RESUMEN
Antigen-specific class-switched antibodies are detected at the same time or even before IgM in serum of non-vaccinated individuals infected with SARS-CoV-2. These derive from the first wave of plasmablasts formed. Hence, the phenotype and specificity of plasmablasts can reveal information about early B-cell activation. Here we have analyzed B cells and plasmablasts circulating in blood of COVID-19 patients not previously exposed to SARS-CoV-2 during and after disease. We find that during infection with the original Wuhan strain, plasmablasts in blood produce IgA1, IgG1, and IgM, and that most express CCR10 and integrin ß1, only some integrin ß7, while the majority lack CCR9. Plasmablast-secreted antibodies are reactive to the spike (S) and nucleocapsid (N) proteins of the Wuhan strain as well as later variants of concern, but also bind S proteins from endemic and non-circulating betacoronaviruses. In contrast, after recovery, antibodies produced from memory B cells target variants of SARS-CoV-2 and SARS-CoV-1 but compared to previously non-infected individuals do not show increased binding to endemic coronaviruses. This suggests that the early antibody response to a large extent stems from pre-existing cross-reactive class-switched memory B cells, and that although newly formed memory cells target the novel SARS-CoV-2 virus the numbers of broadly cross-reactive memory B cells do not increase extensively. The observations give insight into the role of pre-existing memory B cells in early antibody responses to novel pathogens and may explain why class-switched antibodies are detected early in the serum of COVID-19 patients.
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COVID-19 , SARS-CoV-2 , Humanos , Inmunoglobulina G , Inmunoglobulina M , Anticuerpos Antivirales , Anticuerpos NeutralizantesRESUMEN
The atypical protein kinase C isoform ι (PKCι) is upregulated, which cooperates with mutated KRAS (mu-KRAS) to promote the development of pancreatic cancers. However, the exact role of PKCι in KRAS-mediated pancreatic tumorigenesis is not fully defined. In the present study, we demonstrate that mu-KRAS upregulates and activates PKCι, accompanied by dephosphorylation of large tumor suppressor (LATS), a key member of the growth-inhibiting Hippo signaling pathway. As a result, Yes-associated protein 1 (YAP1; a transcriptional coactivator) is dephosphorylated and translocates to the nucleus, which promotes transcription of downstream target genes to sustain the transformed growth of pancreatic cancer cells. In contrast, when PKCι is suppressed by the chemical inhibitor or small-hairpin RNA, the levels of phosphorylated LATS and YAP1 are elevated and YAP1 is excluded from the nucleus, which enhances the susceptibility of pancreatic cancer cells harboring mu-KRAS to apoptosis. These findings shed new light on the mechanisms underlying the pancreatic tumorigenesis initiated by mu-KRAS, and suggest that the PKCι-YAP1 signaling may potentially be therapeutically targeted for restricting the growth and inducing apoptosis in pancreatic tumors expressing mu-KRAS.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Isoenzimas/metabolismo , Neoplasias Pancreáticas/patología , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Factores de Transcripción/metabolismo , Animales , Apoptosis/fisiología , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias Pancreáticas/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Señalizadoras YAPRESUMEN
There is growing evidence that macroautophagic cargo is not limited to bulk cytosol in response to starvation and can occur selectively for substrates, including aggregated proteins. It remains unclear, however, whether starvation-induced and selective macroautophagy share identical adaptor molecules to capture their cargo. Here, we report that Alfy, a phosphatidylinositol 3-phosphate-binding protein, is central to the selective elimination of aggregated proteins. We report that the loss of Alfy inhibits the clearance of inclusions, with little to no effect on the starvation response. Alfy is recruited to intracellular inclusions and scaffolds a complex between p62(SQSTM1)-positive proteins and the autophagic effectors Atg5, Atg12, Atg16L, and LC3. Alfy overexpression leads to elimination of aggregates in an Atg5-dependent manner and, likewise, to protection in a neuronal and Drosophila model of polyglutamine toxicity. We propose that Alfy plays a key role in selective macroautophagy by bridging cargo to the molecular machinery that builds autophagosomes.
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Autofagia/fisiología , Proteínas de la Membrana/metabolismo , Proteínas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Relacionadas con la Autofagia , Proteínas Portadoras/metabolismo , Humanos , Proteínas de la Membrana/genética , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
The identity of the cells that contribute to brain tumor structure and progression remains unclear. Mesenchymal stem cells (MSCs) have recently been isolated from normal mouse brain. Here, we report the infiltration of MSC-like cells into the GL261 murine glioma model. These brain tumor-derived mesenchymal stem cells (BT-MSCs) are defined with the phenotype (Lin-Sca-1+CD9+CD44+CD166+/-) and have multipotent differentiation capacity. We show that the infiltration of BT-MSCs correlates to tumor progression; furthermore, BT-MSCs increased the proliferation rate of GL261 cells in vitro. For the first time, we report that the majority of GL261 cells expressed mesenchymal phenotype under both adherent and sphere culture conditions in vitro and that the non-MSC population is nontumorigenic in vivo. Although the GL261 cell line expressed mesenchymal phenotype markers in vitro, most BT-MSCs are recruited cells from host origin in both wild-type GL261 inoculated into green fluorescent protein (GFP)-transgenic mice and GL261-GFP cells inoculated into wild-type mice. We show the expression of chemokine receptors CXCR4 and CXCR6 on different recruited cell populations. In vivo, the GL261 cells change marker profile and acquire a phenotype that is more similar to cells growing in sphere culture conditions. Finally, we identify a BT-MSC population in human glioblastoma that is CD44+CD9+CD166+ both in freshly isolated and culture-expanded cells. Our data indicate that cells with MSC-like phenotype infiltrate into the tumor stroma and play an important role in tumor cell growth in vitro and in vivo. Thus, we suggest that targeting BT-MSCs could be a possible strategy for treating glioblastoma patients.
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Neoplasias Encefálicas/patología , Encéfalo/patología , Glioma/patología , Células Madre Mesenquimatosas/patología , Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Animales , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Células Cultivadas , Progresión de la Enfermedad , Citometría de Flujo , Glioma/genética , Glioma/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Receptores de Hialuranos/metabolismo , Inmunofenotipificación , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Transgénicos , Microscopía Confocal , Células Madre Multipotentes/metabolismo , Células Madre Multipotentes/patología , Receptores CXCR/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR6 , Análisis de Supervivencia , Tetraspanina 29/metabolismoRESUMEN
BACKGROUND: The Rh blood group system is highly complex, polymorphic, and immunogenic. The presence of RHD gene variants in RhD negative pregnant women is a challenge in fetal RHD genotyping as it may influence the antenatal management of anti-D prophylaxis. The aim of this study was to determine the efficiency of a non-invasive single-exon approach in the obstetric population of Western Sweden in a 31-month follow up. The frequency and type of maternal RHD variants were explored and the relation to the ethnicity was elucidated. Discrepant results between fetal RHD genotyping and serological blood group typing of newborns were investigated and clarified. MATERIALS AND METHODS: RHD exon 4 was analysed with quantitative real-time PCR technique in a total of 6,948 blood samples from RhD negative women in early pregnancy. All cases with suspected maternal RHD gene and discrepant results observed in newborn samples, were further investigated using both serological and molecular technologies. RESULTS: A total of 43 samples (0.6%) had inconclusive fetal genotyping result due the presence of a maternal RHD gene. These findings were in most cases (>66%) observed in pregnant women of non-European ancestry. Additionally, two novel RHD alleles were found. Seven discrepant results between fetal RHD genotype and serological RhD type of the newborns, were shown to be related to D antigen variants in newborns. Assay sensitivity was 99.95%, specificity 100%, and accuracy 99.97%. DISCUSSION: The single-exon approach for fetal RHD screening early in pregnancy is an appropriate choice in the population of Western Sweden, with a very low frequency of inconclusive results caused by the presence of maternal RHD gene variants. Due to the high sensitivity, specificity, and accuracy of the test, serological typing of neonates born to RhD negative women has no longer been performed at our laboratory since June 2023.
RESUMEN
BACKGROUND: RhD immunization is still the major cause of hemolytic disease of the fetus and newborn. Fetal RHD genotyping during pregnancy followed by tailored anti-D prophylaxis for pregnant RhD-negative women carrying an RHD-positive fetus to prevent RhD immunization is a well-established practice in many countries. This study aimed to validate a platform for high-throughput, non-invasive, single-exon, fetal RHD genotyping consisting of automated DNA extraction and PCR set-up, and a novel system for electronic data transfer to the real-time PCR instrument. We also investigated the effect of storage conditions of fresh or frozen samples on the outcome of the assay. MATERIALS AND METHODS: Blood samples from 261 RhD-negative pregnant women collected in Gothenburg, Sweden, between November 2018 and April 2020 during gestation week 10-14 were either tested as fresh after storage for 0-7 days at room temperature or as thawed plasma samples previously separated and stored for up to 13 months at -80°C. Extraction of cell-free fetal DNA and PCR set-up were performed in a closed automated system. Fetal RHD genotyping was determined by real-time PCR amplification of the RHD gene exon 4. RESULTS: The outcome of RHD genotyping was compared with either the results obtained with serological RhD typing of newborns or with the results of RHD genotyping performed by other laboratories. No difference was observed in genotyping results when using fresh or frozen plasma during short- and long-term storage, revealing high stability of cell-free fetal DNA. The assay has shown high sensitivity (99.37%), specificity (100%), and accuracy (99.62%). DISCUSSION: These data confirm that the proposed platform for non-invasive, single-exon, RHD genotyping early in pregnancy is accurate and robust. Importantly, we demonstrated the stability of cell-free fetal DNA in fresh and frozen samples after short- and long-term storage.
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Feto , Sistema del Grupo Sanguíneo Rh-Hr , Embarazo , Femenino , Humanos , Recién Nacido , Genotipo , Sistema del Grupo Sanguíneo Rh-Hr/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Exones , ADNRESUMEN
Understanding persistence and evolution of B cell clones after COVID-19 infection and vaccination is crucial for predicting responses against emerging viral variants and optimizing vaccines. Here, we collected longitudinal samples from patients with severe COVID-19 every third to seventh day during hospitalization and every third month after recovery. We profiled their antigen-specific immune cell dynamics by combining single-cell RNA-Seq, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), and B cell receptor-Seq (BCR-Seq) with oligo-tagged antigen baits. While the proportion of Spike receptor binding domain-specific memory B cells (MBC) increased from 3 months after infection, the other Spike- and Nucleocapsid-specific B cells remained constant. All patients showed ongoing class switching and sustained affinity maturation of antigen-specific cells, and affinity maturation was not significantly increased early after vaccine. B cell analysis revealed a polyclonal response with limited clonal expansion; nevertheless, some clones detected during hospitalization, as plasmablasts, persisted for up to 1 year, as MBC. Monoclonal antibodies derived from persistent B cell families increased their binding and neutralization breadth and started recognizing viral variants by 3 months after infection. Overall, our findings provide important insights into the clonal evolution and dynamics of antigen-specific B cell responses in longitudinally sampled patients infected with COVID-19.
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COVID-19 , SARS-CoV-2 , Humanos , Linfocitos B , Células Plasmáticas , Células ClonalesRESUMEN
Treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid and/or arsenic trioxide represents a paradigm in targeted cancer therapy because these drugs cause clinical remission by affecting the stability of the fusion oncoprotein promyelocytic leukemia (PML)/retinoic acid receptor alpha (RARA). The authors of previous studies have implicated the ubiquitin-proteasome pathway as the main mechanism involved in therapy-induced PML/RARA degradation. Here we have investigated a role of autophagy, a protein degradation pathway that involves proteolysis of intracellular material within lysosomes. We found that both all-trans retinoic acid and arsenic trioxide induce autophagy via the mammalian target of rapamycin pathway in APL cells and that autophagic degradation contributes significantly both to the basal turnover as well as the therapy-induced proteolysis of PML/RARA. In addition, we observed a correlation between autophagy and therapy-induced differentiation of APL cells. Given the central role of the PML/RARA oncoprotein in APL pathogenesis, this study highlights an important role of autophagy in the development and treatment of this disease.
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Autofagia/efectos de los fármacos , Autofagia/fisiología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Receptores de Ácido Retinoico/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Trióxido de Arsénico , Arsenicales/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular Tumoral , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Promielocítica Aguda/patología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Óxidos/farmacología , Proteína de la Leucemia Promielocítica , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Solubilidad , Serina-Treonina Quinasas TOR , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Tretinoina/farmacología , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
Patient-derived scaffolds (PDSs) generated from primary breast cancer tumors can be used to model the tumor microenvironment in vitro. Patient-derived scaffolds are generated by repeated detergent washing, removing all cells. Here, we analyzed the protein composition of 15 decellularized PDSs using liquid chromatography-mass spectrometry/mass spectrometry. One hundred forty-three proteins were detected and their relative abundance was calculated using a reference sample generated from all PDSs. We performed heatmap analysis of all the detected proteins to display their expression patterns across different PDSs together with pathway enrichment analysis to reveal which processes that were connected to PDS protein composition. This protein dataset together with clinical information is useful to investigators studying the microenvironment of breast cancers. Further, after repopulating PDSs with either MCF7 or MDA-MB-231 cells, we quantified their gene expression profiles using RNA sequencing. These data were also compared to cells cultured in conventional 2D conditions, as well as to cells cultured as xenografts in immune-deficient mice. We investigated the overlap of genes regulated between these different culture conditions and performed pathway enrichment analysis of genes regulated by both PDS and xenograft cultures compared to 2D in both cell lines to describe common processes associated with both culture conditions. Apart from our described analyses of these systems, these data are useful when comparing different experimental model systems. Downstream data analyses and interpretations can be found in the research article "Patient-derived scaffolds uncover breast cancer promoting properties of the microenvironment" [1].
RESUMEN
Tumor cells interact with the microenvironment that specifically supports and promotes tumor development. Key components in the tumor environment have been linked to various aggressive cancer features and can further influence the presence of subpopulations of cancer cells with specific functions, including cancer stem cells and migratory cells. To model and further understand the influence of specific microenvironments we have developed an experimental platform using cell-free patient-derived scaffolds (PDSs) from primary breast cancers infiltrated with standardized breast cancer cell lines. This PDS culture system induced a series of orchestrated changes in differentiation, epithelial-mesenchymal transition, stemness and proliferation of the cancer cell population, where an increased cancer stem cell pool was confirmed using functional assays. Furthermore, global gene expression profiling showed that PDS cultures were similar to xenograft cultures. Mass spectrometry analyses of cell-free PDSs identified subgroups based on their protein composition that were linked to clinical properties, including tumor grade. Finally, we observed that an induction of epithelial-mesenchymal transition-related genes in cancer cells growing on the PDSs were significantly associated with clinical disease recurrences in breast cancer patients. Patient-derived scaffolds thus mimics in vivo-like growth conditions and uncovers unique information about the malignancy-inducing properties of tumor microenvironment.
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Neoplasias de la Mama , Línea Celular Tumoral , Proliferación Celular , Transición Epitelial-Mesenquimal , Humanos , Recurrencia Local de Neoplasia , Células Madre Neoplásicas , Microambiente TumoralRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a fatal malignant disease with 5-year survival rate of less than 6%. Activating mutations of Kras (mu-Kras) are often detected in most of PDAC patients. Although it has been known that oncogenic Kras is the driver of pancreatic cancer initiation and development, the underlying mechanisms by which mu-Kras promotes PDAC remain poorly understood. Here, we identify that PKCι is one of the crucial factors for supporting the survival of pancreatic cancer cells expressing mu-Kras. Our study demonstrates that after the knockdown of PKCι, the expression of the transcriptional co-activator YAP1 is decreased, which hinders the expression of the downstream target gene Mcl-1, and subsequently sensitizes pancreatic cancer MiaPaCa and PANC-1 cells experssing mu-Kras to apoptosis. In comparison, the suppression of PKCι has little impact on the viability of non-neoplastic pancreatic HPDE6-C7 cells. Moreover, the transient overexpression of oncogenic Kras in HPDE6-C7 elevates the expression of PKCι and YAP1 concomitantly. The upregulated YAP1 in HPDE6-C7/ mu-Kras cells is abolished once PKCι is suppressed, suggesting the linear relationship among mu-Kras, PKCι and YAP1. This phenomenon is further proven by the co-upregulation of PKCι and YAP1 in HPDE6-C7 cells stably transfected with mu-Kras. Taken together, our findings suggest that PKCι acts through promoting YAP1 function to promote the survival of pancreatic cancer cells expressing mu-Kras. It appears that targeting PKCι-YAP1 signaling is a feasible strategy for developing new therapeutics for treating pancreatic cancer patients.
RESUMEN
Acute myeloid leukemia (AML) is a malignancy of myeloid progenitor cells that are blocked in differentiation. Acute promyelocytic leukemia (APL) is a rare form of AML, which generally presents with a t(15;17) translocation causing expression of the fusion protein PML-RARA. Pharmacological doses of all-trans retinoic acid (ATRA) induce granulocytic differentiation of APL cells leading to cure rates of >80% if combined with conventional chemotherapy. Autophagy is a lysosomal degradation pathway for the removal of cytoplasmic content and recycling of macromolecules. ATRA induces autophagy in ATRA-sensitive AML and APL cells and autophagy inhibition attenuates ATRA-triggered differentiation. In this study, we aimed at identifying if the autophagy-linked FYVE-domain containing protein (ALFY/WDFY3) is involved in autophagic degradation of protein aggregates contributes to ATRA therapy-induced autophagy. We found that ALFY mRNA levels increase significantly during the course of ATRA-induced differentiation of APL and AML cell lines. Importantly ALFY depletion impairs ATRA-triggered granulocytic differentiation of these cells. In agreement with its function in aggrephagy, knockdown of ALFY results in reduced ATRA-induced proteolysis. Our data further suggest that PML-RARα is an autophagy substrate degraded with the help of ALFY. In summary, we present a crucial role for ALFY in retinoid triggered maturation of AML cells.
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Autofagia , Diferenciación Celular , Leucemia Mieloide Aguda/patología , Proteínas de la Membrana/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Autofagia/efectos de los fármacos , Proteínas Relacionadas con la Autofagia , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Regulación Leucémica de la Expresión Génica , Células HEK293 , Humanos , Leucemia Mieloide Aguda/genética , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Proteínas de la Membrana/genética , MicroARNs/genética , MicroARNs/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , Proteolisis/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Tretinoina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Polymorphic variants of the FTO (fat mass and obesity) gene associate with body mass index in humans, but the underlying molecular mechanisms have not been firmly determined. FTO is linked to energy homeostasis via amino acid sensing and is thought to activate the mammalian target of rapamycin complex 1, a negative regulator of autophagy. FTO localises both to the nucleus and the cytoplasm, and in this study we identify a functional nuclear localisation signal (NLS) in the N-terminus of FTO, as well as nuclear localization information in its very C-terminus. Inhibition of FTO nuclear transport has no effect on autophagy and in contrast to a previously proposed role of FTO in autophagy, we find no difference in starvation-induced autophagy in control cells compared to a panel of cell types depleted of FTO. Future studies that further characterise the cellular functions of FTO will be important to understand why variants in FTO are associated with body weight.
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Transporte Activo de Núcleo Celular , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Autofagia , Isoformas de Proteínas/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Animales , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Señales de Localización Nuclear , Isoformas de Proteínas/genéticaRESUMEN
Upon completion of cytokinesis, the midbody ring is transported asymmetrically into one of the two daughter cells where it becomes a midbody ring derivative that is degraded by autophagy. In this study we showed that the ubiquitin-binding autophagy receptor SQSTM1/p62 and the interacting adaptor protein WDFY3/ALFY form a complex with the ubiquitin E3 ligase TRAF6 and that these proteins, as well as NBR1, are important for efficient clearance of midbody ring derivatives by autophagy. The number of ubiquitinated midbody ring derivatives decreases in TRAF6-depleted cells and we showed that TRAF6 mediates ubiquitination of the midbody ring localized protein KIF23/MKLP1. We conclude that TRAF6-mediated ubiquitination of the midbody ring is important for its subsequent recognition by ubiquitin-binding autophagy receptors and degradation by selective autophagy.
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Autofagia/genética , Citocinesis/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Factor 6 Asociado a Receptor de TNF/fisiología , Ubiquitinación/genética , Proteínas Adaptadoras Transductoras de Señales/fisiología , Células Cultivadas , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas/fisiología , Proteolisis , Proteína Sequestosoma-1 , Ubiquitina/metabolismoRESUMEN
Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Drosophila/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Animales Modificados Genéticamente , Autofagia , Citoplasma/metabolismo , Proteínas de Unión al ADN , Detergentes/farmacología , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Humanos , Microscopía Electrónica de Transmisión/métodos , Modelos Biológicos , Modelos Genéticos , Mutación , Proteína Sequestosoma-1 , Factores de TiempoRESUMEN
Accumulation of ubiquitinated proteins in cytoplasmic and/or nuclear inclusions is a hallmark of several diseases associated with premature cell death. SQSTM1/p62 is known to bind ubiquitinated substrates and aid their aggregation and degradation by macroautophagy. We show here that p62 is required to recruit the large phosphoinositide-binding protein ALFY to cytoplasmic p62 bodies generated upon amino acid starvation or puromycin-treatment. ALFY, as well as p62, is required for formation and autophagic degradation of cytoplasmic ubiquitin-positive inclusions. Moreover, both p62 and ALFY localize to nuclear promyleocytic leukemia (PML) bodies. The Drosophila p62 homologue Ref(2) P accumulates in ubiquitinated inclusions in the brain of flies carrying mutations in the ALFY homologue Blue cheese, demonstrating that ALFY is required for autophagic degradation of p62-associated ubiquitinated proteins in vivo. We conclude that p62 and ALFY interact to organize misfolded, ubiquitinated proteins into protein bodies that become degraded by autophagy.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia/fisiología , Cuerpos de Inclusión/metabolismo , Proteínas de la Membrana/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Relacionadas con la Autofagia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Inhibidores Enzimáticos/metabolismo , Células HeLa , Humanos , Macrólidos/metabolismo , Proteínas de la Membrana/genética , Complejos Multiproteicos/metabolismo , Pliegue de Proteína , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína Sequestosoma-1 , Factores de Transcripción/genética , Proteínas Ubiquitinadas/metabolismoRESUMEN
Autophagy is involved with the turnover of intracellular components and the management of stress responses. Genetic studies in mice have shown that suppression of neuronal autophagy can lead to the accumulation of protein aggregates and neurodegeneration. However, no study has shown that increasing autophagic gene expression can be beneficial to an aging nervous system. Here we demonstrate that expression of several autophagy genes is reduced in Drosophila neural tissues as a normal part of aging. The age-dependent suppression of autophagy occurs concomitantly with the accumulation of insoluble ubiquitinated proteins (IUP), a marker of neuronal aging and degeneration. Mutations in the Atg8a gene (autophagy-related 8a) result in reduced lifespan, IUP accumulation and increased sensitivity to oxidative stress. In contrast, enhanced Atg8a expression in older fly brains extends the average adult lifespan by 56% and promotes resistance to oxidative stress and the accumulation of ubiquitinated and oxidized proteins. These data indicate that genetic or age-dependent suppression of autophagy is closely associated with the buildup of cellular damage in neurons and a reduced lifespan, while maintaining the expression of a rate-limiting autophagy gene prevents the age-dependent accumulation of damage in neurons and promotes longevity.