RESUMO
Gametophyte development is a pre-requisite for plant reproduction and seed yield; therefore, studies of gametophyte development help us understand fundamental biological questions and have potential applications in agriculture. The biogenesis and dynamics of endomembrane compartments are critical for cell survival, and their regulatory mechanisms are just beginning to be revealed. Here, we report that the Arabidopsis thaliana SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) protein YKT61 is essential for both male and female gametogenesis. By using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based genome editing, we demonstrated that male and female gametophytes carrying YKT61 loss-of-function alleles do not survive. Specifically, loss of YKT61 function resulted in the arrest of male gametophytic development at pollen mitosis I and the degeneration of female gametophytes. A three-base-pair deletion in YKT61 in the ykt61-3 mutant resulted in a single-amino acid deletion in the longin domain of YKT61; the resulting mutant protein does not interact with multiple SNAREs and showed substantially reduced membrane association, suggesting that the N-terminal longin domain of YKT61 plays multiple roles in its function. This study demonstrates that Arabidopsis YKT61 is essential for male and female gametogenesis and sets an example for functional characterization of essential genes with the combination of Cas9-mediated editing and expression from a Cas9-resistant transgene.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Pólen/metabolismo , Proteínas R-SNARE/metabolismo , Alelos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Pólen/genética , Tubo Polínico/genética , Tubo Polínico/metabolismo , Proteínas R-SNARE/genéticaRESUMO
Increasing evidences reveal that autophagy inhibitor could enhance the effect of chemotherapy to cancer. However, few autophagy inhibitors are currently approved for clinical application in humans. Berbamine (BBM) is a natural compound extracted from traditional Chinese medicine that is widely used for treatment of a variety of diseases without any obvious side effects. Here we found that BBM is a novel auophagy inhibitor, which potently induced the accumulation of autophagosomes by inhibiting autophagosome-lysosome fusion in human breast cancer cells. Mechanistically, we found that BBM blocked autophagosome-lysosome fusion by inhibiting the interaction of SNAP29 and VAMP8. Furthermore, BBM induced upregulation of BNIP3 and the interaction between SNAP29 and BNIP3. BNIP3 depletion or SNAP29 overexpression abrogated BBM-mediated blockade of autophagosome-lysosome fusion through the interaction between SNAP29 and VAMP8, whereas BNIP3 overexpression blocked autophagosome-lysosome fusion through inhibition of the interaction between SNAP29 and VAMP8. These findings suggest that upregulation of BNIP3 and interaction between BNIP3 and SNAP29 could be involved in BBM-mediated blockade of autophagosome-lysosome fusion through inhibition of the interaction between SNAP29 and VAMP8. Our findings identify the critical role of BNIP3 in blockade of autophagosome-lysosome fusion mediated by BBM, and suggest that BBM could potentially be further developed as a novel autophagy inhibitor, which could enhance the effect of chemotherapy to cancer.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Autofagia/efeitos dos fármacos , Benzilisoquinolinas/farmacologia , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Proteínas R-SNARE/genética , Células A549 , Autofagossomos/metabolismo , Autofagossomos/virologia , Autofagia/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Lisossomos/metabolismo , Lisossomos/virologia , Células MCF-7 , Fusão de Membrana/efeitos dos fármacos , Proteínas de Membrana/agonistas , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas/agonistas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de SinaisRESUMO
Niemann-Pick disease type C (NPC) is an inherited lysosomal storage disease characterised by accumulation of cholesterol and glycosphingolipids. NPC patients suffer a progressive neurodegenerative phenotype presenting with motor dysfunction, mental retardation and cognitive decline. To examine the onset and progression of neuropathological insults in NPC we have systematically examined the CNS of a mouse model of NPC1 (Npc1(-/-) mice) at different stages of the disease course. This revealed a specific spatial and temporal pattern of neuropathology in Npc1(-/-) mice, highlighting that sensory thalamic pathways are particularly vulnerable to loss of NPC1 resulting in neurodegeneration in Npc1(-/-) mice. Examination of markers of astrocytosis and microglial activation revealed a particularly pronounced reactive gliosis in the thalamus early in the disease, which subsequently also occurred in interconnected cortical laminae at later ages. Our examination of the precise staging of events demonstrate that the relationship between glia and neurons varies between brain regions in Npc1(-/-) mice, suggesting that the cues causing glial reactivity may differ between brain regions. In addition, aggregations of pre-synaptic markers are apparent in white matter tracts and the thalamus and are likely to be formed within axonal spheroids. Our data provide a new perspective, revealing a number of events that occur prior to and alongside neuron loss and highlighting that these occur in a pathway dependent manner.
Assuntos
Córtex Cerebral/patologia , Neuroglia/patologia , Doenças de Niemann-Pick/genética , Doenças de Niemann-Pick/patologia , Proteínas/genética , Sinapses/patologia , Tálamo/patologia , Fatores Etários , Análise de Variância , Animais , Antígenos CD/metabolismo , Axônios/patologia , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica/genética , Glutamato Descarboxilase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais/patologia , Neuroglia/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Proteína C1 de Niemann-Pick , Proteínas/metabolismo , Proteínas R-SNARE/metabolismoRESUMO
BACKGROUND: In neurons, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25, and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual protein-protein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To bridge this gap, we have used purified synaptic vesicles, together with artificial membranes containing core-constituted SNAREs as reaction partners, in fusion assays. RESULTS: By using complementary experimental approaches, we show that synaptic vesicles fuse constitutively, and with high efficiency, with proteoliposomes containing the plasma membrane proteins syntaxin 1 and SNAP-25. Fusion is inhibited by clostridial neurotoxins and involves the formation of SNARE complexes. Despite the presence of endogenous synaptotagmin, Ca(2+) does not enhance fusion, even if phosphatidylinositol 4,5-bisphosphate is present in the liposome membrane. Rather, fusion kinetics are dominated by the availability of free syntaxin 1/SNAP-25 acceptor sites for synaptobrevin. CONCLUSIONS: Synaptic vesicles are constitutively active fusion machines, needing only synaptobrevin for activity. Apparently, the final step in fusion does not involve the regulatory activities of other vesicle constituents, although these may be involved in regulating earlier processes. This is particularly relevant for the calcium-dependent regulation of exocytosis, which, in addition to synaptotagmin, requires other factors not present in the vesicle membrane. The in vitro system described here provides an ideal starting point for unraveling of the molecular details of such regulatory events.
Assuntos
Fusão de Membrana , Proteínas R-SNARE/metabolismo , Vesículas Sinápticas/fisiologia , Animais , Cálcio/metabolismo , Exocitose/fisiologia , Lipossomos/metabolismo , Ratos , Sinapses/fisiologia , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismoRESUMO
BACKGROUND/AIMS: Electroconvulsive therapy (ECT) is an effective treatment modality for severe psychiatric disorders. Many studies have suggested that the therapeutic efficacy of ECT can be attributed to the structural and functional readjustment of the brain cells, which is mediated by differential gene expression in the brain. The aim of this study is to understand the molecular mechanism of ECT. METHODS: We used microarray-based gene expression profiling technology and real-time quantitative PCR (RT-qPCR) to screen differentially expressed genes in the brain in a rat model of ECT. RESULTS: Four upregulated and three downregulated genes were identified in this study. The 4 upregulated genes are S100 protein, beta polypeptide (S100b), S100 calcium binding protein A13_predicted (S100a13_predicted), diazepam-binding inhibitor (Dbi), and YKT6 homolog (S. Cerevisiae) (Ykt6), respectively; while the 3 downregulated genes are basigin (Bsg), histidine triad nucleotide binding protein 1(Hint 1), and neural precursor cell expressed, developmentally downregulated gene 8 (Nedd8), respectively. CONCLUSION: In view of the neurobiological function of these genes and their relevance to mental disorders, repeated ECS can affect gene expression involved in the neurotransmission and synaptic plasticity, which may account for the clinical effects of ECT.
Assuntos
Eletroconvulsoterapia , Lobo Frontal/metabolismo , Regulação da Expressão Gênica , Animais , Basigina/genética , Basigina/metabolismo , Inibidor da Ligação a Diazepam/metabolismo , Regulação para Baixo , Perfilação da Expressão Gênica , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas S100/genética , Proteínas S100/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Regulação para CimaRESUMO
Although much is known regarding the exocytic responses of mast cells following allergen/IgE-mediated activation, little is currently known of the fate of the activating allergens, many of which are particles. We have found that IgE-bound particulate allergens were phagocytosed by activated mast cells in a lipid raft-dependent manner. The nascent allergen-containing phagosomes were found to transform into granule compartments by acquiring VAMP7 and serotonin and exhibited the capacity to empty their contents upon mast cell activation. When allergen-harboring mast cells were stimulated, the intracellular allergens were expelled intact and shown to activate adjacent mast cells. This capacity of mast cells to phagocytose and retain whole and antigenically intact allergens could potentially contribute to the course of inflammatory diseases such as asthma.
Assuntos
Alérgenos/metabolismo , Imunoglobulina E/metabolismo , Mastócitos/imunologia , Microdomínios da Membrana/imunologia , Fagocitose , Pólen/metabolismo , Receptores de IgE/metabolismo , Alérgenos/imunologia , Alérgenos/ultraestrutura , Animais , Imunoglobulina E/análise , Mastócitos/ultraestrutura , Microdomínios da Membrana/química , Camundongos , Camundongos Endogâmicos C57BL , Fagossomos/química , Fagossomos/imunologia , Pólen/imunologia , Pólen/ultraestrutura , Proteínas R-SNARE/análise , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Receptores de IgE/análise , Serotonina/análise , Serotonina/metabolismoRESUMO
Synaptic core complex formation between syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) on the plasma membrane and synaptobrevin on the vesicle membrane is responsible for membrane fusion and neurotransmitter release. A radiolabeled protein binding assay for synaptic core complex formation was developed. The components of this assay included recombinant glutathione S-transferase (GST)-syntaxin immobilized on glutathione agarose beads, SNAP25 and (125)I-labeled synaptobrevin. Reactions were performed in tubes containing filter inserts to facilitate removal of unbound protein. The radiolabeled protein bound was then quantified by gamma counter. A K(d) of 1.6 microM was determined for the GST-syntaxin/SNAP25/synaptobrevin complex, and a K(d) of 12 microM was determined for the GST-syntaxin/synaptobrevin complex. The assay was used to screen 14 herbal extracts for effectors of core complex formation. Herbs traditionally used to treat neurological conditions such as depression, anxiety, and stress were chosen. A Hypericum perforatum extract was found to have a nonspecific effect via protein complexation, whereas an Albizzia julibrissin extract was found to reduce the level of core complex formation. The assay was used to further investigate the effect of the A. julibrissin extract. The discovery of an inhibitor of core complex formation demonstrates the efficacy of the assay in screening natural products for substances that affect core complex formation.