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 提交时间: 2016-05-11
摘要: Hsp70 chaperone proteins play crucial roles in the cell. Extensive structural and functional studies have been performed for bacterial and mammalian Hsp70s. Ssa1 from Saccharomyces cerevisiae is a member of the Hsp70 family. In vivo and biochemical studies on Ssa1 have revealed that it regulates prion propagation and the cell cycle. However, no structural data has been obtained for Ssa1 up to now. Here we report the almost complete (96 %) H-1, C-13, N-15 backbone and side chain NMR assignment of the 18.8 kDa Ssa1 substrate binding domain. The construct includes residues 382-554, which corresponds to the entire substrate binding domain and two following alpha-helices in homologous structures. The secondary structure predicted from the assigned chemical shifts is consistent with that of homologous Hsp70 substrate binding domains.
分类: 地球科学 >> 空间物理学 提交时间: 2016-12-26
摘要: Derivation of equivalent current systems (ECS) from a global magnetospheric magnetohydrodynamics (MHD) model is very useful in studying magnetosphere-ionosphere coupling, ground induction effects, and space weather forecast. In this study we introduce an improved method to derive the ECS from a global MHD model, which takes account of the obliqueness of the magnetic field lines. By comparing the ECS derived from this improved method and the previous method, we find that the main characteristics of the ECS derived from the two methods are generally consistent with each other, but the eastward-westward component of the geomagnetic perturbation calculated from the ECS derived from the improved method is much stronger than that from the previous method. We then compare the geomagnetic perturbation as a function of the interplanetary magnetic field (IMF) clock angle calculated from the ECS derived from both methods with the observations. The comparison indicates that the improved method can improve the performance of the simulation. Furthermore, it is found that the incomplete counterbalance of the geomagnetic effect produced by the ionospheric poloidal current and field-aligned current (FAC) contributes to most of the eastward-westward component of geomagnetic perturbation.
分类: 地球科学 >> 空间物理学 提交时间: 2016-12-26
摘要: Independent clocks provide time tags for the precision orbit determination (POD) equipment and the radar altimeter onboard the HY-2A satellite, and a bias between POD data' time tag and corresponding range observation's time tag from the HY-2A altimeter exists. The time tag bias contributes a bias in the sea surface height observation due to the nonzero time rate of change of the HY-2A altimeter's height. A transponder for in-orbit radar altimeter calibration provides an approach to estimate the time tag bias. The altimeter receives the responding signals from the transponder and generates ranges. Pertinent reference ranges are obtained fromthe POD data and the transponder's coordinate. Using the ranges from the radar altimeter and the reference ranges, the time tag bias between the POD data and the altimeter observations can be estimated. During an in situ HY-2A altimeter calibration campaign using a reconstructive transponder from August 9, 2012, to July 20, 2014, 17 estimations of the altimeter's time tag bias were obtained. The preliminary results are presented in this letter.
分类: 生物学 >> 生物物理学 >> 衰老生物学 提交时间: 2016-05-05
摘要: microRNA-9 (miR-9) is highly expressed in the nervous system across species and plays essential roles in neurogenesis and axon growth; however, little is known about the mechanisms that link miR-9 with dendrite growth. Using an in vivo model of Drosophila class I dendrite arborization (da) neurons, we show that miR-9a, a Drosophila homolog of mammalian miR-9, downregulates the cadherin protein Flamingo (Fmi) thereby attenuating dendrite development in a non-cell autonomous manner. In miR-9a knockout mutants, the dendrite length of a sensory neuron ddaE was significantly increased. Intriguingly, miR-9a is specifically expressed in epithelial cells but not in neurons, thus the expression of epithelial but not neuronal Fmi is greatly elevated in miR-9a mutants. In contrast, overexpression of Fmi in the neuron resulted in a reduction in dendrite growth, suggesting that neuronal Fmi plays a suppressive role in dendrite growth, and that increased epithelial Fmi might promote dendrite growth by competitively binding to neuronal Fmi. Fmi has been proposed as a G protein-coupled receptor (GPCR), we find that neuronal G protein Gq (Gq), but not Go, may function downstream of Fmi to negatively regulate dendrite growth. Taken together, our results reveal a novel function of miR-9a in dendrite morphogenesis. Moreover, we suggest that Gq might mediate the intercellular signal of Fmi in neurons to suppress dendrite growth. Our findings provide novel insights into the complex regulatory mechanisms of microRNAs in dendrite development, and further reveal the interplay between the different components of Fmi, functioning in cadherin adhesion and GPCR signalling. (c) 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 225-237, 2016
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: Mapping the pattern of connectivity between neurons is widely regarded to be critical for understanding the nervous system. GRASP (GFP reconstitution across synaptic partners) has been used as a promising method for mapping neuronal connectivity, but is currently available in the green color only, limiting its potential applications. Here we demonstrate CRASP (CFP reconstitution across synaptic partners), a cyan colored version of GRASP. We validated the system in HEK 293T cells, and generated transgenic Drosophila lines to show that the system could reliably detect neuronal contacts in the brain. Furthermore, we showed that the CRASP signal could be selectively amplified using standard immunohistochemistry methods. The CRASP system adds to the toolkit available to researchers for mapping neuronal connectivity, and substantially expands the potential application of GRASP-like strategies. (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: 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: 2016-05-15
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 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-11
摘要: Long noncoding RNAs (lncRNAs) are pervasively transcribed in the human genome. Recent studies suggest that the involvement of IncRNAs in human diseases could be far more prevalent than previously appreciated. Here we have identified a lncRNA termed Lnc_bc060912 whose expression is increased in human lung and other tumors. Lnc_bc060912 is 1.2 kb in length and is composed of two exons. The expression of Lncbc060912, was repressed by p53. Lnc_bc060912 suppressed cell apoptosis. Using a recently developed method for RNA-pulldown with formaldehyde cross-linking, we found that Lnc_bc060912 interacted With the two DNA damage repair proteins PARP1 and NPM1. Together, these results suggest that Lnc_bc060912, via PARP1 and NPM1, affects cell apoptosis and may play important roles in tumorigenesis and cancer progression.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: Protein lysine malonylation, a newly identified protein post-translational modification (PTM), has been proved to be evolutionarily conserved and is present in both eukaryotic and prokaryotic cells. However, its potential roles associated with human diseases remain largely unknown. In the present study, we observed an elevated lysine malonylation in a screening of seven lysine acylations in liver tissues of db/db mice, which is a typical model of type 2 diabetes. We also detected an elevated lysine malonylation in ob/ob mice, which is another model of type 2 diabetes. We then performed affinity enrichment coupled with proteomic analysis on liver tissues of both wild-type (wt) and db/db mice and identified a total of 573 malonylated lysine sites from 268 proteins. There were more malonylated lysine sites and proteins in db/db than in wt mice. Five proteins with elevated malonylation were verified by immunoprecipitation coupled with Western blot analysis. Bioinformatic analysis of the proteomic results revealed the enrichment of malonylated proteins in metabolic pathways, especially those involved in glucose and fatty acid metabolism. In addition, the biological role of lysine malonylation was validated in an enzyme of the glycolysis pathway. Together, our findings support a potential role of protein lysine malonylation in type 2 diabetes with possible implications for its therapy in the future.
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 提交时间: 2016-05-12
摘要: One of the major challenges in prostate cancer therapy remains the development of effective treatments for castration-resistant prostate cancer (CRPC), as the underlying mechanisms for its progression remain elusive. Previous studies showed that androgen receptor (AR) is crucially involved in regulation of metabolism in prostate cancer (PCa) cells throughout the transition from early stage, androgen-sensitive PCa to androgen-independent CRPC. AR achieves such metabolic rewiring directively either via its transcriptional activity or via interactions with AMP-activated protein kinase (AMPK). However, due to the heterogeneous expression and activity status of AR in PCa cells, it remains a challenge to investigate the links between AR status and metabolic alterations. To this end, we compared the proteomes of three pairs of androgen-sensitive (AS) and androgen-independent (AI) PCa cell lines, namely, PC3-AR(+)/PC3, 22Rv1/Du145, and LNCaP/C42B, using an iTRAQ labeling approach. Our results revealed that most of the differentially expressed proteins between each pair function in metabolism, indicating a metabolic shift between AS and AT cells, as further validated by multiple reaction monitoring (MRM)-based quantification of nucleotides and relative comparison of fatty acids between these cell lines. Furthermore, increased adenylate kinase isoenzyme 1 (AK1) in AS relative to AT cells may result in activation of AMPK, representing a major regulatory factor involved in the observed metabolic shift in PCa cells.
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: 2016-05-05
摘要: Xeroderma pigmentosum (XP) is a group of genetic disorders caused by mutations of XP-associated genes, resulting in impairment of DNA repair. XP patients frequently exhibit neurological degeneration, but the underlying mechanism is unknown, in part due to lack of proper disease models. Here, we generated patient-specific induced pluripotent stem cells (iPSCs) harboring mutations in five different XP genes including XPA, XPB, XPC, XPG, and XPV. These iPSCs were further differentiated to neural cells, and their susceptibility to DNA damage stress was investigated. Mutation of XPA in either neural stem cells (NSCs) or neurons resulted in severe DNA damage repair defects, and these neural cells with mutant XPA were hyper-sensitive to DNA damage-induced apoptosis. Thus, XP-mutant neural cells represent valuable tools to clarify the molecular mechanisms of neurological abnormalities in the XP patients.