RESUMO
Mastocytosis is a KIT-related myeloproliferative disease characterised by abnormal expansion of neoplastic mast cells (MC) in the skin or virtually any other organ system. The cutaneous form of adult-onset mastocytosis is almost invariably combined with indolent systemic involvement for which curative therapy is yet not available. Here we evaluated a concept of depleting cutaneous MCs in mastocytosis lesions ex vivo by targeting their secretory granules. Skin biopsies from mastocytosis patients were incubated with or without mefloquine, an antimalarial drug known to penetrate into acidic organelles such as MC secretory granules. Mefloquine reduced the number of dermal MCs without affecting keratinocyte proliferation or epidermal gross morphology at drug concentrations up to 40 µM. Flow cytometric analysis of purified dermal MCs showed that mefloquine-induced cell death was mainly due to apoptosis and accompanied by caspase-3 activation. However, caspase inhibition provided only partial protection against mefloquine-induced cell death, indicating predominantly caspase-independent apoptosis. Further assessments revealed that mefloquine caused an elevation of granule pH and a corresponding decrease in cytosolic pH, suggesting drug-induced granule permeabilisation. Extensive damage to the MC secretory granules was confirmed by transmission electron microscopy analysis. Further, blockade of granule acidification or serine protease activity prior to mefloquine treatment protected MCs from apoptosis, indicating that granule acidity and granule-localised serine proteases play major roles in the execution of mefloquine-induced cell death. Altogether, these findings reveal that mefloquine induces selective apoptosis of MCs by targeting their secretory granules and suggest that the drug may potentially extend its range of medical applications.
Assuntos
Mastocitose Cutânea , Mastocitose , Adulto , Humanos , Mastócitos/metabolismo , Mefloquina/metabolismo , Mastocitose Cutânea/metabolismo , Vesículas Secretórias/metabolismo , Vesículas Secretórias/patologia , Apoptose , Caspases/metabolismoRESUMO
Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by the inability to properly terminate an immune response. Familial HLH (FHLH) and related immune dysregulation syndromes are associated with mutations in the genes PRF1, UNC13D, STX11, STXBP2, LYST, AP3B1, and RAB27A, all of which are required for the assembly, exocytosis, and function of cytotoxic granules within CD8+ T cells and natural killer (NK) cells. Loss-of-function mutations in these genes render the cytotoxicity pathway ineffective, thereby failing to eradicate immune stimuli, such as infectious pathogens or malignant cells. The resulting persistent immune system stimulation drives hypercytokinemia, ultimately leading to severe tissue inflammation and end-organ damage. Traditionally, a diagnosis of FHLH requires the identification of biallelic loss-of-function mutations in one of these degranulation pathway genes. However, this narrow definition fails to encompass patients with other genetic mechanisms underlying degranulation pathway dysfunction. In particular, mounting clinical evidence supports a potential digenic mode of inheritance of FHLH in which single loss-of-function mutations in two different degranulation pathway genes cooperate to impair pathway activity. Here, we review the functions of the FHLH-associated genes within the degranulation pathway and summarize clinical evidence supporting a model in which cumulative defects along this mechanistic pathway may underlie HLH.
Assuntos
Degranulação Celular/genética , Hereditariedade , Células Matadoras Naturais/imunologia , Linfo-Histiocitose Hemofagocítica/genética , Herança Multifatorial , Mutação , Vesículas Secretórias/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Predisposição Genética para Doença , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Linfo-Histiocitose Hemofagocítica/imunologia , Linfo-Histiocitose Hemofagocítica/metabolismo , Linfo-Histiocitose Hemofagocítica/patologia , Fenótipo , Prognóstico , Fatores de Risco , Vesículas Secretórias/metabolismo , Vesículas Secretórias/patologia , Transdução de Sinais , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/patologiaRESUMO
OBJECTIVE: Parasympathetic network damage results in facial nerve damage, sublingual ganglion degeneration, sublingual gland dysfunction, and dry mouth. In this study, subarachnoid hemorrhage (SAH) was considered to be the cause of dry mouth. METHODS: We assessed 23 hybrid rabbits, including 5 control (group 1, Control). One milliliter of serum saline was injected into the cisterna magna of 5 animals (group 2). SAH was induced by injecting 1 mL of autologous blood into the cisterna magna of 13 animals (group 3). The animals were killed after 3 weeks of induction. The animals' sublingual ganglion and sublingual gland were excised for histopathological examination. The number of degenerated cells in the sublingual ganglion, secretory vesicles, and secretory granules in the sublingual gland that contain salivary components were estimated using Sequential Window Acquisition of All Theoretical Mass Spectra data analysis. The values were compared by the Mann-Whitney U-test. RESULTS: The numbers of secretory vesicles in the sublingual gland were 5.3 ± 1.1 × 103 (group 1), 4.23 ± 0.45 × 103 (group 2), and 1.56 ± 0.22 × 103 (group 3); the numbers of secretory vesicles containing saliva in the sublingual gland were 324 ± 12.18 (group 1), 263 ± 36.23 (group 2), and 114 ± 23.14 (group 3); and the numbers of degenerated cells in the sublingual ganglion were 11 ± 3/mm3 (group 1), 98.43 ± 15.54/mm3 (group 2), and 346 ± 12.28/mm3 (group 3) (P < 0.05). CONCLUSIONS: Clinical findings in infection and diseases such as Sjögren syndrome, aseptic meningitis, and SAH are similar. However, until now, SAH has not been demonstrated experimentally to cause dry mouth. Discovering that SAH might cause dry mouth might prevent unnecessary use of antibiotics and decrease morbidity due to the wrong or late diagnosis.
Assuntos
Nervo Facial/irrigação sanguínea , Hemorragia Subaracnóidea/complicações , Xerostomia/etiologia , Animais , Modelos Animais de Doenças , Isquemia , Coelhos , Saliva/citologia , Vesículas Secretórias/patologia , Glândula Sublingual/patologiaRESUMO
The efficient induction and long-term persistence of pathogen-specific memory CD8 T cells are pivotal to rapidly curb the reinfection. Recent studies indicated that long-noncoding RNAs expression is highly cell- and stage-specific during T cell development and differentiation, suggesting their potential roles in T cell programs. However, the key lncRNAs playing crucial roles in memory CD8 T cell establishment remain to be clarified. Through CD8 T cell subsets profiling of lncRNAs, this study found a key lncRNA-Snhg1 with the conserved naivehi-effectorlo-memoryhi expression pattern in CD8 T cells of both mice and human, that can promote memory formation while impeding effector CD8 in acute viral infection. Further, Snhg1 was found interacting with the conserved vesicle trafficking protein Vps13D to promote IL-7Rα membrane location specifically. With the deep mechanism probing, the results show Snhg1-Vps13D regulated IL-7 signaling with its dual effects in memory CD8 generation, which not just because of the sustaining role of STAT5-BCL-2 axis for memory survival, but more through the STAT3-TCF1-Blimp1 axis for transcriptional launch program of memory differentiation. Moreover, we performed further study with finding a similar high-low-high expression pattern of human SNHG1/VPS13D/IL7R/TCF7 in CD8 T cell subsets from PBMC samples of the convalescent COVID-19 patients. The central role of Snhg1-Vps13D-IL-7R-TCF1 axis in memory CD8 establishment makes it a potential target for improving the vaccination effects to control the ongoing pandemic.
Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Interleucina-7/imunologia , Proteínas/imunologia , RNA Longo não Codificante/imunologia , SARS-CoV-2/imunologia , Vesículas Secretórias/imunologia , Transdução de Sinais/imunologia , Animais , Transporte Biológico Ativo , Linfócitos T CD8-Positivos/patologia , COVID-19/patologia , Humanos , Memória Imunológica , Camundongos , Vesículas Secretórias/patologiaRESUMO
Lysosome-related organelles (LROs) are a category of secretory organelles enriched with ions such as calcium, which are maintained by ion transporters or channels. Homeostasis of these ions is important for LRO biogenesis and secretion. Hermansky-Pudlak syndrome (HPS) is a recessive disorder with defects in multiple LROs, typically platelet dense granules (DGs) and melanosomes. However, the underlying mechanism of DG deficiency is largely unknown. Using quantitative proteomics, we identified a previously unreported platelet zinc transporter, transmembrane protein 163 (TMEM163), which was significantly reduced in BLOC-1 (Dtnbp1sdy and Pldnpa)-, BLOC-2 (Hps6ru)-, or AP-3 (Ap3b1pe)-deficient mice and HPS patients (HPS2, HPS3, HPS5, HPS6, or HPS9). We observed similar platelet DG defects and higher intracellular zinc accumulation in platelets of mice deficient in either TMEM163 or dysbindin (a BLOC-1 subunit). In addition, we discovered that BLOC-1 was required for the trafficking of TMEM163 to perinuclear DG and late endosome marker-positive compartments (likely DG precursors) in MEG-01 cells. Our results suggest that TMEM163 is critical for DG biogenesis and that BLOC-1 is required for the trafficking of TMEM163 to putative DG precursors. These new findings suggest that loss of TMEM163 function results in disruption of intracellular zinc homeostasis and provide insights into the pathogenesis of HPS or platelet storage pool deficiency.
Assuntos
Plaquetas/patologia , Síndrome de Hermanski-Pudlak/patologia , Proteínas de Membrana/metabolismo , Animais , Plaquetas/metabolismo , Síndrome de Hermanski-Pudlak/metabolismo , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos Endogâmicos C57BL , Vesículas Secretórias/metabolismo , Vesículas Secretórias/patologia , Zinco/metabolismoRESUMO
Asthma is a chronic disease of the airways that has an important inflammatory component. Multiple cells are implicated in asthma pathogenesis (lymphocytes, eosinophils, mast cells, basophils, neutrophils), releasing a wide variety of cytokines. These cells can exert their inflammatory functions throughout extracellular vesicles (EVs), which are small vesicles released by donor cells into the extracellular microenvironment that can be taken up by recipient cells. Depending on their size, EVs can be classified as microvesicles, exosomes, or apoptotic bodies. EVs are heterogeneous spherical structures secreted by almost all cell types. One of their main functions is to act as transporters of a wide range of molecules, such as proteins, lipids, and microRNAs (miRNAs), which are single-stranded RNAs of approximately 22 nucleotides in length. Therefore, exosomes could influence several physiological and pathological processes, including those involved in asthma. They can be detected in multiple cell types and biofluids, providing a wealth of information about the processes that take account in a pathological scenario. This review thus summarizes the most recent insights concerning the role of exosomes from different sources (several cell populations and biofluids) in one of the most prevalent respiratory diseases, asthma.
Assuntos
Asma/etiologia , Exossomos/fisiologia , Inflamação/etiologia , Animais , Asma/patologia , Comunicação Celular/fisiologia , Micropartículas Derivadas de Células/metabolismo , Exossomos/patologia , Humanos , Inflamação/patologia , Vesículas Secretórias/patologia , Vesículas Secretórias/fisiologiaRESUMO
AIMS/HYPOTHESIS: We hypothesised that human beta cells are structurally and functional polarised with respect to the islet capillaries. We set out to test this using confocal microscopy to map the 3D spatial arrangement of key proteins and live-cell imaging to determine the distribution of insulin granule fusion around the cells. METHODS: Human pancreas samples were rapidly fixed and processed using the pancreatic slice technique, which maintains islet structure and architecture. Slices were stained using immunofluorescence for polarity markers (scribble, discs large [Dlg] and partitioning defective 3 homologue [Par3]) and presynaptic markers (liprin, Rab3-interacting protein [RIM2] and piccolo) and imaged using 3D confocal microscopy. Isolated human islets were dispersed and cultured on laminin-511-coated coverslips. Live 3D two-photon microscopy was used on cultured cells to image exocytic granule fusion events upon glucose stimulation. RESULTS: Assessment of the distribution of endocrine cells across human islets found that, despite distinct islet-to-islet complexity and variability, including multi-lobular islets, and intermixing of alpha and beta cells, there is still a striking enrichment of alpha cells at the islet mantle. Measures of cell position demonstrate that most beta cells contact islet capillaries. Subcellularly, beta cells consistently position polar determinants, such as Par3, Dlg and scribble, with a basal domain towards the capillaries and apical domain at the opposite face. The capillary interface/vascular face is enriched in presynaptic scaffold proteins, such as liprin, RIM2 and piccolo. Interestingly, enrichment of presynaptic scaffold proteins also occurs where the beta cells contact peri-islet capillaries, suggesting functional interactions. We also observed the same polarisation of synaptic scaffold proteins in islets from type 2 diabetic patients. Consistent with polarised function, isolated beta cells cultured onto laminin-coated coverslips target insulin granule fusion to the coverslip. CONCLUSIONS/INTERPRETATION: Structural and functional polarisation is a defining feature of human pancreatic beta cells and plays an important role in the control of insulin secretion.
Assuntos
Diabetes Mellitus Tipo 2/patologia , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/patologia , Doadores de Tecidos , Biomarcadores/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Microscopia Confocal , Microscopia de Fluorescência por Excitação Multifotônica , Fenótipo , Vesículas Secretórias/metabolismo , Vesículas Secretórias/patologia , Técnicas de Cultura de TecidosRESUMO
Prader-Willi syndrome (PWS) is a developmental disorder caused by loss of maternally imprinted genes on 15q11-q13, including melanoma antigen gene family member L2 (MAGEL2). The clinical phenotypes of PWS suggest impaired hypothalamic neuroendocrine function; however, the exact cellular defects are unknown. Here, we report deficits in secretory granule (SG) abundance and bioactive neuropeptide production upon loss of MAGEL2 in humans and mice. Unbiased proteomic analysis of Magel2pΔ/m+ mice revealed a reduction in components of SG in the hypothalamus that was confirmed in 2 PWS patient-derived neuronal cell models. Mechanistically, we show that proper endosomal trafficking by the MAGEL2-regulated WASH complex is required to prevent aberrant lysosomal degradation of SG proteins and reduction of mature SG abundance. Importantly, loss of MAGEL2 in mice, NGN2-induced neurons, and human patients led to reduced neuropeptide production. Thus, MAGEL2 plays an important role in hypothalamic neuroendocrine function, and cellular defects in this pathway may contribute to PWS disease etiology. Moreover, these findings suggest unanticipated approaches for therapeutic intervention.
Assuntos
Antígenos de Neoplasias/fisiologia , Hipotálamo/patologia , Neurônios/patologia , Neuropeptídeos/metabolismo , Síndrome de Prader-Willi/fisiopatologia , Proteínas/metabolismo , Proteínas/fisiologia , Vesículas Secretórias/patologia , Animais , Feminino , Humanos , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Fenótipo , Transporte Proteico , Proteínas/genética , Proteoma/análise , Proteoma/metabolismo , Vesículas Secretórias/metabolismoRESUMO
TP53 missense mutations leading to the expression of mutant p53 oncoproteins are frequent driver events during tumorigenesis. p53 mutants promote tumor growth, metastasis and chemoresistance by affecting fundamental cellular pathways and functions. Here, we demonstrate that p53 mutants modify structure and function of the Golgi apparatus, culminating in the increased release of a pro-malignant secretome by tumor cells and primary fibroblasts from patients with Li-Fraumeni cancer predisposition syndrome. Mechanistically, interacting with the hypoxia responsive factor HIF1α, mutant p53 induces the expression of miR-30d, which in turn causes tubulo-vesiculation of the Golgi apparatus, leading to enhanced vesicular trafficking and secretion. The mut-p53/HIF1α/miR-30d axis potentiates the release of soluble factors and the deposition and remodeling of the ECM, affecting mechano-signaling and stromal cells activation within the tumor microenvironment, thereby enhancing tumor growth and metastatic colonization.
Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Complexo de Golgi/patologia , Síndrome de Li-Fraumeni/genética , MicroRNAs/metabolismo , Proteína Supressora de Tumor p53/genética , Animais , Biópsia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Feminino , Fibroblastos , Regulação Neoplásica da Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Síndrome de Li-Fraumeni/patologia , Camundongos , Microtúbulos/metabolismo , Microtúbulos/patologia , Mutação , Cultura Primária de Células , Vesículas Secretórias/metabolismo , Vesículas Secretórias/patologia , Transdução de Sinais/genética , Pele/citologia , Pele/patologia , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: Human patients with Duchenne muscular dystrophy (DMD) commonly exhibit a short stature, but the pathogenesis of this growth retardation is not completely understood. Due to the suspected involvement of the growth hormone/insulin-like growth factor 1 (GH/IGF1) system, controversial therapeutic approaches have been developed, including both GH- administration, as well as GH-inhibition. In the present study, we examined relevant histomorphological and ultrastructural features of adenohypophyseal GH-producing somatotroph cells in a porcine DMD model. METHODS: The numbers and volumes of immunohistochemically labelled somatotroph cells were determined in consecutive semi-thin sections of plastic resin embedded adenohypophyseal tissue samples using unbiased state-of-the-art quantitative stereological analysis methods. RESULTS: DMD pigs displayed a significant growth retardation, accounting for a 55% reduction of body weight, accompanied by a significant 50% reduction of the number of somatotroph cells, as compared to controls. However, the mean volumes of somatotroph cells and the volume of GH-granules per cell were not altered. Western blot analyses of the adenohypophyseal protein samples showed no differences in the relative adenohypophyseal GH-abundance between DMD pigs and controls. CONCLUSION: The findings of this study do not provide evidence for involvement of somatotroph cells in the pathogenesis of growth retardation of DMD pigs. These results are in contrast with previous findings in other dystrophin-deficient animal models, such as the golden retriever model of Duchenne muscular dystrophy, where increased mean somatotroph cell volumes and elevated volumes of intracellular GH-granules were reported and associated with DMD-related growth retardation. Possible reasons for the differences of somatotroph morphology observed in different DMD models are discussed.
Assuntos
Transtornos do Crescimento/patologia , Hormônio do Crescimento/metabolismo , Distrofia Muscular de Duchenne/patologia , Vesículas Secretórias/patologia , Somatotrofos/patologia , Animais , Animais Geneticamente Modificados , Contagem de Células , Modelos Animais de Doenças , Distrofina/genética , Transtornos do Crescimento/complicações , Transtornos do Crescimento/metabolismo , Microscopia Eletrônica , Distrofia Muscular de Duchenne/complicações , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Tamanho do Órgão , Hipófise/patologia , Hipófise/ultraestrutura , Adeno-Hipófise/patologia , Adeno-Hipófise/ultraestrutura , Vesículas Secretórias/ultraestrutura , Somatotrofos/ultraestrutura , SuínosRESUMO
Type 2 diabetes mellitus is a disease characterized by the formation of amyloid fibrillar deposits consisting mainly in human islet amyloid polypeptide (hIAPP), a peptide co-produced and co-secreted with insulin. hIAPP and insulin are synthesized by pancreatic ß cells initially as prehormones resulting after sequential cleavages in the mature peptides as well as the two flanking peptides (N- and C-terminal) and the C-peptide, respectively. It has been suggested that in the secretory granules, the kinetics of hIAPP fibril formation could be modulated by some internal factors. Indeed, insulin is known to be a potent inhibitor of hIAPP fibril formation and hIAPP-induced cell toxicity. Here we investigate whether the flanking peptides could regulate hIAPP fibril formation and toxicity by combining biophysical and biological approaches. Our data reveal that both flanking peptides are not amyloidogenic. In solution and in the presence of phospholipid membranes, they are not able to totally inhibit hIAPP-fibril formation neither hIAPP-membrane damage. In the presence of INS-1 cells, a rat pancreatic ß-cell line, the flanking peptides do not modulate hIAPP fibrillation neither hIAPP-induced cell death while in the presence of human islets, they have a slightly tendency to reduce hIAPP fibril formation but not its toxicity. These data demonstrate that the flanking peptides do not strongly contribute to reduce mature hIAPP amyloidogenesis in solution and in living cells, suggesting that other biochemical factors present in the cells must act on mature hIAPP fibril formation and hIAPP-induced cell death.
Assuntos
Amiloide/química , Morte Celular , Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Neoplasias Pancreáticas/metabolismo , Vesículas Secretórias/metabolismo , Sequência de Aminoácidos , Agonistas dos Receptores da Amilina/farmacologia , Amiloide/efeitos dos fármacos , Animais , Células Cultivadas , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Insulinoma/tratamento farmacológico , Insulinoma/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Ratos , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/patologiaRESUMO
We herein describe a rare case of low-grade endobronchial tumor that exhibited two distinct features of typical carcinoid and acinic cell carcinoma (ACC) by immunohistochemical and ultrastructure study. ACC was suspected on transbronchial biopsy. The resected specimen showed that the tumor surface comprised an acinic cell component (40% of the tumor), and the central area comprised typical carcinoid (60% of the tumor). The acinic cell component was positive for chromogranin A, synaptophysin and alpha-1-antichymotrypsin. Additionally, this component showed focal apical membranous staining for DOG1 and weak positivity for BCL10 and SOX10. Conversely, the carcinoid component was negative for all proteins except for chromogranin A and synaptophysin. Electron microscopy indicated zymogen-type granules (600-800 nm in diameter) in the acinic cell component, whereas neuroendocrine-type granules (200-300 nm in diameter) were observed in the carcinoid component. Nuclear NR4A3 immunostaining, which is highly specific for ACC of the salivary gland, was negative in this case. We conclude that the pulmonary carcinoid tumor with true zymogen-type granules could be seen but showed superficial similarities to ACC based on negative nuclear staining for NR4A3. Pulmonary carcinoids encompass a wide morphological spectrum and may exhibit prominent acinic cell differentiation.
Assuntos
Biomarcadores Tumorais/análise , Tumor Carcinoide/diagnóstico por imagem , Carcinoma de Células Acinares/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico por imagem , Idoso , Tumor Carcinoide/patologia , Carcinoma de Células Acinares/patologia , Diferenciação Celular , Feminino , Humanos , Imuno-Histoquímica , Pulmão/diagnóstico por imagem , Pulmão/patologia , Neoplasias Pulmonares/patologia , Microscopia Eletrônica , Vesículas Secretórias/patologia , Tomografia Computadorizada por Raios XRESUMO
Phosphatidylinositol 4-phosphate (PI4P) is a membrane glycerophospholipid and a major regulator of the characteristic appearance of the Golgi complex as well as its vesicular trafficking, signalling and metabolic functions. Phosphatidylinositol 4-kinases, and in particular the PI4KIIIß isoform, act in concert with PI4P to recruit macromolecular complexes to initiate the biogenesis of trafficking vesicles for several Golgi exit routes. Dysregulation of Golgi PI4P metabolism and the PI4P protein interactome features in many cancers and is often associated with tumour progression and a poor prognosis. Increased expression of PI4P-binding proteins, such as GOLPH3 or PITPNC1, induces a malignant secretory phenotype and the release of proteins that can remodel the extracellular matrix, promote angiogenesis and enhance cell motility. Aberrant Golgi PI4P metabolism can also result in the impaired post-translational modification of proteins required for focal adhesion formation and cell-matrix interactions, thereby potentiating the development of aggressive metastatic and invasive tumours. Altered expression of the Golgi-targeted PI 4-kinases, PI4KIIIß, PI4KIIα and PI4KIIß, or the PI4P phosphate Sac1, can also modulate oncogenic signalling through effects on TGN-endosomal trafficking. A Golgi trafficking role for a PIP 5-kinase has been recently described, which indicates that PI4P is not the only functionally important phosphoinositide at this subcellular location. This review charts new developments in our understanding of phosphatidylinositol 4-kinase function at the Golgi and how PI4P-dependent trafficking can be deregulated in malignant disease.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Complexo de Golgi/enzimologia , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Fosfatos de Fosfatidilinositol/metabolismo , Vesículas Secretórias/enzimologia , Animais , Complexo de Golgi/patologia , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Neoplasias/patologia , Vesículas Secretórias/patologiaRESUMO
Platelet degranulation, a form of regulated exocytosis, is crucial for hemostasis and thrombosis. Exocytosis in platelets is mediated by SNARE proteins, and in most mammalian cells this process is controlled by Munc18 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 18) proteins. Platelets express all Munc18 paralogs (Munc18-1, -2, and -3), but their roles in platelet secretion and function have not been fully characterized. Using Munc18-1, -2, and -3 conditional knockout mice, here we deleted expression of these proteins in platelets and assessed granule exocytosis. We measured products secreted by each type of platelet granule and analyzed EM platelet profiles by design-based stereology. We observed that the removal of Munc18-2 ablates the release of alpha, dense, and lysosomal granules from platelets, but we found no exocytic role for Munc18-1 or -3 in platelets. In vitro, Munc18-2-deficient platelets exhibited defective aggregation at low doses of collagen and impaired thrombus formation under shear stress. In vivo, megakaryocyte-specific Munc18-2 conditional knockout mice had a severe hemostatic defect and prolonged arterial and venous bleeding times. They were also protected against arterial thrombosis in a chemically induced model of arterial injury. Taken together, our results indicate that Munc18-2, but not Munc18-1 or Munc18-3, is essential for regulated exocytosis in platelets and platelet participation in thrombosis and hemostasis.
Assuntos
Plaquetas/metabolismo , Exocitose , Hemostasia , Proteínas Munc18/metabolismo , Vesículas Secretórias/metabolismo , Trombose/metabolismo , Animais , Plaquetas/patologia , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Proteínas Munc18/genética , Vesículas Secretórias/genética , Vesículas Secretórias/patologia , Trombose/genética , Trombose/patologiaRESUMO
Acute pancreatitis is one of the first pathological processes where autophagy has been described in a human tissue. Autophagy, autodigestion, and cell death are early cellular events in acute pancreatitis. Recent advances in understanding autophagy highlight its importance in pathological conditions. However, methods for monitoring autophagic activity during complex diseases, involving highly differentiated secretory cells, are complicated, and the results are sometimes misinterpreted. Here, we describe methods used to identify autophagic structures and to measure autophagic flux in cultured cell models and animal models of pancreatitis. We also briefly describe the pancreas specific autophagy mouse model that was useful to understand the actual role of autophagy in pancreatitis and to identify a novel selective autophagy pathway named zymophagy. Lastly, we describe the immunomagnetic isolation of autophagosomes and the detection of autophagy in pancreatic tissue samples derived from humans.
Assuntos
Autofagossomos/patologia , Autofagia , Precursores Enzimáticos/metabolismo , Pâncreas/patologia , Pancreatite/patologia , Células Acinares , Animais , Autofagossomos/ultraestrutura , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular , Ceruletídeo/toxicidade , Modelos Animais de Doenças , Humanos , Lisossomos/metabolismo , Masculino , Camundongos , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Pâncreas/citologia , Pancreatectomia , Pancreatite/induzido quimicamente , Pancreatite/cirurgia , Ratos , Vesículas Secretórias/patologiaRESUMO
The evolution of peptidergic signaling systems in the central nervous system serves a distinct and crucial role in brain processes and function. The diversity of physiological peptides and the complexity of their regulation and secretion from the dense core vesicles (DCV) throughout the brain is a topic greatly in need of investigation, though recent years have shed light on cellular and molecular mechanisms that are summarized in this review. Here, we focus on the convergence of peptidergic systems onto the Locus Coeruleus (LC), the sole provider of norepinephrine (NE) to the cortex and hippocampus, via large DCV. As the LC-NE system is one of the first regions of the brain to undergo degeneration in Alzheimer's Disease (AD), and markers of DCV have consistently been demonstrated to have biomarker potential for AD progression, here we summarize the current literature linking the LC-NE system with DCV dysregulation and Aß peptides. We also include neuroanatomical data suggesting that the building blocks of senile plaques, Aß monomers, may be localized to DCV of the LC and noradrenergic axon terminals of the prefrontal cortex. Finally, we explore the putative consequences of chronic stress on Aß production and the role that DCV may play in LC degeneration. Clinical data of immunological markers of DCV in AD patients are discussed.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Locus Cerúleo/fisiologia , Vesículas Secretórias/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismo , Humanos , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Norepinefrina/metabolismo , Norepinefrina/fisiologia , Vesículas Secretórias/patologiaRESUMO
Ca2+-dependent secretory granule fusion with the plasma membrane is the final step for the exocytic release of inflammatory mediators, neuropeptides, and peptide hormones. Secretory cells use a similar protein machinery at late steps in the regulated secretory pathway, employing protein isoforms from the Rab, Sec1/Munc18, Munc13/CAPS, SNARE, and synaptotagmin protein families. However, no small-molecule inhibitors of secretory granule exocytosis that target these proteins are currently available but could have clinical utility. Here we utilized a high-throughput screen of a 25,000-compound library that identified 129 small-molecule inhibitors of Ca2+-triggered secretory granule exocytosis in RBL-2H3 mast cells. These inhibitors broadly fell into six different chemical classes, and follow-up permeable cell and liposome fusion assays identified the target for one class of these inhibitors. A family of 2-aminobenzothiazoles (termed benzothiazole exocytosis inhibitors or bexins) was found to inhibit mast cell secretory granule fusion by acting on a Ca2+-dependent, C2 domain-containing priming factor, Munc13-4. Our findings further indicated that bexins interfere with Munc13-4-membrane interactions and thereby inhibit Munc13-4-dependent membrane fusion. We conclude that bexins represent a class of specific secretory pathway inhibitors with potential as therapeutic agents.
Assuntos
Degranulação Celular/efeitos dos fármacos , Exocitose , Leucemia Basofílica Aguda/patologia , Mastócitos/patologia , Proteínas/metabolismo , Vesículas Secretórias/patologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Leucemia Basofílica Aguda/tratamento farmacológico , Leucemia Basofílica Aguda/metabolismo , Mastócitos/efeitos dos fármacos , Fusão de Membrana , Proteínas/genética , Ratos , Vesículas Secretórias/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The resurgence of Group A Streptococcus (GAS) infections in the past two decades has been a rising major public health concern. Due to a large number of GAS infections occurring in the skin, mast cells (MCs), innate immune cells known to localize to the dermis, could play an important role in controlling infection. MCs can exert their antimicrobial activities either early during infection, by degranulation and release of antimicrobial proteases and the cathelicidin-derived antimicrobial peptide LL-37, or by forming antibacterial MC extracellular traps (MCETs) in later stages of infection. We demonstrate that MCs do not directly degranulate in response to GAS, reducing their ability to control bacterial growth in early stages of infection. However, MC granule components are highly cytotoxic to GAS due to the pore-forming activity of LL-37, while MC granule proteases do not significantly affect GAS viability. We therefore confirmed the importance of MCETs by demonstrating their capacity to reduce GAS survival. The data therefore suggests that LL-37 from MC granules become embedded in MCETs, and are the primary effector molecule by which MCs control GAS infection. Our work underscores the importance of a non-traditional immune effector cell, utilizing a non-conventional mechanism, in the defense against an important human pathogen.
Assuntos
Degranulação Celular/imunologia , Armadilhas Extracelulares/imunologia , Mastócitos/imunologia , Dermatopatias Bacterianas/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Linhagem Celular , Armadilhas Extracelulares/microbiologia , Humanos , Mastócitos/microbiologia , Mastócitos/patologia , Vesículas Secretórias/imunologia , Vesículas Secretórias/microbiologia , Vesículas Secretórias/patologia , Dermatopatias Bacterianas/microbiologia , Dermatopatias Bacterianas/patologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/patologia , CatelicidinasRESUMO
Platelets have long been recognized as key players in hemostasis and thrombosis; however, growing evidence suggests that they are also significantly involved in cancer, the second leading cause of mortality worldwide. Preclinical and clinical studies showed that tumorigenesis and metastasis can be promoted by platelets through a wide variety of crosstalk between platelets and cancer cells. For example, cancer changes platelet behavior by directly inducing tumor-platelet aggregates, triggering platelet granule and extracellular vesicle release, altering platelet phenotype and platelet RNA profiles, and enhancing thrombopoiesis. Reciprocally, platelets reinforce tumor growth with proliferation signals, antiapoptotic effect, and angiogenic factors. Platelets also activate tumor invasion and sustain metastasis via inducing an invasive epithelial-mesenchymal transition phenotype of tumor cells, promoting tumor survival in circulation, tumor arrest at the endothelium, and extravasation. Furthermore, platelets assist tumors in evading immune destruction. Hence, cancer cells and platelets maintain a complex, bidirectional communication. Recently, aspirin (acetylsalicylic acid) has been recognized as a promising cancer-preventive agent. It is recommended at daily low dose by the US Preventive Services Task Force for primary prevention of colorectal cancer. The exact mechanisms of action of aspirin in chemoprevention are not very clear, but evidence has emerged that suggests a platelet-mediated effect. In this article, we will introduce how cancer changes platelets to be more cancer-friendly and highlight advances in the modes of action for aspirin in cancer prevention. We also discuss the opportunities, challenges, and opposing viewpoints on applying aspirin and other antiplatelet agents for cancer prevention and treatment.
Assuntos
Aspirina/uso terapêutico , Plaquetas , Comunicação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias , Inibidores da Agregação Plaquetária/uso terapêutico , Plaquetas/metabolismo , Plaquetas/patologia , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/prevenção & controle , Vesículas Secretórias/metabolismo , Vesículas Secretórias/patologiaRESUMO
Autologous submandibular gland transplantation is an effective treatment for severe dry eye syndrome. However, the protein secretion in transplanted gland is altered by a mechanism that remains to be elucidated. In the present study, we found that ß1-adrenoceptor (ß1-AR) and ß2-AR expression and the phosphorylation of the downstream molecule protein kinase A (PKA) were elevated in transplanted submandibular glands obtained from epiphora patients. Synaptobrevin/vesicle-associated membrane protein 2 (VAMP-2) interacted with syntaxin-4 and actin in human submandibular gland. The contents of syntaxin-4 and actin interacting with VAMP-2 were increased in transplanted gland. Moreover, VAMP-2 and syntaxin-4 expression in the secretory granule fraction, and VAMP-2 expression in the membrane protein fraction were increased in isoproterenol-treated and transplanted glands. Isoproterenol increased F-actin polymerization in the apical and lateral regions of the cytoplasm in both control and transplanted glands. Inhibiting PKA activity and/or F-actin formation abolished the isoproterenol-enhanced expression of VAMP-2 and syntaxin-4 in the secretory granule fraction and the isoproterenol-enhanced expression of VAMP-2 in the membrane protein fraction. Taken together, these results indicate that the activation of ß-ARs induces secretory granules and cell membrane fusion via the interaction of VAMP-2 and syntaxin-4 in a PKA- and F-actin-dependent manner in human submandibular gland. Up-regulated ß-ARs might participate in altering protein secretion in transplanted submandibular gland by promoting the interaction of VAMP-2 with syntaxin-4.