分类: 生物学 >> 生物物理学 >> 神经科学 提交时间: 2016-06-06
摘要: Previous studies have shown that the Hippo pathway effector yes-associated protein (YAP) plays an important role in maintaining stem cell proliferation. However, the precise molecular mechanism of YAP in regulating murine embryonic neural stem cells (NSCs) remains largely unknown. Here, we show that bone morphogenetic protein-2 (BMP2) treatment inhibited the proliferation of mouse embryonic NSCs, that YAP was critical for mouse NSC proliferation, and that BMP2 treatment-induced inhibition of mouse NSC proliferation was abrogated by YAP knockdown, indicating that the YAP protein mediates the inhibitory effect of BMP2 signaling. Additionally, we found that BMP2 treatment reduced YAP nuclear translocation, YAP-TEAD interaction, and YAP-mediated transactivation. BMP2 treatment inhibited YAP/TEAD-mediated Cyclin D1 (ccnd1) expression, and knockdown of ccnd1 abrogated the BMP2-mediated inhibition of mouse NSC proliferation. Mechanistically, we found that Smad1/4, effectors of BMP2 signaling, competed with YAP for the interaction with TAED1 and inhibited YAP's cotranscriptional activity. Our data reveal mechanistic cross talk between BMP2 signaling and the Hippo-YAP pathway in murine NSC proliferation, which may be exploited as a therapeutic target in neurodegenerative diseases and aging.
分类: 生物学 >> 生物物理学 >> 免疫学 提交时间: 2016-05-15
摘要: The lineage commitment of HSCs generates balanced myeloid and lymphoid populations in hematopoiesis. However, the underlying mechanisms that control this process remain largely unknown. Here, we show that insulin-insulin receptor (InsR) signaling is required for lineage commitment of multipotent progenitors (MPPs). Deletion of Insr in murine bone marrow causes skewed differentiation of MPPs to myeloid cells. mTOR acts as a downstream effector that modulates MPP differentiation. mTOR activates Stat3 by phosphorylation at serine 727 under insulin stimulation, which binds to the promoter of Ikaros, leading to its transcription priming. Our findings reveal that the insulin-InsR signaling drives MPP differentiation into lymphoid lineages in early lymphopoiesis, which is essential for maintaining a balanced immune system for an individual organism.
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: 2016-05-15
摘要: The cyclic nucleotide-binding (CNB)-like protein (CNB-L) from Brucella abortus shares sequence homology with CNB domain-containing proteins. We determined the crystal structure of CNB-L at 2.0 angstrom resolution in the absence of its C-terminal helix and nucleotide. The 3D structure of CNB-L is in a twofold symmetric form. Each protomer shows high structure similarity to that of cGMP-binding domain-containing proteins, and likely mimics their nucleotide-free conformation. A key residue, Glu17, mediates the dimerization and prevents binding of cNMP to the canonical ligand-pocket. The structurally observed dimer of CNB-L is stable in solution, and thus is likely to be biologically relevant. (C) 2015 Elsevier Inc. All rights reserved.
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 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-12
摘要: Translationally controlled tumor protein (TCTP) is an abundant protein that is highly conserved in eukaryotes. However, its primary function is still not clear. Human TCTP interacts with the metazoan-specific eukaryotic elongation factor 1B delta (eEF1B delta) and inhibits its guanine nucleotide exchange factor (GEF) activity, but the structural mechanism remains unknown. The interaction between TCTP and eEF1B delta was investigated by NMR titration, structure determination, paramagnetic relaxation enhancement, site-directed mutagenesis, isothermal titration calorimetry, and HADDOCK docking. We first demonstrated that the catalytic GEF domain of eEF1B delta is not responsible for binding to TCTP but rather a previously unnoticed central acidic region (CAR) domain in eEF1B delta. The mutagenesis data and the structural model of the TCTP-eEF1B delta CAR domain complex revealed the key binding residues. These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1B delta, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1B delta CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP.
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: The transcription factor nuclear factor kB (NF-kappa B) is crucial for innate immune defense against viral infections, and its activation requires the ubiquitylation of upstream proteins, including the adaptor protein NEMO (NF-kappa B essential modulator). Many infectious pathogens, including hepatitis C virus (HCV), inhibit NF-kappa B signaling in host cells, which promotes pathogen survival. Frequently, HCV-infected individuals develop a chronic infection, which suggests that HCV can subvert host antiviral responses. We found that HCV infection and replication inhibited the activation of NF-kappa B by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), which was mediated by the viral protein NS3 and, to a lesser extent, NS5B. NS3 directly interacted with linear ubiquitin chain assembly complex (LUBAC), competed with NEMO for binding to LUBAC, and inhibited the LUBAC-mediated linear ubiquitylation of NEMO and the subsequent activation of NF-kappa B. Together, our results highlight an immune evasion strategy adopted by HCV to modulate host antiviral responses and enhance virus survival and persistence.
分类: 生物学 >> 生物物理学 >> 肿瘤学 提交时间: 2016-05-12
摘要: The signal transduction pathway initiated by vascular endothelial growth factor-vascular endothelial growth factor receptor 2 (VEGF-VEGFR2) plays an important role in the angiogenesis of tumors. The effective antagonists of VEGFR2 would behave as potent drugs for the treatment of malignant cancers. In our study, specific binding peptides with high affinity to VEGFR2 were obtained through bacterial display technology. Conserved motif (FF/YEXWGVK) among those peptide sequences was discovered. One of the selected peptides, VRBP1 (YDGNSFYEMWGVKPASES) was identified by screening the biased bacterial peptide library and its physiochemical feature was further characterized. The results of surface plasmon resonance (SPR) assay indicated that the dissociation constant (K-D) value of VRBP1 was 228.3 nM and this peptide competed with VEGF binding to VEGFR2. Particles conjugated with VRBP1 could recognize the human umbilical vein endothelial cells (HUVEC) which express VEGFR2 on the surface. Further therapeutic effect of VRBP1 was examined by in vivo experiments. VRBP1 could result in a significant decrease in tumor size of H460 xenografts. The results from the immunohistochemical assay showed that CD31 positive signals in VRBP1-treated group were fewer than those in the control ones. These data highlighted the potential of VEGFR2-binding peptides as effective molecules for cancer diagnosis and therapy.
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: The lipid droplet (LD) is a cellular organelle that stores neutral lipids in cells and has been linked with metabolic disorders. Caenorhabditis elegans has many characteristics which make it an excellent animal model for studying LDs. However, unlike in mammalian cells, no LD structure-like/resident proteins have been identified in C. elegans, which has limited the utility of this model for the study of lipid storage and metabolism. Herein based on three lines of evidence, we identified that MDT-28 and DHS-3 previously identified in C. elegans LD proteome were two LD structure-like/resident proteins. First, MDT-28 and DHS-3 were found to be the two most abundant LD proteins in the worm. Second, the proteins were specifically localized to LDs and we identified the domains responsible for this targeting in both proteins. Third and most importantly, the depletion of MDT-28 induced LD clustering while DHS-3 deletion reduced triacylglycerol content (TAG). We further characterized the proteins finding that MDT-28 was ubiquitously expressed in the intestine, muscle, hypodermis, and embryos, whereas DHS-3 was expressed mainly in intestinal cells. Together, these two LD structure-like/resident proteins provide a basis for future mechanistic studies into the dynamics and functions of LDs in C. elegans. (C) 2015 Elsevier B.V. All rights reserved.
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: The human influenza A (H3N2) virus dominated the 2014-2015 winter season in many countries and caused massive morbidity and mortality because of its antigenic variation. So far, very little is known about the antigenic patterns of the recent H3N2 virus. By systematically mapping the antigenic relationships of H3N2 strains isolated since 2010, we discovered that two groups with obvious antigenic divergence, named SW13 (A/Switzerland/9715293/2013-like strains) and HK14 (A/Hong Kong/5738/2014-like strains), co-circulated during the 2014-2015 winter season. HK14 group co-circulated with SW13 in Europe and the United States during this season, while there were few strains of HK14 in mainland China, where SW13 has dominated since 2012. Furthermore, we found that substitutions near the receptor-binding site on hemagglutinin played an important role in the antigenic variation of both the groups. These findings provide a comprehensive understanding of the recent antigenic evolution of H3N2 virus and will aid in the selection of vaccine strains.
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 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-12
摘要: The architecture and structural details of chromatin assembler, chromatin modifier, and chromatin remodeler, and their complexes with nucleosome and chromatin, are critical for revealing the molecular mechanisms for the establishment, maintenance and regulation of epigenetic information. The fast-developing cryo-electron microscopy (cryo-EM) provides an indispensible tool for the structure determiantion of the huge, multi-subunits, and flexible epigenetic regulation related macromolecular assemblies. In this review, we summarized the recent progresses on EM structural studies of macromolecules and complexes relating to epigenetic regulation.
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: Tandem-arranged PDZ [PSD-95 (postsynaptic density-95), Dlg (discs large homologue) and ZO-1 (zonula occludens-1)] domains often form structural and functional supramodules with distinct target-binding properties. In the present study, we found that the two PDZ domains within the PDZ34 tandem of Scribble, a cell polarity regulator, tightly pack in a 'front-to-back' mode to form a compact supramodule. Although PDZ4 contains a distorted alpha B/beta B pocket, the attachment of PDZ4 to PDZ3 generates an unexpected interdomain pocket that is adjacent to and integrates with the canonical aB/aB pocket of PDZ3 to form an expanded target-binding groove. The structure of the PDZ34-target peptide complex further demonstrated that the peptide binds to this expanded target-binding groove with its upstream residues anchoring into the interdomain pocket directly. Mutations of the interdomain pocket and disruptions of the PDZ34 supramodule both interfere with its target-binding capacity. Therefore, the interdomain interface between the PDZ34 supramodule is intrinsically required for its target recognition and determines its target-binding specificity. This interdomain interface-mediated specific recognition may represent a novel mode of target recognition and would broaden the target-binding versatility for PDZ supramodules. The supramodular nature and target recognitionmode of the PDZ34 tandem found in the present study would also help to identify the new binding partners of Scribble and thus may direct further research on the PDZ domain-mediated assembly of Scribble polarity complexes.
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: Talin, as the activator of integrin and the adaptor between the cytoskeleton and integrin, plays a key role in a series of processes such as cell adhesion and migration. The activation of integrin involves F3 subdomain of Talin-FERM domain binding the cytoplasmic tail of integrin beta-subunit. Talin has two states: auto-inhibited and activated. We previously reported the auto-inhibition complex structure of Talin F2F3/R9, in which the integrin binding site F3 interacts with R9(1654 similar to 1822 a.a.) of Talin-ROD, such that integrin cannot be activated. However, besides F3 and R9, it remains unclear what structural or functional roles the other domains of the 270 ku Talin play in the regulation of its activation. Here we solved the crystal structures of Talin R9-R10 (1654 similar to 1973 a.a.) and R10-R11 (1815 similar to 2140 a.a.), respectively. R9, R10 and R11 are all 5-helix bundles. R9 and R10 is joined together by a long alpha-helix instead of a flexible loop, and the two bundles are located at the opposite sides of the long helix with an angle of about 150 degrees. The linker between R10 and R11 is stabilized by neighboring hydrogen bonds, forming an angle of about 120 between the two bundles. These angles observed in our crystal structures are consistent with the previously reported SAXS and EM results. After superimposition of R9-10, R10-11 with previously reported structures of R7-8 and R11-12, a model of R7-12 was acquired, which adopts an elongated linear conformation, except that R8 protrudes from the ROD. According to this model, R10-12 does not intrude the interaction between F3 and R9, whereas R8 not only masks the F3 binding site of R9, but also might electrostatically hinders F2F3 approaching via its unique positively charged surface. This hypothesis was further verified by the results of size exclusion chromatography. Our work provides a new structural basis for studying the mechanism of Talin auto-inhibition.
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: Talin is an integrin-binding protein located at focal adhesion site and serves as both an adapter and a force transmitter. Its integrin binding activity is regulated by the intramolecular autoinhibition interaction between its F3 and RS domains. Here, we used atomic force microscopy to measure the strength of talin autoinhibition complex. Our results suggest that the lifetime of talin autoinhibition complex shows weak catch bond behavior and does not change significantly at smaller forces, while it drops rapidly at larger forces (>10 pN). Moreover, besides the complex conformation revealed by crystal structure, our molecular dynamics (MD) simulations indicate the possible existence of another stable conformation. Further analysis indicates that forces may regulate the equilibrium of the two stable binding states and result in the non-exponential force dependence of the binding lifetime. Our findings reveal a negative regulation mechanism on talin activation and provide a new point of view on the function of talin in focal adhesion.
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 >> 神经科学 提交时间: 2016-05-12
摘要: Orientation, the spatial organization of animal behavior, is an essential faculty of animals. Bacteria and lower animals such as insects exhibit taxis, innate orientation behavior, directly toward or away from a directional cue. Organisms can also orient themselves at a specific angle relative to the cues. In this study, using Drosophila as a model system, we established a visual orientation conditioning paradigm based on a flight simulator in which a stationary flying fly could control the rotation of a visual object. By coupling aversive heat shocks to a fly's orientation toward one side of the visual object, we found that the fly could be conditioned to orientate toward the left or right side of the frontal visual object and retain this conditioned visual orientation. The lower and upper visual fields have different roles in conditioned visual orientation. Transfer experiments showed that conditioned visual orientation could generalize between visual targets of different sizes, compactness, or vertical positions, but not of contour orientation. Rut-Type I adenylyl cyclase and Dnc-phosphodiesterase were dispensable for visual orientation conditioning. Normal activity and scb signaling in R3/R4d neurons of the ellipsoid body were required for visual orientation conditioning. Our studies established a visual orientation conditioning paradigm and examined the behavioral properties and neural circuitry of visual orientation, an important component of the insect's spatial navigation.
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: NLRP10 is a special member of NOD-like receptors (NLRs) family that lacks the leucine-rich repeats, suggesting that NLRP10 may act as an immunity regulator rather than directly receptor recognizing intracellular pathogen products. Previous studies on NLRP10 show that NLRP10 can interact with several components of NOD1 pathway thereby enhancing NOD1-mediated innate immune responses. In particular models, NLRP10 also negatively affects the activation of NLRP3 inflammasome. It has been proposed that NLRP10 oligomers interact with ASC to form a multi-protein platform for the recruitment of caspase-1 or other signaling components. Here, we show that pyrin-like domain (PYD) degradation induce the formation of NLRP10 oligomers, which present stick-shaped and circular structure. With the NLRP10 mutant G173A made by means of site-directed mutagenesis, we successfully obtain homogeneous full-length NLRP10 preparations. Corresponding gel filtration analysis and electron microscope (EM) data further proved that the PYD domain is important in protecting NLRP10 against aggregation.