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Two different RNAi methods were used to inhibit the expression of prohormone convertase 1 (PC1) in At-T20 cells. Transient transfection of double stranded RNA and stable expression of a vector expressing hairpin-loop RNA targeting PC1 reduced cholecystokinin (CCK) secretion from At-T20 cells. PC1 mRNA and protein were also decreased in the vector transfected cells. This treatment caused a shift in the forms of cholecystokinin (CCK) secreted, decreasing CCK 22 and increasing CCK 8. Stable expression of RNAi effectively decreased PC1 expression. The observed decrease in CCK seen with these RNAi treatments further supports a role for PC1 in CCK processing in these cells.

The prohormone convertase 2 (PC2) is hypothesized to convert multiple pro-neuropeptides into active peptides that function as neurotransmitters. To examine the in vivo role of PC2 in neuropeptide production, the tissue contents of six different neuropeptides in brain and peripheral nervous tissues were examined in PC2 deficient mice. Specific neuropeptide radioimmunoassays and RP-HPLC (reverse-phase HPLC) provided evaluation of processed, active neuropeptides in brain and neuroendocrine tissues of PC2 deficient mice. Results demonstrated three features with regard to the selective roles of PC2 in determining the production of NPY, somatostatin-28, enkephalin, VIP, galanin, and CRF in neuroendocrine tissues. Firstly, PC2 deficient mice showed changes in several neuropeptides, but not all neuropeptides examined. The absence of active PC2 resulted in altered cellular levels of NPY, somatostatin-28, and (Met)enkephalin; few changes in VIP or galanin occurred in the tissues examined. CRF content was not altered in brains of PC2 deficient mice. Secondly, comparison of a single neuropeptide among different tissues of PC2 deficient mice demonstrated tissue-selective roles for PC2 in production of the neuropeptide. For example, NPY levels were decreased in ileum of PC2 deficient mice, but NPY content was not altered in hypothalamus that is abundant in NPY. In addition, (Met)enkephalin levels in hypothalamus and cortex were decreased in PC2 deficient mice, but no changes were observed in adrenal or intestine. Thirdly, a single tissue region often showed selective alterations among different neuropeptides. For example, the neuropeptide-rich hypothalamus region showed decreased (Met)enkephalin in PC2 deficient mice, but NPY, VIP, galanin, and CRF were not altered. These results demonstrate the selective role of PC2 in neuropeptide production that provides active peptide neurotransmitter or hormones for biological functions in brain and neuroendocrine systems.

Endocrine tumor cells in culture and in vitro cleavage assays have shown that PC1 and PC2 are capable of processing pro-CCK into smaller, intermediate and final, bioactive forms. Similar studies have shown that PC5 has the ability to process a number of propeptides. Here, we use GT1–7 (mouse hypothalamic) and SK-N-MC and SK-N-SH (human neuroblastoma) tumor cell lines to study the ability of PC5 to process pro-CCK. RT-PCR and Western blot analysis showed that the cells express PC5 mRNA and protein, but not PC1 or PC2. They were engineered to stably overexpress CCK and cell media was analyzed for pro-CCK expression and cleavage of the prohormone. Radioimmunoassays showed that pro-CCK was expressed, but no amidated CCK was detected. Lack of production of amidated CCK may be due to the lack of the appropriate carboxypeptidase and amidating enzymes. Production of glycine-extended CCK processing products was evaluated by treatment of media with carboxypeptidase B followed by analysis with a CCK Gly RIA. Glycine-extended forms of the peptide were found in the media. The predominant forms co-eluted with CCK 12 Gly and CCK 22 Gly on gel filtration chromatography. The results demonstrate that these cell lines which express PC5 and not PC1 or PC2 have the ability to process pro-CCK into intermediate, glycine-extended forms more closely resembling pro-CCK products in intestine than in brain.

Nonstructural protein 3 (NS3) from hepatitis C virus (HCV) is a serine protease that provides an essential function in maturation of the virus by cleaving the nonstructural regions of the viral polyprotein. The goal of this work was to isolate RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide DeltaNS3. RNA aptamers were selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). The RNA pool for SELEX had a 30-nucleotide randomized core region. After nine selection cycles, a pool of DeltaNS3-specific RNA aptamers were obtained. This RNA pool included 45 clones that divided into three main classes (G9-I, II and III). These classes include the conserved sequence GA(A/U)UGGGAC. These aptamers bind to DeltaNS3 with a binding constant of about 10 nM and inhibit approximately 90% of the protease activity of DeltaNS3 and MBP-NS3 (full-length of NS3 fused with maltose binding protein). In addition, these aptamers inhibited approximately 70% of the MBP-NS3 protease activity in the presence of the NS4A peptide P41. G9-I aptamer appeared to be a noncompetitive inhibitor for DeltaNS3 with a Ki approximately 100 nM in the presence of P41. These results suggest that the pool of selected aptamers have potential as anti-HCV compounds. Mutational analysis of the G9-I aptamer demonstrated that the sequences required for protease inhibition are in stem I, stem III and loop III of the aptamer. These regions include the conserved sequence GA(A/U)UGGGAC.

Analysis of CCK content in extracts of whole forebrain from PC2 and 7B2 null mouse brain showed a significant decrease relative to wild-type brains. More detailed analysis revealed that CCK 8 amide levels in cerebral cortex and forebrain regions were more decreased than in hypothalamus. CCK 8 content in PC2 null mouse intestines was identical to control. Null mutant brains contained less CCK 8 than wild type and no other forms were seen when analyzed by gel filtration chromatography. No brain area examined was completely devoid of CCK, suggesting that other enzymes can partially compensate for the loss of PC2. This is the first demonstration that any endoprotease is important for CCK processing but also suggest the presence of a redundant system to ensure production of active CCK in the brain.

Ultraviolet, fluorescence and CD spectral analysis suggested unusual structural features of Rhodotorula gracilis ATCC 90950 DNA. R. gracilis DNA exhibited 13% hyperchromicity at 260 nm as against 26% shown by calf thymus DNA. The biphasic melting curve, one phase between 88-92 degrees C and the other between 92-97 degrees C, was attributed to different unwinding pattern of R. gracilis DNA as a function of rise in temperature. The binding affinity of ethidium bromide to R. gracilis DNA determined was almost the same as that of calf thymus DNA. Fluorescence spectra with rise in temperature showed decrease in the quanta of fluorescence intensity after transition temperature, suggesting the quenching due to variation in structure. The CD spectra of R. gracilis DNA did not resemble the spectra of any of the known DNA forms and it showed increase in the magnitude of negative band with rise in temperature suggesting a B-C transition. Disruption of intermolecular and higher order structures by sonication and salt concentration did not change this behaviour implicating the influence of sequence and base composition of R. gracilis DNA on thermal melting transition.

Prohormone convertase 2 is widely co-localized with cholecystokinin in rodent brain. To examine its role in cholecystokinin processing, cholecystokinin levels were measured in dissected brain regions from prohormone convertase 2 knock-out mice. Cholecystokinin levels were lower in hippocampus, septum, thalamus, mesencephalon, and pons in knock-out mice than wild-type mice. In cerebral cortex, cortex-related structures and olfactory bulb, cholecystokinin levels were higher than wild type. Female mice were more affected by the loss of prohormone convertase 2 than male mice. The decrease in cholecystokinin levels in these brain regions shows that prohormone convertase 2 is important for cholecystokinin processing. Quantitative polymerase chain reaction measurements were performed to examine the relationship between peptide levels and cholecystokinin and enzyme expression. They revealed that cholecystokinin and prohormone convertase 1 mRNA levels in cerebral cortex and olfactory bulb were actually lower in knock-out than wild type, whereas their expression in other brain regions of knock-out mouse brain was the same as wild type. Female mice frequently had higher expression of cholecystokinin and prohormone convertase 1, 2, and 5 mRNA than male mice. The loss of prohormone convertase 2 alters CCK processing in specific brain regions. This loss also appears to trigger compensatory mechanisms in cerebral cortex and olfactory bulb that produce elevated levels of cholecystokinin but do not involve increased expression of cholecystokinin, prohormone convertase 1 or 5 mRNA.

Cholecystokinin (CCK) is expressed in the central and peripheral nervous systems and functions as a neurotransmitter and neuroendocrine hormone. The in vivo forms of CCK include CCK-83, -58, -39, -33, -22, -12, and -8. Tissues in the periphery produce the larger forms of CCK, such as CCK-58, whereas the brain primarily produces CCK-8. The different biologically active forms of CCK observed in vivo may result from cell-specific differences in endoproteolytic cleavage during post-translational processing. Evidence suggests that cleavages of pro-CCK occur in a specific sequential order. To further delineate the progression of cleavages during pro-CCK maturation, mutagenesis was used to disrupt putative mono- and dibasic cleavage sites. AtT-20 cells transfected with wild-type rat prepro-CCK secret CCK-22 and -8. Mutagenesis of the cleavage sites of pro-CCK had profound effects on the products that were produced. Substitution of basic cleavage sites with nonbasic amino acids inhibits cleavage and leads to the secretion of pathway intermediates such as CCK-83, -33, and -12. These results suggest that CCK-58 is cleaved to both CCK-33 and -22. Furthermore, CCK-8 and -12 are likely derived from cleavage of CCK-33 but not CCK-22. Alanine substitution at the same site completely blocked production of amidated products, whereas serine substitution did not. The cleavages observed at nonbasic residues in this study may represent the activity of enzymes other than PC1 and carboxypeptidase E, such as the enzyme SKI-1. A model for the progression of pro-CCK processing in AtT-20 cells is proposed. The findings in this study further supports the hypothesis that pro-CCK undergoes parallel pathways of proteolytic cleavages.

The role of P-glycoprotein (P-gp) on the distribution of the benzodiazepine triazolam (TRZ) and the azole antifungal agent ketoconazole (KET), and on the TRZ-KET interaction, was studied using mdr1a(-) or mdr1a/b(-/-) mice (P-gp-deficient mice) and matched controls. TRZ and KET also were studied in Caco-2 cells in Transwell culture. After single i.p. injections of TRZ or KET in separate groups of control mice, brain concentrations of TRZ exceeded those in serum [brain/serum area under the concentration curve (AUC) ratio, 5.0], whereas brain/serum AUC ratios for KET were approximately 0.5. On the basis of single time points, brain concentrations of TRZ, or brain/serum ratios, were similar in P-gp-deficient animals compared with controls, whereas P-gp-deficient animals had significantly higher KET brain concentrations and brain/serum ratios. Coadministration of KET with TRZ increased TRZ concentrations in serum, liver, and brain, both in controls and in P-gp-deficient animals, probably attributable to impairment by KET of CYP3A-mediated clearance of TRZ. However, KET did not increase brain/serum ratios of TRZ in either group. In Caco-2 cells, basal-to-apical flux of TRZ was higher than apical-to-basal flux. However, verapamil (100 microM) did not alter flux in either direction. KET inhibited basal-to-apical transport of rho-damine-123, with a 50% inhibitory concentration of 2.7 microM. Thus, TRZ does not appear to undergo measurable blood-brain barrier efflux transport by P-gp in this animal model. KET impairs clearance of TRZ but does not increase tissue uptake. However, KET itself may be a substrate for efflux transport at the blood-brain barrier.

Prohormone convertase (PC1) is found in endocrine cell lines that express cholecystokinin (CCK) mRNA and process pro CCK to biologically active products. Other studies have demonstrated that PC1 may be a one of the enzymes responsible for the endoproteolytic cleavages that occur in pro CCK during its biosynthesis and processing. Prohormone convertase 1 (PC1) has a distribution that is similar to cholecystokinin (CCK) in rat brain. A moderate to high percentage of CCK mRNA-positive neurons express PC1 mRNA. CCK levels were measured in PC1 knockout and control mice to assess the degree to which loss of PC1 changed CCK content. CCK levels were decreased 62% in hippocampus, 53% in amygdala and 57% in pons-medulla in PC1 knockout mice as compared to controls. These results are highly correlated with the colocalization of CCK and PC1. The majority of CCK mRNA-positive neurons in the pyramidal cell layer of the hippocampus express PC1 mRNA and greater than 50% of CCK mRNA-positive neurons in several nuclei of the amygdala also express PC1. These results demonstrate that PC1 is important for CCK processing. PC2 and PC5 are also widely colocalized with CCK. It may be that PC2, PC5 or another non-PC enzyme are able to substitute for PC1 and sustain production of some amidated CCK. Together these enzymes may represent a redundant system to insure the production of CCK.

During posttranslational processing to generate CCK 8, pro-cholecystokinin (CCK) undergoes endoproteolytic cleavage at three sites. Several studies using endocrine and neuronal tumor cells in culture and recombinant enzymes and synthetic substrates in vitro have pointed to the subtilisin/kexin-like enzymes prohormone convertase (PC) 1, PC2, and PC5 as potential candidates for these endoproteolytic cleavages. In these experimental models, they all appear to be able to cleave pro-CCK to make the correct products. One rodent model has provided information about the role of PC2. PC2 knockout mouse brains had less CCK 8 than wild-type, although a substantial amount of CCK was still present. The degree to which CCK levels were reduced in these mice was regionally specific. These data indicated that PC2 is important for normal production of CCK but that it is not the only endoprotease that is involved in CCK processing. To evaluate whether PC1 and PC5 are possible candidates for the other enzymes involved in CCK processing, the distribution of PC1, PC2, and PC5 mRNA was studied in rat brain. Their colocalization with CCK mRNA was examined using double-label in situ hybridization. PC2 was the most abundant of these enzymes in terms of the intensity and number of cells labeled. It was widely colocalized with CCK. PC1 and PC5 mRNA-positive cells were less abundant, but they were also widely distributed and strongly colocalized with CCK in the cerebral cortex, hippocampus, amygdala, ventral tegmental area, and substantia nigra zona compacta. The degree of colocalization of the enzymes with CCK was regionally specific. It is clear that PC1 and PC5 are extensively colocalized with CCK and could be participating in CCK processing in the rat brain and may be able to substitute for PC2 in its absence. These three enzymes may represent a redundant system to ensure production of biologically active CCK.

The discovery of the prohormone convertase (PC) family of enzymes has provided several good candidates (PC1, PC2, and PC5) for the enzymes responsible for the endoproteolytic cleavage of procholecystokinin (pro-CCK). Determination of the role of individual pro-hormone convertases in the processing of pro-CCK is complicated because several of these enzymes are found in endocrine tumor cells expressing CCK mRNA and in identified neurons in the brain. Production of active recombinant PC5 permits the determination of its ability to cleave substrates related to pro-CCK. Active PC5, secreted from baculovirus-infected Sf9 cells, was partially purified by ion-exchange chromatography. Western blot analysis confirmed the presence of the active form of the enzyme in infected cell media and its absence from uninfected cell media. The enzyme is most active at acidic pH 6.5 and is maximally activated by 5 mM calcium. PC5 was able to cleave both monobasic and dibasic substrates without a requirement for a basic residue at P-4 and it displayed a Km in the micromolar range. The enzyme was inhibited by EDTA, 1,10-phenanthroline, and p-CMS, as well as by two specific PC inhibitors. This is the first reported preparation of active recombinant PC5. Like the other members of its family, it has the correct catalytic characteristics in vitro to play a role in the processing of neuropeptide precursor proteins into their final bioactive forms.

Lovastatin prevents isoprene synthesis thereby affecting the structural organization of proteins involved in protein transport and secretion. Lovastatin at 1 microM decreases CCK 8 secretion by over 50% in WE cells and in CCK 8 expressing AtT20 cells. At 10 microM CCK 8 secretion was inhibited by two thirds and at 100 microM, cytotoxic effects were observed in both cell types. Addition of mevalonate does not restore CCK secretion and stimulation of secretion by forskolin is also partially inhibited. Cellular content of CCK 8 and pro-CCK were not altered in either of these cell lines except at 100 microM lovastatin. Our results clearly demonstrate that lovastatin at 1 microM strongly inhibits CCK 8 secretion at multiple levels while having little or no effect on its synthesis. This effect on secretion may be partly responsible for the adverse gastrointestinal side effects of lovastatin in patients.

Publisher Summary Studies using antisense inhibition have been criticized because of the possible nonspecific cellular effects complicating interpretation of the results. In light of these concerns, several controls have been performed to ensure that the effects of antisense expression on the processing of pro-cholecystokinin (CCK) are specific. The two most important controls performed were to measure the effects of antisense expression on target messenger ribonucleic acid (mRNA) and target protein. Another control that was performed was to measure the effect of polycystin 2 (PC2) message and protein level in antisense PC1-transfected cells and vice versa. Although PC1 and PC2 are homologous genes within the same family of endoproteases, the effect of antisense is specific for each. These observations suggest that the effect of antisense on the RNA level is complex but specific and may occur at multiple levels to produce inhibition of the protein product. Knowledge of the actual enzymes responsible for prohormone cleavages in specific tissues lags behind progress in other areas of the peptide hormone field owing to the technical difficulty of identification, isolation, characterization, and inhibition of these enzymes. As important as the PC enzymes appear to be, it is possible that proteases other than the PCs are involved in prohormone processing.

A high-affinity RNA aptamer to human activated protein C (APC) was selected from a pool of random sequences using in vitro selection. Activated protein C, a trypsin-like serine protease plays an important role along with thrombin as a regulator in blood clotting cascade. After seven rounds of selection and amplification, a single predominant nucleic acid sequence APC-167, a 167-base oligonucleotide with a random sequence core of 120 bases, was obtained. The selected aptamer did not bind to thrombin or factor Xa and thus demonstrated specificity to APC. Furthermore, this aptamer was a non-competitive inhibitor to the cleavage reaction of a fluorogenic substrate catalyzed by APC. The inhibition constant (Ki) of APC-167 was 83 nM. The 99-base oligonucleotide (APC-99) derived from APC-167 by deleting both primer binding sites, was also found to inhibit APC strongly (Ki = 137 nM). Two stem-loop structures and at least one G x U wobble base pair in the stem were elucidated as important structural motifs for binding.

Hepatitis C virus (HCV) is a single-stranded RNA virus and its genome is translated into a single large polyprotein. The viral-encoded NS3 protein possesses protease, nucleoside triphosphatase, and helicase activities. Since these activities appear to be important for viral replication, efforts are being made to identify compounds that might inhibit the enzymatic activities of NS3 and serve as potential anti-HCV agents. We used a genetic selection strategyin vitroto isolate, from a pool of completely random RNA (120 random bases), those RNA aptamers that could bind to NS3. After six cycles of selection and amplification, 14% of the pooled RNAs could bind specifically to the NS3 protein. When the aptamers in the pool (cycle 6) were analyzed for binding and inhibition of the proteolytic activity of NS3 with the NS5A/NS5B peptide as substrate (S1), two aptamers, designated G6–16 and G6–19 RNA, were found to inhibit NS3in vitro.Kinetic studies of the inhibition revealed that the aptamer G6–16 inhibited the NS3 protease with an inhibitory constant (Ki) of 3 μM.We also analyzed aptamers G6–16 and G6–19 for their action with a longer protein substrate (amino acid region 2203–2506) and found that these aptamers efficiently inhibited the proteolytic activity of NS3. In addition, both G6–16 and G6–19 aptamers were found to inhibit the helicase activity of NS3. Since these aptamers possesses dual inhibitory function for NS3, they could prove to be useful as anti-HCV drug leads.

The serine protease domain of HCV comprising amino acids 1027-1218 (deltaNS3) was expressed in E. coli with a His tag at its N-terminal end. The protease was purified to apparent homogeneity by a single step affinity chromatography resulting in high yields (approximately 3 mg/l of cultured cells). The deltaNS3 efficiently cleaves a 17-mer peptide corresponding to the NS5A-NS5B junction with kcat/Km = 160 x 10(-3) min(-1) microM(-1) in the presence of NS4A peptide. Our deltaNS3 represents the minimal domain possessing highly active protease of NS3 constructed so far. The deltaNS3 protein also efficiently processed a longer substrate corresponding to NS5A/5B junction (2203-2506 amino acids) that was synthesized by in vitro transcription and translation system.