分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-12
摘要: UNC-13-Munc13s have a central function in synaptic-vesicle priming through their MUN domains. However, it is unclear whether this function arises from the ability of the MUN domain to mediate the transition from the Munc18-1-closed syntaxin-1 complex to the SNARE complex in vitro. The crystal structure of the rat Munc13-1 MUN domain now reveals an elongated, arch-shaped architecture formed by a-helical bundles, with a highly conserved hydrophobic pocket in the middle. Mutation of two residues (NF) in this pocket abolishes the stimulation caused by the Munc13-1 MUN domain on SNARE-complex assembly and on SNARE-dependent proteoliposome fusion in vitro. Moreover, the same mutation in UNC-13 abrogates synaptic-vesicle priming in Caenorhabditis elegans neuromuscular junctions. These results support the notion that orchestration of syntaxin-1 opening and SNARE-complex assembly underlies the central role of UNC-13-Munc13s in synaptic-vesicle priming.
分类: 生物学 >> 生物物理学 >> 生物力学与生物流变学 提交时间: 2016-05-12
摘要: Previously reported studies have demonstrated the involvement of p21(Waf1/CIP1) in radiation-induced bystander effects (RIBE). Mouse embryonic fibroblasts (MEFs) lacking Hus1 fail to proliferate in vitro, but inactivation of p21 allows for the continued growth of Hus1-deficient cells, indicating the close connection between p21 and Hus1 cells. In this study, wildtype MEFs, Hus1(+/+)p21(-/-) MEFs and p21(-/-)Hus1(-/-) MEFs were used in a series of radiation-induced bystander effect experiments, the roles of p21 and Hus1 in the induction and transmission of radiation-induced damage signals were investigated. Our results showed that after 5 cGy alpha particle irradiation, wild-type MEFs induced significant increases in gamma-H2AX foci and micronuclei formation in bystander cells, whereas the bystander effects were not detectable in p21(-/-)Hus1(+/+) MEFs and were restored again in p21(-/-)Hus1(-/-) MEFs. Media transfer experiments showed that p21(-/-)Hus1(+/+) MEFs were deficient in the production bystander signals, but could respond to bystander signals. We further investigated the mitogen-activated protein kinases (MAPKs) that might be involved in the bystander effects. It was found that although knocking out p21 did not affect the expression of connexin43 and its phosphorylation, it did result in inactivation of some MAPK signal pathway kinases, including JNK1/2, ERK1/2 and p38, as well as a decrease in reactive oxygen species (ROS) levels in irradiated cells. However, the activation of MAPK kinases and the ROS levels in irradiated cells were restored in the cell line by knocking out Hus1. These results suggest that p21(Waf1/CIP1) and Hus1 play crucial roles in the generation and transmission of bystander damage signals after low-dose a particle irradiation. (C) 2015 by Radiation Research
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-12
摘要: Mutations in Fused in sarcoma (FUS) gene cause a subset of familial amyotrophic lateral sclerosis (ALS), a fatal motor neuron degenerative disease. Wild-type FUS is largely localized in the nucleus, but mutant FUS accumulates in the cytoplasm and forms inclusions. It is unclear whether FUS depletion from the nucleus or FUS inclusions in the cytoplasm triggers motor neuron degeneration. In this study, we revealed that the nuclear and cytoplasmic FUS proteins form distinct local distribution patterns. The nuclear FUS forms oligomers and appears granular under confocal microscope. In contrast, the cytoplasmic FUS forms inclusions with no oligomers detected. These patterns are determined by the subcellular localization of FUS, regardless of wild-type or mutant protein. Moreover, mutant FUS remained or re-directed in the nucleus can oligomerize and behave similarly to the wild-type FUS protein. We further found that nuclear RNAs are critical to its oligomerization. Interestingly, the formation of cytoplasmic FUS inclusions is also dependent on RNA binding. Since the ALS mutations disrupt the nuclear localization sequence, mutant FUS is likely retained in the cytoplasm after translation and interacts with cytoplasmic RNAs. We therefore propose that local RNA molecules interacting with the FUS protein in different subcellular compartments play a fundamental role in determining FUS protein architecture and function.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 angstrom, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-11
摘要: Lysine formylation is a newly discovered post-translational modification (PTM) in histones and other nuclear proteins; it has a well-recognized but poorly defined role in chromatin conformation modulation and gene expression. To date, there is no general method to site-specifically incorporate N-epsilon-formyllysine at a defined site of a protein. Here we report the highly efficient genetic incorporation of the unnatural amino acid N-epsilon-formyllysine into proteins produced in Escherichia coli and mammalian cells, by using an orthogonal N-epsilon-formyllysine tRNAsynthetase/tRNA(CUA) pair. This technique can be applied to study the role of lysine formylation in epigenetic regulation.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-11
摘要: In Alzheimer's disease (AD), activated microglia invade and surround beta-amyloid plaques, possibly contributing to the aggregation of amyloid beta (A beta), which affect the survival of neurons and lead to memory loss. Phosphodiesterase-5 (PDE-5) inhibitors have recently been shown a potential therapeutic effect on AD. In this study, the effects of yonkenafil (yonk), a novel PDE-5 inhibitor, on cognitive behaviors as well as the pathological features in transgenic AD mice were investigated. Seven-month-old APP/PSI transgenic mice were treated with yonk (2, 6, or 18 mg/kg, intraperitoneal injection (i.p.)) or sildenafil (slid) (6 mg/kg, i.p.) daily for 3 months and then behavioral tests were performed. The results demonstrated that yonk improved nesting-building ability, ameliorated working memory deficits in the Y-maze tasks, and significantly improved learning and memory function in the Morris water maze (MWM) tasks. In addition, yonk reduced the area of A beta plaques, and inhibited over-activation of microglia and astrocytes. Furthermore, yonk increased neurogenesis in the dentate granule brain region of APP/PSI mice, indicated by increased BrdU(+)/NeuN(+) and BrdU(+)/DCX+ cells compared to vehicle-treated transgenic mice. These results suggest that yonk could rescue cognitive deficits by ameliorated amyloid burden through regulating APP processing, inhibited the over-activation of microglia and astrocytes as well as restored neurogenesis. (C) 2015 Elsevier Ireland Ltd. All rights reserved.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-11
摘要: Brown adipose tissue (BAT) maintains animal body temperature by non-shivering thermogenesis, which is through uncoupling protein 1 (UCP1) that uncouples oxidative phosphorylation and utilizes beta-oxidation of fatty acids released from triacylglycerol (TAG) in lipid droplets (LDs). Increasing BAT activity and "browning" other tissues such as white adipose tissue (WAT) can enhance the expenditure of excess stored energy, and in turn reduce prevalence of metabolic diseases. Although many studies have characterized the biology of BAT and brown adipocytes, BAT LDs especially their activation induced by cold exposure remain to be explored. We have isolated LDs from mouse interscapular BAT and characterized the full proteome using mass spectrometry. Both morphological and biochemical experiments showed that the LDs could tightly associate with mitochondria. Under cold treatment mouse BAT started expressing LD structure protein PLIN-2/ADRP and increased expression of PLIN1 Both hormone sensitive lipase (HSL) and adipose TAG lipase (ATGL) were increased in LDs. In addition, isolated BAT LDs showed increased levels of the mitochondrial protein UCP1, and prolonged cold exposure could stimulate BAT mitochondrial cristae biogenesis. These changes were in agreement with the data from transcriptional analysis. Our results provide the BAT LD proteome for the first time and show that BAT LDs facilitate heat production by coupling increasing TAG hydrolysis through recruitment of ATGL and HSL to the organelle and expression of another LD resident protein PLIN2/ADRP, as well as by tightly associating with activated mitochondria. These findings will benefit the study of BAT activation and the interaction between LDs and mitochondria. (C) 2015 Elsevier B.V. All rights reserved.
分类: 生物学 >> 生物物理学 >> 神经科学 提交时间: 2016-05-05
摘要: Background: Mutations in the fused in sarcoma (FUS) gene have been linked to amyotrophic lateral sclerosis (ALS). ALS patients with FUS mutations exhibit neuronal cytoplasmic mislocalization of the mutant FUS protein. ALS patients' fibroblasts or induced pluripotent stem cell (iPSC)-derived neurons have been developed as models for understanding ALS-associated FUS (ALS-FUS) pathology; however, pathological neuronal signatures are not sufficiently present in the fibroblasts of patients, whereas the generation of iPSC-derived neurons from ALS patients requires relatively intricate procedures. Results: Here, we report the generation of disease-specific induced neurons (iNeurons) from the fibroblasts of patients who carry three different FUS mutations that were recently identified by direct sequencing and multi-gene panel analysis. The mutations are located at the C-terminal nuclear localization signal (NLS) region of the protein (p.G504Wfs*12, p.R495*, p.Q519E): two de novo mutations in sporadic ALS and one in familial ALS case. Aberrant cytoplasmic mislocalization with nuclear clearance was detected in all patient-derived iNeurons, and oxidative stress further induced the accumulation of cytoplasmic FUS in cytoplasmic granules, thereby recapitulating neuronal pathological features identified in mutant FUS (p.G504Wfs*12)-autopsied ALS patient. Importantly, such FUS pathological hallmarks of the patient with the p.Q519E mutation were only detected in patient-derived iNeurons, which contrasts to predominant FUS (p.Q519E) in the nucleus of both the transfected cells and patient-derived fibroblasts. Conclusions: Thus, iNeurons may provide a more reliable model for investigating FUS mutations with disrupted NLS for understanding FUS-associated proteinopathies in ALS.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-05
摘要: Autofocusing is a fundamental step towards automated microscopic screening of Caenorhabditis elegans. Determining the optimal focus in an optical microscope is based on a clarity-evaluation function that is applied to images acquired from different focuses of the same field. The maximum value of the function is considered as the point of optimal focus. In this paper, 16 autofocus algorithms which were collected from well-known algorithms as well as the most recently proposed focusing algorithms have been evaluated. Through these evaluations, an optimal algorithm was found for C. elegants lipid droplets to set up an automatic screening system. Many features were assessed in this paper, for instance accuracy, computational time, addition of noise, and focusing curve. Our results have shown that most of the algorithms show an overall high performance for this type of image, and absolute Tenengrad algorithm will be our first choice for its best performance considering accuracy.