Expression of RARRES1 and AGBL2 and progression of conventional renal cell carcinoma.

BACKGROUND: Approximately 15% of clinically localised conventional renal cell carcinoma (RCC) will develop metastasis within 5 years of follow-up. The aim of this study was to identify biomarkers predicting the postoperative tumour relapse. METHODS: Tissue microarrays of conventional RCC from a cohort of 691 patients without metastasis at the time of operation were analysed by immunohistochemistry for the expression of carboxypeptase inhibitor RARRES1 and its substrate carboxypeptidase AGBL2. Univariate and multivariate Cox regression models were addressed to postoperative tumour relapse and the metastasis-free survival time was estimated by Kaplan-Meier analysis. RESULTS: In multivariate analysis, the lack of staining or cytoplasmic staining of RARRES1 was a significant risk factor indicating five times higher risk of cancer relapse. Combining its co-expression with AGBL2, we found that RARRES1 cytoplasmic/negative and AGBL2-positive/negative staining is a significant risk factor for tumour progression indicating 11-15 times higher risk of cancer relapse, whereas the membranous RARRES1 expression, especially its co-expression with AGBL2, associated with excellent disease outcome. CONCLUSIONS: RARRES1 and AGBL2 expression defines groups of patients at low and high risk of tumour progression and may direct an active surveillance to detect metastasis as early as possible and to apply adjuvant therapy.

Zebrafish cytosolic carboxypeptidases 1 and 5 are essential for embryonic development.

The cytosolic carboxypeptidases (CCPs) are a subfamily of metalloenzymes within the larger M14 family of carboxypeptidases that have been implicated in the post-translational modification of tubulin. It has been suggested that at least four of the six mammalian CCPs function as tubulin deglutamylases. However, it is not yet clear whether these enzymes play redundant or unique roles within the cell. To address this question, genes encoding CCPs were identified in the zebrafish genome. Analysis by quantitative polymerase chain reaction indicated that CCP1, CCP2, CCP5, and CCP6 mRNAs were detectable between 2 h and 8 days postfertilization with highest levels 5-8 days postfertilization. CCP1, CCP2, and CCP5 mRNAs were predominantly expressed in tissues such as the brain, olfactory placodes, and pronephric ducts. Morpholino oligonucleotide-mediated knockdown of CCP1 and CCP5 mRNA resulted in a common phenotype including ventral body curvature and hydrocephalus. Confocal microscopy of morphant zebrafish revealed olfactory placodes with defective morphology as well as pronephric ducts with increased polyglutamylation. These data suggest that CCP1 and CCP5 play important roles in developmental processes, particularly the development and functioning of cilia. The robust and similar defects upon knockdown suggest that each CCP may have a function in microtubule modification and ciliary function and that other CCPs are not able to compensate for the loss of one.

Baculoviral expression and characterization of human recombinant PGCP in the form of an active mature dimer and an inactive precursor protein.

The human-blood plasma glutamate carboxypeptidase (PGCP) is a proteinase that acts on the unsubstituted N- and C-termini of dipeptides. It has been suggested that this PGCP is involved in the release of thyroxine. Furthermore, research has suggested that its activity is up-regulated in hepatitis-C-virus-infected patients with hepatocellular carcinoma. In this study expressed human PGCP in the baculovirus expression system was produced by a Sf9 insect cell line with aim to prepare sufficient amounts of active recombinant enzyme for a subsequent biological characterization. Recombinant PGCP was expressed and secreted into the medium in the form of an inactive proenzyme. It was gradually converted into an active form in the medium after three days, with the highest expression of the active form on day six. The protein was sequentially purified by a combination of various liquid chromatographies, such as hydroxyapatite, ion exchange, and gel chromatography, and as final step with affinity chromatography on Phe-Leu-Sepharose. The human PGCP was purified as an active enzyme in the dimer form and as inactive precursor protein. The dipeptidase activity was confirmed by measuring the hydrolysis of the Ser-Met dipeptide at a slightly acidic pH.

Secretory granule proteases in rat mast cells. Cloning of 10 different serine proteases and a carboxypeptidase A from various rat mast cell populations.

Two of the major rat mast cell proteases, rat mast cell protease 1 (RMCP-1) and RMCP-2, have for many years served as important phenotypic markers for studies of various aspects of mast cell (MC) biology. However, except for these proteases only fragmentary information has been available on the structure and complexity of proteases expressed by different subpopulations of rat MCs. To address these questions, cDNA libraries were constructed from freshly isolated rat peritoneal MCs and from the rat mucosal MC line RBL-1. cDNA clones for 10 different serine proteases (RMCP-1-10), and the MC carboxypeptidase A were isolated and characterized. Six of these proteases have not been isolated previously. Based on their protease content, three separate subpopulations of MCs were identified. Connective tissue MCs (CTMCs) from the ear and peritoneum express the chymases RMCP-1 and -5, the tryptases RMCP-6, and -7 and the carboxypeptidase A. However, based on a large difference in the level of expression of RMCP-7, CTMCs of these two organs may be regarded as two separate subpopulations. RMCP-2 and the three closely related proteases of the RMCP-8 subfamily were identified as the major mucosal MC proteases in rat. In contrast to what has been reported for human MCs, no expression of cathepsin G or cathepsin G-like proteases was detected in any of the rat MC populations. To determine mRNA frequencies for the various proteases expressed by normal tissue MCs, an unamplified peritoneal MC cDNA library was screened with a panel of mono-specific cDNA probes. These results showed that peritoneal MCs are highly specialized effector cells with mRNA frequencies for the major proteases in the range of several percent of the total mRNA pool.

Hormonal stimulation in the exocrine pancreas results in coordinate and anticoordinate regulation of protein synthesis.

24-h intravenous caerulein infusion studies in the rat were combined with in vitro amino acid incorporation studies followed by high-resolution separation of proteins by two-dimensional isoelectric focusing and SDS gel electrophoresis to study the extent to which persistent changes in the biosynthesis of exocrine pancreatic proteins are regulated by cholecystokinin-like peptides. Beginning in the third hour of optimal hormone infusion at 0.25 microgram kg-1 h-1, changes were observed in the synthetic rates of 12 proteins, which progressed over the course of the 24-h study. Based on coordinate response patterns, exocrine proteins could be classified into four distinct groups. Group I (trypsinogen forms 1 and 2) showed progressive increases in synthetic rates reaching a combined 4.3-fold increase over control levels. Group II (amylase forms 1 and 2) showed progressive decreases in synthesis to levels 7.1- and 14.3-fold lower than control levels, respectively. Group III proteins (ribonuclease, chymotrypsinogen forms 1 and 2, procarboxypeptidase forms A and B, and proelastase 1) showed moderate increases in synthesis, 1.4-2.8-fold, and group IV proteins (trypsinogen 3, lipase, proelastase 2, and unidentified proteins 1-4) did not show changes in synthesis with hormone stimulation. Regulation of protein synthesis in response to caerulein infusion was specific for individual isoenzymic forms in the case of both trypsinogen and proelastase. The ratio of biosynthetic rates of trypsinogen forms 1 + 2 to amylase forms 1 + 2 increased from a control value of 0.56 to 24.4 after 24 h of hormonal stimulation (43.5-fold increase). Biosynthetic rates for an unidentified protein (P23) with an Mr = 23,000 and isoelectric point of 6.2 increased 14.2-fold, and the ratio of synthesis of P23 to amylase 2 increased 200-fold during caerulein infusion. During hormone stimulation the anticoordinate response in the synthesis of pancreatic glycosidases (decreased synthesis) and serine protease zymogens (increased synthesis) explain previous observations that showed little change in rates of total protein synthesis under similar conditions.

Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae.

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn decrease mannosylation of proteins and lipids in this compartment. In fact, we found a partial block in O- and N-glycosylation of proteins such as chitinase and carboxypeptidase Y and underglycosylation of invertase. In addition, mannosylinositolphosphorylceramide levels were drastically reduced.

Overexpression of CPXM2 predicts an unfavorable prognosis and promotes the proliferation and migration of gastric cancer.

Carboxypeptidase X, M14 family member 2 (CPXM2), has been associated with several human disorders such as developmental diseases. However, whether CPXM2 is involved in oncogenesis or tumor progression remains unclear. In the present study, we used clinical samples from gastric cancer (GC) patients to investigate potential roles of CPXM2 in GC. We also analyzed datasets from the Oncomine database, The Cancer Genome Atlas (TCGA), and the Kaplan­Meier Plotter to validate these results. We found that CPXM2 was overexpressed in GC and that the overexpression was associated with an unfavorable prognosis, regardless of the Lauren classification and tumor node metastasis staging. In addition, knockdown of CPXM2 in cultured GC cells significantly impeded cell proliferation and migration, as indicated by the cholecystokinin octapeptide, colony formation assay, scratch wound healing assay, and Transwell® migration assay. Furthermore, gene set enrichment analysis using RNA­seq data from TCGA indicated that high CPXM2 expression in GC patients was positively correlated with the HALLMARK_APICAL_JUNCTION and HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION gene sets. Finally, western blotting results revealed that several key molecules involved in the epithelial mesenchymal transition were regulated by CPXM2. Taken together, these results imply an active role for CPXM2 in promoting tumor aggressiveness via epithelial to mesenchymal transition (EMT) modulation in GCs.

Efficient bacterial expression of fusion proteins and their selective processing by a recombinant Kex-1 protease.

A secreted, soluble variant of the Kex-1 endopeptidase from Kluyveromyces lactis has been produced and studied as a novel cleavage enzyme exhibiting high specificity for the Lys-Arg peptide. This highly selective, efficient enzyme is particularly adapted for use in manufacturing when a recombinant therapeutic protein, possessing its native N-terminus, has to be released in vitro from a bacterially-expressed fusion protein. In this paper, we describe the preparation of a Kex-1 variant using Saccharomyces cerevisiae and its application in the production of important therapeutic recombinant proteins such as human growth hormone, granulocyte colony-stimulating factor and interferon-alpha-2b.

Increased secretory demand rather than a defect in the proinsulin conversion mechanism causes hyperproinsulinemia in a glucose-infusion rat model of non-insulin-dependent diabetes mellitus.

Hyperproinsulinemia in non-insulin-dependent diabetes mellitus (NIDDM) is due to an increased release of proinsulin from pancreatic beta cells. This could reside in increased secretory demand placed on the beta cell by hyperglycemia or in the proinsulin conversion mechanism. In this study, biosynthesis of the proinsulin conversion enzymes (PC2, PC3, and carboxypeptidase-H [CP-H]) and proinsulin, were examined in islets isolated from 48-h infused rats with 50% (wt/vol) glucose (hyperglycemic, hyperinsulinemic, and increased pancreatic proinsulin to insulin ratio), 20% (wt/vol) glucose (normoglycemic but hyperinsulinemic), and 0.45% (wt/vol) saline (controls). A decrease in the islet content of PC2, PC3, and CP-H from hyperglycemic rats was observed. This reduction did not correlate with any deficiency in mRNA levels or biosynthesis of PC2, PC3, CP-H, or proinsulin. Furthermore, proinsulin conversion rate was comparable in islets from hyperglycemic and control rats. However, in islets from hyperglycemic rats an abnormal increased proportion of proinsulin was secreted, that was accompanied by an augmented release of PC2, PC3 and CP-H. Stimulation of the beta cell's secretory pathway by hyperglycemia, resulted in proinsulin being prematurely secreted from islets before its conversion could be completed. Thus, hyperproinsulinemia induced by chronic hyperglycemia likely results from increased beta cell secretory demand, rather than a defect in the proinsulin processing enzymes per se.

Continued functioning of the secretory pathway is essential for ribosome synthesis.

To explore the regulatory elements that maintain the balanced synthesis of the components of the ribosome, we isolated a temperature-sensitive (ts) mutant of Saccharomyces cerevisiae in which transcription both of rRNA and of ribosomal protein genes is defective at the nonpermissive temperature. Temperature sensitivity for growth is recessive and segregates 2:2. A gene that complements the ts phenotype was cloned from a genomic DNA library. Sequence analysis revealed that this gene is SLY1, encoding a protein essential for protein and vesicle transport between the endoplasmic reticulum and the Golgi apparatus. In the strain carrying our ts allele of SLY1, accumulation of the carboxypeptidase Y precursor was detected at the nonpermissive temperature, indicating that the secretory pathway is defective. To ask whether the effect of the ts allele on ribosome synthesis was specific for sly1 or was a general result of the inactivation of the secretion pathway, we assayed the levels of mRNA for several ribosomal proteins in cells carrying ts alleles of sec1, sec7, sec11, sec14, sec18, sec53, or sec63, representing all stages of secretion. In each case, the mRNA levels were severely depressed, suggesting that this is a common feature in mutants of protein secretion. For the mutants tested, transcription of rRNA was also substantially reduced. Furthermore, treatment of a sensitive strain with brefeldin A at a concentration sufficient to block the secretion pathway also led to a decrease of the level of ribosomal protein mRNA, with kinetics suggesting that the effect of a secretion defect is manifest within 15 to 30 min. We conclude that the continued function of the entire secretion pathway is essential for the maintenance of ribosome synthesis. The apparent coupling of membrane synthesis and ribosome synthesis suggest the existence of a regulatory network that connects the production of the various structural elements of the cell.

Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae.

Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane. To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked. Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth. On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway. On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine. Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential. In psd1Delta strains or cho1Delta strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting. Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells. Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p. Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics. Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.

Yeast Vps55p, a functional homolog of human obesity receptor gene-related protein, is involved in late endosome to vacuole trafficking.

The Saccharomyces cerevisiae VPS55 (YJR044c) gene encodes a small protein of 140 amino acids with four potential transmembrane domains. VPS55 belongs to a family of genes of unknown function, including the human gene encoding the obesity receptor gene-related protein (OB-RGRP). Yeast cells with a disrupted VPS55 present normal vacuolar morphology, but exhibit an abnormal secretion of the Golgi form of the soluble vacuolar carboxypeptidase Y. However, trafficking of the membrane-bound vacuolar alkaline phosphatase remains normal. The endocytosis of uracil permease, used as an endocytic marker, is normal in vps55Delta cells, but its degradation is delayed and this marker transiently accumulates in late endosomal compartments. We also found that Vps55p is mainly localized in the late endosomes. Collectively, these results indicate that Vps55p is involved in late endosome to vacuole trafficking. Finally, we show that human OB-RGRP displays the same distribution as Vps55p and corrects the phenotypic defects of the vps55Delta strain. Therefore, the function of Vps55p has been conserved throughout evolution. This study highlights the importance of the multispanning Vps55p and OB-RGRP in membrane trafficking to the vacuole/lysosome of eukaryotic cells.

Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases.

vps35 mutants of Saccharomyces cerevisiae exhibit severe defects in the localization of carboxypeptidase Y, a soluble vacuolar hydrolase. We have cloned the wild-type VPS35 gene by complementation of the vacuolar protein sorting defect exhibited by the vps35-17 mutant. Sequence analysis revealed an open reading frame predicted to encode a protein of 937 amino acids that lacks any obvious hydrophobic domains. Subcellular fractionation studies indicated that 80% of Vps35p peripherally associates with a membranous particulate cell fraction. The association of Vps35p with this fraction appears to be saturable; when overproduced, the vast majority of Vps35p remains in a soluble fraction. Disruption of the VPS35 gene demonstrated that it is not essential for yeast cell growth. However, the null allele of VPS35 results in a differential defect in the sorting of vacuolar carboxypeptidase Y (CPY), proteinase A (PrA), proteinase B (PrB), and alkaline phosphatase (ALP). proCPY was quantitatively missorted and secreted by delta vps35 cells, whereas almost all of proPrA, proPrB, and proALP were retained within the cell and converted to their mature forms, indicating delivery to the vacuole. Based on these observations, we propose that alternative pathways exist for the sorting and/or delivery of proteins to the vacuole.

Tissue expression of the novel serine carboxypeptidase Scpep1.

We previously identified a novel gene designated retinoid-inducible serine carboxypeptidase (RISC or Scpep1). Here we characterize a polyclonal antibody raised to Scpep1 and assess its localization in mouse cells and tissues. Western blot analysis revealed an immunospecific approximately 35-kDa protein corresponding to endogenous Scpep1. This protein is smaller than the predicted approximately 51-kDa, suggesting that Scpep1 is proteolytically cleaved to a mature enzyme. Immunohistochemical studies demonstrate Scpep1 expression in embryonic heart and vasculature as well as in adult aortic smooth muscle cells and endothelial cells. Scpep1 displays a broad expression pattern in adult tissues with detectable levels in epithelia of digestive tract and urinary bladder, islet of Langerhans, type II alveolar cells and macrophages of lung, macrophage-like cells of lymph nodes and spleen, Leydig cells of testis, and nerve fibers in brain and ganglia. Consistent with previous mRNA studies in kidney, Scpep1 protein is restricted to proximal convoluted tubular epithelium (PCT). Immunoelectron microscopy shows enriched Scpep1 within lysosomes of the PCT, and immunofluorescence microscopy colocalizes Scpep1 with lysosomal-associated membrane protein-2. These results suggest that Scpep1 is a widely distributed lysosomal protease requiring proteolytic cleavage for activity. The highly specific Scpep1 antibody characterized herein provides a necessary reagent for elucidating Scpep1 function.

Murine six-transmembrane epithelial antigen of the prostate, prostate stem cell antigen, and prostate-specific membrane antigen: prostate-specific cell-surface antigens highly expressed in prostate cancer of transgenic adenocarcinoma mouse prostate mice.

To identify genes that are differentially up-regulated in prostate cancer of transgenic adenocarcinoma mouse prostate (TRAMP) mice, we subtracted cDNA isolated from mouse kidney and spleen from cDNA isolated from TRAMP-C1 cells, a prostate tumor cell line derived from a TRAMP mouse. Using this strategy, cDNA clones that were homologous to human six-transmembrane epithelial antigen of the prostate (STEAP) and prostate stem cell antigen (PSCA) were isolated. Mouse STEAP (mSteap) is 80% homologous to human STEAP at both the nucleotide and amino acid levels and contains six potential membrane-spanning regions similar to human STEAP. Mouse PSCA (mPsca) shares 65% homology with human PSCA at the nucleotide and amino acid levels. mRNA expression of mSteap and mPsca is largely prostate-specific and highly detected in primary prostate tumors and metastases of TRAMP mice. Both mSteap and mPsca map to chromosome 5. Another known gene coding for mouse prostate-specific membrane antigen (mPsma) is also highly expressed in both primary and metastatic lesions of TRAMP mice. These results indicate that the TRAMP mouse model can be used to effectively identify genes homologous to human prostate-specific genes, thereby allowing for the investigation of their functional roles in prostate cancer. mSteap, mPsca, and mPsma constitute new tools for preventative and/or therapeutic vaccine construction and immune monitoring in the TRAMP mouse model that may provide insights into the treatment of human prostate cancer.

In vitro targeted killing of prostate tumor cells by a synthetic amoebapore helix 3 peptide modified with two gamma-linked glutamate residues at the COOH terminus.

Prostate-specific membrane antigen (PSMA) is a trans-membrane protein specifically expressed in LNCaP cells, malignant human prostate tissues, and the surrounding neovasculature. PSMA is a unique exopeptidase with reactivity toward poly-gamma-glutamated folates. It can sequentially remove the poly-gamma-glutamyl termini. To target prostate tumor cells, a novel procytolytic peptide was designed with a backbone consisting of an amoebapore H3 domain modified by two gamma-linked glutamate residues at the epsilon-amino group of the COOH-terminal lysine residue. The strategy behind the design of this prolytic peptide was to inactivate the lytic amoebapore H3 peptide by replacing its functionally important COOH-terminal positive charge with negatively charged groups, which in turn might be selectively removed by the PSMA exopeptidase. This peptide exhibited little cytolytic activity toward PSMA-negative cells, such as PC-3 cells. On the other hand, this peptide exhibited strong cytolytic activity toward PSMA-positive LNCaP cells in a concentration-dependent manner. The carboxypeptidase inhibitor 4,4'-phosphonicobis (butane-1,3-dicarboxylic acid) can inhibit this activity. Moreover, this peptide also exhibited cytolytic activity toward PSMA cDNA-transfected PC-3 cells.

Prostate-specific membrane antigen directed selective thrombotic infarction of tumors.

Prostate-specific membrane antigen (PSMA), a glutamyl preferring carboxypeptidase, is found in prostate and other carcinomas present on both tumor cells and associated microvascular lining cells. We find that the channel structures delineated by PSMA-expressing cells in human and rat prostate tumors are in functional continuity with the vasculature and thus form part of tumor microvasculature. The PSMA-positive cell-outlined channels are CD31 negative and mutually exclusive of CD31-positive cell-lined channels elsewhere in the tumor consistent with tumor cells adapted to a pseudoendothelial phenotype in vasculogenic mimicry. To assess the functional potential of such PSMA-lined microvasculature to selectively direct infarctive tumor therapy, we coupled the soluble extracellular domain of tissue factor to a PSMA catalytic site inhibitor to create a PSMA-directed selective tumor vascular thrombogen (STVT). This protein induced selective local in vivo infarctive necrosis of the rat Mat Lu prostate tumor when administered i.v. The combined administration of this STVT with low-dose doxorubicin produced a profound tumoricidal effect, resulting in complete eradication of some tumors. This is consistent with the therapeutic potential for a PSMA-directed STVT and expands the potential for selective infarctive ablation of tumors.

Carboxypeptidase A3 (CPA3): a novel gene highly induced by histone deacetylase inhibitors during differentiation of prostate epithelial cancer cells.

Butyrate and its structural analogues have recently entered clinical trials as a potential drug for differentiation therapy of advanced prostate cancer. To better understand the molecular mechanism(s) involved in prostate cancer differentiation, we used mRNA differential display to identify the gene(s) induced by butyrate. We found that the androgen-independent prostate cancer cell line PC-3 undergoes terminal differentiation and apoptosis after treatment with sodium butyrate (NaBu). A novel cDNA designated carboxypeptidase A3 (CPA3), which was up-regulated in NaBu-treated PC-3 cells, was identified and characterized. This gene expresses a 2795-bp mRNA encoding a protein with an open reading frame of 421 amino acids. CPA3 has 37-63% amino acid identity with zinc CPs from different mammalian species. It also shares 27-43% amino acid similarity with zinc CPs from several nonmammalian species, including Escherichia coli, yeast, Caenorhabditis elegans, and Drosophila. The structural similarity between CPA3 and its closest homologues indicates that the putative CPA3 protein contains a 16-residue signal peptide sequence, a 95-residue NH2-terminal activation segment, and a 310-residue CP enzyme domain. The consistent induction of CPA3 by NaBu in several prostate cancer cell lines led us to investigate the signaling pathway involved in the induction of CPA3 mRNA. Trichostatin A, a potent and specific inhibitor of histone deacetylase, also induced CPA3 mRNA expression, suggesting that CPA3 gene induction is mediated by histone hyperacetylation. We demonstrated that CPA3 induction was a downstream effect of the treatment with butyrate or trichostatin A, but that the induction of p21(WAF1/CIP1) occurred immediately after these treatments. We also demonstrated that the induction of CPA3 mRNA by NaBu was inhibited by p21(WAF1/CIP1) antisense mRNA expression, indicating that p21 transactivation is required for the induction of CPA3 by NaBu. Our data demonstrate that the histone hyperacetylation signaling pathway is activated during NaBu-mediated differentiation of PC-3 cells, and the new gene, CPA3, is involved in this pathway.

Vacuolar proteases from Candida glabrata: Acid aspartic protease PrA, neutral serine protease PrB and serine carboxypeptidase CpY. The nitrogen source influences their level of expression.

BACKGROUND: The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. AIMS: The present paper is the first report on proteolytic activity in the C. glabrata vacuole. METHODS: Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. RESULTS: Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. CONCLUSIONS: The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen.

Dietary regulation of pancreatic protein synthesis. I. Rapid and specific modulation of enzyme synthesis by changes in dietary composition.

Dietary adaptation of pancreatic protein synthesis and of pancreatic enzyme concentration, was studied over the first 24 h of exposure to a new diet. Rats were adapted to a carbohydrate-rich (G) or to a protein-rich diet (P) and were switched to the opposite regime after a 15 h fast. The evolution of the relative rate of synthesis of amylase, chymotrypsinogen and trypsinogen and of the pancreatic concentration of amylase and chymotrypsinogen were followed. Fasting caused important modifications in the relative rate of synthesis of the three enzymes in rats adapted to a P diet. Adaptative changes in the relative rate of synthesis of amylase, chymotrypsinogen and trypsinogen were seen within 2 h after the beginning of refeeding. These changes were followed by corresponding adaptative modifications in pancreatic contents 4 h after the beginning of refeeding. After 24 h of refeeding, significant adaptative changes had occurred in both the relative rates of synthesis and in enzyme concentrations. Thus exocrine pancreatic protein synthesis can be modulated as early as 2 h after refeeding and this modulation is followed by adaptative changes in pancreatic enzyme content.