Protein sorting upon exit from the endoplasmic reticulum dominates Golgi biogenesis in budding yeast.

Cells sense and control the number and quality of their organelles, but the underlying mechanisms of this regulation are not understood. Our recent research in the yeast Saccharomyces cerevisiae has shown that long acyl chain ceramides in the endoplasmic reticulum (ER) membrane and the lipid moiety of glycosylphosphatidylinositol (GPI) anchor determine the sorting of GPI-anchored proteins in the ER. Here, we show that a mutant strain, which produces shorter ceramides than the wild-type strain, displays a different count of Golgi cisternae. Moreover, deletions of proteins that remodel the lipid portion of GPI anchors resulted in an abnormal number of Golgi cisternae. Thus, our study reveals that protein sorting in the ER plays a critical role in maintaining Golgi biogenesis.

Clinical Profiling and Biomarkers for Post-Operative Atrial Fibrillation Prediction in Patients Undergoing Cardiac Surgery.

Post-operative atrial fibrillation (POAF) is the most common arrhythmia in the post-operative period after cardiac surgery. We aim to investigate the main clinical, local, and/or peripheral biochemical and molecular predictors for POAF in patients undergoing coronary and/or valve surgery. Between August 2020 and September 2022, consecutive patients undergoing cardiac surgery without previous history of AF were studied. Clinical variables, plasma, and biological tissues (epicardial and subcutaneous fat) were obtained before surgery. Pre-operative markers associated with inflammation, adiposity, atrial stretch, and fibrosis were analyzed on peripheral and local samples with multiplex assay and real-time PCR. Univariate and multivariate logistic regression analyses were performed in order to identify the main predictors for POAF. Patients were followed-up until hospital discharge. Out of 123 consecutive patients without prior AF, 43 (34.9%) developed POAF during hospitalization. The main predictors were cardiopulmonary bypass time (odds ratio (OR) 1.008 (95% confidence interval (CI), 1.002-1.013), p = 0.005), and plasma pre-operative orosomucoid levels (OR 1.008 (1.206-5.761). After studying differences regarding sex, orosomucoid was the best predictor for POAF in women (OR 2.639 (95% CI, 1.455-4.788), p = 0.027) but not in men. The results support the pre-operative inflammation pathway as a factor involved in the risk of POAF, mainly in women.

Impact of sphingolipids on protein membrane trafficking.

Membrane trafficking is essential to maintain the spatiotemporal control of protein and lipid distribution within membrane systems of eukaryotic cells. To achieve their functional destination proteins are sorted and transported into lipid carriers that construct the secretory and endocytic pathways. It is an emerging theme that lipid diversity might exist in part to ensure the homeostasis of these pathways. Sphingolipids, a chemical diverse type of lipids with special physicochemical characteristics have been implicated in the selective transport of proteins. In this review, we will discuss current knowledge about how sphingolipids modulate protein trafficking through the endomembrane systems to guarantee that proteins reach their functional destination and the proposed underlying mechanisms.

GPI anchors: Regulated as needed.

GPI anchoring is an essential post-translational modification in eukaryotes that links proteins to the plasma membrane. In this issue, Liu et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202208159) suggest, for the first time, a regulation on demand of the GPI glycolipid precursor biosynthesis.

The P4-ATPase Drs2 interacts with and stabilizes the multisubunit tethering complex TRAPPIII in yeast.

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.

Relación entre la autoeficacia para dejar de fumar, la voluntad propia y quién envía a los pacientes / Relationship between self-efficacy to quit smoking, own will and who referred patients

Objetivo: Comprobar si el grado de autoeficacia (AE) para dejar de fumar es diferente según quién remita al fumador a la consulta de tabaquismo y evaluar si el grado de AE medida según procedencia tiene impacto en el resultado (éxito o fracaso). Método: Estudio observacional, longitudinal (cohorte prospectiva), multicéntrico, en consultas para dejar de fumar de España y Argentina. Para valorar la motivación, fueron utilizados test cuantitativos (Richmond, Henry Mondor de Paris y Khimji Watts) y una escala semicuantitativa. La AE se midió con una pregunta de dos cuestionarios distintos (pregunta 4 del cuestionario de Richmond y pregunta 3 del cuestionario de Khimji-Watts). El análisis estadístico incluyó modelos descriptivos, de asociación y de regresión. Resultados: Muestra de 182 sujetos (90 [49,5%] mujeres, edad media 51,4 años [DE: 11; IC 26-77]). Con la pregunta 3 de AE del test Khimji-Watts se encontró asociación con la procedencia en los varones: acudir por voluntad propia incrementa la AE frente a los que acuden enviados desde atención primaria o desde otras especialidades. Ninguna de las variables de AE presentó asociación con el resultado en dejar de fumar, pero las mujeres que acudieron por voluntad propia o enviadas desde atención primaria tienen más probabilidades de dejar de fumar. Los varones procedentes de otras especialidades y con edades medias tienen más probabilidades de fracaso. Conclusiones: Los varones que acuden a dejar de fumar por voluntad propia presentan puntuaciones más altas de AE que aquellos que acuden enviados por atención primaria y otras especialidades. No hemos encontrado una mayor abstinencia final según la puntuación de AE y la procedencia.(AU)

Objective: To check whether the degree of self-efficacy to quit smoking is different depending on who refers the smoker to the smoking cessation clinics, considering as origin: primary care, from another medical specialist or by the patient’s own volition, and to assess whether the degree of self-efficacy measured by who refers the patient has an impact on the ­outcome (success or failure). Methods: Observational, longitudinal (prospective cohort), multicentre study in smoking cessation clinics in Spain and Argentina (daily clinical practice). Quantitative tests (Richmond, Henry Mondor de Paris and Khimji Watts) and one semiquantitative test were used to assess motivation. Self-efficacy was measured with one question from two different questionnaires (question 4 of the Richmond questionnaire and question 3 of the Khimji-Watts questionnaire). Statistical analysis included descriptive, association, and regression models. Results: 182 subjects [90 (49.5%) women, mean age 51.4 years (11; 25-77)]. With question three of the auto-efficacy of the Khimji-Watts test an association was found with the origin in men: attending of their own free will increases self-efficacy compared to those who attend sent from primary care or from other specialties. None of the self-efficacy variables was associated with the outcome of quitting smoking, but women who attended of their own free will or referred from primary care are more likely to quit smoking. Men from other specialties and with middle ages are more likely to fail. Conclusions: Men referred for QS by his OV have higher SE scores (KWT3 question) than those referred by PC and OS. We did not find a higher final abstinence according to SE score and source.(AU)

Comorbilidades asociadas a mortalidad o enfermedad severa en pacientes hospitalizados con COVID-19 / Comorbidities associated with mortality or severe disease in hospitalized patients with COVID-19

Objetivo: Conocer las comorbilidades de los pacientes hospitalizados con COVID-19 e identificar cuales se asocian a mayor severidad y/o mortalidad intrahospitalaria. Métodos: Estudio de cohortes retrospectivo en el que se incluyeron todos los pacientes ingresados con COVID-19 desde 1 de marzo del 2020 hasta el 31 mayo de 2020. Se realizó un análisis descriptivo de las comorbilidades y se vio cuales se asocian a una mayor mortalidad intrahospitalaria y/o severidad de la enfermedad mediante un modelo de regresión logística binaria. Resultados: Un total de 336 pacientes fueron incluidos en el estudio de los cuales 52 (15,5%) fallecieron durante el ingreso. Un 58% eran varones, la edad media fue 66 años y el índice Charlson fue de 1. En el análisis multivariante se identificaron como comorbilidades asociadas a mortalidad la edad > 65 años (OR 2,65; p 0,021), el sexo masculino (OR 3,26; p 0,004), la enfermedad cardiovascular ateroesclerótica (OR 2,11; p<0,040) y no ateroesclerótica (OR 6,40; p<0,001) y la neoplasia (OR 5,09; p<0,001). Se asociaron a mayor severidad de la COVID-19 la edad> 65 años (OR 1,87; p 0,033), el sexo masculino (OR 2,86; p <0,001), la obesidad (OR 1,82; p 0,034) y SAOS (OR 5,26; p 0,006). Conclusiones: La enfermedad cardiovascular previa y la neoplasia se asocian a mortalidad intrahospitalaria mientras que la obesidad y el SAOS se asocian a severidad de la enfermedad en pacientes hospitalizados con COVID-19. La edad >65 años y el sexo masculino se asocian a una mayor severidad y mortalidad intrahospitalaria. (AU)

Objective: To evaluate the comorbidities in hospitalized patients with COVID-19 and identify which ones are associated with severe COVID-19 disease and/or in-hospital mortality. Methods: A retrospective cohort study was performed. All patients admitted with confirmed COVID-19 from March 1, 2020 to May 31, 2020 were included. A descriptive analysis of comorbidities was made. We evaluated what comorbidities are associated with in-hospital mortality and/or severe COVID-19 disease using a binary logistic regression model. Results: A total of 336 patients were included in the study: 52 (15,5%) died during hospitalization. Mean age was 66 + 14 years, 58% were men and the Charlson Comorbidity Index was 1. In multivariate analysis, age >65 years (HR 2,65; p 0,021), male sex (HR 3,26; p 0,004), atherosclerotic cardiovascular disease (HR 2,11; p 0,040), non-atherosclerotic cardiovascular disease (HR 6,40; p<0,001) and malignancy (HR 5,09; p< 0,001), were identified as comorbidities associated with in hospital-mortality. Age >65 years (HR 1,87; p 0,033), male sex (HR 2,86; p<0,001), obesity (HR 1,82; p 0,034) and obstructive sleep apnea (HR 5,26; p 0,006) were associated with severe COVID-19 disease. Conclusions: Previous cardiovascular disease and malignancy are risk factors of in-hospital mortality while obesity and obstructive sleep apnea are associated with severe COVID-19 disease in hospitalized patients. Age >65 years and male sex are associated with both. (AU)

Quality-controlled ceramide-based GPI-anchored protein sorting into selective ER exit sites.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) exit the endoplasmic reticulum (ER) through a specialized export pathway in the yeast Saccharomyces cerevisiae. We have recently shown that a very-long acyl chain (C26) ceramide present in the ER membrane drives clustering and sorting of GPI-APs into selective ER exit sites (ERES). Now, we show that this lipid-based ER sorting also involves the C26 ceramide as a lipid moiety of GPI-APs, which is incorporated into the GPI anchor through a lipid-remodeling process after protein attachment in the ER. Moreover, we also show that a GPI-AP with a C26 ceramide moiety is monitored by the GPI-glycan remodelase Ted1, which, in turn, is required for receptor-mediated export of GPI-APs. Therefore, our study reveals a quality-control system that ensures lipid-based sorting of GPI-APs into selective ERESs for differential ER export, highlighting the physiological need for this specific export pathway.

CD4+ and CD8+ T-cell responses in bone marrow to fatty acids in high-fat diets.

Obesity is associated with disruptions in the adaptive immune system; however, dietary fatty acids in high-fat diets (HFDs) that induce obesity have consequences that are currently unclear regarding T-cell maintenance in bone marrow (BM). C57BL/6J mice were randomly assigned to isocaloric HFDs formulated with dietary fats rich in saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), or MUFAs supplemented with eicosapentaenoic and docosahexaenoic acids for 20 weeks, followed by an analysis of the immunophenotypic feature of lymphocytes (CD3+) T and their subsets CD4+ and CD8+ T cells in spleen and BM, identification of fatty acids in BM extracellular fluid and analysis of the correspondence between fatty acids with the frequency of T-cell subsets in BM. Splenic CD3+ T cells were reduced irrespective of HFDs. In BM, CD3+ T cells were reduced after HFD-SFAs, while CD4+ T cells were increased after HFDs enriched in MUFAs and CD8+ T cells were reduced irrespective of HFDs. In BM extracellular fluid, the content of palmitic and myristic acids increased after HFD-SFAs and that of oleic acid increased after HFDs enriched in MUFAs. There was a statistical correspondence between HFD-induced changes in fatty acids in BM extracellular fluid and HFD-induced changes in the frequency of CD3+ and CD4+ T cells in BM. These findings reveal an undervalued critical role for dietary fatty acids in the selective acquisition of T-cell subsets in BM, highlighting that oleic acid existing in the surroundings of T-cell niches during HFD-induced obesity could be instrumental in the maintenance of CD4+ T cells.

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway.

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to stabilize the transient and/or weak protein interactions. Here, we describe a protocol of protein crosslinking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.

[Positioning Document of Spanish Society of Pulmonology and Thoracic Surgery (SEPAR) on Tobacco Harm Reduction Strategies]. / Documento de posicionamiento de la Sociedad Española de Neumología y Cirugía Torácica (SEPAR) ante las estrategias de reducción del daño del tabaco.

In the last decade, the appearance of electronic cigarettes and new tobacco products (heated tobacco or smokeless tobacco) has generated a growing interest in harm reduction methods that are defined as mechanisms that seek to reduce the harmful consequences of tobacco without give up the pleasant effects of it by replacing it with these new electronic devices. However, these products are addictive and not safe as they contain nicotine. Harm reduction is a false solution as it represents a commercial strategy of the tobacco industry to increase its sales, making it difficult to control smoking, since it keeps smokers from consuming tobacco and prevents them from making serious attempts to quit. In addition, these products are a gateway to adolescents in tobacco.

A Role for Lipids in Protein Sorting?

Lipid and protein diversity provides structural and functional identity to the membrane compartments that define the eukaryotic cell. This compositional heterogeneity is maintained by the secretory pathway, which feeds newly synthesized proteins and lipids to the endomembrane systems. The precise sorting of lipids and proteins through the pathway guarantees the achievement of their correct delivery. Although proteins have been shown to be key for sorting mechanisms, whether and how lipids contribute to this process is still an open discussion. Our laboratory, in collaboration with other groups, has recently addressed the long-postulated role of membrane lipids in protein sorting in the secretory pathway, by investigating in yeast how a special class of lipid-linked cell surface proteins are differentially exported from the endoplasmic reticulum. Here we comment on this interdisciplinary study that highlights the role of lipid diversity and the importance of protein-lipid interactions in sorting processes at the cell membrane.

Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).

Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into different endoplasmic reticulum (ER) exit sites (ERES) for their differential ER export to the Golgi apparatus. We used an optimized protocol that combines a live cell dual-cargo ER export system with a 3D simultaneous multi-color high-resolution live cell microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking COPII function upon incubation of the thermo-sensitive COPII allele sec31-1 at restrictive temperature (37°C). ER export is restored by shifting down to permissive temperature (24°C) and progressive incorporation of the two different types of cargos into the fluorescently labelled ERES can be then simultaneously captured at 3D high spatial resolution by SCLIM microscopy. By using this protocol, we have shown that newly synthesized glycosylphosphatidylinositol (GPI)-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized ERES that are distinct from those used by transmembrane secretory proteins. Furthermore, we showed that the chain length of the ceramide present in the ER membrane is critical for this sorting selectivity. Therefore, thanks to the presented method we could obtain the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective ERES.

Structural analysis of the GPI glycan.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes a specialized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis and analysis of the peptides bearing GPI glycans by mass spectrometry after trypsin digestion. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm predicted GPI-anchoring of individual proteins.