Animals ; Aquaporins/metabolism* ; Disease Models, Animal ; Glycoproteins/pharmacology* ; Humans ; Lung/metabolism* ; Protective Agents/pharmacology* ; Respiratory Distress Syndrome/physiopathology* ; Sus scrofa ; Trypsin Inhibitors/pharmacology* ; Up-Regulation

In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5-10 mmol/L calcium ion concentration and pH value 8.0-8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.
Amino Acid Sequence ; Chromatography, Liquid ; Chymotrypsin/metabolism* ; Digestion ; Humans ; Membrane Proteins ; Tandem Mass Spectrometry ; Trypsin ; Vitamin K Epoxide Reductases

Proteomics is a fast-growing discipline that aims at systematic identification, quantification of proteins and their post-translational modifications in cells. Mass spectrometry-based shotgun proteomics technology is currently one of the mainstream methods for proteomics research. With this method, proteins need to be digested to peptides by site-specific proteases before they can be detected with mass spectrometry. Therefore, site-specific proteases played key roles in this process and so far, a variety of specific proteases have been developed and used in proteomics study. Particularly, the identification, characterization and development of proteases that cleave at the N-termini of corresponding amino acid residues, which are just mirrors to those of typical C-termini proteases, provide novel tools for proteomics analysis. In this review, we summarized the proprieties of LysargiNase, a most recently identified mirror trypsin, and its applications in proteomics research to promote its more widespread usage.
Mass Spectrometry ; Metalloproteases ; chemistry ; metabolism ; Protein Processing, Post-Translational ; Proteomics ; Trypsin ; chemistry

PURPOSE: Early application of protease inhibitors through the intestinal lumen could increase survival following experimental shock by blocking the pancreatic digestive enzymes. Hence, it was hypothesized that two-route injection (intraintestinal + intravenous) of ulinastatin (UTI), a broad-spectrum protease inhibitor, could better alleviate intestinal injury than single-route injection (either intravenous or intraintestinal). METHODS: A sepsis model induced by lipopolysaccharide on rats was established. The rats were randomly divided into five groups: sham, sepsis, UTI intravenous injection (Uiv), UTI intraintestinal injection (Uii), and UTI intraintestinal + intravenous injection (Uii + Uiv) groups. The mucosal barrier function, enzyme-blocking effect, levels of systemic inflammatory cytokines, and 5-day survival rate were compared among groups. The small intestinal villus height (VH), crypt depth (CD), and two components of mucosal barrier (E-cadherin and mucin-2) were measured to evaluate the mucosal barrier function. The levels of trypsin and neutrophil elastase (NE) in the intestine, serum, and vital organs were measured to determine the enzyme-blocking effect. RESULTS: Compared with the single-route injection group (Uiv or Uii), the two-route injection (Uii + Uiv) group displayed: (1) significantly higher levels of VH, VH/CD, E-cadherin, and mucin-2; (2) decreased trypsin and NE levels in intestine, plasma, and vital organs; (3) reduced systemic inflammatory cytokine levels; and (4) improved survival of septic rats. CONCLUSION Two-route UTI injection was superior to single-route injection in terms of alleviating intestinal injury, which might be explained by extensive blockade of proteases through different ways.
Animals ; Cadherins ; metabolism ; Cytokines ; metabolism ; Disease Models, Animal ; Glycoproteins ; administration & dosage ; pharmacology ; Inflammation Mediators ; metabolism ; Injections, Intralesional ; Injections, Intravenous ; Intestinal Diseases ; drug therapy ; etiology ; metabolism ; Intestinal Mucosa ; metabolism ; pathology ; Intestines ; Leukocyte Elastase ; metabolism ; Male ; Mucin-2 ; metabolism ; Rats, Wistar ; Sepsis ; complications ; Trypsin ; metabolism ; Trypsin Inhibitors ; administration & dosage ; pharmacology

The aim of the present study was to observe the effect of trypsin digestion on the purity of in vitro cultured astrocytes and optimize the culture methods. The cerebral cortical tissue from newborn Sprague Dawley (SD) rats was isolated and digested with 0.25% trypsin for 20, 30, or 40 min. The obtained single cell suspension was then cultured. Once reaching confluence, the cells were shaken at a constant temperature. Then, each of 20 and 30 min groups was subdivided into two groups, the control group with normal digestion and two-time-digestion group, and the cells were passaged and purified. Through inverted phase contrast microscope and MTT assay, cell growth and proliferation were observed, respectively. Immunofluorescence for glial fibrillary acidic protein (GFAP) was used to observe the morphology of astrocytes and to assess their purity in different stages. Flow cytometric analysis was used to detect the apoptotic rates of purified astrocytes. The results showed that, the cells being digested for 20 min usually reached confluence at 9 d after seeding. When the digestion time was extended to 30 min, the cells grew faster and reached confluence at 7 d after seeding, meanwhile the morphology of astrocytes was normal, GFAP positive rate (70.2 ± 4.0)% being much higher than that of the 20 min group (P < 0.05). Compared with 20 min group, 40 min group showed higher GFAP positive rate, whereas the cell proliferation was slower, and cell injury was more obvious. After shaking at constant temperature, two times of trypsin digestion could decrease the number of contaminated cells after passage. The GFAP positive rates of two-time-digestion groups in passage 1 (P1) were higher than those of corresponding control groups, and the GFAP positive rate of 30 min + two-time-digestion group in P1 reached (98.1 ± 1.7)%, which was equivalent to that of the 20 min + control group in P3. However, the apoptotic rate showed no significant difference between these two groups. Based on above mentioned results, we conclude that 30 min + two-time of trypsin digestion effectively improves the purity of astrocytes and shortens the time of primary culture and purification, suggesting that it is a rapid and effective method to obtain astrocytes with high purity in vitro.
Animals ; Astrocytes ; cytology ; Cell Culture Techniques ; Cell Proliferation ; Cell Separation ; methods ; Glial Fibrillary Acidic Protein ; metabolism ; Rats ; Rats, Sprague-Dawley ; Trypsin

OBJECTIVETo study the expression and prognostic significance of ERG and SPINK1 expression in endocrine-treated prostatic cancer.

METHODSProstatic needle biopsies from 118 prostatic cancer patients primarily treated with endocrine therapy were reviewed. Immunohistochemical study for ERG and SPINK1 protein was carried out.

RESULTSCo-expression of ERG and SPINK1 was not observed. The frequency of ERG protein expression in the 118 biopsies studied was 9.3% (11/118). The positive expression correlated with T stage (P=0.04) but not with patient age at diagnosis, prostatic specific antigen level, Gleason's score, M stage, tumor area and progression-free survival. Positive expression of SPINK1 was demonstrated in 11.0% (13/118) of the biopsies. SPINK1-positive cases carried a significantly shorter progression-free survival, as compared with SPINK1-negative cases (P=0.022). The expression was not associated with any other clinicopathologic variables. The following expression pattern showed statistically significant correlation with the clinical progress (P=0.029): ERG+/SPINK1- (11/118, 9.3%), ERG-/SPINK1+ (13/118, 11.0%) and ERG-/SPINK1- (94/118, 79.7%).

CONCLUSIONSERG and SPINK1 proteins are mutually exclusive.SPINK1 expression is associated with more aggressive clinical behavior and can serve as a prognostic biomarker in prostatic cancer.

Aged ; Aged, 80 and over ; Antineoplastic Agents, Hormonal ; therapeutic use ; Carrier Proteins ; metabolism ; Disease-Free Survival ; Follow-Up Studies ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Neoplasm Grading ; Neoplasm Staging ; Prognosis ; Prostatic Neoplasms ; drug therapy ; metabolism ; pathology ; Trans-Activators ; metabolism ; Transcriptional Regulator ERG ; Trypsin Inhibitor, Kazal Pancreatic

Trypsin as an important serine protease has been widely used in food, pharmaceutical and tanning industries. In this study, we successfully expressed trypsin (cloning from Streptomyces griseus ATCC10137) in Streptomyces lividans TK24 and comparatively investigated its enzymatic properties. Specifically, applying S. griseus ATCC 10137 genome as template, we obtained the sprT gene and sub-cloned it into the expression plasmid pIJ86, generating the recombinant strain S. lividans TK24/pIJ86-sprT. When cultivated in R2YE and SELF, the activity of rSGT reached 9.21 U/mL and 8.61 U/mL, respectively. Meanwhile, the results of the enzymatic analysis showed that rSGT exhibited a higher acid tolerance and a higher specificity to hydrolyze amide bonds compared with bovine trypsin (BT). In addition, Zn2+ and organic solvents up-regulated esterase and amidase of rSGT. Taken together, the results obtained herein provide meaningful information for further modification of rSGT and its industrial application.
Fermentation ; Recombinant Proteins ; biosynthesis ; genetics ; Streptomyces griseus ; enzymology ; Streptomyces lividans ; genetics ; metabolism ; Trypsin ; biosynthesis ; genetics

Antigens, Neoplasm ; metabolism ; Antigens, Surface ; metabolism ; Autoantibodies ; metabolism ; Biomarkers, Tumor ; metabolism ; Carrier Proteins ; metabolism ; Glutamate Carboxypeptidase II ; metabolism ; Humans ; Kallikreins ; metabolism ; Male ; MicroRNAs ; metabolism ; Oncogene Proteins, Fusion ; metabolism ; PTEN Phosphohydrolase ; metabolism ; Prostate-Specific Antigen ; metabolism ; Prostatic Neoplasms ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Trypsin Inhibitor, Kazal Pancreatic ; Urokinase-Type Plasminogen Activator ; metabolism

OBJECTIVETo identify the ileum tissue proteins differentially expressed in Pi-qi deficiency syndrome rats using proteomic approach.

METHODSThirty-seven rats were randomly divided into 3 groups, i. e., the normal control group (Group 1), the Pi-qi deficiency syndrome model group (Group 2), and the reserpine group (Group 3). The Pi-qi deficiency syndrome model was established using excessive exerting combined with irregular diet, and peritoneally injecting Reserpine Injection (1 mg/mL) respectively. The ileum tissues were separated after identified fuzzy method. The differentially expressed proteins were separated with two dimensional electrophoresis (2-DE), analyzed by Mass Spectrometry, and identified by MASCOT Software.

RESULTSThree proteins were differentially expressed in Group 2 and nine proteins were differentially expressed in Group 3 (P < 0.05).

CONCLUSIONPi-qi deficiency syndrome was closely related with decreased expression of albumin as well as increased expressions of trypsin and glucose-regulated protein 78.

Albumins ; metabolism ; Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Heat-Shock Proteins ; metabolism ; Ileum ; drug effects ; metabolism ; Male ; Medicine, Chinese Traditional ; Proteome ; analysis ; Proteomics ; Qi ; Rats ; Rats, Sprague-Dawley ; Syndrome ; Trypsin ; metabolism

Huwentoxin-XI (HWTX-XI) is a protein isolated from the crude venom of spider Ornithoctonus huwena. It has 55 amino acid residues containing 6 cysteine residues forming 3 disulfide bonds. It shows potent inhibitory effect on trypsin and voltage-gated potassium channels in rat dorsal root ganglion cells. According to the structure-function relationship of HWTX-XI, we designed two mutants through mutation of potassium channel inhibition related amino acid residues (R5I, R10T,R25A and R5I,R25A) and then expressed them with high purity by using the vector pVT102U on Saccharamyces cerevisiae strain S78; The two mutants had the same trypsin inhibition activity as HWTX-XI, whereas their potassium channel inhibition activity and animal toxicity were much lower than those of HWTX-XI. This study is helpful for designing drugs of trypsin related diseases based on HWTX-XI.
Amino Acid Sequence ; Animals ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Mutant Proteins ; biosynthesis ; genetics ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Rats ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Saccharomyces cerevisiae ; genetics ; metabolism ; Spider Venoms ; biosynthesis ; genetics ; pharmacology ; Spiders ; Trypsin Inhibitors ; pharmacology