分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification that plays essential roles in many cellular pathways. Research in this field, however, is hampered by the lack of suitable probes to identify, accumulate, and purify the O-GlcNAcylated proteins. We have previously reported the identification of a lectin from the mushroom Agrocybe aegerita, i.e., Agrocybe aegerita lectin 2, or AAL2, that could bind terminal N-acetylglucosamine with higher affinities and specificity than other currently used probes. In this paper, we report the crystal structures of AAL2 and its complexes with GlcNAc and GlcNAc beta 1-3Gal beta 1-4GlcNAc and reveal the structural basis of GlcNAc recognition by AAL2 and residues essential for the binding of terminal N-acetylglucosamine. Study on AAL2 may enable us to design a protein probe that can be used to identify and purify O-GlcNAcylated proteins more efficiently.
分类: 生物学 >> 生物物理学 >> 神经科学 提交时间: 2016-05-05
摘要: Postoperative cognitive dysfunction (POCD) is an important complication following major surgery and general anesthesia in older patients. However, the etiology of POCD remains largely to be determined. It is unknown how surgical stress and psychological stress affect the postoperative learning and memory function in geriatric patients. We therefore established a pre-clinical model in aged C57BL/6 mice and aimed to investigate the effects of surgical stress and psychological stress on learning and memory function and the possible roles of the protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway. The surgical stress was induced by abdominal surgery under local anesthesia, and the psychological stress was induced by a communication box. Cognitive functions and markers of the AKT/mTOR pathway were assessed at 1, 3 and 7 days following the stress. The impairments of learning and memory function existed for up to 7 days following surgical stress and surgical stress plus psychological stress, whereas the psychological stress did not affect the cognitive function alone or combined with surgical stress. Analysis of brain tissue revealed a significant involvement of the AKT/mTOR pathway in the impairment of cognition. These data suggested that surgical stress could induce cognitive impairment in aged mice and perioperative psychological stress is not a constitutive factor of POCD. The AKT/mTOR pathway is likely involved as one of the underlying mechanisms of the development of POCD. (C) 2016 IBRO. Published by Elsevier 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: 2016-05-05
摘要: Oxidative stress is a major cause of sporadic Parkinson's disease (PD). Here, we demonstrated that c-Abl plays an important role in oxidative stress-induced neuronal cell death. C-Abl, a nonreceptor tyrosine kinase, was activated in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP)-induced acute PD model. Conditional knockout of c-Abl in neurons or treatment of mice with STI571, a c-Abl family kinase inhibitor, reduced the loss of dopaminergic neurons and ameliorated the locomotive defects induced by short-term MPTP treatment. By combining the SILAC (stable isotope labeling with amino acids in cell culture) technique with other biochemical methods, we identified p38 alpha as a major substrate of c-Abl both in vitro and in vivo and c-Abl-mediated phosphorylation is critical for the dimerization of p38 alpha. Furthermore, p38 alpha inhibition mitigated the MPTP-induced loss of dopaminergic neurons. Taken together, these data suggested that c-Abl-p38 alpha signaling may represent a therapeutic target for PD.
分类: 生物学 >> 生物物理学 >> 神经科学 提交时间: 2016-05-11
摘要: All neurodegenerative diseases are associated with oxidative stress-induced neuronal death. Forkhead box O3a (FOXO3a) is a key transcription factor involved in neuronal apoptosis. However, how FOXO3a forms complexes and functions in oxidative stress processing remains largely unknown. In the present study, we show that histone deacetylase 2 (HDAC2) forms a physical complex with FOXO3a, which plays an important role in FOXO3a-dependent gene transcription and oxidative stress-induced mouse cerebellar granule neuron (CGN) apoptosis. Interestingly, we also found that HDAC2 became selectively enriched in the promoter region of the p21 gene, but not those of other target genes, and inhibited FOXO3a-mediated p21 transcription. Furthermore, we found that oxidative stress reduced the interaction between FOXO3a and HDAC2, leading to an increased histone H4K16 acetylation level in the p21 promoter region and upregulated p21 expression in a manner independent of p53 or E2F1. Phosphorylation of HDAC2 at Ser 394 is important for the HDAC2-FOXO3a interaction, and we found that cerebral ischemia/reperfusion reduced phosphorylation of HDAC2 at Ser 394 and mitigated the HDAC2-FOXO3a interaction in mouse brain tissue. Our study reveals the novel regulation of FOXO3a-mediated selective gene transcription via epigenetic modification in the process of oxidative stress-induced cell death, which could be exploited therapeutically.
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 提交时间: 2016-05-11
摘要: Influenza A viruses (IAVs) rely on host factors to support their life cycle, as viral proteins hijack or interact with cellular proteins to execute their functions. Identification and understanding of these factors would increase our knowledge of the molecular mechanisms manipulated by the viruses. In this study, we searched for novel binding partners of the influenza A virus NS2 protein, the nuclear export protein responsible for overcoming host range restriction, by a yeast two-hybrid screening assay and glutathione S-transferase-pulldown and coimmunoprecipitation assays and identified AIMP2, a potent tumor suppressor that usually functions to regulate protein stability, as one of the major NS2-binding candidates. We found that the presence of NS2 protected AIMP2 from ubiquitin-mediated degradation in NS2-transfected cells and AIMP2 functioned as a positive regulator of IAV replication. Interestingly, AIMP2 had no significant effect on NS2 but enhanced the stability of the matrix protein M1. Further, we provide evidence that AIMP2 recruitment switches the modification of M1 from ubiquitination to SUMOylation, which occurs on the same attachment site (K242) on M1 and thereby promotes M1-mediated viral ribonucleoprotein complex nuclear export to increase viral replication. Collectively, our results reveal a new mechanism of AIMP2 mediation of influenza virus replication. IMPORTANCE Although the ubiquitination of M1 during IAV infection has been observed, the precise modification site and the molecular consequences of this modification remain obscure. Here, we demonstrate for the first time that ubiquitin and SUMO compete for the same lysine (K242) on M1 and the interaction of NS2 with AIMP2 facilitates the switch of the M1 modification from ubiquitination to SUMOylation, thus increasing viral replication.
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: Calcium (Ca2+) oscillations play a central role in varieties of cellular processes including fertilization and immune response, but controversy over the regulation mechanisms still exists. It has been known that nitric oxide (NO) dependently regulates Ca2+ signaling in most physiopathological processes. Previous study indicated that eNOS translocation during some pathological process influences intracellular Ca2+ homeostasis. In this study, we investigated the role and mechanism of NO on Ca2+ release by overexpressing eNOS in cytoplasm (Cyto-eNOS) and endoplasmic reticulum (ER-eNOS) of HeLa cells. We found that the properties of Ca2+ release were altered by the overexpression of eNOS. The amplitude and frequency of extracellular ATP (eATP)-induced Ca2+ oscillation were enhanced in both Cyto-eNOS and ER-eNOS cells, respectively. Especially, the enhancement of the amplitude and frequency of the Ca2+ oscillation was much more significant in the ER-eNOS cells than that of Cyto-eNOS cells. Further study indicated that this effect was abrogated by NO inhibitor, 1..-NAME, suggesting it was not an artificial result induced by ER stress. Furthermore, an up-regulated phosphorylation of phospholamban (PLB) was observed and the sarco-endoplasmic reticulum Ca2tATPase (SERCA) function was activated followed by the significant increase in the ER Ca2+ load. Taken together, we revealed a novel regulatory mechanism of Ca2+ oscillation. (C) 2014 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: MDC1 is a key factor of DNA damage response in mammalian cells. It possesses two phospho-binding domains. In its C terminus, a tandem BRCA1 C-terminal domain binds phosphorylated histone H2AX, and in its N terminus, a forkhead-associated (FHA) domain mediates a phosphorylation-enhanced homodimerization. The FHA domain of the Drosophila homolog of MDC1, MU2, also forms a homodimer but utilizes a different dimer interface. The functional importance of the dimerization of MDC1 family proteins is uncertain. In the fission yeast Schizosaccharomyces pombe, a protein sharing homology with MDC1 in the tandem BRCA1 C-terminal domain, Mdb1, regulates DNA damage response and mitotic spindle functions. Here, we report the crystal structure of the N-terminal 91 amino acids of Mdb1. Despite a lack of obvious sequence conservation to the FHA domain of MDC1, this region of Mdb1 adopts an FHA-like fold and is therefore termed Mdb1-FHA. Unlike canonical FHA domains, Mdb1-FHA lacks all the conserved phospho-binding residues. It forms a stable homodimer through an interface distinct from those of MDC1 and MU2. Mdb1-FHA is important for the localization of Mdb1 to DNA damage sites and the spindle midzone, contributes to the roles of Mdb1 in cellular responses to genotoxins and an antimicrotubule drug, and promotes in vitro binding of Mdb1 to a phospho-H2A peptide. The defects caused by the loss of Mdb1-FHA can be rescued by fusion with either of two heterologous dimerization domains, suggesting that the main function of Mdb1-FHA is mediating dimerization. Our data support that FHA-mediated dimerization is conserved for MDC1 family proteins.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (F-19-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific beta-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, b-arrestin-1 'reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by F-19-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of b-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific b-arrestin-1 conformations. The identified receptor-phosphoselective mechanism for arrestin conformation and the spacing of the multiple phosphatebinding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors.