Sini Decoction (SNT) is a traditional formula recognized for its efficacy in warming the spleen and stomach and dispersing cold. However, elucidating the mechanism of action of SNT remains challenging due to its complex multiple components. This study utilized a synergistic approach combining two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE)-based drug affinity responsive target stability (DARTS) with label-free quantitative proteomics techniques to identify the direct and indirect protein targets of SNT in myocardial infarction. The analysis identified 590 proteins, with 30 proteins showing significant upregulation and 51 proteins showing downregulation when comparing the SNT group with the model group. Through the integration of 2D-DIGE DARTS with proteomics data and pharmacological assessments, the findings indicate that protein disulfide-isomerase A3 (PDIA3) may serve as a potential protein target through which SNT provides protective effects on myocardial cells during myocardial infarction.
Myocardial Infarction/genetics* ; Proteomics/methods* ; Drugs, Chinese Herbal/chemistry* ; Animals ; Protein Disulfide-Isomerases/genetics* ; Male ; Two-Dimensional Difference Gel Electrophoresis/methods* ; Humans ; Rats ; Rats, Sprague-Dawley ; Electrophoresis, Gel, Two-Dimensional

The protein encoded by TXNDC5 is a member the protein disulfide isomerase family, which has disulfide isomerase activity and can act as the molecular chaperone to reduce the synthesis of abnormal proteins. Its biological functions include anti-oxidation, promoting angiogenesis, taking part in cellular inflammation, and energy metabolism, etc. Studies have demonstrated that the expression of TXNDC5 is increased in many types of tumors including cervical carcinoma, gastric carcinoma and colorectal cancer. Moreover, TXNDC5 is also closely associated with rheumatoid arthritis, diabetes, hepatic steatosis and vitiligo. This paper aims to summarize the latest progress in research on TXNDC5 in terms of biochemical function, relationship with diseases and the underlying mechanism.
Animals ; Arthritis, Rheumatoid ; enzymology ; genetics ; Diabetes Mellitus ; enzymology ; genetics ; Humans ; Neoplasms ; enzymology ; genetics ; Protein Disulfide-Isomerases ; genetics

OBJECTIVETo investigate the role of TXNDC5 in serum starvation-induced proliferation inhibition of HeLa cell.

METHODSTXNDC5 was either over-expressed or knocked down by small interfering RNA (siRNA) in HeLa cells which were then cultured in conventional medium or serum starvation medium. The protein level of TXNDC5 was evaluated by Western blot analysis. The mRNA level of TXNDC5 was measured by quantitative real-time PCR. Cell growth rate was determined by cell proliferation assay kit (MTS method). Cell cycle distribution and apoptosis were detected by flow cytometry.

RESULTSSerum starvation mildly reduced the mRNA level of TXNDC5 (P<0.05), but dramatically increased the protein level of TXNDC5 in HeLa cells. The stability of TXNDC5 mRNA remained unchanged. Cycloheximide abolished the serum starvation-induced up-regulation of TXNDC5 protein. Over-expression of TXNDC5 had no effect on cell proliferation. However, suppression of TXNDC5 attenuated the proliferation inhibition of HeLa cell induced by serum starvation (P<0.05), increased the proportion of cells in S phase (P<0.05), but had no effect on cell apoptosis.

CONCLUSIONTXNDC5 mediates serum starvation-induced proliferation inhibition of HeLa cell.

Apoptosis ; Cell Cycle ; Cell Proliferation ; Culture Media ; chemistry ; Gene Knockdown Techniques ; HeLa Cells ; Humans ; Protein Disulfide-Isomerases ; genetics ; metabolism ; Serum ; chemistry

OBJECTIVETo investigate the relationship and significance between endoplasmic reticulum protein 57 (ERp57) gene promoter region methylation with the pathogenesis of cervical lesions in Uighur women.

METHODSThe special software was used to design specific primers of CpG island fragments of ERp57 gene promoter and bisulfite-modified SiHa cancer cell DNA for PCR amplification, cloning and sequencing the target fragments to obtain relevant information of CpG methylation in the gene base sequencs. Seventy-eight fresh tissues of CIN, CSCC and normal control were collected, and the methylation level of ERp57 gene promoter regions in different cervical lesions were identified using Sequenom MassARRAY(DNA) technology.

RESULTSERp57 gene corresponding target fragment contained the 18 CpG sites. All of the CpG sites methylation occurred in SiHa cervical cancer cell genomic DNA. The analysis of the data resulted from the quantitative analysis of single CpG site methylation by Sequenom MassARRAY platform showed that the methylation level between three CpG sites (CpG_1, CpG_5 and CpG_7) from CpG_1, CpG_2, CpG_3.4, CpG_5, CpG_6, CpG_7, CpG_8 and CpG_ 9 had significant differences in the CSCC, CIN or control groups.

CONCLUSIONSAlthough the global methylation level of the ERp57 gene promoter is higher in CSCC than that in CIN and normal control tissues in Uighur women, hypermethylation occurs only in certain CpG islands and sites. This indicates that the regulation of expression by DNA methylation is not CpG island-specific, but varies for individual CpG sites, and may explain to a certain extent the epigenetic mechanisms regulated by Erp57 gene expression.

Adult ; Aged ; Asian Continental Ancestry Group ; ethnology ; genetics ; Carcinoma, Squamous Cell ; genetics ; Cell Line, Tumor ; Cervical Intraepithelial Neoplasia ; genetics ; CpG Islands ; genetics ; DNA Methylation ; Female ; Humans ; Middle Aged ; Promoter Regions, Genetic ; genetics ; Protein Disulfide-Isomerases ; genetics ; Uterine Cervical Neoplasms ; genetics

OBJECTIVE: To determine the screening of the expression of membrane proteins in androgen-dependent prostate cancer (ADPC) and androgen-independent prostate cancer (AIPC) and to explore the mechanism of membrane proteins in these two cancers. METHODS: Serum samples were collected from 3 patients with ADPC and another 3 patients with AIPC. The serum was incubated with ADPC cell line LNCaP and/or AIPC cell line PC-3 and detected by immunoprecipitation and Western blot. Differentially expressed proteins between ADPC and AIPC identified by mass spectrometry were compared and their expression level and location were analyzed by immunofluorescence. RESULTS: Altogether 11 membrane proteins were identifited, such as the Neural-Cadherin precursor, ER60 precursor, Claudin-4, and so on. Immunofluorescence revealed that the expression level of Claudin-4 in PC-3 cells was higher than in LNCaP cells. CONCLUSION We can use the screening method to study membrane proteins in prostate cancer. Claudin-4 may play an important role in the pathogenesis and the development of AIPC.
Aged ; Androgens ; genetics ; metabolism ; Cell Line, Tumor ; Claudin-4 ; genetics ; metabolism ; Gene Expression Profiling ; Humans ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Protein Disulfide-Isomerases ; genetics ; metabolism

BACKGROUNDIt has been confirmed that defective expression of human leukocyte antigen class I (HLA-I) molecules can contribute to the immune evasion of cancer cells in some types of cancer. The aim of this study was to examine the expression of HLA class I antigen and the antigen-processing machinery (APM) components in esophageal squamous cell carcinoma (ESCC) and their role in high risk human papillomavirus (HPV) infection, and to analyze their association with histopathological characteristics in the Kazak ethnic group.

METHODSA total of 50 formalin-fixed, paraffin-embedded ESCC lesions were collected from the First Affiliated Hospital of Xinjiang Medical University, China. The expression levels of HLA-I antigen and APM components were determined by immunohistochemistry; the HPV DNA were detected using polymerase chain reaction (PCR).

RESULTSA high frequency of down-regulation or loss of expression of HLA and APM components were found in esophageal cancer in Kazak people. HLA-I, TAP1, CNX, LMP7, Erp57, Tapasin and ERAP1 were down-regulated in 68%, 44%, 48%, 40%, 52%, 32% and 20% of ESCC lesions then, respectively. The loss of expression of HLA-I antigen was significantly correlated with part of the APM components and positively correlated with high risk HPV16 infection. TAP1, CNX, LMP7, Erp57 and Tapasin loss were significantly associated with tumor grading, lymph node metastasis and depth of invasion (P < 0.05).

CONCLUSIONOur results suggest that APM component defects are a mechanism underlying HLA-I antigen down-regulation in ESCC lesions, and indicate that the loss expression of HLA-I and APM components will become an important marker of ESCC and analysis of HLA-I and APM component expression can provide useful prognostic information for patients with ESCC from the Kazak ethnic group.

ATP-Binding Cassette Sub-Family B Member 2 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; Adult ; Aged ; Aminopeptidases ; genetics ; metabolism ; Antigen Presentation ; genetics ; physiology ; Calnexin ; genetics ; metabolism ; Esophageal Neoplasms ; metabolism ; Female ; Histocompatibility Antigens Class I ; genetics ; metabolism ; Human papillomavirus 16 ; genetics ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Male ; Membrane Transport Proteins ; genetics ; metabolism ; Middle Aged ; Minor Histocompatibility Antigens ; Polymerase Chain Reaction ; Proteasome Endopeptidase Complex ; genetics ; metabolism ; Protein Disulfide-Isomerases ; genetics ; metabolism

To test the hypothesis that in vitro protein cross-linking could be accomplished in three concerted steps: (1) a change in protein conformation; (2) formation of interchain disulfide bonds; and (3) formation of interchain isopeptide cross-links, we amplified wild and Cys/Ser mutant genes with PCR technique from E. coli BL21 cells and subcloned them into expression plasmid pTrcHisC. Recombinant proteins, which were associated with formation of inclusion bodies induced by IPTG, were purified by immobilized metal affinity chromatography (IMAC) and refolded by dialysis. In thermal unfolding and oxidative refolding experiment, wild TtdB was proved to form cross-linked dimmers/oligomers as revealed by SDS-PAGE; cross-linking intensity was obviously weakened when the loading buffer contained the reducing agent dithiothreitol (DTT). The residual cross-linking was isopeptide bonds; no dimmers/oligomers were detected when the refolding and unfolding solution contained DTT. In addition, Cys/Ser point mutation abrogated its ability to cross-link into homodimers, which showed disulfide bonds could facilitate the following formation of isopeptide bonds.
Cross-Linking Reagents ; chemistry ; Escherichia coli ; enzymology ; genetics ; Escherichia coli Proteins ; chemistry ; Hydro-Lyases ; chemistry ; genetics ; Mutation ; Protein Disulfide-Isomerases ; chemistry ; Protein Folding

Disulfide bond formation protein A, DsbA, is one of the important proteins located in E. coli periplasm, which is a foldase facilitating the folding of nascent secreted proteins, especially for those with many pairs of disulfide bonds. The crystal structure and phylogenetic analysis of DsbA and DsbA-mediated protein folding, alternatively in vivo and in vitro, are summarized. Both the extremely low pKa of Cys30 , about 3.5, and the destabilizing effect of the active site disulfide contribute to its strong oxidizing power. The Cys30 is also considered as the most important residue closely related to its activity using site-directed mutagenesis methodology. DsbA could effectively assist proteins folding, both in vivo coexpressed with the target protein, and in vitro replenished as foldases. Moreover, DsbA also has the chaperone-like activity in the assistant refolding of genetically engineered inclusion bodies.
Amino Acid Sequence ; Disulfides ; chemistry ; metabolism ; Escherichia coli ; enzymology ; genetics ; Escherichia coli Proteins ; chemistry ; classification ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Protein Disulfide-Isomerases ; chemistry ; classification ; metabolism ; Protein Folding ; Protein Structure, Tertiary ; Sequence Homology, Amino Acid

The formation of disulfide bonds in secreted proteins of E. coli is a synergetic process depending on a series of Dsb proteins containing DsbA, DsbB, DsbC, DsbD, DsbE and DsbG. DsbA functions as an oxidant to form a disulfide bond between two -SH- in vivo and DsbB reactivates DsbA by reoxidizing it. Both DsbC and DsbG, two periplasmic proteins with isomerase activity, can correct mis-paired disulfide bonds introduced by DsbA although they recognize different substrates. DsbD, an inner membrane protein, plays a role in reducing DsbC and DsbG in vivo. It is regarded that DsbE has the similar function with DsbD. All DsbA, DsbC and DsbG have chaperone activity besides involving in the formation of disulfide bonds. Furthermore, their chaperone activity can promote the formation of protein disulfide bonds. There are a few reports dealing with soluble expression of heterologous proteins containing disulfide bonds assisted by DsbA and DsbC in E. coli. So far there has been no reports about the soluble expression of heterologous proteins promoted by DsbG. Our experiments first demonstrated that both DsbC and DsbG can improve the expression of single chain antibodies as soluble and functional forms in E. coli, and DsbG has additive effects with DsbC.
Bacterial Proteins ; chemistry ; physiology ; Escherichia coli ; genetics ; Escherichia coli Proteins ; Genetic Engineering ; methods ; Membrane Proteins ; chemistry ; physiology ; Molecular Chaperones ; physiology ; Oxidoreductases ; chemistry ; physiology ; Periplasmic Proteins ; Protein Disulfide-Isomerases ; chemistry ; physiology ; Recombinant Proteins ; biosynthesis

Using the large scale sequencing, a novel human cDNA of 1,645 bp was screened from the cDNA library of human fetal brain. The cDNA contains an ORF encoding a 296-aa protein with a calculated molecular weight of 34.0 KD. Compared with that of current sequence databases, the putative protein was found to have 36% homology with protein-disulfide isomerase (PDI). So this cDNA was named PDI like (PDI-L) gene. Multiple tissue Northern blot analysis shows that PDI-L cDNA is expressed in heart, brain, liver, kidneys and so on. The ORF fragment of PDI-L cDNA was inserted into reconstructed pBV220 expression vehicle and its predicted expressive protein was obtained.
Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Gene Library ; Humans ; Protein Disulfide-Isomerases ; biosynthesis ; genetics