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BACKGROUND: Aging is a prominent risk factor for diverse diseases; therefore, an in-depth understanding of its physiological mechanisms is required. Nonhuman primates, which share the closest genetic relationship with humans, serve as an ideal model for exploring the complex aging process. However, the potential of the nonhuman primate animal model in the screening of human aging markers is still not fully exploited. Multiomics analysis of nonhuman primate peripheral blood offers a promising approach to evaluate new therapies and biomarkers. This study explores aging-related biomarker through multilayer omics, including transcriptomics (mRNA, lncRNA, and circRNA) and proteomics (serum and serum-derived exosomes) in rhesus monkeys (Macaca mulatta). RESULTS: Our findings reveal that, unlike mRNAs and circRNAs, highly expressed lncRNAs are abundant during the key aging period and are associated with cancer pathways. Comparative analysis highlighted exosomal proteins contain more types of proteins than serum proteins, indicating that serum-derived exosomes primarily regulate aging through metabolic pathways. Finally, eight candidate aging biomarkers were identified, which may serve as blood-based indicators for detecting age-related brain changes. CONCLUSIONS: Our results provide a comprehensive understanding of nonhuman primate blood transcriptomes and proteomes, offering novel insights into the aging mechanisms for preventing or treating age-related diseases.
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Envejecimiento , Biomarcadores , Exosomas , Macaca mulatta , Proteómica , Animales , Envejecimiento/genética , Biomarcadores/sangre , Exosomas/metabolismo , Exosomas/genética , Proteómica/métodos , Transcriptoma , Perfilación de la Expresión Génica , ARN Largo no Codificante/genética , ARN Largo no Codificante/sangre , ARN Largo no Codificante/metabolismo , Modelos Animales , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteoma/metabolismo , Genómica/métodosRESUMEN
Lamin A/C is involved in macrophage activation and premature aging, also known as progeria. As the resident macrophage in brain, overactivation of microglia causes brain inflammation, promoting aging and brain disease. In this study, we investigated the role of Lamin A/C in microglial activation and its impact on progeria using Lmna-/- mice, primary microglia, Lmna knockout (Lmna-KO) and Lmna-knockdown (Lmna-KD) BV2 cell lines. We found that the microglial activation signatures, including cell proliferation, morphology changes, and proinflammatory cytokine secretion (IL-1ß, IL-6, and TNF-α), were significantly suppressed in all Lamin A/C-deficient models when stimulated with LPS. TMT-based quantitative proteomic and bioinformatic analysis were further applied to explore the mechanism of Lamin A/C-regulated microglia activation from the proteome level. The results revealed that immune response and phagocytosis were impaired in Lmna-/- microglia. Stat1 was identified as the hub protein in the mechanism by which Lamin A/C regulates microglial activation. Additionally, DNA replication, chromatin organization, and mRNA processing were also altered by Lamin A/C, with Ki67 fulfilling the main hub function. Lamin A/C is a mechanosensitive protein and, the immune- and proliferation-related biological processes are also regulated by mechanotransduction. We speculate that Lamin A/C-mediated mechanotransduction is required for microglial activation. Our study proposes a novel mechanism for microglial activation mediated by Lamin A/C.
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Lamina Tipo A , Progeria , Animales , Ratones , Proliferación Celular , Activación de Macrófagos , Mecanotransducción Celular , Microglía , Fagocitosis , ProteómicaRESUMEN
PURPOSE: Mucinous adenocarcinoma (MC) is a distinct pathological subtype of colon adenocarcinoma, which is associated with a worse prognosis compared with non-mucinous adenocarcinoma (AC). However, clear distinctions between MC and AC remain unknown. Extracellular vesicles (EVs) are a class of enclosed vesicles containing proteins, lipids, and nucleic acids that are secreted by cells into surrounding tissues or into serum. The EVs could facilitate tumorigenesis by regulating tumor cell proliferation, invasiveness, metastasis, angiogenesis, and evasion of immune surveillance. METHODS: Quantitative proteomics analysis was performed to determine the characterization and biological differences of serum-derived EVs in two subtypes of colon adenocarcinoma (MC and AC). Serum-derived EVs from patients with MC, AC, and healthy volunteers were included in this study. The role of PLA2G2A in cell migration and invasion were evaluate with transwell assay, and its prognostic predictive value was further assessed based on TCGA database. RESULTS: Quantitative proteomics analysis revealed 846 differentially expressed proteins (DEPs) in EVs from MC patients compared with those from AC patients. Bioinformatics analysis revealed that the most prominent protein cluster included those involved in cell migration and the tumor microenvironment. Overexpression of PLA2G2A, one of the key EV proteins upregulated in patients with MC, in colon cancer cell line SW480 promoted the cell invasion and migration ability. In addition, the high level of PLA2G2A is associated with poor prognosis of colon cancer patients harboring BRAF mutations. Further, after EV stimulation, proteomic analysis of recipient SW480 cells showed that MC-derived EVs activated multiple cancer-related pathways, including the Wnt/ß-Catenin signaling pathway, and might promote the malignancy of mucinous adenocarcinoma through these pathways. CONCLUSIONS: The identification of differential protein profiles between MC and AC helps to elucidate the underlying molecular mechanisms of MC pathogenesis. The PLA2G2A in EVs is a potential prognostic predictive marker for those patients harboring with BRAF mutations.
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Adenocarcinoma Mucinoso , Adenocarcinoma , Neoplasias del Colon , Vesículas Extracelulares , Humanos , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Proteómica , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas/metabolismo , Adenocarcinoma Mucinoso/patología , Línea Celular Tumoral , Vesículas Extracelulares/metabolismo , Microambiente TumoralRESUMEN
The hippocampus and entorhinal cortex (EC), the earliest affected areas, are considered relative to early memory loss in Alzheimer's disease (AD). The hippocampus is composed of heterogeneous subfields that are affected in a different order and varying degrees during AD pathogenesis. In this study, we conducted a comprehensive proteomic analysis of the hippocampal subfields and EC region in human postmortem specimens obtained from the Chinese human brain bank. Bioinformatics analysis identified region-consistent differentially expressed proteins (DEPs) which associated with astrocytes, and region-specific DEPs which associated with oligodendrocytes and the myelin sheath. Further analysis illuminated that the region-consistent DEPs functioned as connection of region-specific DEPs. Moreover, in region-consistent DEPs, the expression level of S100A10, a marker of protective astrocytes, was increased in both aging and AD patients. Immunohistochemical analysis confirmed an increase in the number of S100A10-positive astrocytes in all hippocampal subfields and the EC region of AD patients. Dual immunofluorescence results further showed that S100A10-positive astrocytes contained apoptotic neuron debris in AD patients, suggesting that S100A10-positive astrocytes may protect brain through phagocytosis of apoptotic neurons. In region-specific DEPs, the proteome showed a specific reduction of oligodendrocytes and myelin markers in CA1, CA3, and EC regions of AD patients. Immunohistochemical analysis confirmed the loss of myelin in EC region. Above all, these results highlight the role of the glial cells in AD and provide new insights into the pathogenesis of AD and potential therapeutic strategies.
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Enfermedad de Alzheimer , Enfermedad de Alzheimer/patología , Astrocitos/patología , Hipocampo/patología , Humanos , Trastornos de la Memoria/patología , ProteómicaRESUMEN
OBJECTIVE: Synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome is a rare chronic inflammatory disorder and the underlying pathogenesis is unclear. In this study, 88 SAPHO patients and 118 healthy controls were recruited to investigate the role of serum-derived extracellular vesicles (SEVs) in SAPHO syndrome. METHODS: Quantitative proteomics was applied for SEVs proteome identification, and ELISA and Western blotting was performed to verify the results of mass spectrum data. In vitro osteoclastogenesis and osteogenesis assay was used to confirm the effects of SEVs on bone metabolism. RESULTS: Tandem mass tagging-based quantitative proteomic analysis of SAPHO SEVs revealed differential expressed proteins involved in bone metabolism. Of these, serum amyloid A-1 (SAA1) and C-reactive protein (CRP) were upregulated. Higher SAA1 levels in SAPHO patients were confirmed by ELISA. In addition, SAA1 levels were positively correlated with CRP, an inflammatory marker related to the condition of patients. In vitro celluler studies confirmed that SAPHO SEVs inhibited osteoclastogenesis in patients mainly in the active phase of the disease. Further analysis demonstrated that Nucleolin was upregulated in osteoclasts of active-phase patients under SAPHO SEVs stimulation. CONCLUSION: In this study, we identified SAA1 as an additional inflammation marker that can potentially assist the diagnosis of SAPHO syndrome, and speculated that Nucleolin is a key regulator of osteoclastogenesis in active-phase patients.
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Osteoporosis is characterized by a decrease in bone mass and strength, rendering people prone to osteoporotic fractures caused by low-energy forces. The primary treatment strategy for osteoporotic fractures is surgery; however, the compromised and comminuted bones in osteoporotic fracture sites are not conducive to optimum reduction and rigid fixation. In addition, these patients always exhibit accompanying aging-related disorders, including high inflammatory status, decreased mechanical loading and abnormal skeletal metabolism, which are disadvantages for fracture healing around sites that have undergone orthopedic procedures. Since the incidence of osteoporosis is expected to increase worldwide, orthopedic surgeons should pay more attention to comprehensive strategies for improving the poor prognosis of osteoporotic fractures. Herein, we highlight the molecular basis of osteoimmunology and bone mechanosensation in different healing phases of elderly osteoporotic fractures, guiding perioperative management to alleviate the unfavorable effects of insufficient mechanical loading, high inflammatory levels and pathogen infection. The well-informed pharmacologic and surgical intervention, including treatment with anti-inflammatory drugs and sufficient application of antibiotics, as well as bench-to-bedside strategies for bone augmentation and hardware selection, should be made according to a comprehensive understanding of bone biomechanical properties in addition to the remodeling status of osteoporotic bones, which is necessary for creating proper biological and mechanical environments for bone union and remodeling. Multidisciplinary collaboration will facilitate the improvement of overall osteoporotic care and reduction of secondary fracture incidence.
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The failure of remodeling process that constantly regenerates effete, aged bone is highly associated with bone nonunion and degenerative bone diseases. Numerous studies have demonstrated that aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) activate cytokines and mediators on osteoclasts, osteoblasts and their constituent progenitor cells located around the remodeling area. These cells contribute to a complex metabolic scenario, resulting in degradative or synthetic functions for bone mineral tissues. The spatiotemporal effects of aspirin and NSAIDs in the bone remodeling are controversial according the specific therapeutic doses used for different clinical conditions. Herein, we review in vitro, in vivo, and clinical studies on the dose-dependent roles of aspirin and NSAIDs in bone remodeling. Our results show that low-dose aspirin (< 100 µg/mL), which is widely recommended for prevention of thrombosis, is very likely to be benefit for maintaining bone mass and qualities by activation of osteoblastic bone formation and inhibition of osteoclast activities via cyclooxygenase-independent manner. While, the roles of high-dose aspirin (150-300 µg/mL) and other NSAIDs in bone self-regeneration and fracture-healing process are difficult to elucidate owing to their dual effects on osteoclast activity and bone formation of osteoblast. In conclusion, this study highlighted the potential clinical applications of low-dose aspirin in abnormal bone remodeling as well as the risks of high-dose aspirin and other NSAIDs for relieving pain and anti-inflammation in fractures and orthopedic operations.
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Exosomes are secreted into the blood by various types of cells. These extracellular vesicles are involved in the contribution of exosomal proteins to osteoblastic or osteoclastic regulatory networks during the failure of bone remodeling, which results in age-related bone loss. However, the molecular changes in serum-derived exosomes (SDEs) from aged patients with low bone density and their functions in bone remodeling remain to be fully elucidated. We present a quantitative proteomics analysis of exosomes purified from the serum of the elderly patients with osteoporosis/osteopenia and normal volunteers; these data are available via Proteome Xchange with the identifier PXD006463. Overall, 1,371 proteins were identified with an overlap of 1,160 Gene IDs among the ExoCarta proteins. Bioinformatics analysis and in vitro studies suggested that protein changes in SDEs of osteoporosis patients are not only involved in suppressing the integrin-mediated mechanosensation and activation of osteoblastic cells, but also trigger the differentiation and resorption of osteoclasts. In contrast, the main changes in SDEs of osteopenia patients facilitated both activation of osteoclasts and formation of new bone mass, which could result in a compensatory elevation in bone remodeling. While the SDEs from aged normal volunteers might play a protective role in bone health through facilitating adhesion of bone cells and suppressing aging-associated oxidative stress. This information will be helpful in elucidating the pathophysiological functions of SDEs and aid in the development of senile osteoporosis diagnostics and therapeutics.
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Enfermedades Óseas Metabólicas/genética , Remodelación Ósea/genética , Exosomas/genética , Osteoclastos/metabolismo , Osteoporosis/genética , Proteínas/metabolismo , Anciano , HumanosRESUMEN
Osteoclasts are absorptive cells that play a critical role in homeostatic bone remodeling and pathological bone resorption. Emerging evidence suggests an important role of epigenetic regulation in osteoclastogenesis. In this study, we investigated the role of DOT1L, which regulates gene expression epigenetically by histone H3K79 methylation (H3K79me), during osteoclast formation. Using RANKL-induced RAW264.7 macrophage cells as an osteoclast differentiation model, we found that DOT1L and H3K79me2 levels were upregulated during osteoclast differentiation. Small molecule inhibitor- (EPZ5676 or EPZ004777) or short hairpin RNA-mediated reduction in DOT1L expression promoted osteoclast differentiation and resorption. In addition, DOT1L inhibition increased osteoclast surface area and accelerated bone-mass reduction in a mouse ovariectomy (OVX) model of osteoporosis without alter osteoblast differentiation. DOT1L inhibition increase reactive oxygen species (ROS) generation and autophagy activity, and cell migration in pre-osteoclasts. Moreover, it strengthened expression of osteoclast fusion and resorption-related protein CD9 and MMP9 in osteoclasts derived from RAW264.7. Our findings support a new mechanism of DOT1L-regulated, H3K79me2-mediated, epigenetic regulation of osteoclast differentiation, implicating DOT1L as a new therapeutic target for osteoclast dysregulation-induced disease.
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Histona Metiltransferasas/genética , Metiltransferasas/genética , Osteoclastos/metabolismo , Osteogénesis/genética , Osteoporosis/genética , Animales , Histona Metiltransferasas/metabolismo , N-Metiltransferasa de Histona-Lisina , Metiltransferasas/metabolismo , Ratones , TransfecciónRESUMEN
Lysosomes play vital roles in both innate and adaptive immunity. It is widely accepted that lysosomes do not function exclusively as a digestive organelle. It is also involved in the process of immune cells against pathogens. However, the changes in the lysosomal proteome caused by infection with various microbes are still largely unknown, and our understanding of the proteome of the purified lysosome is another obstacle that needs to be resolved. Here, we performed a proteomic study on lysosomes enriched from THP1 cells after infection with Listeria monocytogenes (L.m), Herpes Simplex Virus 1 (HSV-1) and Vesicular Stomatitis Virus (VSV). In combination with the gene ontology (GO) analysis, we identified 284 lysosomal-related proteins from a total of 4560 proteins. We also constructed the protein-protein interaction networks for the differentially expressed proteins and revealed the core lysosomal proteins, including SRC in the L. m treated group, SRC, GLB1, HEXA and HEXB in the HSV-1 treated group and GLB1, CTSA, CTSB, HEXA and HEXB in the VSV treated group, which are involved in responding to diverse microbial infections. This study not only reveals variable lysosome responses depending on the bacterial or virus infection, but also provides the evidence based on which we propose a novel approach to proteome research for investigation of the function of the enriched organelles.
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Infecciones Bacterianas/genética , Proteoma/genética , Virosis/genética , Células Cultivadas , Perfilación de la Expresión Génica/métodos , Humanos , Lisosomas/genética , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas/genética , Proteómica/métodosRESUMEN
Phagocytosis and autophagy in macrophages have been shown to be essential to both innate and adaptive immunity. Lysosomes are the main catabolic subcellular organelles responsible for degradation and recycling of both extracellular and intracellular material, which are the final steps in phagocytosis and autophagy. However, the molecular mechanisms underlying lysosomal functions after infection remain obscure. In this study, we conducted a quantitative proteomics analysis of the changes in constitution and glycosylation of proteins in lysosomes derived from murine RAW 264.7 macrophage cells treated with different types of pathogens comprising examples of bacteria (Listeria monocytogenes, L. m), DNA viruses (herpes simplex virus type-1, HSV-1) and RNA viruses (vesicular stomatitis virus, VSV). In total, 3,704 lysosome-related proteins and 300 potential glycosylation sites on 193 proteins were identified. Comparative analysis showed that the aforementioned pathogens induced distinct alterations in the proteome of the lysosome, which is closely associated with the immune functions of macrophages, such as toll-like receptor activation, inflammation and antigen-presentation. The most significant changes in proteins and fluctuations in glycosylation were also determined. Furthermore, Western blot analysis showed that the changes in expression of these proteins were undetectable at the whole cell level. Thus, our study provides unique insights into the function of lysosomes in macrophage activation and immune responses.
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Glicoproteínas/metabolismo , Lisosomas/metabolismo , Activación de Macrófagos , Macrófagos/metabolismo , Proteínas/metabolismo , Proteómica/métodos , Animales , Western Blotting , Cromatografía Líquida de Alta Presión , Técnica del Anticuerpo Fluorescente , Glicoproteínas/inmunología , Glicosilación , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/patogenicidad , Interacciones Huésped-Patógeno , Listeria monocytogenes/inmunología , Listeria monocytogenes/patogenicidad , Lisosomas/inmunología , Lisosomas/microbiología , Lisosomas/virología , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/virología , Ratones , Proteínas/inmunología , Células RAW 264.7 , Espectrometría de Masas en Tándem , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismo , Vesiculovirus/inmunología , Vesiculovirus/patogenicidadRESUMEN
Tumor cells of colorectal cancer (CRC) release exosomes into the circulation. These exosomes can mediate communication between cells and affect various tumor-related processes in their target cells. We present a quantitative proteomics analysis of the exosomes purified from serum of patients with CRC and normal volunteers; data are available via ProteomeXchange with identifier PXD003875. We identified 918 proteins with an overlap of 725 Gene IDs in the Exocarta proteins list. Compared with the serum-purified exosomes (SPEs) of normal volunteers, we found 36 proteins upregulated and 22 proteins downregulated in the SPEs of CRC patients. Bioinformatics analysis revealed that upregulated proteins are involved in processes that modulate the pretumorigenic microenvironment for metastasis. In contrast, differentially expressed proteins (DEPs) that play critical roles in tumor growth and cell survival were principally downregulated. Our study demonstrates that SPEs of CRC patients play a pivotal role in promoting the tumor invasiveness, but have minimal influence on putative alterations in tumor survival or proliferation. According to bioinformatics analysis, we speculate that the protein contents of exosomes might be associated with whether they are involved in premetastatic niche establishment or growth and survival of metastatic tumor cells. This information will be helpful in elucidating the pathophysiological functions of tumor-derived exosomes, and aid in the development of CRC diagnostics and therapeutics.
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Adenocarcinoma/sangre , Neoplasias Colorrectales/sangre , Exosomas/química , Proteínas de Neoplasias/sangre , Proteómica/métodos , Adenocarcinoma/química , Adenocarcinoma/genética , Anciano , Estudios de Casos y Controles , División Celular , Movimiento Celular , Neoplasias Colorrectales/química , Neoplasias Colorrectales/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Ontología de Genes , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Células Neoplásicas Circulantes , Células Tumorales CultivadasRESUMEN
Surgical resection supplemented with adjuvant chemotherapy is the current preferred treatment for Stage III colorectal cancer (CRC). However, as many as 48% of patients who undergo curative resection eventually suffer from incurable distant recurrence. To investigate the molecular mechanisms involved in Stage III CRC post-surgical distant recurrence, we identified a total of 146 differentially expressed proteins (DEPs) associated with distant recurrence in Stage III CRC using TMT-based quantitative mass spectrometry. Among these DEPs, the altered expressions of R-Ras and Transgelin were then validated in 192 individual specimens using immunohistochemistry (IHC). Furthermore, Kaplan-Meier analysis revealed that the levels of R-Ras and Transgelin were significantly associated with 5-year overall survival (OS) and disease-free survival (DFS), and multivariate Cox-regression analyses revealed that R-Ras and Transgelin were independent prognostic factors for OS and DFS, respectively. In conclusion, this study identified potential biochemical players involved in distant recurrence and indicates that R-Ras and Transgelin are potential post-surgical prognostic biomarkers for Stage III CRC. This proteomics data have been submitted to Proteome Xchange under accession number PXD002903.
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Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/patología , Proteínas de Microfilamentos/biosíntesis , Proteínas Musculares/biosíntesis , Recurrencia Local de Neoplasia/patología , Proteínas ras/biosíntesis , Adulto , Anciano , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/mortalidad , Supervivencia sin Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Proteínas de Microfilamentos/análisis , Persona de Mediana Edad , Proteínas Musculares/análisis , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/mortalidad , Pronóstico , Modelos de Riesgos Proporcionales , Proteómica/métodos , Proteínas ras/análisisRESUMEN
The hippocampus appears commonly affected by aging and various neurologic disorders in humans, whereas little is known about age-related change in overall protein expression in this brain structure. Using the 4-plex tandem mass tag labeling, we carried out a quantitative proteomic study of the hippocampus during normal aging using postmortem brains from Chinese subjects. Hippocampal samples from 16 subjects died of non-neurological/psychiatric diseases were divided into 4 age groups: 22-49, 50-69, 70-89, and >90. Among 4582 proteins analyzed, 35 proteins were significantly elevated, whereas 25 proteins were downregulated, along with increasing age. Several upregulated proteins, including transgelin, vimentin, myosin regulatory light polypeptide 9, and calcyphosin, were further verified by quantitative Western blot analysis of hippocampal tissues from additional normal subjects. Bioinformatic analysis showed that the upregulated and downregulated proteins were largely involved in several important protein-protein interaction networks. Proteins in the electron transport chain and synaptic vesicle fusion pathway were consistently downregulated with aging, whereas proteins associated with Alzheimer's disease showed little change. Our study demonstrates substantial protein profile changes in the human hippocampus during aging, which could be of relevance to age-related loss of hippocampal functions.
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Envejecimiento/genética , Envejecimiento/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Expresión Génica/genética , Hipocampo/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Vimentina/genética , Vimentina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Pueblo Asiatico , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Regulación hacia Abajo/genética , Transporte de Electrón/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Dominios y Motivos de Interacción de Proteínas , Vesículas Sinápticas/genética , Espectrometría de Masas en Tándem , Regulación hacia Arriba/genética , Adulto JovenRESUMEN
The temporal lobe is a portion of the cerebral cortex with critical functionality. The age-related protein profile changes in the human temporal lobe have not been previously studied. This 4-plex tandem mass tag labeled proteomic study was performed on samples of temporal lobe from Chinese donors. Tissue samples were assigned to four age groups: Group A (the young, age: 34±13 years); Group B (the elderly, 62±5 years); Group C (the aged, 84±4 years) and Group D (the old, 95±1 years). Pooled samples from the different groups were subjected to proteomics and bioinformatics analysis to identify age-related changes in protein expression and associated pathways. We isolated 5072 proteins, and found that 67 proteins were downregulated and 109 proteins were upregulated in one or more groups during the aging process. Western blotting assays were performed to verify the proteomic results. Bioinformatic analysis identified proteins involved in neuronal degeneration, including proteins involved in neuronal firing, myelin sheath damage, and cell structure stability. We also observed the accumulation of extracellular matrix and lysosomal proteins which imply the occurrence of fibrosis and autophagy. Our results suggest a series of changes across a wide range of proteins in the human temporal lobe that may relate to aging and age-related neurodegenerative disorders.
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Envejecimiento/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Lóbulo Temporal/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Western Blotting/métodos , Cromatografía Líquida de Alta Presión/métodos , Biología Computacional/métodos , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Proteómica/métodos , Transducción de Señal/fisiología , Espectrometría de Masas en Tándem/métodos , Bancos de Tejidos , Adulto JovenRESUMEN
Bone metabolism is a lifelong process for maintaining skeletal system homeostasis, which is regulated by bone-resorbing osteoclasts and bone-forming osteoblasts. Aberrant differentiation of osteoclasts and osteoblasts leads to imbalanced bone metabolism, resulting in ossification and osteolysis diseases. MicroRNAs (miRNAs) are pivotal factors in regulating bone metabolism via post-transcriptional inhibition of target genes. Recent studies have revealed that miR-223 exerts multiple effects on bone metabolism, especially in the processes of osteoclast and osteoblasts differentiation. In this review, we highlight the roles of miR-223 during the processes of osteoclast and osteoblast differentiation, as well as the potential clinical applications of miR-223 in bone metabolism disorders.
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MicroARNs/fisiología , Interferencia de ARN , Animales , Desarrollo Óseo , Remodelación Ósea , Diferenciación Celular , Expresión Génica , Humanos , Factores de Transcripción NFI/genética , Factores de Transcripción NFI/metabolismo , Osteoblastos/fisiología , Osteoclastos/fisiología , Transducción de SeñalRESUMEN
Estrogen plays an important role in inhibiting osteoclast differentiation and protecting against bone loss from osteoporosis, especially in postmenopausal women. However, the precise mechanisms underlying the effect of estrogen on osteoclasts are not well known. In the present study, we performed proteomics analysis and bioinformatics analysis to comprehensively compare the differential expression of proteins in receptor activator of nuclear factor-κB ligand RANKL-induced osteoclasts in the presence and absence of estrogen. We identified 6403 proteins, of which 124 were upregulated and 231 were downregulated by estrogen. Bioinformatics analysis showed that estrogen treatment interfered with 77 intracellular pathways, including both confirmed canonical and unconfirmed pathways of osteoclast formation. Our findings validate the inhibitory effect of estrogen on osteoclasts via the promotion of apoptosis and suppression of differentiation and polarization and suggest that estrogen might inhibit osteoclast formation via other pathways, which requires further investigation and verification.
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Estrógenos/metabolismo , Osteoclastos/metabolismo , Proteómica , Ligando RANK/biosíntesis , Animales , Resorción Ósea/genética , Resorción Ósea/fisiopatología , Diferenciación Celular/genética , Estrógenos/administración & dosificación , Femenino , Humanos , Ratones , Osteoclastos/patología , Osteoporosis/metabolismo , Osteoporosis/fisiopatología , Ligando RANK/genética , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Cultivation of osteoclasts is a basic tool for investigating osteolytic bone diseases. Fetal bovine serum (FBS) is the standard supplement used for in vitro cell culture medium. Typically, the serum volume fraction used for osteoclast cultivation is 10%. In this study, we investigated the use of a low serum (1% FBS) model for culturing osteoclasts. RESULTS: To confirm the validity of this model for use in osteoclast research, we compared the capacity for osteoclastogenesis and bone resorption of RANKL-induced RAW 264.7 cells cultured in medium supplemented with 10% FBS and 1% FBS. Osteoclasts were successfully generated in medium supplemented with 1% FBS, and exhibited prolonged longevity and similar bone resorbing ability to those generated in medium supplemented with 10% FBS, although the osteoclasts were smaller in size. Proteomics and bioinformatics analyses were performed to assess the suitability of osteoclasts formed in low serum-containing medium for use in research focusing on osteoclast differentiation and function. Our study demonstrated that a total of 100 proteins were differentially expressed in cells cultured in medium containing 1% FBS, of which 29 proteins were upregulated, and 71 proteins were downregulated. Bioinformatics analysis showed that the electron transport chain and oxidative phosphorylation pathways were downregulated obviously; however, the osteoclast signaling pathway was unaffected. The data have been deposited to the ProteomeXchange with identifier PXD001935. CONCLUSION: Our study provides clear evidence of the validity of the low serum model for use in studying RANKL-dependent osteoclasts differentiation and bone resorption with the advantage of prolonged survival time.
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Signal transduction from toll-like receptors (TLRs) is important for innate immunity against infections, but deregulated TLR signalling contributes to inflammatory disorders. Here we show that myeloid cell-specific ablation of TRAF2 greatly promotes TLR-stimulated proinflammatory cytokine expression in macrophages and exacerbates colitis in an animal model of inflammatory bowel disease. TRAF2 deficiency does not enhance upstream signalling events, but it causes accumulation of two transcription factors, c-Rel and IRF5, known to mediate proinflammatory cytokine induction. Interestingly, TRAF2 controls the fate of c-Rel and IRF5 via a proteasome-dependent mechanism that also requires TRAF3 and the E3 ubiquitin ligase cIAP. We further show that TRAF2 also regulates inflammatory cytokine production in tumour-associated macrophages and facilitates tumour growth. These findings demonstrate an unexpected anti-inflammatory function of TRAF2 and suggest a proteasome-dependent mechanism that limits the proinflammatory TLR signalling.
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Colitis/inmunología , Citocinas/metabolismo , Enfermedades Inflamatorias del Intestino/inmunología , Macrófagos/metabolismo , Transducción de Señal/inmunología , Factor 2 Asociado a Receptor de TNF/metabolismo , Receptores Toll-Like/metabolismo , Animales , Inmunoprecipitación de Cromatina , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Immunoblotting , Ratones , Ratones Noqueados , Proteolisis , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Lysosomes perform a range of functions, some of which, such as degradation, are common to all cell types. Others, such as secretion or lysosomal exocytosis, are more specialised and tend to involve fusion of this organelle with the cell surface to release its contents. This review describes lysosomal regulation of the inflammatory glucocorticoid signaling pathways, and summarizes the roles of lysosomes in negatively or positively modulating the production of inflammatory cytokines. We also review the characteristic changes in lysosomal hydrolases and membrane proteins in common autoimmune diseases. Finally, future directions in lysosome research are proposed, with it being suggested that the role of lysosomes will continue to be of growing interest in immunity research.