Abstract:With the rapid development of high throughput DNA sequencing, genomes of more and more species have been sequenced. Identifying and determining the structures of coding genes are the basic tasks of genome annotation. To understand the sequenced genome precisely, it is necessary to integrate muilti-"omics" data to annotate genomes. However, the annotation methods developed in the past decade are mainly based on genome and transcriptome data. Recently, mass spectrometry based proteomics has come of age, which can cover proteomes nearly completely and make it possible to use mass spectrometry data to annotate genomes. Mass spectrometry data can verify annotated genes on one hand, and refine annotated genes, discover novel genes on the other, which achieve the goal of re-annotating genomes. This is exactly the research content of proteogenomics, and using proteogenomic techniques to systematically annotate the sequenced genome is becoming increasingly important. This article reviews the research content, methods and recent trends of proteogenomics.

Abstract:Protein post-translational modifications (PTM) are widespread in eukaryotic cells and have a significant influence on the structure and function of proteins. The rapid development of tandem mass spectrometry (MS/MS) has provided a sensitive and accurate platform in high throughput PTM identification. However, the traditional database search engines could not meet the needs of modification data analysis, which has made the unrestricted modification search become one of the major methods/strategies to identify modifications in proteomics. Without requirement to specify the type of modifications in advance, unrestricted modification search can detect a large number of known and unanticipated modifications from the samples, which is of great significance to improve the identification rate of tandem mass spectra and reveal the biological function of proteins. In this paper, we first described the definition and development of unrestricted modification search. Then we discussed the algorithms of unrestricted PTM identification based on two major approaches, sequence matching and spectral matching，as well as the quality control of modification identification. Finally, we summarized several applications of unrestricted PTM identification, and the challenges and strategies discussed here could benefit the future research.

Abstract:Nasopharyngeal carcinoma (NPC) is a polygenic inheritance disorder. Both environmental and genetic risk factors play important roles in NPC carcinogenesis and progression, and there are complex interactions between genes and environmental carcinogens. Whole-genome loss of heterozygosity analyses and comparative genomic hybridization analyses identified that the short arm of chromosome 3 were high-frequency deleted in NPC biopsies, and genetic linkage analyses of human NPC families identified chromosome 3p21 as a new susceptibility gene region. Using phenotype cloning strategy, we identified LTF as a tumor suppressor and susceptibility gene of NPC on chromosome 3p21. Lactotransferrin, encoded by LTF gene, is an innate immune protein that widespreadly secreted into most biological fluids including mammal milk, nasal secretions, tears, etc. LTF was highly expressed in normal nasopharynx and significantly down-regulated in NPC tissues and down-regulation of LTF was correlated with progression, invasion and metastasis of NPC. A case-control study implied that two single nucleotide polymorphisms (SNPs) in LTF gene affect the expression level of LTF, and associated with NPC risk. We found that lactotransferrin could protect human primary B lymphocytes and nasopharyngeal epithelial cells from Epstein-Barr virus (EBV) infection by binding to CD21, the EBV receptor on B lymphocyte surface, blocking the infection of EBV into B cells and inhibiting the transfer of EBV from B cells to nasopharyngeal epithelial cells. In addition, lactotransferrin could reduce the EBV stimulated inflammatory responses and protect nasopharyngeal epithelial cells from carcinogenesis. We also found that lactotransferrin could inhibiting the proliferation and metastasis through MAPK and AKT pathways. These results implied that as a natural component of milk, lactotransferrin will has important application potential in the prevention and therapy of NPC and other EBV related diseases.

Abstract:Ubiquitination plays very important roles in regulating protein degradation and transportation. Many RING finger proteins act as E3 ligase in these processes. A new typeⅠ transmembrane protein with unknown function, named RNF148 was studied. A RFP -IRES- TSPANs-EGFP/pCI vector was constructed to screen the substrate of RNF148. By using FACS, co-IP and confocal test, RNF148 was identified to interact with TSPAN15 which belongs to a tetraspanin family. RNF148 could down-regulate TSPAN15 by its E3 ligase activity. The RING domain of RNF148 was a key structure of its ubiquitin-ligase activity and was required for the poly-ubiquitination of TSPAN15. The mutation of Lys-21 and Lys-278 to arginine in the cytosolic fragment of TSPAN15 led to low ubiquitination density compared with the wild-type. RNF148-mediated ubiquitination promoted proteasomal degradation and downregulation of TSPAN15 via Lys-29 linkages. RNF148 also ubiquitinated TSPAN15 via generating Lys-63 polymerize ubiquitin molecules. These findings demonstrate for the first time that RNF148 is a RING finger ubiquitin E3 ligase and TSPAN15 is one of its substrates.

Abstract:Glycosyltransferases are involved in the biosynthesis of oligosaccharides and polysaccharides. They are presented in both prokaryotes and eukaryotes, and play significant roles in biological processes. We deleted the glycosyltransferase homologue gene, sll1466, from Synechocystis PCC 6803. Thin-section electron micrographs showed that the mutant Synechocystis contained more carboxysomes when grown in the absence of CO₃^2- and while more glycogen granules when grown in the presence of 0.5 mol/L NaCl than the wild type Synechocystis. Absorption spectra of the mutant grown under different light intensities differed significantly from wild type Synechocystis. At the molecular level, the mutant differed from the wild type in 3 aspects: (1) glycosylation of two carbohydrate-selective OprB porins in the thylakoid membrane; (2) phosphorylation of the rod-core linker, CpcG1 (Slr2051), in the supernatant of the thylakoid membrane; and (3) transcriptional variations of genes related to the above changes. These results suggested a profound role for Sll1466 in regulating physiology, metabolism, and energy transfer.

Abstract:Simple repetitive sequences are widely present in genomes of most organisms. They are closely related to molecular evolution, genetic diversity, molecular marker and some hereditary diseases. In this article, all of the 18 bp trinucleotide repetitive double strand sequences were used, and their expansion by DNA polymerase was researched systematically. We discussed the factors including reaction temperature and sequence which influence the amplification efficiency and the length of product. The result showed that almost all of the repeats can be elongated, and the variance in sequence had great effect on the amplification efficiency. The repeats with more GC, especially those with G and C in one of the strands were much easier to grow. Duplexes with one strand containing only A and C were amplified a lot as well. The optimum reaction temperature and the GC content of the duplexes had a positive correlation. Agarose and polyacrylamide gel electrophoresis showed that the product has narrow size distribution of molecules, and the expansion has a linear dependence of the length of products on the reaction time. Differentiation of the products occurred more at higher reaction temperatures. At last, a slippage model demonstrating the mechanism of duplex expansion was discussed, and it is promising to be used for explanation of molecular evolution and unusual expansion of repetitive sequences during gene amplification and detection.

Abstract:WRKY transcription factors, one of the largest gene families in rice genome, are reported to play important roles during disease resistance and other signaling pathways. We chose five WRKY proteins to survey their expression profiles in the process of leaf growth and the Xa21-mediated Xanthomonas oryzae pv. oryzae (Xoo) resistance responses by Western blotting. The results indicated that OsWRKY13, OsWRKY23 and OsWRKY71 were expressed increasingly with leaf growth till the flowering stage and their expression were declined slightly at filling stage. However, the expression of OsWRKY45 and OsWRKY53 were not detected. In the Xa21-mediated resistance response, OsWRKY45, OsWRKY53 and OsWRKY71 proteins were induced, while the expression of OsWRKY13 and OsWRKY23 proteins were stable. Comparison among the resistance reaction, susceptible reaction and mock treatment revealed that the expression of OsWRKY45, OsWRKY53 and OsWRKY71 proteins had a similar pattern between resistance and susceptible reactions. This result suggested that OsWRKY13 and OsWRKY23 may play roles in rice leaf growth, OsWRKY45 and OsWRKY53 may play roles in rice-Xoo interactions and OsWRKY71 is functioning in both situations.

Abstract:Cortical neurons that are tuned to sound intensity (non-monotonic neurons) are very important for processing auditory information. Considering the fact that all auditory nerve fibers have monotonical responses, inhibition in the primary auditory cortex (AI) is essential for intensity tuning. By using free field sound stimulation and in vivo extracellular recording, the present study investigated the intensity-tuning properties in AI neurons of mouse (Mus musculus, Km). We also examined the effect of cortical application of the GABAa receptor antagonist bicuculline on AI intensity tuning in order to indentify the possible source of inhibition. The intensity-tuning curves were recorded in 72 AI neurons among which 28 showed monotonic responses and 44 showed non-monotonic responses. In non-monotonic neurons, there was no change in the first spike latency but a decrease in the firing duration (P < 0.01) when the sound intensity increased from best intensity to highest intensity. After AI application of bicuculline by electrophoresis (n = 28), the non-monotonic responses did not change in 39.3% neurons, was weakened in 42.9% neurons and was enhanced in 17.9% neurons. It suggested that GABAergic inhibition must be involved in AI intensity tuning but was not the only source. Non-monotonic excitatory input and non-GABAergic inhibition should also be considered. A postulated model was presented to explain the mechanism of intensity-tuning in AI neurons.

Abstract:Fibroblast growth factor 21 (FGF-21) is a member of FGF family. Recently, it is discovered as a non-insulin-dependent cytokine to regulate blood glucose and a potential drug candidate for treatment of type 2 diabetes mellitus. However, the stability of FGF-21 is poor and its half-life in vivo is short, which severely affect its application in clinical practice. In order to solve this problem, the N-terminus of mFGF-21 was PEGylated in a site-specific manner by methoxy poly-ethylene glycol (mPEG) propionaldehydes with an average molecular mass of 20 ku for improving its biological properties including increasing half-life in vivo, and decreasing immunogenicity. The effects of pH, reaction time, protein concentrations and mass ratio between the reactants on the PEGylation of mFGF-21 were analyzed. PEG-mFGF-21 was isolated by Capto Q anion exchange chromatography or Superdex 75 gel filtration chromatography. As a result, the optimal reaction method for mFGF-21 PEGylation and purification processes of PEG-mFGF-21 were established. Then we study the physical and chemical properties, immunogenicity, in vivo half-life, in vitro biological activity and in vivo hypoglycemic effects of PEG-mFGF-21. We found that the temperature stability and anti-protease ability of mFGF-21 were significantly improved after PEGylation. The indirect ELISA results for detection of serum antibody levels against mFGF-21 and the target protein concentration showed that PEGlyation of mFGF-21 significantly reduced its immunogenicity and increased its half-life in vivo. The glucose uptake assay results in HepG2 cells demonstrated that the in vitro activity of PEG-mFGF-21 did not decline. However, with the increment of stimulating time, the glucose uptake in cells treated with PEG-mFGF-21 significantly increased than mFGF-21. The experimental results of short-term blood glucose regulation in type 2 diabetic animals showed that the hypoglycemic speed of mFGF-21 was faster than PEG-mFGF-21, but it lasted shorter. The long-term blood glucose regulation experimental results showed that PEG-mFGF-21 had better hypoglycemic effect than mFGF-21. Surprisingly, blood glucose of PEG-mFGF-21-treated mice remained at low level for several days after the drug was withdrawn. In conclusion, under the premise of remaining its biological activity in vitro, PEGylation of mFGF-21 can improve its physical stability and anti-protease ability, decrease its immunogenicity and increase its in vivo half-life. It can also prolong the hypoglycemic effect of mFGF-21 in diabetic animals. This study provides an important technology platform for drug development of FGF-21.

Abstract:An important way for RNA to implement its biological function in cells is the binding to proteins. Thus the analysis of RNA binding proteins in cells is necessary for the investigation of RNA's functions. So far, there have been various methods to identify RNA binding proteins. In this study, we describe a novel method, i.e. to immobilize an A/U-rich RNA to a solid medium amine M-270 magnetic beads by coupling them in a direct condensation reaction using the soluble carbodiimide reagent EDC, and then to isolate RNA binding proteins from cell lysate by RNA affinity chromatography using the immobilized RNA. The RNA specific binding proteins were then identified by SDS-PAGE, mass spectrometry and Western blotting, such as hnRNPC which bound specifically to the A/U-rich RNA of C/EBPβ 3'UTR. Colocalization in vivo between A/U-rich RNA and the identified protein hnRNPC was shown by fluorescence in situ hybridization using RNA molecular beacon and anti-hnRNPC antibody and confocal microscopy. Our results indicated that the direct immobilization of RNA with EDC is highly efficient, simple and easy.