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Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.
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Autofagia , Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestructura , Línea Celular Tumoral , Microdominios de Membrana/metabolismo , Exosomas/metabolismo , Exosomas/ultraestructura , Tetraspanina 30/metabolismo , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
Antiphospholipid antibody syndrome is an autoimmune disease characterized by thrombosis and/or pregnancy morbidity in association with circulating antiphospholipid antibodies, mainly anti-ß2 glycoprotein 1 antibodies (anti-ß2-GPI antibodies). Previous studies demonstrated that the signaling pathway may involve lipid rafts, plasma membrane microdomains enriched in glycosphingolipid and cholesterol. In this study, we analyzed the signaling pathway of LRP8/ApoER2, a putative receptor of anti-ß2-GPI antibodies, through lipid rafts in human endothelial cells. LRP8, Dab2 and endothelial nitric oxide synthase (e-NOS) phosphorylation were evaluated using Western blot, Nitric Oxide (NO) production with cytofluorimetric analysis, LRP8 enrichment in lipid rafts via sucrose gradient fractionation, and scanning confocal microscopy analysis of its association with ganglioside GM1 was also conducted. The analyses demonstrated that affinity-purified anti-ß2-GPI antibodies induced LRP8 and Dab-2 phosphorylation, together with a significant decrease in e-NOS phosphorylation, with consequent decrease in NO intracellular production. These effects were almost completely prevented by Methyl-ß-cyclodextrin (MßCD), indicating the involvement of lipid rafts. It was supported with the observation of LRP8 enrichment in lipid raft fractions and its association with ganglioside GM1, detected with scanning confocal microscopy. These findings demonstrate that LRP8 signaling triggered by anti-ß2-GPI antibodies in endothelial cells occurs through lipid rafts. It represents a new task for valuable therapeutic approaches, such as raft-targeted therapy, including cyclodextrins and statins.
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Cell outer membranes contain glycosphingolipids and protein receptors, which are integrated into glycoprotein domains, known as lipid rafts, which are involved in a variety of cellular processes, including receptor-mediated signal transduction and cellular differentiation process. In this study, we analyzed the lipidic composition of human Dental Pulp-Derived Stem Cells (DPSCs), and the role of lipid rafts during the multilineage differentiation process. The relative quantification of lipid metabolites in the organic fraction of DPSCs, performed by Nuclear Magnetic Resonance (NMR) spectroscopy, showed that mono-unsaturated fatty acids (MUFAs) were the most representative species in the total pool of acyl chains, compared to polyunsatured fatty acids (PUFAs). In addition, the stimulation of DPSCs with different culture media induces a multilineage differentiation process, determining changes in the gangliosides pattern. To understand the functional role of lipid rafts during multilineage differentiation, DPSCs were pretreated with a typical lipid raft affecting agent (MßCD). Subsequently, DPSCs were inducted to differentiate into osteoblast, chondroblast and adipoblast cells with specific media. We observed that raft-affecting agent MßCD prevented AKT activation and the expression of lineage-specific mRNA such as OSX, PPARγ2, and SOX9 during multilineage differentiation. Moreover, this compound significantly prevented the tri-lineage differentiation induced by specific stimuli, indicating that lipid raft integrity is essential for DPSCs differentiation. These results suggest that lipid rafts alteration may affect the signaling pathway activated, preventing multilineage differentiation.
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Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, characterized by persistent joint inflammation, leading to cartilage and bone destruction. Autoantibody production is directed to post-translational modified (PTM) proteins, i.e., citrullinated or carbamylated. Autophagy may be the common feature in several types of stress (smoking, joint injury, and infections) and may be involved in post-translational modifications (PTMs) in proteins and the generation of citrullinated and carbamylated peptides recognized by the immune system in RA patients, with a consequent breakage of tolerance. Interestingly, autophagy actively provides information to neighboring cells via a process called secretory autophagy. Secretory autophagy combines the autophagy machinery with the secretion of cellular content via extracellular vesicles (EVs). A role for exosomes in RA pathogenesis has been recently demonstrated. Exosomes are involved in intercellular communications, and upregulated proteins and RNAs may contribute to the development of inflammatory arthritis and the progression of RA. In RA, most of the exosomes are produced by leukocytes and synoviocytes, which are loaded with PTM proteins, mainly citrullinated proteins, inflammatory molecules, and enzymes that are implicated in RA pathogenesis. Microvesicles derived from cell plasma membrane may also be loaded with PTM proteins, playing a role in the immunopathogenesis of RA. An analysis of changes in EV profiles, including PTM proteins, could be a useful tool for the prevention of inflammation in RA patients and help in the discovery of personalized medicine.
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Artritis Reumatoide , Exosomas , Vesículas Extracelulares , Humanos , Artritis Reumatoide/etiología , Autofagia , InflamaciónRESUMEN
BACKGROUND: Heparanase (HPSE) is an endo-ß-glucuronidase that cleaves heparan sulfate side chains, leading to the disassembly of the extracellular matrix, facilitating cell invasion and metastasis dissemination. In this research, we investigated the role of a new HPSE inhibitor, RDS 3337, in the regulation of the autophagic process and the balance between apoptosis and autophagy in U87 glioblastoma cells. METHODS: After treatment with RDS 3337, cell lysates were analyzed for autophagy and apoptosis-related proteins by Western blot. RESULTS: We observed, firstly, that LC3II expression increased in U87 cells incubated with RDS 3337, together with a significant increase of p62/SQSTM1 levels, indicating that RDS 3337 could act through the inhibition of autophagic-lysosomal flux of LC3-II, thereby leading to accumulation of lipidated LC3-II form. Conversely, the suppression of autophagic flux could activate apoptosis mechanisms, as revealed by the activation of caspase 3, the increased level of cleaved Parp1, and DNA fragmentation. CONCLUSIONS: These findings support the notion that HPSE promotes autophagy, providing evidence that RDS 3337 blocks autophagic flux. It indicates a role for HPSE inhibitors in the balance between apoptosis and autophagy in U87 human glioblastoma cells, suggesting a potential role for this new class of compounds in the control of tumor growth progression.
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Glioblastoma , Humanos , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia , Línea Celular Tumoral , Glioblastoma/metabolismo , Glucuronidasa/antagonistas & inhibidores , Glucuronidasa/metabolismoRESUMEN
Background: Several viral and bacterial infections, including COVID-19, may lead to both thrombotic and hemorrhagic complications. Previously, it has been demonstrated an "in vitro" pathogenic effect of "antiphospholipid" antibodies (aPLs), which are able to activate a proinflammatory and procoagulant phenotype in monocytes, endothelial cells and platelets. This study analyzed the occurrence of aPL IgG in patients with acute ischemic stroke (AIS) during COVID-19, evaluating the effect of Ig fractions from these patients on signaling and functional activation of platelets. Materials and methods: Sera from 10 patients with AIS during COVID-19, 10 non-COVID-19 stroke patients, 20 COVID-19 and 30 healthy donors (HD) were analyzed for anti-cardiolipin, anti-ß2-GPI, anti-phosphatidylserine/prothrombin and anti-vimentin/CL antibodies by ELISA. Platelets from healthy donors were incubated with Ig fractions from these patients or with polyclonal anti-ß2-GPI IgG and analyzed for phospho-ERK and phospho-p38 by western blot. Platelet secretion by ATP release dosage was also evaluated. Results: We demonstrated the presence of aPLs IgG in sera of patients with AIS during COVID-19. Treatment with the Ig fractions from these patients or with polyclonal anti-ß2-GPI IgG induced a significant increase of phospho-ERK and phospho-p38 expression. In the same vein, platelet activation was supported by the increase of adenyl nucleotides release induced by Ig fractions. Conclusions: This study demonstrates the presence of aPLs in a subgroup of COVID-19 patients who presented AIS, suggesting a role in the mechanisms contributing to hypercoagulable state in these patients. Detecting these antibodies as a serological marker to check and monitor COVID-19 may contribute to improve the risk stratification of thromboembolic manifestations in these patients.
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Síndrome Antifosfolípido , COVID-19 , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Humanos , Células Endoteliales , COVID-19/complicaciones , Anticuerpos Antifosfolípidos , beta 2 Glicoproteína I , Activación Plaquetaria , Accidente Cerebrovascular/complicaciones , Transducción de Señal , Inmunoglobulina GRESUMEN
The pathological features of antiphospholipid syndrome (APS) are related to the activity of circulating antiphospholipid antibodies (aPLs) associated with vascular thrombosis and obstetric complications. Indeed, aPLs are not only disease markers, but also play a determining pathogenetic role in APS and exert their effects through the activation of cells and coagulation factors and inflammatory mediators for the materialization of the thromboinflammatory pathogenetic mechanism. Cellular activation in APS necessarily involves the interaction of aPLs with target receptors on the cell membrane, capable of triggering the signal transduction pathway(s). This interaction occurs at specific microdomains of the cell plasma membrane called lipid rafts. In this review, we focus on the key role of lipid rafts as signaling platforms in the pathogenesis of APS, and propose this pathogenetic step as a strategic target of new therapies in order to improve classical anti-thrombotic approaches with "new" immunomodulatory drugs.
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OBJECTIVES: To investigate the expression of citrullinated and carbamylated proteins in extracellular microvesicles (EMVs) from RA patients. METHODS: We enrolled 24 RA naïve for biological therapy and 20 healthy donors (HD), matched for age and sex. For each patient, laboratory and clinical data were recorded and clinical indexes were measured (Clinical Disease Activity Index, Simplified Disease Activity Index, DAS28). EMVs in RA patients and HD were purified from plasma and measured by nanoparticle tracking analysis (NanoSight). Further, EMVs were incubated with anti-citrullinated/carbamylated proteins antibodies and processed by flow cytometry and western blot to evaluate the expression of citrullinated/carbamylated antigens. RESULTS: NanoSight revealed a significant increase of EMVs in RA compared with HD. Moreover, cytofluorimetric analysis showed a significative higher expression of citrullinated antigens on EMVs' surface in RA than donors, while no substantial difference was found in the expression of carbamylated antigens. These data were confirmed by western blot which identified vimentin, glycolytic enzyme alpha-enolase 1 and collagen type II as the main citrullinated and carbamylated proteins carried by EMVs. Finally, a relevant correlation between the expression of citrullinated antigens and disease activity was found. CONCLUSIONS: The results of this study suggest an involvement of EMVs in the pathogenesis of RA by inducing autoimmunity.
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Artritis Reumatoide , Autoanticuerpos , Humanos , Autoantígenos , Western Blotting , Colágeno Tipo IIRESUMEN
The scenario of proteogenomics is rapidly evolving and novel technologies are enabling comprehensive molecular exploration down to single cells. Likewise, digital (immuno-)assays are revolutionizing the field of biomarker detection and have reached the grade for population-level screenings with single-molecule sensitivity. Nonetheless, cost- and time-effective, high-throughput targeted phospho-proteomics at a preclinical stage still relies on ad hoc microarray platforms, such as the Reverse-Phase Protein microArrays (RPPA). Although this technique requires specific knowledge and equipment and different laboratories worldwide have implemented alternative methodological strategies, the application of RPPA to biomarker discovery has proven successful on diverse types of samples, including tissues and biological fluids as well as nanovesicles and in vitro cultured lines. Among these, cancer stem(-like) cells (CSC) represent an ideal experimental model system for preclinical discovery and definition of novel drug targets. The present methodological article provides the basic knowledge and steps on how to deploy an RPPA analysis with specific reference to an ideal experimental setup of drug testing on CSC.
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Neoplasias , Análisis por Matrices de Proteínas , Biomarcadores , Neoplasias/metabolismo , Células Madre Neoplásicas , Análisis por Matrices de Proteínas/métodos , Proteómica/métodosRESUMEN
Antiphospholipid syndrome (APS), characterized by artherial and/or venous thrombosis, pregnancy morbidity and "antiphospholipid" antibodies (aPLs), is more common in women than in men, with a female to male ratio of about 3.5:1. Only few studies have investigated the clinical differences between male and female patients with APS. Therefore, this study was aimed to analyze the differences of clinical manifestations and laboratory tests, at diagnosis, between female and male APS patients and the clinical outcome. We enrolled 191 consecutive APS patients (125 with primary APS, PAPS, and 66 with secondary APS, SAPS) with a female predominant ratio of approximately 3:1 (142 vs 49). The prevalence of PAPS was higher in males than females (p<0.001). The analysis of aPL profile revealed that high IgM anti-cardiolipin (aCL) and high-medium IgG aCL titers were more frequent in males. In thrombotic APS peripheral arterial thrombosis was more common in male than female patients (p=0.049), as well as myocardial infarction (p=0.031). Multivariate analysis to correct for cardiovascular risk factors, high titer of aPLs and triple positivity for aPLs, revealed that the odds ratio for myocardial infarction in male was 3.77. Thus, APS may be considered as a disease in which serological (IgM titer) and clinical profiles are influenced by gender.
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Síndrome Antifosfolípido , Infarto del Miocardio , Trombosis , Anticuerpos Antifosfolípidos , Síndrome Antifosfolípido/complicaciones , Síndrome Antifosfolípido/diagnóstico , Síndrome Antifosfolípido/epidemiología , Cardiolipinas , Femenino , Humanos , Inmunoglobulina M , Masculino , Infarto del Miocardio/complicaciones , Embarazo , Factores SexualesRESUMEN
Objective: Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, a novel syndrome known as a multisystem inflammatory syndrome in children (MIS-C) was reported in previously healthy children. A possible pro-inflammatory molecule, high-mobility group box 1 (HMGB1), may be assumed to play an important role in the pathogenesis and clinical presentation of MIS-C. We described the clinical picture of patients with MIS-C and we also aimed to test and compare HMGB1 serum levels of MIS-C patients with those of patients with previous SARS-CoV2 infection and healthy children. Study design: We determined HMGB1 levels by Western blot in 46 patients and divided them into three groups, namely, five patients with MIS-C (median age: 8.36 years), 20 children with a history of SARS-CoV-2 infection (median age: 10.45 years), and 21 healthy children (controls) (median age: 4.84 years), without evidence of respiratory infection in the last 3 months. Results: The median level of HMGB1 in the serum of five patients with MIS-C was found to be significantly higher compared with both patients with a recent history of COVID-19 (1,151.38 vs. 545.90 densitometric units (DU), p = 0.001) and control (1,151.38 vs. 320.33 DU, p = 0.001) groups. The HMGB1 level in MIS-C patients with coronary involvement had a slightly higher value with respect to patients without coronary dilatation (1,225.36 vs. 1,030.49 DU, p = 0.248). In two of the five children with MIS-C that performed a follow-up, the HMGB1 value decreased to levels that were superimposable to the ones of the control group. Conclusion: The significantly high level of HMGB1 protein found in the serum of COVID-19 and patients with MIS-C supports its involvement in inflammatory manifestations, suggesting HMGB1 as a potential biomarker and therapeutic target in patients with severe illness.
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In this study we analyzed whether anti-ß2-GPI antibodies from patients with APS induce the endothelial cell expression of Tissue Factor (TF) by a LRP6 signal transduction pathway involving lipid rafts. HUVEC were stimulated with affinity purified anti-ß2-GPI antibodies. Both LRP6 and ß-catenin phosphorylation, as well as TF expression, were evaluated by western blot. Results demonstrated that triggering with affinity purified anti-ß2-GPI antibodies induced LRP6 phosphorylation with consequent ß-catenin activation, leading to TF expression on the cell surface. Interestingly, the lipid rafts affecting agent methyl-ß-cyclodextrin as well as the LRP6 inhibitor Dickkopf 1 (DKK1) partially reduced the anti-ß2-GPI antibodies effect, indicating that the anti-ß2-GPI effects on TF expression may depend on a signalling transduction pathway involving both lipid rafts and LRP6. An interaction between ß2-GPI, LRP6 and PAR-2 within these microdomains was demonstrated by gradient fractionation and coimmunoprecipitation experiments. Thus, anti-ß2-GPI antibodies react with their target antigen likely associated to LRP6 and PAR-2 within plasma membrane lipid rafts of the endothelial cell. Anti-ß2-GPI binding triggers ß-catenin phosphorylation, leading to a procoagulant phenotype characterized by TF expression. These findings deal with a novel signal transduction pathway which provides new insight in the APS pathogenesis, improving the knowledge of valuable therapeutic target(s).
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Síndrome Antifosfolípido , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Microdominios de Membrana , Transducción de Señal , Tromboplastina , Células Endoteliales/metabolismo , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Microdominios de Membrana/metabolismo , Tromboplastina/metabolismo , beta 2 Glicoproteína I , beta Catenina/metabolismoRESUMEN
In this article, we present data on the proteome of human neuroblastoma cells stably overexpressing Neuroglobin (NGB). The neuroprotective role of NGB is clearly established, nevertheless the related mechanistic processes, which are dependent on NGB overexpression, are not known. To address this question, we performed shotgun label-free quantification (LFQ) proteomics using an SH-SY5Y cell model of neuroblastoma that overexpresses an NGB-FLAG construct, and wild type cells transfected with an empty vector as control (CTRL). The proteomes from six biological samples per condition were digested using the S-Trap sample preparation followed by LC-MS/MS analysis with a LTQ-Orbitrap XL mass spectrometer. The quantitative analysis was performed using the LFQ algorithm of MaxQuant, leading to 1654 correctly quantified proteins over 2580 identified proteins. Finally, the statistic comparison of the two analyzed groups within Perseus platform identified 178 differential proteins (107 up- and 71 down-regulated). In addition, multivariate statistical analysis was carried out using MetaboAnalyst 5.0 software. MS proteomics data are available via ProteomeXchange with the dataset identifier PXD029012.
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OBJECTIVES: Antiphospholipid syndrome (APS) is a prothrombotic condition defined by recurrent thrombosis, pregnancy complications and circulating antiphospholipid antibodies (aPL), including anti-ß2-glycoprotein I (ß2-GPI). In clinical practice it is possible to find patients with APS persistently negative for the aPL tests according to Sydney criteria ('seronegative APS', SN-APS). Recently, several autoimmune responses have been described as a consequence of post-translational modifications of their target autoantigens. This study was undertaken to test carbamylated-ß2-GPI (Carb-ß2-GPI) as a new autoantigen of APS. METHODS: ß2-GPI was carbamylated by potassium cyanate and used to investigate its effect on monocyte-derived dendritic cell (moDC) phenotype and function. Sera from 114 SN-APS patients, 60 APS, 20 patients with RA, 20 non-APS thrombosis and 50 healthy donors were analysed for anti-Carb-ß2-GPI by ELISA. RESULTS: Carb-ß2-GPI is able to activate moDCs, inducing upregulation of CD80, CD86 and CD40, activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase and nuclear factor-κB, and IL-12p70 release. Serological results showed that both 37/114 SN-APS (32.46%) and 23/60 APS (38.33%) patients resulted positive for anti-Carb-ß2-GPI. Interestingly, SN-APS patients who tested positive for anti-Carb-ß2-GPI showed a higher prevalence of thrombocytopenia (P = 0.04, likelihood positive ratio of 3.9). CONCLUSION: Data obtained from both functional tests on moDCs and immunological approaches prompted identification of Carb-ß2-GPI as a 'new' antigenic target in APS. In particular, anti-Carb-ß2-GPI revealed a potential usefulness in identification of a significant proportion of SN-APS patients. Moreover, since patients who tested positive for anti-Carb-ß2-GPI reported a high risk of thrombocytopenia, this test may be considered a suitable approach in the clinical evaluation of SN-APS.
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Síndrome Antifosfolípido , Trombocitopenia , Trombosis , Anticuerpos Antifosfolípidos , Síndrome Antifosfolípido/complicaciones , Autoantígenos , Quinasas MAP Reguladas por Señal Extracelular , Femenino , Humanos , FN-kappa B , Embarazo , Carbamilación de Proteína , Trombocitopenia/complicaciones , Trombosis/etiología , beta 2 Glicoproteína I , Proteínas Quinasas p38 Activadas por MitógenosAsunto(s)
Plaquetas , Tromboplastina , Células Endoteliales , Glucuronidasa , beta 2 Glicoproteína IAsunto(s)
COVID-19 , Trombocitopenia , Vacunas contra la COVID-19 , Humanos , Activación Plaquetaria , SARS-CoV-2 , Transducción de Señal , VacunaciónRESUMEN
Neuroglobin (NGB) is an O2-binding globin mainly expressed in the central and peripheral nervous systems and cerebrospinal fluid. Previously, it was demonstrated that NGB overexpression protects cells from hypoxia-induced death. To investigate processes promoted by NGB overexpression, we used a cellular model of neuroblastoma stably overexpressing an NGB-FLAG construct. We used a proteomic approach to identify the specific profile following NGB overexpression. To evaluate the role of NGB overexpression in increasing energetic metabolism, we measured oxygen consumption rate (OCR) and the extracellular acidification rate through Seahorse XF technology. The effect on autophagy induction was evaluated by analyzing SQSTM1/p62 and LC3-II expression. Proteomic analysis revealed several differentially regulated proteins, involved in oxidative phosphorylation and integral mitochondrial proteins linked to energy metabolism. The analysis of mitochondrial metabolism demonstrated that NGB overexpression increases mitochondrial ATP production. Indeed, NGB overexpression enhances bioenergetic metabolism, increasing OCR and oxygen consumption. Analysis of autophagy induction revealed an increase of LC3-II together with a significant decrease of SQSTM1/p62, and NGB-LC3-II association during autophagosome formation. These results highlight the active participation of NGB in several cellular processes that can be upregulated in response to NGB overexpression, playing a role in the adaptive response to stress in neuroblastoma cells.
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Autofagia/genética , Proteínas Asociadas a Microtúbulos/genética , Neuroblastoma/genética , Neuroglobina/genética , Proteína Sequestosoma-1/genética , Adenosina Trifosfato/genética , Línea Celular Tumoral , Metabolismo Energético/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Mitocondrias/genética , Neuroblastoma/patología , Consumo de Oxígeno/genética , Proteoma/genéticaRESUMEN
ER lipid raft-associated protein 1 (ERLIN1) and 2 (ERLIN2) are 40 kDa transmembrane glycoproteins belonging to the family of prohibitins, containing a PHB domain. They are generally localized in the endoplasmic reticulum (ER), where ERLIN1 forms a heteroligomeric complex with its closely related ERLIN2. Well-defined functions of ERLINS are promotion of ER-associated protein degradation, mediation of inositol 1,4,5-trisphosphate (IP3) receptors, processing and regulation of lipid metabolism. Until now, ERLINs have been exclusively considered protein markers of ER lipid raft-like microdomains. However, under pathophysiological conditions, they have been described within mitochondria-associated endoplasmic reticulum membranes (MAMs), tethering sites between ER and mitochondria, characterized by the presence of specialized raft-like subdomains enriched in cholesterol and gangliosides, which play a key role in the membrane scrambling and function. In this context, it is emerging that ER lipid raft-like microdomains proteins, i.e., ERLINs, may drive mitochondria-ER crosstalk under both physiological and pathological conditions by association with MAMs, regulating the two main processes underlined, survival and death. In this review, we describe the role of ERLINs in determining cell fate by controlling the "interchange" between apoptosis and autophagy pathways, considering that their alteration has a significant impact on the pathogenesis of several human diseases.
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Señalización del Calcio , Retículo Endoplásmico/fisiología , Metabolismo de los Lípidos , Microdominios de Membrana/fisiología , Membranas Mitocondriales/fisiología , Proteínas del Tejido Nervioso/metabolismo , Apoptosis , Autofagia , Humanos , Proteínas del Tejido Nervioso/genética , ProhibitinasRESUMEN
Intrinsic disorder is a natural feature of polypeptide chains, resulting in the lack of a defined three-dimensional structure. Conformational changes in intrinsically disordered regions of a protein lead to unstable ß-sheet enriched intermediates, which are stabilized by intermolecular interactions with other ß-sheet enriched molecules, producing stable proteinaceous aggregates. Upon misfolding, several pathways may be undertaken depending on the composition of the amino acidic string and the surrounding environment, leading to different structures. Accumulating evidence is suggesting that the conformational state of a protein may initiate signalling pathways involved both in pathology and physiology. In this review, we will summarize the heterogeneity of structures that are produced from intrinsically disordered protein domains and highlight the routes that lead to the formation of physiological liquid droplets as well as pathogenic aggregates. The most common proteins found in aggregates in neurodegenerative diseases and their structural variability will be addressed. We will further evaluate the clinical relevance and future applications of the study of the structural heterogeneity of protein aggregates, which may aid the understanding of the phenotypic diversity observed in neurodegenerative disorders.
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Enfermedades Neurodegenerativas/genética , Agregado de Proteínas/genética , Agregación Patológica de Proteínas/genética , Conformación Proteica en Lámina beta , Amiloide/genética , Amiloide/ultraestructura , Humanos , Proteínas Intrínsecamente Desordenadas , Enfermedades Neurodegenerativas/patología , alfa-Sinucleína/genética , alfa-Sinucleína/ultraestructura , Proteínas tau/genética , Proteínas tau/ultraestructuraRESUMEN
Anti-phospholipid syndrome (APS) is a systemic autoimmune disorder defined by the simultaneous presence of vascular clinical events, pregnancy morbidity and anti-phospholipid antibodies (aPL). In clinical practice, it is possible to find patients with APS who are persistently negative for the routine aPL tests (seronegative APS; SN-APS). Recently, the identification of aPL immunoglobulin (Ig)A and/or anti-ß2-glycoprotein-I (ß2-GPI) IgA was shown to represent a further test in SN-APS patients. In this study we analyzed the presence of anti-vimentin/cardiolipin (aVim/CL) IgA in a large cohort of patients with SN-APS, evaluating their possible association with clinical manifestations of the syndrome. This study includes 60 consecutive SN-APS patients, 30 patients with APS and 40 healthy donors. aVim/CL IgA were detected by enzyme-linked immunosorbent assay (ELISA). Results show that 12 of 30 APS patients (40%) and 16 of 60 SN-APS patients (26.7%) resulted positive for aVim/CL IgA. Interestingly, SN-APS patients who tested positive for aVim/CL IgA showed a higher prevalence of arterial thrombosis (p = 0.017, likelihood positive ratio = 5.7). This study demonstrates for the first time, to our knowledge, the presence of aVim/CL IgA in sera of patients with APS. In particular, they revealed a potential usefulness in identification of a significant proportion of SN-APS patients. Moreover, as patients tested positive for aVim/CL IgA reported a high likelihood ratio to have the clinical features of APS, this test may be considered a suitable approach in the clinical evaluation of SN-APS.