Xiaolan Yu

Toward an understanding of the protein interaction network of the human liver.

Proteome‐scale protein interaction maps are available for many organisms, ranging from bacteria, yeast, worms and flies to humans. These maps provide substantial new insights into systems biology, disease research and drug discovery. However, only a small fraction of the total number of human protein–protein interactions has been identified. In this study, we map the interactions of an unbiased selection of 5026 human liver expression proteins by yeast two‐hybrid technology and establish a human liver protein interaction network (HLPN) composed of 3484 interactions among 2582 proteins. The data set has a validation rate of over 72% as determined by three independent biochemical or cellular assays. The network includes metabolic enzymes and liver‐specific, liver‐phenotype and liver‐disease proteins that are individually critical for the maintenance of liver functions. The liver enriched proteins had significantly different topological properties and increased our understanding of the functional relationships among proteins in a liver‐specific manner. Our data represent the first comprehensive description of a HLPN, which could be a valuable tool for understanding the functioning of the protein interaction network of the human liver.

Two novel reassortants of avian influenza A (H5N6) virus in China.

Eight avian influenza A (H5N6) viruses were isolated from live poultry markets (LPMs) in Sichuan and Jiangxi Provinces in China in 2014, including those close to the county where the human H5N6 infection occurred. Genetic and phylogenetic analyses revealed that these H5N6 viruses were novel reassortants between H5N1 clade 2.3.4 and H6N6 viruses, and had evolved into two distinct lineages (Sichuan and Jiangxi). Moreover, the human H5N6 virus was closely related to the avian-source viruses of Sichuan lineage. Notably, H5N6 viruses contained a T160A substitution in the haemagglutinin protein and an 11 aa deletion in the neuraminidase stalk, which may aid in enhancing viral affinity for human-like receptors and virulence in mammals. As the H5N1 virus infects humans through direct contact, infection with the novel H5N6 virus raised significant concerns that the H5 subtype was a likely candidate for a pandemic. Therefore, extensive and long-term surveillance of avian influenza viruses in LPMs is essential.

Anti-tumor activity of metformin: from metabolic and epigenetic perspectives.

Metformin has been used to treat type 2 diabetes for over 50 years. Epidemiological, preclinical and clinical studies suggest that metformin treatment reduces cancer incidence in diabetes patients. Due to its potential as an anti-cancer agent and its low cost, metformin has gained intense research interest. Its traditional anti-cancer mechanisms involve both indirect and direct insulin-dependent pathways. Here, we discussed the anti-tumor mechanism of metformin from the aspects of cell metabolism and epigenetic modifications. The effects of metformin on anti-cancer immunity and apoptosis were also described. Understanding these mechanisms will shed lights on application of metformin in clinical trials and development of anti-cancer therapy.

High-level expression of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and its application for the deglycosylation of glycoproteins.

Endo-β-N-acetylglucosaminidase H (Endo H, EC3.2.1.96) is a glycohydrolase that is widely used in the study of glycoproteins. The present study aimed to assess the effect of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris. The DNA coding sequence of this enzyme was optimized based on the codon usage bias of Pichia pastoris and synthesized through overlapping PCR. This novel gene was cloned into a pHBM905A vector and introduced into Pichia pastoris GS115 for secretary expression. The yield of the target protein reached approximately 397 mg/l after a 6-d induction with 1% (v/v) methanol in shake flasks, which is much higher than that observed upon heterologous expression in Escherichia coli and silkworm. This recombinant enzyme was purified and its enzymatic features were studied. Its specific activity was 461573 U/mg. Its optimum pH and temperature were pH 5.5 and 37°C, respectively. Moreover, our study showed that the N-linked glycan side-chains of several recombinant proteins expressed in Pichia pastoris can be efficiently removed through either the co-fermentation of this recombinant strain with strains expressing substrates or by mixing the cell culture supernatants of the endo-β-N-acetylglucosaminidase H expressing strain with strains expressing substrates after fermentation. This is the first report of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris and the application of this enzyme in the deglycosylation of raw glycoproteins heterologously expressed in Pichia pastoris using simplified methods.

Regulation of SESAME-mediated H3T11 phosphorylation by glycolytic enzymes and metabolites.

Cancer cells prefer aerobic glycolysis, but little is known about the underlying mechanism. Recent studies showed that the rate-limiting glycolytic enzymes, pyruvate kinase M2 (PKM2) directly phosphorylates H3 at threonine 11 (H3T11) to regulate gene expression and cell proliferation, revealing its non-metabolic functions in connecting glycolysis and histone modifications. We have reported that the yeast homolog of PKM2, Pyk1 phosphorylates H3T11 to regulate gene expression and oxidative stress resistance. But how glycolysis regulates H3T11 phosphorylation remains unclear. Here, using a series of glycolytic enzyme mutants and commercial available metabolites, we investigated the role of glycolytic enzymes and metabolites on H3T11 phosphorylation. Mutation of glycolytic genes including phosphoglucose isomerase (PGI1), enolase (ENO2), triosephosphate isomerase (TPI1), or folate biosynthesis enzyme (FOL3) significantly reduced H3T11 phosphorylation. Further study demonstrated that glycolysis regulates H3T11 phosphorylation by fueling the substrate, phosphoenonylpyruvate and the coactivator, FBP to Pyk1. Thus, our results provide a comprehensive view of how glycolysis modulates H3T11 phosphorylation.

Deep sequencing reveals the viral adaptation process of environment-derived H10N8 in mice.

The H10N8 virus was isolated from the water of Dongting Lake, China. Mice were infected while showing no obvious symptoms and replication was restricted to the lungs. When the wild-type virus was serially passaged in the lungs of mice, the resulting viruses became lethal and capable of replication in many other organs. This offered an applicable model for the exploration of viral genome gradual mutation during adaptation in mice. The different passage viruses from mice lung lavage were named P1, P3, P5, and P7, respectively. We sequenced the four viruses using next-generation sequencing (NGS) to analyze the dynamics of the H10N8 viral genome, polymorphism, and amino acid mutation of related proteins. We aimed to demonstrate how a mutant strain of low pathogenicity could become lethal to mice. Using Illumina high-throughput data, we detected the gradual mutations of F277S, C278Q, F611S and L653P in the polymerase acidic (PA) protein, and of L207V and E627K in the PB2 protein during adaptation. Interestingly, many amino acid sites mutated quickly; the others did so more slowly and remained in a heterozygous state for several generations. The PA amino acids S277 and Q278 have previously been found in clinical wild-type strains, including the human-H10N8 isolate in 2013. This demonstrates that the wild-type H10N8 virus had mutated to adapt to mammalian hosts. These data provide important reference information for influenza virus research.

Expression and Characterization of the RKOD DNA Polymerase in Pichia pastoris

The present study assessed high-level expression of the KOD DNA polymerase in Pichia pastoris. Thermococcus kodakaraensis KOD1 is a DNA polymerase that is widely used in PCR. The DNA coding sequence of KOD was optimized based on the codon usage bias of P. pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus was fused to the C-terminus of KOD. The resulting novel gene was cloned into a pHBM905A vector and introduced into P. pastoris GS115 for secretory expression. The yield of the target protein reached approximately 250 mg/l after a 6-d induction with 1% (v/v) methanol in shake flasks. This yield is much higher than those of other DNA polymerases expressed heterologously in Escherichia coli. The recombinant enzyme was purified, and its enzymatic features were studied. Its specific activity was 19,384 U/mg. The recombinant KOD expressed in P. pastoris exhibited excellent thermostability, extension rate and fidelity. Thus, this report provides a simple, efficient and economic approach to realize the production of a high-performance thermostable DNA polymerase on a large scale. This is the first report of the expression in yeast of a DNA polymerase for use in PCR.

Secretory expression of organophosphorus hydrolase OPHC2 in Yarrowia lipolytica Polg

In the present study, recombinant organophosphorus hydrolase OPHC2 was successfully produced by Yarrowia lipolytica and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses showed a major polypeptide band of 36 kDa. The purified enzyme was optimally active at 65°C and pH 8.5 and also displayed good thermal and pH stability using methyl parathion (O,O-dimethyl-O-4-p-nitrophenyl phosphorothioate) as a substrate. Moreover, as Y. lipolytica is a non-pathogenic, generally regarded as safe (GRAS) yeast, the cell culture supernatant can be used directly on vegetables and fruits that are contaminated by organophosphorus pesticides.

A single-step mixing cloning method for assembly of lentiviral short hairpin RNA expression vectors for gene silencing.

Lentiviral expression vectors encoding short hairpin RNA (shRNA) are widely used for RNAi-based gene silencing in mammalian cells. However, current methods for the construction of shRNA expression vectors require multiple steps, which are expensive, time-consuming, and error-prone. Here, we developed a single-step mixing cloning method for the generation of lentiviral shRNA expression vectors. With this method, a pair of short oligonucleotides (∼50 nt) is required and a lentiviral shRNA vector can be constructed with only one step. This method has been used to construct 30 lentiviral shRNA expression vectors successfully.

Linear DNA vaccine prepared by large-scale PCR provides protective immunity against H1N1 influenza virus infection in mice

Linear DNA vaccines provide effective vaccination. However, their application is limited by high cost and small scale of the conventional polymerase chain reaction (PCR) generally used to obtain sufficient amounts of DNA effective against epidemic diseases. In this study, a two-step, large-scale PCR was established using a low-cost DNA polymerase, RKOD, expressed in Pichia pastoris. Two linear DNA vaccines encoding influenza H1N1 hemagglutinin (HA) 1, LEC-HA, and PTO-LEC-HA (with phosphorothioate-modified primers), were produced by the two-step PCR. Protective effects of the vaccines were evaluated in a mouse model. BALB/c mice were immunized three times with the vaccines or a control DNA fragment. All immunized animals were challenged by intranasal administration of a lethal dose of influenza H1N1 virus 2 weeks after the last immunization. Sera of the immunized animals were tested for the presence of HA-specific antibodies, and the total IFN-γ responses induced by linear DNA vaccines were measured. The results showed that the DNA vaccines but not the control DNA induced strong antibody and IFN-γ responses. Additionally, the PTO-LEC-HA vaccine effectively protected the mice against the lethal homologous mouse-adapted virus, with a survival rate of 100% versus 70% in the LEC-HA-vaccinated group, showing that the PTO-LEC-HA vaccine was more effective than LEC-HA. In conclusion, the results indicated that the linear H1N1 HA-coding DNA vaccines induced significant immune responses and protected mice against a lethal virus challenge. Thus, the low-cost, two-step, large-scale PCR can be considered a potential tool for rapid manufacturing of linear DNA vaccines against emerging infectious diseases.