The functional performance of the alpha I domain alpha(7) helix in beta(2) integrin activation depends on the allostery of the alpha(7) helix, which axially slides down; therefore, it is critical to elucidate what factors regulate the allostery. In this study, we determined that there were two conservative salt bridge interaction pairs that constrain both the upper and bottom ends of the alpha(7) helix. Molecular dynamics (MD) simulations for three beta(2) integrin members, lymphocyte function-associated antigen-1 (LFA-1; alpha(L)beta(2)), macrophage-1 antigen (Mac-1; alpha(M)beta(2)) and alpha(x)beta(2), indicated that the magnitude of the salt bridge interaction is related to the stability of the alpha I domain and the strength of the corresponding force-induced allostery. The disruption of the salt bridge interaction, especially with double mutations in both salt bridges, significantly reduced the force-induced allostery time for all three members. The effects of salt bridge interactions of the alpha I domain alpha(7) helix on beta(2) integrin conformational stability and allostery were experimentally validated using Mac-1 constructs. The results demonstrated that salt bridge mutations did not alter the conformational state of Mac-1, but they did increase the force-induced ligand binding and shear resistance ability, which was consistent with MD simulations. This study offers new insight into the importance of salt bridge interaction constraints of the alpha I domain alpha(7) helix and external force for beta(2) integrin function.
Circular RNAs (circRNAs) are a newly discovered type of RNA generated by back-splicing of precursor mRNA and found in many species. They are, expressed in a tissue-specific manner and fulfill regulatory activities in many biological processes. Recent research has revealed that circRNAs play critical roles in the development and pathologies of the cardiovascular system. Some of these circRNAs show aberrant expression and regulatory activities during heart disease including heart failure and cardiac infarction and hypertrophy. These findings suggest that circRNAs might be a suitable target for the treatment and prevention of heart disease. In this review, we summarize the latest research on the biogenesis and functions of circRNAs with emphasis on the regulatory roles of circRNAs in the development and pathologies of the cardiovascular system.
Human embryonic stem cells (hESCs) are extremely delicate, and survive poorly under suboptimal culture conditions, severelyrestricting long-term studies and practical applications. Thus, a protective agent that promotes stem cell survival is urgently needed. In this study, we evaluated the protective effects of stemazole in single-cell and starved hESC cultures. Colony formation was quantified by alkaline phosphatase and immunofluorescence staining, while apoptosis was assessed by flow cytometry and TUNEL assay. Expression of hESC and other stem cell markers was evaluated by western blot, RT-PCR, and qPCR. We found that stemazole enhanced clonal expansion from single cells in dose-dependent fashion and clearly decreased apoptosis from 54.1% to 25.2%. Furthermore, the drug reduced apoptosis from 43.6% to 8.4% over 15h of starvation, with 66% of stemazole-treated cells remaining viable after 2weeks of starvation. Importantly, starved cells protected with stemazole retained the same proliferation and differentiation properties as cells in normal culture. In conclusion, stemazole significantly promotes survival of stem cells in single-cell or starvation cultures without compromising stemness and pluripotency.
Hyperlipidemia is a chronic disorder which plays an important role in the development of cardiovascular diseases, type 2 diabetes, atherosclerosis, hypertension, and nonalcoholic fatty liver disease. Genipin (GNP) is a metabolite from genipioside, which is an active component of the traditional Chinese medicine Gardenia jasminoides Ellis, and has been recognized as a beneficial compound against metabolic disorders. However, whether it can correct overnutrition-induced dyslipidemia is still unknown. In this study, the effects of GNP on attenuating hyperlipidemia and hepatic lipid accumulation were investigated using normal and obese mice induced with a high-fat diet (HFD) and primary hepatocytes treated with free fatty acids. We also sought to identify potential targets of GNP to mediate its effects in the liver. We found that obese mice treated with GNP showed a decrease in the body weight, serum lipid levels, as well as hepatic lipid accumulation. Besides, GNP regulated hepatic expression levels of lipid metabolic genes, which are important in maintaining systemic lipid homeostasis. At the molecular level, GNP increased the expression levels of miR-142a-5p, which bound to 3 untranslated region of Srebp-1c, an important regulator of lipogenesis, which thus led to the inhibition of lipogenesis. Collectively, our data demonstrated that GNP effectively antagonized HFD-induced hyperlipidemia and hepatic lipid accumulation in mice. Such effects were achieved by regulating miR-142a-5p/SREBP-1c axis.
Oligosaccharyltransferases (OSTs) mediate the enbloc transfer of N-glycan intermediates onto the asparagine residue in glycosylation sequons (N-X-S/T, XP). These enzymes are typically heteromeric complexes composed of several membrane-associated subunits, in which STT3 is highly conserved as a catalytic core. Metazoan organisms encode two STT3 genes (STT3A and STT3B) in their genome, resulting in the formation of at least two distinct OST isoforms consisting of shared subunits and complex specific subunits. The STT3A isoform of OST primarily glycosylates substrate polypeptides cotranslationally, whereas the STT3B isoform is involved in cotranslational and post-translocational glycosylation of sequons that are skipped by the STT3A isoform. Here, we describe mutant constructs of monomeric enhanced green fluorescent protein (mEGFP), which are susceptible to STT3B-dependent N-glycosylation. The endoplasmic reticulum-localized mEGFP (ER-mEGFP) mutants contained an N-glycosylation sequon at their C-terminus and exhibited increased fluorescence in response to N-glycosylation. Isoform-specific glycosylation of the constructs was confirmed by using STT3A- or STT3B-knockout cell lines. Among the mutant constructs that we tested, the ER-mEGFP mutant containing the N-185-C-186-T-187 sequon was the best substrate for the STT3B isoform in terms of glycosylation efficiency and fluorescence change. Our results suggest that the mutant ER-mEGFP is useful for monitoring STT3B-dependent post-translocational N-glycosylation in cells of interest, such as those from putative patients with a congenital disorder of glycosylation.
The tumor suppressor p14arf interacts, in response to oncogenic signals, with the p53 E3-ubiquitin ligase HDM2, thereby resulting in p53 stabilization and activation. In addition, it also exerts tumor-suppressive functions in p53-independent contexts. The activities of p14arf are regulated by the nucleolar chaperone nucleophosmin (NPM1), which controls its levels and cellular localization. In acute myeloid leukemia with mutations in the NPM1 gene, mutated NPM1 aberrantly translocates in the cytosol carrying with itself p14arf that is subsequently degraded, thus impairing the p14arf-HDM2-p53 axis. In this work we investigated the complex between these two proteins by means of NMR and other techniques. We identified a novel NPM1-interacting motif in the C-terminal region of p14arf, which corresponds to its predicted nucleolar localization signal. This motif recognizes a specific region of the NPM1 N-terminal domain and, upon binding, the two proteins form soluble high molecular weight complexes. By NMR, we identified critical residues on both proteins involved in the interaction. Collectively, our data provide a structural framework to rationalize the overall assembly of the p14arf-NPM1 supramolecular complexes. A number of p14arf cancer-associated mutations cluster in this motif and their effect on the interaction with NPM1 was also analyzed.
Vascular endothelial growth factor (VEGF) A and vascular endothelial growth factor receptor 1 (VEGFR1) signaling is crucial for angiogenesis and progression of osteosarcoma (OS). However, the regulation of the VEGF/VEGFR1 expression is still unclear in OS. Here, we show lower levels of miRNA-134 (miR-134) in OS tissues and cells. Induction of miR-134 overexpression significantly reduced the proliferation of Saos-2 cells and their secretion of pro-angiogenic factors, but increased the frequency of apoptotic Saos-2 cells. Treatment with conditioned medium from the cells transfected with miR-134 reduced the tube formation in human umbilical vein endothelial cells, which was abrogated by a combination of VEGF and conditioned medium. Furthermore, miR-134 significantly inhibited the growth of implanted OS tumors invivo and attenuated the VEGFA and VEGFR1 expression and angiogenesis in the tumors. In addition, higher levels of VEGFA and VEGFR1 were detected and miR-134 inhibited the expression of VEGFA and VEGFR1 in Saos-2 cells and OS tumors. Bioinformatic analysis indicated that the 3-UTR of VEGFA and VEGFR1 contained the motif for miR-134 binding. Co-transfection with the luciferase reporter containing the wild-type, but not the mutant, of the 3-UTR of VEGFA or VEGFR1 together with miR-134 decreased the luciferase activity in Saos-2 cells. Finally, miR-134 dramatically inhibited AKT activation and proliferating cell nuclear antigen expression in Saos-2 cells. Collectively, these findings indicate that miR-134 is a potential tumor suppressor by targeting VEGFA/VEGFR1 signaling to attenuate the progression and angiogenesis in OS. Therefore, miR-134 may be a novel biomarker for the prognosis of OS and a target for the design of new therapies for OS.
Tools that allow inducible and reversible depletion of target proteins are critical for biological studies. The plant-derived auxin-inducible degradation system (AID) enables the degradation of target proteins tagged with the AID motif. This system has been recently employed in mammalian cells as well as in Caenorhabditis elegans and Drosophila. To test the utility of the AID approach in the nervous system, we used circadian locomotor rhythms as a model and applied the AID method to temporally and spatially degrade PERIOD (PER), a critical pacemaker protein in Drosophila. We found that the period locus can be efficiently tagged with the AID motif by CRISPR/Cas9-based genome editing without disrupting PER function. Moreover, we demonstrated that the AID system could be used to induce rapid and efficient protein degradation in the nervous system as shown by effects on circadian and sleep behaviors. Furthermore, the protein degradation by AID was rapidly reversible after auxin removal. Together, our results show that the AID system provides a powerful tool for behavior studies in Drosophila.
As a reader of di-methylated arginine on various proteins, such as histone, RNA polymerase II, PIWI and Fragile X mental retardation protein, the Tudor domain of Tudor domain-containing protein 3 (TDRD3) mediates transcriptional activation in nucleus and formation of stress granules in the cytoplasm. Despite the TDRD3 implication in cancer cell proliferation and invasion, warheads to block the di-methylated arginine recognition pocket of the TDRD3 Tudor domain have not yet been uncovered. Here we identified 14 small molecule hits against the TDRD3 Tudor domain through NMR fragment-based screening. These hits were further cross-validated by using competitive fluorescence polarization and isothermal titration calorimetry experiments. The crystal structure of the TDRD3 Tudor domain in complex with hit 1 reveals a distinct binding mode from the nature substrate. Hit 1 protrudes into the aromatic cage of the TDRD3 Tudor domain, where the aromatic residues are tilted to accommodate a sandwich-like - interaction. The side chain of the conserved residue N596 swings away 3.1 angstrom to form a direct hydrogen bond with hit 1. Moreover, this compound shows a decreased affinity against the single Tudor domain of survival motor neuron protein, but no detectable binding to neither the tandem Tudor domain of TP53-binding protein 1 nor the extended Tudor domain of staphylococcal nuclease domain-containing protein 1. Our work depicts the structural plasticity of the TDRD3 Tudor domain and paves the way for the subsequent structure-guided discovery of selective inhibitors targeting Tudor domains. DatabaseStructural data are available in the PDB under the accession number .
The Arf family of small GTP-binding and -hydrolyzing proteins are some of the most important intracellular regulators of membrane dynamics. In this study, we identified the Golgi-localized Arf family G protein Arf1 in Candida albicans and confirmed its conserved function in regulating the secretory pathway. Interestingly, deletion of ARF1 resulted in intracellular reactive oxygen species (ROS) accumulation, and induced formation of the endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES). Moreover, N-acetylcysteine-mediated ROS scavenging in the arf1/ strain attenuated ERMES formation, indicating that intracellular ROS accumulation resulting from ARF1 deletion facilitated ERMES formation. In addition, Arf1 regulated many key physiological processes in C. albicans, including cell cycle progression, morphogenesis and virulence. This study uncovers a role for Arf family G proteins in regulating ERMES formation and sheds new light on the crucial contribution of ROS to membrane dynamics.
Activation of the histamine-3 receptor (H3R) is involved in memory processes and cognitive action, while blocking H3R activation can slow the progression of neurological disorders, such as Alzheimer's disease, schizophrenia and narcolepsy. To date, however, no direct way to examine the activation of H3R has been utilized. Here, we describe a novel biosensor that can visualize the activation of H3R through an intramolecular fluorescence resonance energy transfer (FRET) signal. To achieve this, we constructed an intramolecular H3R FRET sensor with cyan fluorescent protein (CFP) attached at the C terminus and yellow fluorescent protein (YFP) inserted into the third intracellular loop. The sensor was found to internalize normally on agonist treatment. We measured FRET signals between the donor CFP and the acceptor YFP in living cells in real time, the results of which indicated that H3R agonist treatment (imetit or histamine) increases the FRET signal in a time- and concentration-dependent manner with Kon and Koff values consistent with published data and which maybe correlated with decreasing cAMP levels and the promotion of ERK1/2 phosphorylation. The FRET signal was inhibited by H3R antagonists, and the introduction of mutations at F419A, F423A, L426A and L427A, once again, the promotion of ERK1/2 phosphorylation, was diminished. Thus, we have built a H3R biosensor which can visualize the activation of receptor through real-time structure changes and which can obtain pharmacological kinetic data at the same time. The FRET signals may allow the sensor to become a useful tool for screening compounds and optimizing useful ligands.
The information obtained from crystallized complexes of the Na+,K+-ATPase with cardiotonic steroids (CTS) is not sufficient to explain differences in the inhibitory properties of CTS such as stereoselectivity of CTS binding or effect of glycosylation on the preference to enzyme isoforms. The uncertainty is related to the spatial organization of the hydrophilic cavity at the entrance of the CTS-binding site. Therefore, there is a need to supplement the crystallographic description with data obtained in aqueous solution, where molecules have significant degree of flexibility. This work addresses the applicability of the electron paramagnetic resonance (EPR) method for the purpose. We have designed and synthesized spin-labeled compounds based on the cinobufagin steroid core. The length of the spacer arms between the steroid core and the nitroxide group determines the position of the reporting group (N-O) confined to the binding site. High affinity to Na+,K+-ATPase is inferred from their ability to inhibit enzymatic activity. The differences between the EPR spectra in the absence and presence of high ouabain concentrations identify the signature peaks originating from the fraction of the spin labels bound within the ouabain site. The degree of perturbations of the EPR spectra depends on the length of the spacer arm. Docking of the compounds into the CTS site suggests which elements of the protein structure might be responsible for interference with the spin label (e.g., steric clashes or immobilization). Thus, the method is suitable for gathering information on the cavity leading to the CTS-binding site in Na+,K+-ATPase in all conformations with high affinity to CTS.
The prognosis for patients with malignant glioma is very poor and thus the identification of new potential therapeutic targets is critically important. In this work, we report a previously unknown role for the homeobox transcription factor HOXC10 in regulating immunosuppressive gene expression in glioma cell lines and their proliferative and invasive capacities. Although HOXC10 expression is dysregulated in several types of tumors, its potential function in glioma was not known. We found that HOXC10 expression was upregulated in glioma compared with normal tissue, and that HOXC10 expression positively associated with high grading of glioma. In three independent datasets (REMBRANDT glioma, The Cancer Genome Atlas glioblastoma multiforme and GSE4412), HOXC10 upregulation was associated with short overall survival. In two glioma cell lines, HOXC10 knock-down inhibited cell proliferation, colony formation, migration and invasion, and promoted apoptosis. In addition, HOXC10 knock-down suppressed the expression of genes that are involved in tumor immunosuppression, including those for transforming growth factor- 2, PD-L2, CCL2 and TDO2. A ChIP assay showed that HOXC10 directly bound to the PD-L2 and TDO2 promoter regions. In summary, our results suggest that HOXC10 upregulation in glioma promotes an aggressive phenotype and induces immunosuppressive gene expression, supporting further investigation of the potential of HOXC10 as a therapeutic target in glioma.
The critical and ubiquitous enzyme adenylate kinase (ADK) catalyzes the nucleotide phosphoryl exchange reaction: 2ADP ATP + AMP. The ADK3 in the chloroplasts of the green alga Chlamydomonas reinhardtii, bears an unusual C-terminal extension that is similar to the C-terminal end of the intrinsically disordered protein CP12. In this study, we report that this enzyme, when oxidized but not when reduced, is able to interact with the chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forming a stable complex as shown by native electrophoresis and mass spectrometry. In this bienzyme complex, the activity of ADK3 is unchanged while the NADPH-dependent activity of GAPDH is significantly inhibited. Moreover ADK3, like CP12, can protect GAPDH against thermal inactivation and aggregation. The ADK3-GAPDH bienzyme complex is unable to recruit phosphoribulokinase (PRK), in contrast with the ternary complex formed between GAPDH-CP12 and PRK. The interaction between ADK3 and GAPDH might be a mechanism to regulate the crucial ATP: NADPH ratio within chloroplasts to optimize the Calvin-Benson cycle during rapid fluctuation in environmental resources. EnzymesAdenylate kinase (), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ), phosphoribulokinase (PRK, EC 188.8.131.52).).
Septins generally function as scaffolds and as cortical barriers to restrict the diffusion of membrane proteins. In the fission yeast Schizosaccharomyces pombe, septins form a ring structure at the septum after spindle breakdown during the constriction of the contractile actomyosin ring (CAR) and serve as a scaffold to recruit glucanases to mediate ultimate daughter cell separation. Despite this, it remains unclear if septins play any significant roles before the cell separation during cytokinesis. Employing live cell microscopy, we carefully examined SIN (Septation Initiation Network) signaling and glucan synthases, two key factors ensuring proper function of the CAR. In the absence of the core septin component Spn1p, the formation of a compact CAR is advanced and the CAR constriction rate is slightly but significantly decreased. Moreover, the SIN kinase Sid2p and the glucan synthases Bgs1p and Ags1p form an equatorial ring quite prematurely, but their maintenance at the equatorial region is diminished spn1 cells. These findings suggest that septins act as key players in an accurate establishment and the maintenance of CAR by orchestrating the equatorial dynamics of Sid2p and glucan synthases. Hence, this work demonstrates that, in addition to their function during ultimate cell septation, septins have important roles in regulating earlier cytokinetic events, including CAR assembly and constriction, SIN signaling, and the cortical dynamics of the glucan synthases.