Regulation of Carboxypeptidase E by Membrane Depolarization in PC12 Pheochromocytoma Cells: Comparison with mRNAs Encoding Other Peptide- and Catecholamine-Biosynthetic Enzymes

PC12 cells, a rat pheochromocytoma cell line, have been found to express carboxypeptidase E (CPE) enzymatic activity and CPE, furin, and peptidylglycine alpha-amidating monooxygenase (PAM) mRNAs. PC12 cells secrete CPE activity in response to depolarization induced by 50 mM KCl. Short-term (1- to 3-h) treatments of PC12 cells with KCl stimulates the secretion of CPE but does not appear to stimulate the synthesis of new CPE protein, based on the measurement of CPE activity and incorporation of [35S]-Met into CPE. Also, CPE mRNA is not altered by 2-h treatments with KCl. In contrast, prolonged treatment (24-48 h) of PC12 cells with 50 mM KCl continues to stimulate the secretion of CPE activity, without altering the cellular level of CPE. Levels of CPE mRNA are significantly elevated after long-term treatment of the cells with KCl, with increases of 35% after 5 h and 55-75% after 24 to 72 h of treatment. The level of PAM mRNA is also elevated approximately 70% after 24 h of stimulation with KCl. In contrast, the mRNA levels of furin and dopamine beta-hydroxylase (DBH) do not change on treatment of PC12 cells with KCl. These findings indicate that long-term depolarization, which leads to a prolonged stimulation of PC12 cells to secrete CPE, also stimulates the synthesis of CPE and PAM but not furin or DBH.     

Identification of a Neuropeptide and Neuropeptide-Processing Enzymes in Aqueous Humor Confers Neuroendocrine Features to the Human Ocular Ciliary Epithelium

Abstract: The ocular ciliary epithelium, the site of aqueous humor secretion in the mammalian eye, is believed to play a key function in signaling mechanisms that regulate the rate of secretion, and thus intraocular pressure. One possible way of mediating these signaling functions is through neuropeptides and hormones secreted into the aqueous humor and acting on target tissues. We recently identified a cDNA clone sharing 100% identity with carboxypeptidase E (CPE), a neuropeptide-processing enzyme. Utilizing polymerase chain reaction, we further identified and characterized another processing enzyme, the peptidylglycine α-amidating monooxygenase (PAM), and the neuropeptide secretogranin II, a molecular marker restricted to neuroendocrine tissues. Using specific probes, we found that the nonpigmented ciliary epithelial cells express CPE, PAM, and secretogranin II mRNA, and protein. We also found that CPE and secretogranin II are abundant in aqueous humor. Treatment of cultured ciliary epithelial cells with veratridine and phorbol ester up-regulates CPE and PAM. Secretogranin II was found to be induced by veratridine, whereas phorbol ester had little effect, suggesting different mechanisms for secretion. The results demonstrate that secretogranin II, CPE, and PAM represent a specialized group of neuropeptide and neuropeptide-processing enzymes secreted by the ciliary epithelial cells which may confer to them neuroendocrine functions in cell-cell communication or cell signaling.     

Secretion of Carboxypeptidase E from Cultured Astrocytes and from AtT-20 Cells, a Neuroendocrine Cell Line: Implications for Neuropeptide Biosynthesis

Cultured astrocytes have recently been shown to produce certain neuropeptides, as well as neuropeptide processing enzymes. To characterize the secretory pathway in cultured astrocytes, we used the neuropeptide processing enzyme carboxypeptidase E (CPE) as a marker for neuropeptide secretion. Cultured astrocytes and AtT-20 cells, a mouse pituitary-derived neuroendocrine cell line, were labeled with [35S]Met for 15 min and then chased with unlabeled Met. CPE was isolated from either medium or cell extracts using a substrate affinity column. The time course of secretion of radiolabeled CPE was significantly different for cultured astrocytes as compared with AtT-20 cells. CPE was rapidly secreted from the astrocytes after a 30-min lag time, presumably reflecting transport through the endoplasmic reticulum and Golgi apparatus, followed by constitutive secretion. The secretion of radiolabeled CPE was essentially complete by 2 h. In contrast, only a portion of the radiolabeled CPE was secreted from AtT-20 cells over a 2-3-h period, indicating that the majority of newly synthesized CPE is stored, presumably in secretory granules within the AtT-20 cells. The regulation of CPE secretion from astrocytes was also examined. CPE secretion is stimulated two- to threefold by prolonged treatment (3-48 h) with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) but not by treatment with other secretagogues that stimulate CPE secretion from AtT-20 cells (forskolin, isoproterenol, A23187, and vasoactive intestinal peptide) or short (less than 3 h) exposure to TPA. Taken together, these results indicate that the secretory pathway for CPE, and presumably neuropeptides, is substantially different in astrocytes than the secretory pathway for CPE in neuroendocrine cells.     

Activation and routing of membrane-tethered prohormone convertases 1 and 2.

Many peptide hormones and neuropeptides are processed by members of the subtilisin-like family of prohormone convertases (PCs), which are either soluble or integral membrane proteins. PC1 and PC2 are soluble PCs that are primarily localized to large dense core vesicles in neurons and endocrine cells. We examined whether PC1 and PC2 were active when expressed as membrane-tethered proteins, and how tethering to membranes alters the biosynthesis, enzymatic activity, and intracellular routing of these PCs. PC1 and PC2 chimeras were constructed using the transmembrane domain and cytoplasmic domain of the amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The membrane-tethered PCs were rerouted from large dense core vesicles to the Golgi region. In addition, the chimeras were transiently expressed at the cell surface and rapidly internalized to the Golgi region in a fashion similar to PAM. Membrane-tethered PC1 and PC2 exhibited changes in pro-domain maturation rates, N-glycosylation, and in the pH and calcium optima required for maximal enzymatic activity against a fluorogenic substrate. In addition, the PC chimeras efficiently cleaved endogenous pro-opiomelanocortin to the correct bioactive peptides. The PAM transmembrane domain/cytoplasmic domain also prevented stimulated secretion of pro-opiomelanocortin products in AtT-20 cells.     

Genomic organization and splicing variants of a peptidylglycine alpha-hydroxylating monooxygenase from sea anemones

Cnidarians are primitive animals that use neuropeptides as their transmitters. All the numerous cnidarian neuropeptides isolated, so far, have a carboxy-terminal amide group that is essential for their actions. This strongly suggests that alpha-amidating enzymes are essential for the functioning of primitive nervous systems. In mammals, peptide amidation is catalyzed by two enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) that act sequentially. These two activities are contained within one bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), which is coded for by a single gene. In a previous paper (F. Hauser et al., Biochem. Biophys. Res. Commun. 241, 509-512, 1997) we have cloned the first known cnidarian PHM from the sea anemone Calliactis parasitica. In the present paper we have determined the structure of its gene (CP1). CP1 is >12 kb in size and contains 15 exons and 14 introns. The last coding exon (exon 15) contains a stop codon, leaving no room for PAL and, thereby, for a bifunctional PAM enzyme as in mammals. Furthermore, we found a CP1 splicing variant (CP1-B) that contains exon-9 instead of exon-8, which was present in the previously characterized PHM cDNA (CP1-A). CP1-A and -B have 97% amino acid sequence identity, whereas both splicing variants have around 42% sequence identity with the PHM part of rat PAM. Essential amino acid residues for the catalytic activity and the 3D structure of PHM are conserved between CP1-A, -B and the PHM part of rat PAM. Furthermore, eight introns in CP1 occur in the same positions and have the same intron phasing as eight introns in the rat PAM gene, showing that the sea anemone PHM is not only structurally, but also evolutionarily related to the PHM part of rat PAM.     

Impaired prohormone convertases in Cpe(fat)/Cpe(fat) mice

A spontaneous point mutation in the coding region of the carboxypeptidase E (CPE) gene results in a loss of CPE activity that correlates with the development of late onset obesity (Nagert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Nat. Genet. 10, 135-142). Examination of the level of neuropeptides in these mice showed a decrease in mature bioactive peptides as a result of a decrease in both carboxypeptidase and prohormone convertase activities. A defect in CPE is not expected to affect endoproteolytic processing. In this report we have addressed the mechanism of this unexpected finding by directly examining the expression of the major precursor processing endoproteases, prohormone convertases PC1 and PC2 in Cpe(fat) mice. We found that the levels of PC1 and PC2 are differentially altered in a number of brain regions and in the pituitary. Since these enzymes have been implicated in the generation of neuroendocrine peptides (dynorphin A-17, beta-endorphin, and alpha- melanocyte-stimulating hormone) involved in the control of feeding behavior and body weight, we compared the levels of these peptides in Cpe(fat) and wild type animals. We found a marked increase in the level of dynorphin A-17, a decrease in the level of alpha-melanocyte-stimulating hormone, and an alteration in the level of C-terminally processed beta-endorphin. These results suggest that the impairment in the level of these and other peptides involved in body weight regulation is mainly due to an alteration in carboxypeptidase and prohormone convertase activities and that this may lead to the development of obesity in these animals.     

Changes in levels of translatable mRNA for neuron-specific enolase and non-neuronal enolase during development of rat brain and liver

Neuron-specific enolase (NSE), and non-neuronal enolase (NNE) which exists in many tissues including liver but is localized in glial cells within the nervous system, were synthesized in the rabbit reticulocyte cell-free translation system programmed with brain mRNAs. The in vitro synthesized NSE and NNE were indistinguishable from the two enzymes purified from rat brains. NSE mRNA activity was found only in brain RNAs, while NNE mRNA activity existed in brain RNAs as well as liver RNAs. In developing brains, the level of translatable NSE mRNA was low at the embryonic stage and at birth, increased rapidly from about 10 days postnatal, and reached the adult level, while that of NNE mRNA was high at the embryonic stage and at birth, followed by a slight decrease then a gradual rise to adult levels. These changes correlated with the developmentally regulated appearance and accumulation pattern of each of the two enzymes. These results suggest that the levels of NSE and NNE are controlled primarily by the level of each of the two translatable mRNAs. In developing livers, only the NNE mRNA activity was detected and its level generally paralleled the changes in the level of NNE.     

Development of CAPS and dCAPS markers for <Emphasis Type="Italic">CYP82E4</Emphasis>, <Emphasis Type="Italic">CYP82E5v2</Emphasis> and <Emphasis Type="Italic">CYP82E10</Emphasis> gene mutants reducing nicotine to nornicotine conversion in tobacco

Nornicotine accumulation in tobacco is of concern because nornicotine is a precursor of N-nitrosonornicotine (NNN), a tobacco constituent recognized as a carcinogen by the health community. Nornicotine is derived from nicotine through a demethylation process catalyzed by nicotine demethylase enzymes. Three genes (CYP82E4, CYP82E5v2, and CYP82E10) have currently been identified that encode for these enzymes. Ethyl methane sulfonate has been used to introduce mutations into each of these genes to prevent production of functional gene products. These mutants represent a valuable tool for reducing nornicotine and NNN levels in cured tobacco leaves and their derived products. Methods are currently needed to rapidly and efficiently develop new cultivars possessing these mutant alleles. The objective of this study was to develop efficient, user-friendly DNA markers to identify these mutations based on single nucleotide polymorphisms (SNPs). Four dCAPS (derived cleaved amplified polymorphic sequence) markers were designed for a truncation mutation in CYP82E4, and a single marker was developed for a similar mutation in CYP82E5v2. Two CAPS (cleaved amplified polymorphic sequence) markers were designed for a missense mutation in CYP82E10. Because of the co-dominant nature of the CAPS and dCAPS markers, heterozygous and homozygous plants can be easily differentiated. Genotypes determined by the CAPS and dCAPS marker methods were validated by DNA sequencing and phenotypic analysis of plants carrying various mutant combinations. These markers can be used in marker-assisted selection programs to quickly introgress the desired mutations into commercial varieties in order to reduce nornicotine and NNN levels in tobacco leaves.     

<Emphasis Type="SmallCaps">l</Emphasis>-Dopa decarboxylase expression profile in human cancer cells

l-Dopa decarboxylase (DDC) catalyses the decarboxylation of l-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5′-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5′UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.     

Cas9蛋白变体VQR高效识别水稻NGAC前间区序列邻近基序

成簇的规律间隔短回文重复序列及CRISPR相关蛋白(clustered regularly interspaced short palindromic repeats/CRISPR-associated 9, CRISPR/Cas9)系统是近年来发展起来并被广泛应用的第三代基因组编辑工具。但是,该系统的酿脓链球菌Cas9(Streptococcus pyogenes, SpCas9)仅能识别NGG前间区序列邻近基序(protospacer adjacent motif, PAM),极大地限制了基因组编辑的范围。SpCas9变体VQR(D1135V/R1335Q/T1337R)在水稻中可识别NGAA、NGAG和NGAT PAM,但尚不清楚是否能识别NGAC PAM。本研究利用改进后的CRISPR/VQR系统对水稻中3个相对低效的VQR靶位点NAL1-Q1、NAL1-Q2和LPA1-Q进行了编辑,结果表明改进后的CRISPR/VQR系统可以高效编辑这3个靶位点,编辑效率分别为9.75%、43.90%和29.26%。为了明确改进后的CRISPR/VQR系统对NGAC PAM的识别情况,本研究选择水稻叶片宽度调控基因NARROW LEAF 1 (NAL1)中的NAL-C位点和蜡质合成基因GLOSSY1 (GL1)中的GL1-C位点进行基因编辑,并获得57株转基因水稻。靶位点PCR扩增及测序结果表明,NAL1-C和GL1-C靶标位点突变的植株分别为27株和44株,突变率分别为47.36%和77.19%;其中NAL1-C/GL1-C双突变植株为26株,双突变率为45.61%。进一步分析表明,CRISPR/VQR系统造成的突变有4种类型,分别为杂合突变、双等位突变、嵌合体突变和纯合突变,其中以杂合突变和双等位突变为主。这些结果表明,改进的CRISPR/VQR系统可以高效编辑水稻NGAC PAM位点,并产生丰富的突变类型。本研究为水稻及其他植物相关基因NGAC PAM位点的编辑提供了理论依据。     

Manipulation of neuropeptide biosynthesis through the expression of antisense RNA for peptidylglycine alpha-amidating monooxygenase.

Stable cell lines with significantly elevated or diminished levels of a key neuropeptide processing enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), were generated by transfection of a mouse pituitary cell line with expression vectors containing PAM cDNA in the sense or antisense orientation. By evaluating the ability of these cell lines to alpha-amidate endogenous neuropeptides, a rate-limiting role for PAM in neuropeptide alpha-amidation was demonstrated. Overexpression of either the full-length PAM precursor with its trans-membrane domain or a soluble protein containing only the monooxygenase domain of PAM led to increased alpha-amidation of endogenous neuropeptides. Overexpression of the full-length PAM led to an unexpected decrease in the endoproteolytic processing of endogenous prohormone; conversely, underexpression of PAM led to significantly enhanced endoproteolytic processing of endogenous prohormone. These data suggest that PAM may have additional functions in peptide processing.     

Mammalian peptidylglycine alpha-amidating monooxygenase mRNA expression can be modulated by the La autoantigen

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal alpha-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3' untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3' UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3' UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3' UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules.     

Expression of individual forms of peptidylglycine alpha-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules

Peptidylglycine alpha-amidating monooxygenase (PAM: EC 1.14.17.3) is a bifunctional protein which catalyzes the COOH-terminal amidation of bioactive peptides; the NH2-terminal monooxygenase and mid-region lyase act in sequence to perform the peptide alpha-amidation reaction. Alternative splicing of the single PAM gene gives rise to mRNAs generating PAM proteins with and without a putative transmembrane domain, with and without a linker region between the two enzymes, and forms containing only the monooxygenase domain. The expression, endoproteolytic processing, storage, and secretion of this secretory granule-associated protein were examined after stable transfection of AtT-20 mouse pituitary cells with naturally occurring and truncated PAM proteins. The transfected proteins were examined using enzyme assays, subcellular fractionation, Western blotting, and immunocytochemistry. Western blots of crude membrane and soluble fractions of transfected cells demonstrated that all PAM proteins were endoproteolytically processed. When the linker region was present between the monooxygenase and lyase domains, monofunctional soluble enzymes were generated from bifunctional PAM proteins; without the linker region, bifunctional enzymes were generated. Soluble forms of PAM expressed in AtT-20 cells and soluble proteins generated through selective endoproteolysis of membrane-associated PAM were secreted in an active form into the medium; secretion of the transfected proteins and endogenous hormone were stimulated in parallel by secretagogues. PAM proteins were localized by immunocytochemistry in the perinuclear region near the Golgi apparatus and in secretory granules, with the greatest intensity of staining in the perinuclear region in cell lines expressing integral membrane forms of PAM. Monofunctional and bifunctional PAM proteins that were soluble or membrane-associated were all packaged into regulated secretory granules in AtT-20 cells.     

Complex transcriptional regulation of citrate metabolism in Clostridium perfringens

A Gram-positive, spore-forming bacterium, Clostridium perfringens, possesses genes for citrate metabolism, which might play an important role in the utilization of citrate as a sole carbon source. In this study, we identified a chromosomal citCDEFX-mae-citS operon in C. perfringens strain 13, which is transcribed on three mRNAs of different sizes. Expression of the cit operon was significantly induced when 5 mM extracellular citrate was added to the growth medium. Most interestingly, three regulatory systems were found to be involved in the regulation of the expression of cit genes: 1) the two upstream divergent genes citG and citI; 2) two different two-component regulatory systems, CitA/CitB (TCS6 consisted of CPE0531/CPE0532) and TCS5 (CPE0518/CPE0519); and 3) the global two-component VirR/VirS-VR-RNA regulatory system known to regulate various genes for toxins and degradative enzymes. Our results suggest that in C. perfringens the citrate metabolism might be strictly controlled by a complex regulatory system.     

人源多巴脱羧酶的表达、纯化以及高通量抑制剂筛选模型的建立

帕金森病是一种常见的神经系统退行性疾病。多巴脱羧酶（DDC）是帕金森病研究的靶点蛋白之一,但是目前没有高通量的测活模型。因此,需要构建一种高通量多巴脱羧酶抑制剂的筛选模型,用于发现新型抑制剂。采用克隆表达纯化得到多巴脱羧酶和用于酶偶联反应的磷酸烯醇式丙酮酸羧化酶（PEPC）。基于一系列酶联反应将CO2固定,检测其含量,从而测定多巴脱羧酶的活性。结果得到人源多巴脱羧酶和磷酸烯醇式丙酮酸羧化酶的体外纯酶,建立了一种高通量筛选模型,并且从70个天然化合物中,筛选得到2个多巴脱羧酶的抑制剂。成功构建了一种基于体外纯酶高通量多巴脱羧酶抑制剂的筛选模型。     

Pituitary adenylate cyclase activating polypeptide regulates the basal production of nitric oxide in PC12 cells

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), two members of the VIP/secretin/glucagon family, modulate neurotransmission via stimulation of protein kinases including cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) in the central and peripheral nervous systems. They are reported to co-exist with nitric oxide synthases (NOSs) and other neuropeptides within the nervous system and peripheral tissues. In the present study, we investigated the neuronal role of these peptides in NO production in PC12 cells. We showed that PACAP decreased NO production in a dose-dependent manner, and the activators of protein kinase A and C also inhibited the NO production in PC12 cells. RT-PCR experiments demonstrated that PC12 cells constitutively express the mRNAs for neuronal NOS and the PACAP-specific (PAC1) receptor, and we concluded that PACAP plays an important role in the regulation of nNOS activity through PAC1 receptor in PC12 cells.