The Proteasomal Deubiquitinating Enzyme PSMD14 Regulates Macroautophagy by Controlling Golgi-to-ER Retrograde Transport

Ubiquitination regulates several biological processes, however the role of specific members of the ubiquitinome on intracellular membrane trafficking is not yet fully understood. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1187 genes of the human “ubiquitinome” using amyloid precursor protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 with Capzimin (CZM) caused a robust increase in APP levels at the Golgi apparatus and the swelling of this organelle. We showed that this phenotype is the result of rapid inhibition of Golgi-to-ER retrograde transport, a pathway implicated in the early steps of the autophagosomal formation. Indeed, we observed that inhibition of PSMD14 with CZM acts as a potent blocker of macroautophagy by a mechanism related to the retention of Atg9A and Rab1A at the Golgi apparatus. As pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and the K63-Ub chains, act as a crucial regulatory factor for macroautophagy by controlling Golgi-to-ER retrograde transport

Synthesis of silica chitosan oligosaccharides nanoparticles

Objective: To obtain chitosan oligosaccharides (COS) and evaluate COS uses for the obtention of nanosystem based on silica as vehicle and compare the COS-silica nanosystem with the chitosan (Chi) precursor system as Chitosan-silica nanosystems. Design/methodology/approach: A combination of hydrolysis chemical and mechanical (microwave assisted) were used to obtain COS with the oxidative action of hydrogen peroxide. Sol-Gel adapted method was used to synthetize silica nanoparticles (SiNPs) from sodium metasilicate and the electrostatic interactions between SiNPs and Chi/COS were used to functionalize the SiNPs surface with Chi/COS.   Results: Nanosystem composed from COS and SiNPs were obtained successful as A COS-SiNPs and C COS-SiNPs with particle size of 139.35 nm and 251.8 nm and zeta potential of 30.40 mV and 34.67 mV respectively with antimicrobial activity against Escherichia coli and Staphylococcus aureus. Limitations on study/implications: Stabilize the systems compound of chitosan-silica nanoparticles due to the molecular weight of chitosan which loss the stabilized the SiNPs suspension and due the incompatibility of both systems pH. Findings/conclusions: COS and COS-SiNPs stable systems were obtained with an improvement of the antimicrobial activity of the system in contrast of Chi-SiNPs systems

Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein <i>via </i>the MVB/lysosomal pathway

© FASEB. Brain regions affected by Alzheimer disease (AD) displaywell-recognized early neuropathologic features in the endolysosomal and autophagy systems of neurons, including enlargement of endosomal compartments, progressive accumulation of autophagic vacuoles, and lysosomal dysfunction.Although the primary causes of these disturbances are still under investigation, a growing body of evidence suggests that the amyloid precursor protein (APP) intracellular C-terminal fragment b (C99), generated by cleavage of APP by b-site APP cleaving enzyme 1 (BACE-1), is the primary cause of the endosome enlargement inADand the earliest initiator of synaptic plasticity and long-termmemory impairment. The aimof the present study was to evaluate the possible relationship between the endolysosomal degradation pathway and autophagy on the proteolytic processing and turnover of C99. We found that pharmacologic treatments that either inhibit autophagosomeformationorblock the fusionof autophagosomes to

Increased dietary methionine, lysine and histidine supply modulated the heat stress-induced metabolic remodeling of dairy cows

Increasing dietary Met, Lys, and His supply without increasing the dietary protein content was reported to partially alleviate the productive and physiological impact of heat stress. Nevertheless, the metabolic pathways involved are yet to be identified. Thus, we aimed to explore the metabolic pathways associated with these positive effects and develop new metabolomics-based hypotheses. Twelve lactating Holstein cows (primiparous, n = 6; multiparous, n = 6; 42.2 ± 10.6 kg/d milk yield; 83 ± 28 days in milk) were enrolled in two 3×3 replicated Latin squares consisting of 14-day treatment periods: heat stress [HS; max. Temperature Humidity Index (THI) 84, 16.8% crude protein (CP), 1,741 g/d metabolizable protein (MP), 108 Lys, 33 Met, and 37 His (g/d)], pair feeding in thermo-neutrality (TN; max. THI 64, same diet as HS), and HS with increased Lys, Met and His supply [HS+AA; max. THI 84; 17.0% CP, 1,730 g/d MP, 179 Lys, 58 Met, and 45 His (g/d)]. Blood plasma and milk were sampled on day 14 for metabolomics profiling. Several amino acids (AA) and derivatives differed between the treatments. Plasma and milk Met, Val, Trp and α-amino adipic acid concentrations were highest in HS+AA (false discovery rate-P (FDR) &lt; 0.05). Moreover, only plasma Lys and milk His were highest in HS+AA (FDR &lt; 0.05). Some phosphatidylcholines (PC) and diglycerides had lower concentrations in HS than TN (FDR &lt; 0.05), while HS+AA had similar concentrations as TN. The pathway enrichment analysis revealed that the AA-related pathways were more significantly affected in multiparous than in primiparous cows. Our results suggest that increased supply of Met stimulated PC synthesis in HS+AA to similar concentrations as in TN. Increased Lys supply likely elevated the oxidation rate of Lys and downregulated the catabolism of other essential AA (EAA) such as Val and Trp, stimulating milk protein synthesis. No clear associations were found related to His availability. In conclusion, partial amelioration of productive and physiological effects of heat stress associated with increased dietary Met and Lys supply were likely explained by stimulated PC synthesis and increased plasma and milk concentrations of other EEA

El papel de la enseñanza de la historia en el desarrollo comunicativo del profesional de la salud / The Role of History teaching in the communicative development of the Health Professional

El presente trabajo introduce el tema en cuestión a partir del análisis de diferentes postulados teóricos sobre la comunicación y la importancia de la enseñanza de la Historia con estos propósitos, en el contexto de la formación médica, que sirvió de base para el análisis documental y la elaboración de dos instrumentos que se aplicaron corroborando la necesidad de crear vías para el desarrollo de la comunicación, y la efectividad de la experiencia aplicada desde el Programa de Historia de Cuba III en 4to año de la carrera de Medicina respectivamente. Para el desarrollo de estos propósitos se fundamentaron  teóricamente las potencialidades de la enseñanza de la asignatura y se ofreció un modelo recurrente que constituyó el estilo metodológico con que se impartieron las 16 actividades docentes  del programa para dar cumplimiento al objetivo propuesto, demostrando que el diseño de las estrategias de enseñanza aprendizaje debe estar encaminado a construir eslabones para el desarrollo de la comunicación, donde la enseñanza de la Historia constituya un fuerte puntal en este propósito, además de contribuir a solucionar los problemas de comunicación que afloren en el período de formación médica e incorpore habilidades, métodos y conocimientos al desarrollo del método clínico, cuyos resultados se evidenciaron en el análisis cualitativo y cuantitativo del instrumento aplicado .Palabras Clave: HISTORIA, ESTUDIANTES DE MEDICINA, COMUNICACIÓN, CUBA. ABSTRACT The present work is based on the teaching of History  from the analysis of different  theoretical postulates on communication  and the importance of  History teaching in the medical context,  which was used as a base  for the documented analysis and the creation of two instruments which were applied,  demonstrating the necessity of means  for the development of communication  and the effectiveness of the applied training  from the Cuban History III in the 4 academic year of Medical major. With these purposes the possibilities of the subject teaching were based theoretically  and it was offered a recurrent model which  constituted  the methodological style used for teaching 16 teaching activities of the program  fulfilling the proposed objective  which demonstrated  that the design of the teaching- learning strategies must be directed to create links  for the development of  communication  where the History teaching is a strong  support  and it also contributes to solve  the communication problems  which emerge in the medical formation period  and gives training , methods and knowledges of the clinical method development whose results  were evidenced in the qualitative and quantitative analysis of the applied instrument. Key words: HISTORY, MEDICAL STUDENTS, COMMUNICATION, CUBA

El papel de la enseñanza de la historia en el desarrollo comunicativo del profesional de la salud / The Role of History teaching in the communicative development of the Health Professional

El presente trabajo introduce el tema en cuestión a partir del análisis de diferentes postulados teóricos sobre la comunicación y la importancia de la enseñanza de la Historia con estos propósitos, en el contexto de la formación médica, que sirvió de base para el análisis documental y la elaboración de dos instrumentos que se aplicaron corroborando la necesidad de crear vías para el desarrollo de la comunicación, y la efectividad de la experiencia aplicada desde el Programa de Historia de Cuba III en 4to año de la carrera de Medicina respectivamente. Para el desarrollo de estos propósitos se fundamentaron  teóricamente las potencialidades de la enseñanza de la asignatura y se ofreció un modelo recurrente que constituyó el estilo metodológico con que se impartieron las 16 actividades docentes  del programa para dar cumplimiento al objetivo propuesto, demostrando que el diseño de las estrategias de enseñanza aprendizaje debe estar encaminado a construir eslabones para el desarrollo de la comunicación, donde la enseñanza de la Historia constituya un fuerte puntal en este propósito, además de contribuir a solucionar los problemas de comunicación que afloren en el período de formación médica e incorpore habilidades, métodos y conocimientos al desarrollo del método clínico, cuyos resultados se evidenciaron en el análisis cualitativo y cuantitativo del instrumento aplicado .Palabras Clave: HISTORIA, ESTUDIANTES DE MEDICINA, COMUNICACIÓN, CUBA. ABSTRACT The present work is based on the teaching of History  from the analysis of different  theoretical postulates on communication  and the importance of  History teaching in the medical context,  which was used as a base  for the documented analysis and the creation of two instruments which were applied,  demonstrating the necessity of means  for the development of communication  and the effectiveness of the applied training  from the Cuban History III in the 4 academic year of Medical major. With these purposes the possibilities of the subject teaching were based theoretically  and it was offered a recurrent model which  constituted  the methodological style used for teaching 16 teaching activities of the program  fulfilling the proposed objective  which demonstrated  that the design of the teaching- learning strategies must be directed to create links  for the development of  communication  where the History teaching is a strong  support  and it also contributes to solve  the communication problems  which emerge in the medical formation period  and gives training , methods and knowledges of the clinical method development whose results  were evidenced in the qualitative and quantitative analysis of the applied instrument. Key words: HISTORY, MEDICAL STUDENTS, COMMUNICATION, CUBA

Negative Modulation of Macroautophagy by Stabilized HERPUD1 is Counteracted by an Increased ER-Lysosomal Network With Impact in Drug-Induced Stress Cell Survival

Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER membrane protein with low expression and known to be degraded by the proteasome under normal conditions. Contrary, under ER stress, levels of HERPUD1 increased rapidly due to a blockage in its proteasomal degradation. Thus, we explored whether HERPUD1 stability could work as a negative regulator of autophagy. In this work, we expressed a version of HERPUD1 with its ubiquitin-like domain (UBL) deleted, which is known to be crucial for its proteasome degradation. In comparison to HERPUD1-WT, we found the UBL-deleted version caused a negative role on basal and induced macroautophagy. Unexpectedly, we found stabilized HERPUD1 promotes ER remodeling independent of unfolded protein response activation observing an increase in stacked-tubular structures resembling previously described tubular ER rearrangements. Importantly, a phosphomimetic S59D mutation within the UBL mimics the phenotype observed with the UBL-deleted version including an increase in HERPUD1 stability and ER remodeling together with a negative role on autophagy. Moreover, we found UBL-deleted version and HERPUD1-S59D trigger an increase in cellular size, whereas HERPUD1-S59D also causes an increased in nuclear size. Interestingly, ER remodeling by the deletion of the UBL and the phosphomimetic S59D version led to an increase in the number and function of lysosomes. In addition, the UBL-deleted version and phosphomimetic S59D version established a tight ER-lysosomal network with the presence of extended patches of ER-lysosomal membrane-contact sites condition that reveals an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival

Association of combined anti-Ro52/TRIM21 and anti-Ro60/SSA antibodies with increased Sjögren disease severity through interferon pathway activation

Objective: The biologic diagnosis of primary Sjögren disease (SjD) mainly relies on anti-Ro60/SSA antibodies, whereas the significance of anti-Ro52/TRIM21 antibodies currently remains unclear. The aim of this study was to characterize the clinical, serological, biologic, transcriptomic, and interferon profiles of patients with SjD according to their anti-Ro52/TRIM21 antibody status. Methods: Patients with SjD from the European PRECISESADS (n = 376) and the Brittany Diagnostic Suspicion of primitive Sjögren's Syndrome (DIApSS); (n = 146) cohorts were divided into four groups: double negative (Ro52‾/Ro60‾), isolated anti-Ro52/TRIM21 positive (Ro52+), isolated anti-Ro60/SSA positive (Ro60+), and double-positive (Ro52+/Ro60+) patients. Clinical information; EULAR Sjögren Syndrome Disease Activity Index, a score representing systemic activity; and biologic markers associated with disease severity were evaluated. Transcriptome data obtained from whole blood by RNA sequencing and type I and II interferon signatures were analyzed for PRECISESADS patients. Results: In the DIApSS cohort, Ro52+/Ro60+ patients showed significantly more parotidomegaly (33.3% vs 0%?11%) along with higher β2-microglobulin (P =0.0002), total immunoglobulin (P <0.0001), and erythrocyte sedimentation rate levels (P =0.002) as well as rheumatoid factor (RF) positivity (66.2% vs 20.8%?25%) compared to other groups. The PRECISESADS cohort corroborated these observations, with increased arthritis (P =0.046), inflammation (P =0.005), hypergammaglobulinemia (P <0.0001), positive RF (P <0.0001), leukopenia (P =0.004), and lymphopenia (P =0.009) in Ro52+/Ro60+ patients. Cumulative EULAR Sjögren Syndrome Disease Activity Index results further confirmed these disparities (P =0.002). Transcriptome analysis linked anti-Ro52/TRIM21 antibody positivity to interferon pathway activation as an underlying cause for these clinical correlations. Conclusion: These results suggest that the combination of anti-Ro52/TRIM21 and anti-Ro60/SSA antibodies is associated with a clinical, biologic, and transcriptional profile linked to greater disease severity in SjD through the potentiation of the interferon pathway activation by anti-Ro52/TRIM21 antibodies.Funding: Supported by the Innovative Medicines Initiative Joint Undertaking (grant 115565 [PRECISESADS project]), resources of which include financial contribution from the European Union’s Seventh Framework Program (grant FP7/2007–2013) and EFPIA companies’ in-kind contribution. LBAI laboratory (Lymphocytes B, Auto-immunité et Immunothérapies) was supported by the Agence Nationale de la Recherche under the “Investissement d’Avenir” program (reference ANR-11-LABX-0016-001 [Labex IGO])

Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.</p