Altered Activity and Expression of Cytosolic Peptidases in Colorectal Cancer.

BACKGROUND AND OBJECTIVE: The role of peptidases in carcinogenic processes and their potential usefulness as tumor markers in colorectal cancer (CRC) have been classically attributed to cell-surface enzymes. The objective of the present study was to analyze the activity and mRNA expression of three cytosolic peptidases in the CRC and to correlate the obtained results with classic histopathological parameters for tumor prognosis and survival. METHODS: The activity and mRNA levels of puromycin-sensitive aminopeptidase (PSA), aminopeptidase B (APB) and pyroglutamyl-peptidase I (PGI) were measured by fluorimetric and quantitative RT-PCR methods in colorectal mucosa and tumor tissues and plasma samples from CRC patients (n=81). RESULTS: 1) PSA and APB activity was higher in adenomas and carcinomas than in the uninvolved mucosa. 2) mRNA levels of PSA and PGI was lower in tumors. 3) PGI activity in CRC tissue correlated negatively with histological grade, tumor size and 5-year overall survival of CRC patients. 4) Higher plasmatic APB activity was independently associated with better 5-year overall survival. CONCLUSIONS: Data suggest that cytosolic peptidases may be involved in colorectal carcinogenesis and point to the determination of this enzymes as a valuable method in the determination of CRC prognosis.

Altered levels of acid, basic, and neutral peptidase activity and expression in human clear cell renal cell carcinoma.

Peptides play important roles in cell regulation and signaling in many tissues and are regulated by peptidases, most of which are highly expressed in the kidney. Several peptide convertases have a function in different tumor stages, and some have been clearly characterized as diagnostic and prognostic markers for solid tumors, including renal cancer; however, little is known about their in vivo role in kidney tumors. The present study compares the activity of a range of peptidases in human tumor samples and nontumor tissue obtained from clear cell renal cell carcinoma (CCRCC) patients. To cover the complete spectrum and subcellular distribution of peptide-converting activity, acid, neutral, basic, and omega activities were selected. CCRCC displays a selective and restricted pattern of peptidase activities. Puromycin-sensitive aminopeptidase activity in the tumor increases [tumor (t) = 10,775 vs. nontumor (n) = 7,635 units of peptidase (UP)/mg protein; P < 0.05], whereas aminopeptidase N decreases (t = 6,664 vs. n = 33,381 UP/mg protein; P < 0.001). Aminopeptidase B activity of the particulate fraction in tumors decreases (t = 2,399 vs. n = 13,536 UP/mg protein; P < 0.001) compared with nontumor tissues, and aspartyl-aminopeptidase activity decreases significantly in CCRCC (t = 137 vs. n = 223 UP/mg protein; P < 0.05). Soluble and particulate pyroglutamyl peptidase I activities, aminopeptidase A activity, and soluble aminopeptidase B activity do not vary in renal cancer. The relative expression for the aforementioned peptidases, assayed using quantitative RT-PCR, increases in CCRCC for aminopeptidases B (1.5-fold) and A (19-fold), aspartyl-aminopeptidase (3.9-fold), puromycin-sensitive aminopeptidase (2.5-fold), and pyroglutamyl peptidase I (7.6-fold). Only aminopeptidase N expression decreases in tumors (1.3-fold). This peptidase activity profile in the neoplastic kidney suggests a specific role for the studied convertases and the possible involvement of an intracrine renin-angiotensin system in the pathogenesis of CCRCC.

The biosynthetic incorporation of selenomethionine and telluromethionine into pyrrolidone carboxyl peptidase (PYRase) from S. aureus.

Heavy-atom derivatives of PYRase proteins prepared in the past have been unsuitable for x-ray diffraction analysis. Thus, we propose utilizing unnatural metalloid-containing amino acids as an alternative to heavy-atom derivatization. Selenomethionine-containing proteins analyzed by multiwavelength anomalous diffraction provides a facile means of addressing the phase problem, whose solution is necessary to determine protein structures by X-ray Crystallography [Hendrickson, et al., 1991 and references therein]. Telluromethionine-containing proteins offer the same investigational potential, and additionally allow further simplification of the data collection technique by requiring only traditional methods of phase analysis [Boles et al., 1995 and references therein]. We sought to introduce the required Se and Te atoms into Staphylococcus aureus Pyrrolidone Carboxyl Peptidase (PYRase) via selenomethionine (SeMet) and telluromethionine (TeMet). Complete incorporation of SeMet into S. aureus PYRase was succeeded with little change in enzymatic properties. Incomplete incorporation (75%) of TeMet was accomplished in preparing TeMet-PYRase, however, representing the highest incorporation to date of a tellurium-containing amino acid. Enzymatic properties remained unchanged when TeMet was incorporated. We report herein the biosynthetic substitution and expression, protein purification and comparative biochemistry of SeMet-PYRase and TeMet-PYRase.

Characterization of the pcp gene of Pseudomonas fluorescens and of its product, pyrrolidone carboxyl peptidase (Pcp).

The gene pcp, encoding pyrrolidone carboxyl peptidase (Pcp), from Pseudomonas fluorescens MFO was cloned and its nucleotide sequence was determined. This sequence contains a unique open reading frame (pcp) coding for a polypeptide of 213 amino acids (M(r) 22,441) which has significant homology to the Pcps from Streptococcus pyogenes, Bacillus subtilis, and Bacillus amyloliquefaciens. Comparison of the four Pcp sequences revealed two highly conserved motifs which may be involved in the active site of these enzymes. The cloned Pcp from P. fluorescens was purified to homogeneity and appears to exist as a dimer. This enzyme displays a Michaelis constant of 0.21 mM with L-pyroglutamyl-beta-naphthylamide as the substrate and an absolute substrate specificity towards N-terminal pyroglutamyl residues. Studies of inhibition by chemical compounds revealed that the cysteine and histidine residues are essential for enzyme activity. From their conservation in the four enzyme sequences, the Cys-144 and His-166 amino acids are proposed to form a part of the active site of these enzymes.

Purification and characterization of recombinant pyrrolidone carboxyl peptidase of Bacillus subtilis.

Bacillus subtilis pyrrolidone carboxyl peptidase (Pcp) overexpressed in Escherichia coli was purified to homogeneity in less than 12 h using ammonium sulphate precipitation and hydrophobic interaction chromatography. The enzyme, which removes amino-terminal L-pyroglutamic acid from peptides, appears to be a tetramer of 25,200 molecular mass subunits. The protein cross-reacted with polyclonal antibodies raised against Pcp from Streptococcus pyogenes. The overexpressed enzyme exhibits an absolute substrate specificity towards N-terminal pyroglutamyl residues with a Michaelis constant of 1.04 mM for L-pyroglutamyl-beta-naphthylamide. The enzyme could be used for the removal of pyroglutamyl residues that block amino termini of proteins and peptides before performing Edman sequential degradation.

Inhibition of pyroglutamyl peptidase II synthesis by phorbol ester in the Y-79 retinoblastoma cell.

Pyroglutamyl peptidase II (EC 3.4.19.-), a highly specific membrane-bound TRH-degrading enzyme, is inactivated in Y-79 human retinoblastoma cells by exposure to 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a biphasic manner. We have previously demonstrated a rapid decrease in pyroglutamyl peptidase II activity to 10% of the control level within 15 min, which returns to 70% of the control level by 1 h. This decrease results from enzyme phosphorylation by TPA-activated protein kinase-C. We now report a second phase of inactivation after longer exposure of cells to TPA. After 1 h, enzymatic activity slowly and progressively declined. By 7 h, only 15% of control activity remained. Cotreatment of cells with H-7, a protein kinase-C inhibitor, prevented this second phase of inactivation. Immunoblot experiments demonstrated a reduction in the amount of pyroglutamyl peptidase II in Y-79 membranes after long term exposure to TPA. Y-79 cells were labeled with [35S]methionine, and pyroglutamyl peptidase II was immunoprecipitated. A decreased incorporation of [35S]methionine paralleled the decrease in enzyme activity. These studies demonstrate that the second phase of inactivation after exposure to TPA is due to an inhibition of enzyme synthesis.

Regulation of a thyrotropin-releasing hormone-degrading enzyme in GH3 cells: induction of pyroglutamyl peptidase I by 3,5,3'-triiodothyronine.

The effect of exposure of GH3 cells to T3 on the TRH-degrading enzymes pyroglutamyl peptidase I (EC 3.4.19.3) and prolyl endopeptidase (EC 3.4.21.26) was studied. T3 produced a dose-dependent increase in the specific activity of pyroglutamyl peptidase I after 3 days of exposure. The EC50 for T3 was 5 X 10(-10) M. The specific activity of prolyl endopeptidase was unaffected by exposure to T3. The increase in pyroglutamyl peptidase I activity was dependent upon the time of exposure of the cells to this hormone. A maximal effect occurred at 72 h. The stimulation of pyroglutamyl peptidase I by T3 was totally blocked by cycloheximide, indicating that this enzyme is induced in GH3 cells by T3. The effect of T3 on the two TRH-degrading enzymes was also studied in the ACTH-secreting cell line AtT20. T3 had no effect on these enzymes in the AtT20 cell, suggesting that the effect of T3 on pyroglutamyl peptidase I may be cell specific. These studies indicate that the induction of pyroglutamyl peptidase I by T3 may contribute to the negative feedback regulation of T3 levels.