G protein-coupled receptors (GPCRs) mediate transmembrane signaling. Before ligand binding, GPCRs exist in a basal state. Crystal structures of several GPCRs bound with antagonists or agonists have been solved. However, the crystal structure of the ligand-free basal state of a GPCR, the starting point of GPCR activation and function, has not been determined. Here we report the X-ray crystal structure of the first ligand-free basal state of a GPCR in a lipid membrane-like environment. Oligomeric turkey β1-adrenergic receptors display two alternating dimer interfaces. One interface involves the transmembrane domain (TM) 1, TM2, the C-terminal H8, and the extracellular loop 1. The other interface engages residues from TM4, TM5, the intracellular loop 2 and the extracellular loop 2. Structural comparisons show that this ligand-free state is in an inactive conformation. This provides the structural information regarding GPCR dimerization and oligomerization.
Influenza A virus is an important pathogenic virus known to induce host cell cycle arrest in G0/G1 phase and create beneficial conditions for viral replication. However, how the virus achieves arrest remains unclear. We investigated the mechanisms underlying this process and found that the nonstructural protein 1 (NS1) is required. Based on this finding, we generated a viable influenza A virus (H1N1) lacking the entire NS1 gene to study the function of this protein in cell cycle regulation. In addition to some cell cycle regulators that were changed, the concentration and activity of RhoA protein, which is thought to be pivotal for G1/S phase transition, were also decreased with overexpressing NS1. And in the meantime, the phosphorylation level of cell cycle regulator pRb, downstream of RhoA kinase, was decreased in an NS1-dependent manner. These findings indicate that the NS1 protein induces G0/G1 cell cycle arrest mainly through interfering with the RhoA/pRb signaling cascade, thus providing favorable conditions for viral protein accumulation and replication. We further investigated the NS1 protein of avian influenza virus (H5N1) and found that it can also decrease the expression and activity of RhoA, suggesting that the H5N1 virus may affect the cell cycle through the same mechanism. The NS1/RhoA/pRb cascade, which can induce the G0/G1 cell cycle arrest identified here, provides a unified explanation for the seemingly different NS1 functions involved in viral replication events. Our findings shed light on the mechanism of influenza virus replication and open new avenues for understanding the interaction between pathogens and hosts.
Platelet-rich plasma (PRP) therapy is a recently developed technique that uses a concentrated portion of autologous blood to try to improve and accelerate the healing of various tissues. There is a considerable interest in using these PRP products for the treatment used in bone deficiency healing. Because PRP products are safe and easy to prepare and administer, there has been increased attention toward using PRP in numerous clinical settings. The benefits of PRP therapy appear to be promising, and many investigators are exploring the ways in which this therapy can be used in the clinical setting. At present, the molecular mechanisms of bone defect repair studies have focused on three aspects of the inflammatory cytokines, growth factors and angiogenic factors. The role of PRP works mainly through these three aspects of bone repair. The purpose of this paper is to review the current evidence on the mechanism of the effect of PRP in bone deficiency healing.
AS160 (Akt substrate of 160 kDa) is a Rab GTPase-activating protein implicated in insulin control of GLUT4 (glucose transporter 4) trafficking. In humans, a truncation mutation (R363X) in one allele of AS160 decreased the expression of the protein and caused severe postprandial hyperinsulinaemia during puberty. To complement the limited studies possible in humans, we generated an AS160-knockout mouse. In wild-type mice, AS160 expression is relatively high in adipose tissue and soleus muscle, low in EDL (extensor digitorum longus) muscle and detectable in liver only after enrichment. Despite having lower blood glucose levels under both fasted and random-fed conditions, the AS160-knockout mice exhibited insulin resistance in both muscle and liver in a euglycaemic clamp study. Consistent with this paradoxical phenotype, basal glucose uptake was higher in AS160-knockout primary adipocytes and normal in isolated soleus muscle, but their insulin-stimulated glucose uptake and overall GLUT4 levels were markedly decreased. In contrast, insulin-stimulated glucose uptake and GLUT4 levels were normal in EDL muscle. The liver also contributes to the AS160-knockout phenotype via hepatic insulin resistance, elevated hepatic expression of phosphoenolpyruvate carboxykinase isoforms and pyruvate intolerance, which are indicative of increased gluconeogenesis. Overall, as well as its catalytic function, AS160 influences expression of other proteins, and its loss deregulates basal and insulin-regulated glucose homoeostasis, not only in tissues that normally express AS160, but also by influencing liver function.
Akt substrate of 160 kDa (AS160); glucose transport; insulin resistance; liver; muscle; AS160, Akt substrate of 160 kDa; EDL, extensor digitorum longus; FBP-1, fructose-1,6-bisphosphatase 1; GAP, GTPase-activating protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GIR, glucose infusion rate; GLUT, glucose transporter; GSK3, glycogen synthase kinase 3; MBP, myelin basic protein; PCK/PEPCK, phosphoenolpyruvate carboxykinase; PKB, protein kinase B; PM, plasma membrane; RER, respiratory exchange ratio; TA, tibialis anterior
To explore current status and choices regarding diagnosis and treatment of Parkinson’s disease (PD) among physicians, general neurologists and movement disorders specialists in China via a national survey.
The cross-sectional questionnaire-based survey was conducted from November, 2010 to July, 2011. Six hundreds and twelve doctors from different cities in China were recruited for this study.
68.6% (n=420) and 23.9% (n=146) of doctors have read the national and international guidelines, respectively. There was a larger proportion of movement disorders specialists reading the guidelines, in contrast to physicians and general neurologists (P<0.001). Up to 76.4% (n=465) and 81.8% (n=498) of doctors would choose standard oral levodopa test and conventional MRI(with T1 and T2), respectively; Whereas susceptibility weighed imaging(SWI)(16.1%; n=98), transcranial sonography (TCS) (1.8%; n=11) and functional neuroimaging test, such as single photon emission computed tomography(SPECT) (10.2%; n=62) and positron emission tomography(PET)(13.3%; n=81) were less used for suspected patients with PD in clinical practice. Doctors at different levels or from different hospitals and cities would choose different medication for motor complications and non-motor symptoms of patients with PD, in addition to initial drug selection for newly diagnosed PD. Doctors who had read the guidelines had significantly better knowledge of medication selections for PD under specific circumstances.
Compared with commonly employed standard oral levodopa test and conventional MRI, SWI complements MRI, TCS and functional neuroimaging were less performed for diagnosis of PD in clinical practice in China. The choices of diagnostic methods and therapeutic strategy of PD vary among physicians, general neurologists and movement disorders specialists. Guideline awareness is markedly beneficial to reasonable PD medications strategy in China.
Parkinson’s disease; National survey; Clinical practice guideline; Medication
The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), has an essential role in promoting histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing1–4. This function of EZH2 is important for cell proliferation and inhibition of cell differentiation, and is implicated in cancer progression5–10. Here, we demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) phosphorylate EZH2 at Thr 350 in an evolutionarily conserved motif. Phosphorylation of Thr 350 is important for recruitment of EZH2 and maintenance of H3K27me3 levels at EZH2-target loci. Blockage of Thr 350 phosphorylation not only diminishes the global effect of EZH2 on gene silencing, it also mitigates EZH2-mediated cell proliferation and migration. These results demonstrate that CDK-mediated phosphorylation is a key mechanism governing EZH2 function and that there is a link between the cell-cycle machinery and epigenetic gene silencing.
Histone H3 lysine 27 trimethylation (H3K27me3) catalyzed by the enzymatic subunit EZH2 in the Polycomb repressive complex 2 (PRC 2) is essential for cells to ‘memorize’ gene expression patterns through cell divisions and plays an important role in establishing and maintaining cell identity during development. However, how the epigenetic mark is inherited through cell generations remains poorly understood. Recently, we and others demonstrate that CDK1 and CDK2 phosphorylate EZH2 at threonine 350 (T350) and that T350 phosphorylation is important for the binding of EZH2 to PRC 2 recruiters, such as noncoding RNA s (ncRNAs) HOTA IR and XIST , and for the effective recruitment of PRC 2 to EZH2 target loci in cells. These findings imply that phosphorylation of EZH2 by CDK1 and CD K2 may provide cells a mechanism that enhances EZH2 function during S and G2 phases of the cell cycle, thereby ensuring K27me3 on de novo synthesized H3 incorporated in nascent nucleosomes before sister chromosomes are divided into two daughter cells. Additionally, a potential role of T350 phosphorylation of EZH2 in differing EZH2 from its homolog EZH1 in catalyzing H3K27me3 as well as the interplay between phosphorylation at T350 and other residues [e.g., phosphorylation by p38 at threonine 372 (T372)] in governing EZH2 activity in proliferating versus non-dividing cells are also discussed. Together, CDK phosphorylation of EZH2 at T350 may represent a key regulatory mechanism of EZH2 function that is essential for the maintenance of H3K27me3 marks through cell divisions.
EZH2; PRC2; CDK1; CDK2; cell cycle; epigenetics; cancer
Hundreds of candidate 14-3-3-binding (phospho)proteins have been reported in publications that describe one interaction at a time, as well as high-throughput 14-3-3-affinity and mass spectrometry-based studies. Here, we transcribed these data into a common format, deposited the collated data from low-throughput studies in MINT (http://mint.bio.uniroma2.it/mint), and compared the low- and high-throughput data in VisANT graphs that are easy to analyze and extend. Exploring the graphs prompted questions about technical and biological specificity, which were addressed experimentally, resulting in identification of phosphorylated 14-3-3-binding sites in the mitochondrial import sequence of the iron-sulfur cluster assembly enzyme (ISCU), cytoplasmic domains of the mitochondrial fission factor (MFF), and endoplasmic reticulum-tethered receptor expression-enhancing protein 4 (REEP4), RNA regulator SMAUG2, and cytoskeletal regulatory proteins, namely debrin-like protein (DBNL) and kinesin light chain (KLC) isoforms. Therefore, 14-3-3s undergo physiological interactions with proteins that are destined for diverse subcellular locations. Graphing and validating interactions underpins efforts to use 14-3-3-phosphoproteomics to identify mechanisms and biomarkers for signaling pathways in health and disease.
It has been suggested that pituitary adenoma results from accumulation of multiple genetic and/or epigenetic aberrations, which may be identified through association studies. As pituitary tumor transforming gene-1 (PTTG1)/securin plays a critical role in promoting genomic instability in pituitary neoplasia, the present study explored the association of PTTG1 haplotypes with the risk of pituitary adenoma.
We genotyped five PTTG1 haplotype-tagging SNPs (htSNP) by PCR-RFLP assays in a case-control study, which included 280 Han Chinese patients diagnosed with pituitary adenoma and 280 age-, gender- and geographically matched Han Chinese controls. Haplotypes were reconstructed according to the genotyping data and linkage disequilibrium status of the htSNPs.
No significant differences in allele and genotype frequencies of the htSNPs were observed between pituitary adenoma patients and controls, indicating that none of the individual PTTG1 SNPs examined in this study is associated with the risk of pituitary adenoma. In addition, no significant association was detected between the reconstructed PTTG1 haplotypes and pituitary adenoma cases or the controls.
Though no significant association was found between PTTG1 haplotypes and the risk of pituitary adenoma, this is the first report on the association of individual PTTG1 SNPs or PTTG1 haplotypes with the risk of pituitary adenoma based on a solid study; it will provide an important reference for future studies on the association between genetic alterations in PTTG1 and the risk of pituitary adenoma or other tumors.
Cyclophilin A acts as protein folding chaperones and intracellular transports in many cellular processes. Previous studies have shown that cyclophilin A can interact with HIV-1 (human immunodeficiency virus type 1) gag protein and enhance viral infectivity. Many cyclophilin A inhibitors such as cyclosporin A can inhibit HIV-1 replication in vitro. Here, we report a structure-based identification of novel non-peptidic cyclophilin A inhibitors as anti-HIV lead compounds. Following a computer-aided virtual screening and subsequent surface plasmon resonance (SPR) analysis, 12 low molecular weight cyclophilin A ligands were selected for further evaluation of their in vitro inhibition of peptidyl-prolyl cis-trans isomerase (PPIase) activity of cyclophilin A and HIV-1 replication. Five of these compounds (FD5, FD8, FD9, FD10 and FD12) exhibited inhibition against both PPIase activity and HIV-1 infection. These active compounds will be used as leads for structure and activity relationship (SAR) and optimization studies in order to design more effective anti-HIV-1 therapeutics, and as probes for investigating the effect of cyclophilins on HIV-1 replication.
Cyclophilin A; Cyclosporin A; Human immunodeficiency virus; Peptidyl prolyl cis-trans isomerase
AS160 has emerged as a key player in insulin-mediated glucose transport through controlling GLUT4 trafficking, which is thought to be regulated by insulin-stimulated phosphorylation of sites including the 14-3-3 binding phospho-Thr649 (equivalent to Thr642 in human AS160). To define physiological roles of AS160-Thr649 phosphorylation and 14-3-3 binding in glucose homeostasis, we substituted this residue by a nonphosphorylatable alanine by knockin mutation in mice. The mutant protein was expressed at normal levels, while insulin-stimulated AS160 binding to 14-3-3s was abolished in homozygous knockin mice. These animals displayed impaired glucose disposal and insulin sensitivity, which were associated with decreased glucose uptake in vivo. Insulin-stimulated glucose transport and cell surface GLUT4 content were reduced in isolated muscles, but not in adipocytes. These results provide genetic evidence that insulin-induced AS160-Thr649 phosphorylation and/or its binding to 14-3-3 play an important role in regulating whole-body glucose homeostasis, at least in part through regulating GLUT4 trafficking in muscle.
► AS160 Thr649→Ala knockin mice are made to study roles of AS160/14-3-3 interaction ► The knockin mice display impaired glucose tolerance and reduced insulin sensitivity ► Deregulated GLUT4 trafficking and glucose uptake in muscles underlie this phenotype ► These findings shed new light on understanding of the pathology of type II diabetes
The asymmetric unit of the title compound, C26H36N2O8, comprises two independent molecules. In each molecule, the two pyrrole rings are linked by a –CH2CH2– bridge, with dihedral angles between the two pyrrole rings of 14.5 (3) and 16.4 (3)° in the two molecules. Each pyrrole ring carries 2- and 5-methyl substituents and ethoxycarbonyl groups at the 3- and 5-positions.
Taxol chemotherapy is one of the few therapeutic options for men with castration-resistant prostate cancer (CRPC). However, the working mechanisms for taxol are not fully understood. Here we demonstrated that treatment of 22Rv1, a PTEN-positive CRPC cell line, with paclitaxel and its semisynthetic analogue docetaxel decreases expression of the androgen receptor (AR)-activated genes prostate-specific antigen (PSA) and Nkx3.1 but increases expression of the AR repression gene maspin, suggesting that taxol treatment inhibits AR activity. This was further supported by the observation that the activity of AR luciferase reporter genes was inhibited by paclitaxel. In contrast, paclitaxel treatment failed to inhibit AR activity in the PTEN-null CRPC cell line C4-2. However, pretreatment of C4-2 cells with the PI3K inhibitor LY294002 restored paclitaxel inhibition of the AR. Treatment of 22Rv1 xenografts in mice with docetaxel induced mitotic arrest and a decrease in PSA expression in tumor cells adjacent to vascular vessels. We further demonstrated that paclitaxel induces nuclear accumulation of FOXO1, a known AR suppressive nuclear factor and increases the association of FOXO1 with AR proteins in the nucleus. FOXO1 knockdown with small interference RNA attenuated the inhibitory effect of paclitaxel on AR transcriptional activity, expression of PSA and Nkx3.1 and cell survival. These data reveal a previously uncharacterized, FOXO1-mediated, AR-inhibitory effect of taxol in CRPC cells that may play an important role in taxol-mediated inhibition of CRPC growth.
We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d0/d4) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d0/d4 values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d0/d4 scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser19 of ZNRF2 (RTRAYpS19GS), phospho-Ser90 of SASH1 (RKRRVpS90QD), and phospho- Ser493 of lipolysis-stimulated lipoprotein receptor (RPRARpS493LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways.