification of western blots from the top and three other, independent experiments. CD3δ levels were normalized to loading control. Shown are means +/− standard deviations. P values are from Student T-tests of USP25 compared to vector control. D and E) HEK-293 cells were transfected with the indicated constructs. 48 hours later tagged constructs were immunopurified with bead-bound antibodies and probed as indicated.</p

Development of immunotherapy targeting melanoma stem cells

Diversity, understood as the variety of different elements or configurations that an extensive system has, is a crucial property that allows maintaining the system’s functionality in a changing environment, where failures, random events or malicious attacks are often unavoidable. Despite the relevance of preserving diversity in the context of ecology, biology, transport, finances, etc., the elements or configurations that more contribute to the diversity are often unknown, and thus, they can not be protected against failures or environmental crises. This is due to the fact that there is no generic framework that allows identifying which elements or configurations have crucial roles in preserving the diversity of the system. Existing methods treat the level of heterogeneity of a system as a measure of its diversity, being unsuitable when systems are composed of a large number of elements with different attributes and types of interactions. Besides, with limited resources, one needs to find the best preservation policy, i.e., one needs to solve an optimization problem. Here we aim to bridge this gap by developing a metric between labeled graphs to compute the diversity of the system, which allows identifying the most relevant components, based on their contribution to a global diversity value. The proposed framework is suitable for large multiplex structures, which are constituted by a set of elements represented as nodes, which have different types of interactions, represented as layers. The proposed method allows us to find, in a genetic network (HIV-1), the elements with the highest diversity values, while in a European airline network, we systematically identify the companies that maximize (and those that less compromise) the variety of options for routes connecting different airports.Peer ReviewedPostprint (published version

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