Research progress of small molecule proteasome activators / 药学学报

Proteasome controls the degradation of proteins closely related to life activities and plays a key role in the maintenance of protein homeostasis. Proteasome activities decrease with aging, followed by the overwhelming production of damaged proteins which far exceed the protein consumption. Accumulation of these proteins leads to various diseases including neurodegenerative diseases. Therefore, inducing toxic protein degradation is considered as a promising solution for the treatment of these diseases, while increasing the activity of proteasome is considered as an important strategy. However, the research in this field is still in the preliminary stage, and this review will focus on the discussion of the research progress of various small molecule proteasome activators, including research methods, pharmacological effects, structure-activity relationships and the existing problems.

Multicenter Evaluation of the Molecular Line Probe Assay for Multidrug Resistant Mycobacterium Tuberculosis Detection in China / 生物医学与环境科学（英文）

In order to evaluate the performance of a molecular Hain line probe assay (Hain LPA) for rapid detection of rifampicin and isoniazid resistance of Mycobacterium tuberculosis in China, 1612 smear positive patients were consecutively enrolled in this study. Smear positive sputum specimens were collected for Hain LPA and conventional drug susceptibility testing (DST). The sensitivity and specificity of Hain LPA were analyzed by using conventional DST as golden reference. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for rifampicin resistance detection were 88.33%, 97.66%, 81.54%, and 98.62%, respectively. The sensitivity, specificity, PPV and NPV for isoniazid resistance detection were 80.25%, 98.07%, 87.25%, and 96.78%, respectively. These findings suggested that Hain LPA can be an effective method worthy of broader use in China.

Protein expression and function of gene 2 transregulated by hepatitis B virus pre-s1 protein and its cloning / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To screen proteins in leukocytes interacting with PS1TP2 by yeast-two hybrid and to view their subcellular localization in HepG2 cells.</p><p><b>METHODS</b>The function and structure of PS1TP2 were studied by bioinformatic analysis. PS1TP2 gene was amplified and cloned into plasmid pET32a (+) and pGBKT7 to construct recombinant expression vectors pET32a (+)-PS1TP2 and pGBKT7-PS1TP2. They were transduced into E. coli Rosetta strain and yeast AH109. The transformed yeast mated with yeast Y187 containing leukocyte cDNA library plasmid in a 2xYPDA medium. Diploid yeast cells were plated on a synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) for selecting twice and then screening. Then a green fluorescent protein (GFP) expression vector pEGFP-C1-PS1TP2 was established, transduced into HepG2, and its subcellular localization was studied by fluorescence microscopy and confocal microscopy.</p><p><b>RESULTS</b>Bioinformatic analysis showed that the PS1TP2 gene was located at 6q24.1, the protein was unstable and the aliphatic index was very high. After transformation of the E. coli and yeast AH109, the expression protein showed: (1) the molecular weight of the expressed product was about 41000 Da, and (2) PS1TP2 existed within the cells. Diploid yeast cells were plated on the synthetic dropout nutrient medium containing X-a-gal for selecting twice and then screening. Twenty-six colonies from blue colonies were sequenced, pEGFP-C1-PS1TP2 was successfully expressed in the HepG2 cells, and PS1TP2 was located in the cell plasma.</p><p><b>CONCLUSION</b>A prokaryotic expression vector pET32a(+)-PS1TP2 was constructed successfully and the PS1TP2 was successfully expressed in the yeast system. Genes of PS1TP2 interact with leukocyte proteins. These results bring some new clues for studying the biological functions of HBV.</p>

Expression of human gene 5 transactivated by pre-S1 protein of hepatitis B virus in yeast cell / 中华传染病杂志

Objective To study the exact function of human gene 5 transactivated by pre-S1 protein of hepatitis B virus(PS1TP5)by investigating the gene expression of PS1TP5 in yeast cells. Methods Reverse transcription-polymerase chain reaction(RT-PCR)was performed to amplify the gene of PS1TP5 using the mRNA of HepG2 cells as template and the gene was cloned into pGEM-T vector.The gene of PS1TP5 was cut from pGEM-T-PS1TP5 vector and cloned into yeast expressive plasmid pGBKT7,then pGBKT7-PS1TP5 was transformed into yeast cell AH109.The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western hybridization.Results PS1TP5 gene was successfully amplified and identified by DNA sequencing.The digested fragment was cloned into pGBKT7 vector and transformed into yeast cell AH109.The results of SDS-PAGE and Western assay showed that the relative molecular weight of the expressed product was about 36 950,and PS1TP5 protein existed in yeast cells.Conclusion The findings suggest that PS1TP5 can be successfully expressed in yeast cell.

Screening of genes for proteins interacting with the PS1TP5 protein of hepatitis B virus: probing a human leukocyte cDNA library using the yeast two-hybrid system / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The hepatitis B virus (HBV) genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 (human gene 5 transactivated by pre-S1 protein of HBV) is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory (GenBank accession: AY427953). In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS1TP5 protein.</p><p><b>METHODS</b>The reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then "bait" plasmid pGBKT7-PS1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization. After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH109 with Y187 containing a leukocyte cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for alpha-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis.</p><p><b>RESULTS</b>Forty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes with known functions were obtained, including Homo sapien leukocyte adhesion protein p150, 95, interleukin 2 receptor gamma chain, PALM2-AKAP2 protein (PALM2-AKAP2), eukaryotic translation initiation factor 4A, beta-2-microglobin, solute carrier family 9 (sodium/hydrogen exchanger), calreticulin, asialoglycoprotein receptor 1 (ASGR1), MHC class II lymphocyte antigen, cytochrome c oxidase subunit 1, lymphocyte antigen 86 (LY86) and lymphocyte cytosolic protein 1. One novel gene with unknown function was found and named as PS1TP5BP1. After being electronically spliced, it was deposited in GenBank (accession number: DQ471327).</p><p><b>CONCLUSIONS</b>Genes of proteins interacting with PS1TP5 were successfully screened from leukocyte cDNA library. These results suggested that PS1TP5 was closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, the formation of hepatic fibrosis and initiation and development of tumors and also brought some new clues for further studying the biological functions of the pre-S1 protein.</p>