Abstract:Protein phosphorylation is a wide existing protein post-translational modification in living organisms. This covalently linking mode between a certain amino acid and a phosphate group plays a sophisticated role in the regulation of protein stuctures and functions. Nowadays, nine amino acid residues have been found to have the capacity of phosphorylation, including histidine via a phosphamide bond. Although histidine phosphorylation has a significant regulatory function in both prokaryotic and eukaryotic cells, there has been seldom unimpeachable solution for the biologic research for a long time. Because of the diverse chemical properties of phosphohistidine per se from other phosphorylated amino acids, such as isomerism, chemical lability etc, it hydrolyzes easily during traditional handling methods. Due to the current progress in biochemistry and molecular biology, investigators have established innovative preparation, isolation and characterization strategies for phosphohistidine ad hoc. Therefore, the field begins to rapidly develop. Based on their chemical structures, this article analyzes the main physical and chemical properties as well as their reactivity of two phosphohistidine isomers, followed by the noval chemical biology tools and the main biological progresses.

Abstract:Metabolic diseases are caused by amino acid, glucose and lipid metabolism disorder, chronic inflammation is one of its important characteristics. The Nod-like receptor protein 3 (NLRP3) inflammasome is a protein complexes located in cellular, the main function is to activate caspase -1, and then to indirectly regulate the mature and secretion of interleukin 1β (IL-1β), IL-18 and IL-33. The NLRP3 inflammasome is a hotspot in inflammasome associated studies, a variety of endogenous or exogenous danger signals up-regulated the expression of inflammatory cytokines through the activation of this protein complex, and promoted the occurrence and development of many metabolic diseases. Here we reviewed the structure, function and the regulation of the NLRP3 inflammasome, then discussed its role in metabolic diseases, in order to provide the new targets for the prevention and treatment of metabolic diseases.

Abstract:Many RNAs can undergo post-transcriptional modifications, and recent studies have revealed that the nontemplated 3′-end uridines addition(Uridylation) on RNAs may also be a ubiquitous and conserved modification mode previously underappreciated, which occurs in many RNA substrates, such as polyadenylated mRNA, 5′ fragments of mRNA cleaved by siRNAs or miRNAs, histone mRNAs, most of currently discovered small RNAs, U6 snRNA, transcriptional start site-associated RNAs, spliced introns, and so on, ranging from algaes to humans. These modifications not only play important roles in marking RNA for degradation, promoting or inhibiting RNA biogenesis process, altering RNA activity efficiency and acting as a quality control mechanism of mRNA, but also associate with several human diseases, for example, cancer. In this review, recent achievements of uridylation on small RNAs, mRNA or its cleaved fragments, histone mRNAs and U6 snRNA will be discussed. The applicative perspective of these modifications will be discussed.

Abstract:Aging process is modulated by various genes and signaling pathways. Mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K) not only regulate the physiological functions of cells, but also play an important role in the ageing progression. Recent study showed that inhibiting mTORC1 or S6K can extend the life span of mice. mTOR signaling influences ageing through various ways, including cell autophagy, metabolic by-products accumulation and affects the number of stem cells in tissues. However, the role of S6K in the ageing process remains unclear yet. Studies on the role of these two molecules in the ageing process, which have become a hotspot, are expected to find the method to extend the life span of mammalian and reveal its mechanisms. This review will focus on the role of mTOR and S6K in ageing.

Abstract:FK506 binding protein 12.6 (FKBP12.6), a protein that binds to and regulates the ryanodine receptor type 2 (RyR2) Ca²⁺ release channels, may act as an important regulator of catecholamine secretion. In the present study, the role of FKBP12.6 in the control of chromaffin cell exocytosis has been investigated using FKBP12.6-null mice. The results showed that FKBP12.6 was expressed in mouse chromaffin cells; deletion of FKBP12.6 did not change the depolarization induced Ca² current and exocytosis. However, deletion of FKBP12.6 resulted in an enhanced caffeine-induced global Ca² transient and larger caffeine-induced exocytosis in chromaffin cells of mice. These results indicate that FKBP12.6 is involved in catecholamine secretion through regulation of Ca² release channel in mouse chromaffin cells.

Abstract:The innate immune system is the first line of the multicellular organism against the various pathogens invading. The Notch signaling plays a significant role in maintaining the balance among cell proliferation, differentiation, apoptosis and development. To further investigate the function of Notch signaling pathway in Drosophila innate immunity, Drosophila mutants, E(spl) and Su(H), which are located in the downstream of Notch signaling pathway, were utilized to analyze the survival rate, in vivo phagocytosis and production of antimicrobial peptides after pathogen infections. In addition, the number of circulating plasmatocytes in third instar larvae of mutant and wild type flies was also measured. The results indicate that the E(spl) mutant showed lower survival rate, phagocytosis and the expression of AMP genes after pathogen infections. Furthermore, an abnormal increase in the number of plasmatocytes in E(spl) mutant larvae was also observed. Compared with E(spl) mutant, the mutant of Su(H) only showed sensitive to fungi, however, phagocytic ability of hemocytes was normal in fungi infection. At the same time, the expression of antimicrobial peptides was decreased in Su(H) mutant. These results show that the Notch pathway not only affects the growth and development of individuals, but also plays an important role in regulating innate immunity of Drosophila.

Abstract:In this study, we investigated the characters of residues in IDPs that involved in the interations with other proteins. Firstly, 109 IDPs' chains and 299 IDPs-protein compounds which meet the requirement in database were selected; secondly, 4 822 interface amino acid residues which involving in interaction were extracted from the totally 50 031 amino acid residues in the 109 IDPs chains. The results indicated that the 20 amino acids have different propensities when forming IDPs-protein's interfaces. Therefore, we divided the 20 amino acids into three parts based on their propensities: propensity amino acids (ILE, LEU, ARG, PHE, TYR, MET, TRP), middle amino acids (GLN, GLU, THR, LYS, VAL, ASP, HIS) and non-propensity amino acids(PRO, SER, GLY, ALA, ASN, CYS). Moreover, the results show that the 20 amino acids have different propensities in IDPs' different regions (ordered or disordered regions). For example, TRP, LEU, ILE and CYS are more frequently observed compared with ordered and disordered regions. However, there are no obvious difference for GLU, PHE, HIS and ALA residues between ordered regions and disordered regions. Besides, the amino acids which have larger hydrophobicity, polarizability, side chain's volume, solvent accessible surface area, smaller polarity and net charge index of side chain tend to be IDPs-Protein's interface. The results obtained by principal components analysis showed that the polarizability, side chain's volume and solvent accessible surface area of residues had more affect on the IDPs-Protein's interaction residues.

Abstract:HIV-1 integrase (IN) is an important drug target of current anti-AIDS research and drug development. The drug resistance mutations (DRMs) are the main reason of integrase inhibitor therapy failure, but the drug resistance mechanism remains unclear. We introduced mutations artificially into HIV-1 integrase, tested the activity and drug resistance of individual mutations, and analyzed the integrase drug resistance mechanism preliminarily. The mutations involved contain two single mutations, E92A and N155S, and a double mutation, E92A/N155S. These mutations were obtained using genetic engineering methods, and these recombinant proteins were prepared after prokaryotic expression and protein purification. The strand-transfer activity of the integrases was tested by a magnetic beads based ELISA. S-1360 and Raltegravir were used for drug resistance testing. Besides, molecular docking was performed to study the complex of S-1360 and HIV-1 integrase central catalytic domain (including wild type and mutated type) using Autodock. The main results are as follows: N155S mutation decreases about 80% of the integrase strand transfer activity, while E92A/N155S only decreases about 42%. It indicates that, the E92A mutation on the base of N155S mutation increased the integrase activity observably. Besides, E92A and E92A/N155S mutations exhibit different drug resistance to different inhibitors, and they are more resistant to Raltegravir than to S-1360. A mutation could cause structural change of the integrase catalytic center domain, and the structural change eventually influences the activity and drug resistance. As to E92A, it may reduce the electrostatic interaction to the amino acids around it, and influences D64 and D116 indirectly, which are key amino acids of catalytic center domain. This may give a rational explanation to activity recovery of N155S caused by E92A.

Abstract:The axons of cerebellar Purkinje cells are the sole output of the cerebellar cortex. So it is important to study the spike propagation on the axons of cerebellar Purkinje cells. The morphology and function of cerebellar Purkinje cells mature rapidly in two to three weeks after birth. And the ability to fire spikes increases along with the development in cerebellar Purkinje cells. Whether the capacity of spike propagation on the axons also increase remains elusive. We studied this hypothesis by double-patch recordings in cerebellar Purkinje cells at PND 8(postnatal day) and 15. The spike propagation fidelity on the axons is higher at PND 15 than 8. Afterhyperpolarization(AHP) can increase spike propagation fidelity in cerebellar Purkinje cells at PND 8. Our data indicate that the ability to fire spikes and the capacity of spike propagation on the axons increase correspondingly along with the development.

Abstract:We have previously demonstrated the protective function of hydrogen sulfide (H2S) against the neurotoxicity and oxidative stress of formaldehyde (FA). Paraoxonase-1 (PON-1) is a pivotal endogenous antioxidant. The aim of this present study is to investigate whether PON-1 mediates the protection of H2S against FA-induced neurotoxicity. In the present work, PC12 cells treated with FA were established to the model of FA-induced neurotoxicity. Treatment of PC12 cells with NaHS (a donor of H2S) not only upregulates the activity of PON-1，but also significantly restores FA-induced downregulation of PON-1 activity and expression. A selective inhibitor of PON-1, 2-hydroxyquinoline (2-HQ), markedly attenuated H2S-induced neuroprotection against FA-induced cytotoxicity, apoptosis, and accumulation of intracellular reactive oxygen species (ROS) in PC12 cells. Furthermore, 2-HQ blocks H2S to reverse FA-caused activation of caspase-3 and downregulation of bcl-2 expression in PC12 cells. These results indicate that H2S protects PC12 cells against FA-induced neurotoxicity in a PON-1-dependant manner. Our findings suggest a promising role of PON-1 as a novel therapeutic target for neuronal damage after FA exposure.

Abstract:In this work, we developed a novel fluorescent biosensor that integrates a lateral flow test strip with quantum dot (QD) and a home-made test strip reader for rapid, low-cost, quantitative, and sensitive human chorionic gonadotropin (HCG) detection. The serum HCG level is a reliable marker for detecting persistent gestational trophoblastic neoplasia (GTN) and reflects the clinical course of the disease. The biosensor is designed to combine the rapidness of lateral flow immunoassay and sensitivity of fluorescent immunoassay. The principle of this biosensor is on the basis of a sandwich immunoreaction that was performed on the test strip and the QDs are captured on the test line through antibody-antigen reaction and on the control line through the immobilized secondary antibody. Quantitative detection is realized by recording the intensity of the test line and control line with our home-made test strip reader. The results are determined by the ratio of the fluorescence intensity of the test line and control line (I_T/I_C), which would cancel out the influence of the stability of QDs and antibody. The immunoreaction time and the sample volume are optimized to get a higher sensitivity. Under optimal conditions, the QD-based lateral flow immunoassay system has a wide dynamic range and is capable of detecting a minimum 0.85 IU/L HCG standard analyte in 15 min with 50 μl sample volume. The novel QD-based lateral flow immunoassay biosensor offers a rapid, sensitive and quantitative tool for point-of-care testing of the level of serum HCG and shows great promise in other protein markers.

Abstract:Primary osteosarcoma is the most frequent malignant tumor in bone with early pulmonary metastasis and poor prognosis. Our previous study found that overexpression of IEX-1 in osteosarcoma plays an important role in initiation and development of osteosarcoma. In order to elucidate the mechanism of IEX-1 in osteosarcoma, we have screened its interaction proteins by using yeast two hybrid system.Twelve IEX-1 interacting proteins were identified, including biological oxidation related enzymes, chaperones, signal transduction related protein, etc. The interaction between IEX-1 and CLU was confirmed in vitro through co-immunoprecipiation in combination with Western blotting and immunostaining. Knock down of endogenous CLU expression in osteosarcoma cells significantly inhibit cell proliferation and cell invasion ability in vitro. This preliminary study on the molecular mechanisms that how IEX-1 promote the occurrence and development of osteosarcoma provides novel clues for early-stage diagnosis and target gene therapy of this malignant bone tumor.

Abstract:Calcium imaging has been widely used to monitor the activity of various neurons in C. elegans. However, it is a challenge to determine the calcium transient in a freely moving worm for two reasons. One reason is the challenge in ensuring the co-expression of the genetically encoded calcium indicator and reference fluorescent protein in the same target neurons. Another reason is the common problem with spectrum cross-talk between the fluorescent pair used most often: G-CaMPs (calcium indicator) and DsReds (the reference fluorescent protein). Spectrum cross-talk occasionally introduces artifacts in the calcium transient measurements. Herein, we developed a novel bicistronic expression strategy to ensure co-expression efficiency and simplify the labeling for the target neurons. Moreover, we presented a new fluorescent protein pair for calcium imaging, mKate2 and G-CaMPs, which have less spectrum cross-talk. We successfully tested our new labeling strategy in the C. elegans ASH sensory neuron. We anticipate that this improved technique will facilitate neural circuit studies in C. elegans.