分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-15
摘要: GADD45A is a TP53-regulated and DNA damage-inducible tumor suppressor protein, which regulates cell cycle arrest, apoptosis, and DNA repair, and inhibits tumor growth and angiogenesis. However, the function of GADD45A in autophagy remains unknown. In this report, we demonstrate that GADD45A plays an important role in regulating the process of autophagy. GADD45A is able to decrease LC3-II expression and numbers of autophagosomes in mouse tissues and different cancer cell lines. Using bafilomycin A(1) treatment, we have observed that GADD45A regulates autophagosome initiation. Likely, GADD45A inhibition of autophagy is through its influence on the interaction between BECN1 and PIK3C3. Immunoprecipitation and GST affinity isolation assays exhibit that GADD45A directly interacts with BECN1, and in turn dissociates the BECN1-PIK3C3 complex. Furthermore, we have mapped the 71 to 81 amino acids of the GADD45A protein that are necessary for the GADD45A interaction with BECN1. Knockdown of BECN1 can abolish autophagy alterations induced by GADD45A. Taken together, these findings provide the novel evidence that GADD45A inhibits autophagy via impairing the BECN1-PIK3C3 complex formation.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: Type 2 diabetes mellitus (T2DM) is regarded as one of the serious risk factors for age-related cognitive impairment; however, a causal link between these two diseases has so far not been established. It was recently discovered that, apart from high D-glucose levels, T2DM patients also display abnormally high concentrations of uric D-ribose. Here, we show for the first time that the administration of D-ribose, the most active glycator among monosaccharides, produces high levels of advanced glycation end products (AGEs) and, importantly, triggers hyperphosphorylation of Tau in the brain of C57BL/6 mouse and neuroblastoma N2a cells. However, the administration of D-glucose showed no significant changes in Tau phosphorylation under the same experimental conditions. Crucially, suppression of AGE formation using an AGEs inhibitor (aminoguanidine) effectively prevents hyperphosphorylation of Tau protein. Further study shows AGEs resulted from ribosylation activate calcium-/calmodulin-dependent protein kinase type II (CaMKII), a key kinase responsible for Tau hyperphosphorylation. These data suggest that there is indeed a mechanistic link between ribosylation and Tau hyperphosphorylation. Targeting ribosylation by inhibiting AGE formation may be a promising therapeutic strategy to prevent Alzheimer's disease-like Tau hyperphosphorylation and diabetic encephalopathies.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: The piRNA machinery is known for its role in mediating epigenetic silencing of transposons. Recent studies suggest that this function also involves piRNA-guided cleavage of transposon-derived transcripts. As many piRNAs also appear to have the capacity to target diverse mRNAs, this raises the intriguing possibility that piRNAs may act extensively as siRNAs to degrade specific mRNAs. To directly test this hypothesis, we compared mouse PIWI (MIWI)-associated piRNAs with experimentally identified cleaved mRNA fragments from mouse testes, and observed cleavage sites that predominantly occur at position 10 from the 5' end of putative targeting piRNAs. We also noted strong biases for U and A residues at nucleotide positions 1 and 10, respectively, in both piRNAs and mRNA fragments, features that resemble the pattern of piRNA amplification by the 'ping-pong' cycle. Through mapping of MIWI-RNA interactions by CLIP-seq and gene expression profiling, we found that many potential piRNA-targeted mRNAs directly interact with MIWI and show elevated expression levels in the testes of Miwi catalytic mutant mice. Reporter-based assays further revealed the importance of base pairing between piRNAs and mRNA targets and the requirement for both the slicer activity and piRNA-loading ability of MIWI in piRNA-mediated target repression. Importantly, we demonstrated that proper turnover of certain key piRNA targets is essential for sperm formation. Together, these findings reveal the siRNA-like function of the piRNA machinery in mouse testes and its central requirement for male germ cell development and maturation.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: The mechanisms that specify and maintain the characteristics of germ cells during animal development are poorly understood. In this study, we demonstrated that loss of function of the zinc-finger gene lsy-2 results in various somatic cells adopting germ cells characteristics, including expression of germline-specific P granules, enhanced RNAi activity and transgene silencing. The soma to germ transformation in lsy-2 mutants requires the activities of multiple chromatin remodeling complexes, including the MES-4 complex and the ISW-1 complex. The distinct germline-specific features in somatic cells and the gene expression profile indicate that LSY-2 acts in the Mec complex in this process. Our study demonstrated that lsy-2 functions in the maintenance of the soma-germ distinction.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: The large dense-core vesicle (LDCV), a type of lysosome-related organelle, is involved in the secretion of hormones and neuropeptides in specialized secretory cells. The granin family is a driving force in LDCV biogenesis, but the machinery for granin sorting to this biogenesis pathway is largely unknown. The mu mutant mouse, which carries a spontaneous null mutation on the Muted gene (also known as Bloc1s5), which encodes a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), is a mouse model of Hermansky-Pudlak syndrome. Here, we found that LDCVs were enlarged in mu adrenal chromaffin cells. Chromogranin A (CgA, also known as CHGA) was increased in mu adrenals and muted-knockdown cells. The increased CgA in mu mice was likely due a failure to export this molecule out of immature LDCVs, which impairs LDCV maturation and docking. In mu chromaffin cells, the size of readily releasable pool and the vesicle release frequency were reduced. Our studies suggest that the muted protein is involved in the selective export of CgA during the biogenesis of LDCVs.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: The Hippo signaling pathway restricts organ size by inactivating the Yorkie (Yki)/Yes-associated protein (YAP) family proteins. The oncogenic Yki/YAP transcriptional coactivator family promotes tissue growth by activating target gene transcription, but the regulation of Yki/YAP activation remains elusive. In mammalian cells, we identified Brg1, a major subunit of chromatin-remodeling SWI/SNF family proteins, which interacts with YAP. This finding led us to investigate the in vivo functional interaction of Yki and Brahma (Brm), the Drosophila homolog of Brg1. We found that Brm functions at the downstream of Hippo pathway and interacts with Yki and Scalloped (Sd) to promotes Yki-dependent transcription and tissue growth. Furthermore, we demonstrated that Brm is required for the Crumbs (Crb) dysregulation-induced Yki activation. Interestingly, we also found that crb is a downstream target of Yki-Brm complex. Brm physically binds to the promoter of crb and regulates its transcription through Yki. Together, we showed that Brm functions as a critical regulator of Hippo signaling during tissue growth and plays an important role in the feedback loop between Crb and Yki. (C) 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: The H3 histone variant CENP-A is an epigenetic marker critical for the centromere identity and function. However, the precise regulation of the spatiotemporal deposition and propagation of CENP-A at centromeres during the cell cycle is still poorly understood. Here, we show that CENP-A is phosphorylated at Ser68 during early mitosis by Cdk1. Our results demonstrate that phosphorylation of Ser68 eliminates the binding of CENP-A to the assembly factor HJURP, thus preventing the premature loading of CENP-A to the centromere prior to mitotic exit. Because Cdk1 activity is at its minimum at the mitotic exit, the ratio of Cdk1/PP1 alpha activity changes in favor of Ser68 dephosphorylation, thus making CENP-A available for centromeric deposition by HJURP. Thus, we reveal that dynamic phosphorylation of CENP-A Ser68 orchestrates the spatiotemporal assembly of newly synthesized CENP-A at active centromeres during the cell cycle.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: The fatty alk(a/e)ne biosynthesis pathway found in cyanobacteria gained tremendous attention in recent years as a promising alternative approach for biofuel production. Cyanobacterial aldehyde-deformylating oxygenase (cADO), which catalyzes the conversion of C-n fatty aldehyde to its corresponding Cn-1 alk(a/e)ne, is a key enzyme in that pathway. Due to its low activity, alk(a/e)ne production by cADO is an inefficient process. Previous biochemical and structural investigations of cADO have provided some information on its catalytic reaction. However, the details of its catalytic processes remain unclear. Here we report five crystal structures of cADO from the Synechococcus elongates strain PCC7942 in both its iron-free and iron-bound forms, representing different states during its catalytic process. Structural comparisons and functional enzyme assays indicate that Glu144, one of the iron-coordinating residues, plays a vital role in the catalytic reaction of cADO. Moreover, the helix where Glu144 resides exhibits two distinct conformations that correlates with the different binding states of the di-iron center in cADO structures. Therefore, our results provide a structural explanation for the highly labile feature of cADO di-iron center, which we proposed to be related to its low enzymatic activity. On the basis of our structural and biochemical data, a possible catalytic process of cADO was proposed, which could aid the design of cADO with improved activity.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: The CRISPR/Cas system has proven to be a powerful gene editing tool both in vitro and in vivo. A recent flurry of studies of in vivo gene editing using the CRISPR/Cas system bring bright prospects in creating animal models and targeted gene therapy of human genetic diseases.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: Super-resolution microscopy techniques have overcome the limit of optical diffraction. Recently, the Bayesian analysis of Bleaching and Blinking data (3B) method has emerged as an important tool to obtain super-resolution fluorescence images. 3B uses the change in information caused by adding or removing fluorophores in the cell to fit the data. When adding a new fluorophore, 3B selects a random initial position, optimizes this position and then determines its reliability. However, the fluorophores are not evenly distributed in the entire image region, and the fluorescence intensity at a given position positively correlates with the probability of observing a fluorophore at this position. In this paper, we present a Bayesian analysis of Bleaching and Blinking microscopy method based on fluorescence intensity distribution (FID3B). We utilize the intensity distribution to select more reliable positions as the initial positions of fluorophores. This approach can improve the reconstruction results and significantly reduce the computational time. We validate the performance of our method using both simulated data and experimental data from cellular structures. The results confirm the effectiveness of our method.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: Specific recognition of centromere-specific histone variant CENP-A-containing chromatin by CENP-N is an essential process in the assembly of the kinetochore complex at centromeres prior to mammalian cell division. However, the mechanisms of CENP-N recruitment to centromeres/kinetochores remain unknown. Here, we show that a CENP-A-specific RG loop (Arg80/Gly81) plays an essential and dual regulatory role in this process. The RG loop assists the formation of a compact "ladder-like" structure of CENP-A chromatin, concealing the loop and thus impairing its role in recruiting CENP-N. Upon G1/S-phase transition, however, centromeric chromatin switches from the compact to an open state, enabling the now exposed RG loop to recruit CENP-N prior to cell division. Our results provide the first insights into the mechanisms by which the recruitment of CENP-N is regulated by the structural transitions between compaction and relaxation of centromeric chromatin during the cell cycle.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: Sirtuins with an extended N-terminal domain (NTD), represented by yeast Sir2 and human SIRT1, harbor intrinsic mechanisms for regulation of their NAD-dependent deacetylase activities. Elucidation of the regulatory mechanisms is crucial for understanding the biological functions of sirtuins and development of potential therapeutics. In particular, SIRT1 has emerged as an attractive therapeutic target, and the search for SIRT1-activating compounds (STACs) has been actively pursued. However, the effectiveness of a class of reported STACs (represented by resveratrol) as direct SIRT1 activators is under debate due to the complication involving the use of fluorogenic substrates in in vitro assays. Future efforts of SIRT1-based therapeutics necessitate the dissection of the molecular mechanism of SIRT1 stimulation. We solved the structure of SIRT1 in complex with resveratrol and a 7-amino-4-methylcoumarin (AMC)-containing peptide. The structure reveals the presence of three resveratrol molecules, two of which mediate the interaction between the AMC peptide and the NTD of SIRT1. The two NTD-bound resveratrol molecules are principally responsible for promoting tighter binding between SIRT1 and the peptide and the stimulation of SIRT1 activity. The structural information provides valuable insights into regulation of SIRT1 activity and should benefit the development of authentic SIRT1 activators.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: Sensory dendrites innervate peripheral tissues through cell-cell interactions that are poorly understood. The proprioceptive neuron PVD in C. elegans extends regular terminal dendritic branches between muscle and hypodermis. We found that the PVD branch pattern was instructed by adhesion molecule SAX-7/L1CAM, which formed regularly spaced stripes on the hypodermal cell. The regularity of the SAX-7 pattern originated from the repeated and regularly spaced dense body of the sarcomeres in the muscle. The extracellular proteoglycan UNC-52/Perlecan linked the dense body to the hemidesmosome on the hypodermal cells, which in turn instructed the SAX-7 stripes and PVD dendrites. Both UNC-52 and hemidesmosome components exhibited highly regular stripes that interdigitated with the SAX-7 stripe and PVD dendrites, reflecting the striking precision of subcellular patterning between muscle, hypodermis, and dendrites. Hence, the muscular contractile apparatus provides the instructive cues to pattern proprioceptive dendrites.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: PGE2 elevates IL-23 production in mouse dendritic cells while inhibits IL-23 production in isolated human monocytes. Whether this differential effect of PGE2 on IL-23 production is cell-type-or species-specific has not been investigated in detail. The present study was designed to investigate the effect of PGE2 on IL-23 production in human DCs and the possible underlying mechanisms. Human monocytes derived dendritic cells (Mo-DCs) were pretreated with or without PGE2. Then the cells were incubated with zymosan. Our results demonstrated that PGE2 promoted zymosan-induced IL-23 production in a concentration dependent manner. In addition, it was found that PGE2 is also able to elevate MyD88-mediated IL-23 p19 promoter activity. More importantly, ELISA data demonstrated that db-cAMP, a cAMP analog, and forskolin, an adenylate cyclase activator, can mimic the effect of PGE2 on zymosan-induced IL-23 production, and rp-cAMP, a protein kinase A (PKA) inhibitor, can block the effect of PGE2. Moreover, PGE2 can increase zymosan-induced expression of the mRNA levels of both p19 and p40 subunits, which was mimicked by db-cAMP and forskolin. Our data suggest that PGE2 elevates the production of IL-23 in humanMo-DCs via a cAMP dependent pathway.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: p53 is an important tumor suppressor and stress response mediator. Proper control of p53 level and activity is tightly associated with its function. Posttranslational modifications and the interactions with Mdm2 and Mdm4 are major mechanisms controlling p53 activity and stability. As p53 protein is short-lived and hardly detectable in unstressed situations, less is known on its basal level expression and the corresponding controlling mechanisms in vivo. In addition, it also remains obscure how p53 expression might contribute to its functional regulation. In this study, we established bacterial artificial chromosome transgenic E.coli beta-galactosidase Z gene reporter mice to monitor p53 expression in mouse tissues and identify important regulatory elements critical for the expression in vivo. We revealed preferentially high level of p53 reporter expressions in the proliferating, but not the differentiated compartments of the majority of tissues during development and tissue homeostasis. In addition, tumors as well as regenerating tissues in the p53 reporter mice also expressed high level of beta-gal. Furthermore, both the enhancer box sequence (CANNTG) in the p53 promoter and the 3' terminal untranslated region element were critical in mediating the high-level expression of the reporter. We also provided evidence that cellular myelocytomatosis oncogene was a critical player regulating p53 mRNA expression in proliferating cells and tissues. Finally, we found robust p53 activation preferentially in the proliferating compartment of mouse tissues upon DNA damage and the proliferating cells exhibited an enhanced p53 response as compared with cells in a quiescent state. Together, these results suggested a highly regulated expression pattern of p53 in the proliferating compartment controlled by both transcriptional and posttranscriptional mechanisms, and such regulated p53 expression may impose functional significance upon stress by setting up a precautionary mode in defense of cellular transformation and tumorigenesis.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: Nuclease-based genome editing has proven to be a powerful and promising tool for disease modeling and gene therapy. Recent advances in CRISPR/Cas and TALE indicate that they could also be used as a targeted regulator of gene expression, as well as being utilized for illuminating specific chromosomal structures or genomic regions.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: Nuclease-based gene editing technologies have opened up opportunities for correcting human genetic diseases. For the first time, scientists achieved targeted gene editing of mitochondrial DNA in mouse oocytes fused with patient cells. This fascinating progression may encourage the development of novel therapy for human maternally inherent mitochondrial diseases.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: Neutrophils play an essential role in the innate immune response to infection. Neutrophils migrate from the vasculature into the tissue in response to infection. Recently, a neutrophil cell surface receptor, CD177, was shown to help mediate neutrophil migration across the endothelium through interactions with PECAM1. We examined a publicly available gene array dataset of CD177 expression from human neutrophils following pulmonary endotoxin instillation. Among all 22,214 genes examined, CD177 mRNA was the most upregulated following endotoxin exposure. The high level of CD177 expression is also maintained in airspace neutrophils, suggesting a potential involvement of CD177 in neutrophil infiltration under infectious diseases. To determine the role of CD177 in neutrophils in vivo, we constructed a CD177-genetic knockout mouse model. The mice with homozygous deletion of CD177 have no discernible phenotype and no significant change in immune cells, other than decreased neutrophil counts in peripheral blood. We examined the role of CD177 in neutrophil accumulation using a skin infection model with Staphylococcus aureus. CD177 deletion reduced neutrophil counts in inflammatory skin caused by S. aureus. Mechanistically we found that CD177 deletion in mouse neutrophils has no significant impact in CXCL1/KC- or fMLP-induced migration, but led to significant cell death. Herein we established a novel genetic mouse model to study the role of CD177 and found that CD177 plays an important role in neutrophils.
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.