Proteolytic processing of a precursor protein for a growth-promoting peptide by a subtilisin serine protease in Arabidopsis

Phytosulfokines (PSKs) are secreted, sulfated peptide hormones derived from larger prepropeptide precursors. Proteolytic processing of one of the precursors, AtPSK4, was demonstrated by cleavage of a preproAtPSK4-myc transgene product to AtPSK4-myc. Cleavage of proAtPSK4 was induced by placing root explants in tissue culture. The processing of proAtPSK4 was dependent on AtSBT1.1, a subtilisin-like serine protease, encoded by one of 56 subtilase genes in Arabidopsis. The gene encoding AtSBT1.1 was up-regulated following the transfer of root explants to tissue culture, suggesting that activation of the proteolytic machinery that cleaves proAtPSK4 is dependent on AtSBT1.1 expression. We also demonstrated that a fluorogenic peptide representing the putative subtilase recognition site in proAtPSK4 is cleaved in vitro by affinity-purified AtSBT1.1. An alanine scan through the recognition site peptide indicated that AtSBT1.1 is fairly specific for the AtPSK4 precursor. Thus, this peptide growth factor, which promotes callus formation in culture, is proteolytically cleaved from its precursor by a specific plant subtilase encoded by a gene that is up-regulated during the process of transferring root explants to tissue culture.     

A conserved dibasic site is essential for correct processing of the peptide hormone AtRALF1 in Arabidopsis thaliana

Prohormone proteins in animals and yeast are typically processed at dibasic sites by convertases. Propeptide hormones are also found in plants but little is known about processing. We show for the first time that a dibasic site upstream of a plant peptide hormone, AtRALF1, is essential for processing. Overexpression of preproAtRALF1 causes semi-dwarfism whereas overexpression of preproAtRALF1(R69A), the propeptide with a mutation in the dibasic site, shows a normal phenotype. RALF1(R69A) plants accumulate only the mutated proprotein and not the processed peptide. In vitro processing using microsomal fractions suggests that processing is carried out by a kexin-like convertase.     

Regulation of plant peptide hormones and growth factors by post‐translational modification

The number, diversity and significance of peptides as regulators of cellular differentiation, growth, development and defence of plants has long been underestimated. Peptides have now emerged as an important class of signals for cell‐to‐cell communication over short distances, and also for long‐range signalling. We refer to these signalling molecules as peptide growth factors and peptide hormones, respectively. As compared to remarkable progress with respect to the mechanisms of peptide perception and signal transduction, the biogenesis of signalling peptides is still in its infancy. This review focuses on the biogenesis and activity of small post‐translationally modified peptides. These peptides are derived from inactive pre‐pro‐peptides of approximately 70–120 amino acids. Multiple post‐translational modifications (PTMs) may be required for peptide maturation and activation, including proteolytic processing, tyrosine sulfation, proline hydroxylation and hydroxyproline glycosylation. While many of the enzymes responsible for these modifications have been identified, their impact on peptide activity and signalling is not fully understood. These PTMs may or may not be required for bioactivity, they may inactivate the peptide or modify its signalling specificity, they may affect peptide stability or targeting, or its binding affinity with the receptor. In the present review, we will first introduce the peptides that undergo PTMs and for which these PTMs were shown to be functionally relevant. We will then discuss the different types of PTMs and the impact they have on peptide activity and plant growth and development. We conclude with an outlook on the open questions that need to be addressed in future research.     

Function and solution structure of the Arabidopsis thaliana RALF8 peptide

We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine‐rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well‐resolved nuclear magnetic resonance (NMR) spectra and also biological activity through interaction with a plant receptor kinase, cytoplasmic calcium mobilization, and in vivo root growth suppression. By contrast, RALF1 could only be isolated from inclusion bodies as a construct containing an N‐terminal His‐tag; its poorly resolved NMR spectrum was indicative of aggregation. We prepared samples of the RALF8 peptide labeled with ¹⁵N and ¹³C for NMR analysis and obtained near complete ¹H, ¹³C, and ¹⁵N NMR assignments; determined the disulfide pairing of its four cysteine residues; and examined its solution structure. RALF8 is mostly disordered except for the two loops spanned by each of its two disulfide bridges.     

Receptor Kinase THESEUS1 Is a Rapid Alkalinization Factor 34 Receptor in Arabidopsis

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Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1‐like sequences were observed in most cases; however, RALF27‐like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27‐like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen‐activated protein kinase activation). Gene expression analysis confirmed that a RALF‐encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant–pathogen interactions.     

Effect of recombinant growth hormone on expression of growth hormone receptor,insulin-like growth factor mRNA and serum level of leptin in growing pigs

Sixteen Large White × Landrace castrated male pigs were allotted into treatment and control group. The treatment group was injected intramuscularly with recombinant porcine growth hormone (rpGH, 4 mg d⁻¹) and the control group with vehicle for 28 days. Animals were slaughtered 4 h after final injection for liver, longissimus dorsi (LD) muscle and blood sampling. Serum concentration of insulin-like growth factor 1 (IGF-I) and leptin were determined by RIA. The total RNA was extracted from tissues to measure the abundance of growth hormone receptor (GHR), IGF-I mRNA by RT-PCR with 18S rRNA internal standard. Results showed that rpGH enhanced the average daily weight gain by 26.1% (P < 0.05), the serum IGF-I concentration by 70.94% (P < 0.01), decreased serum leptin by 34.8% (P < 0.01). The relative abundance of GHR and IGF-I mRNA in liver were increased by 24.45% (P < 0.05) and 45.30% (P < 0.01), respectively, but no difference of GHR (P > 0.05) and IGF-I mRNA (P > 0.05) in LD between GH treated and control group was found. These results suggest that rpGH can up-regulate hepatic GHR and IGF-I gene expression and improve animal growth. However the effect of rpGH on GHR and IGF-I gene expression are tissue-specific.     

Effect of recombinant growth hormone on expression of growth hormone receptor, insulin-like growth factor mRNA and serum level of leptin in growing pigs

Growth hormone (GH) plays an important role in regulation of animal growth, metabolism and lactation[1]. Numerous studies have shown that exogenous somatotropin (ST) can increase average daily weight gain, improve feed efficiency, stimulate protein deposition and muscle growth and decrease lipid accretion rate[1]. The original somatomedin hypothesis suggested that the effect of GH on postnatal growth was mediated by insulin-like growth hormone factor 1 (IGF-I) which was thought to be deriv…     

Roles of OsCKI1, a rice casein kinase I, in root development and plant hormone sensitivity

Casein kinases are critical in cell division and differentiation across species. A rice cDNA fragment encoding a putative casein kinase I (CKI) was identified via cDNA macroarray under brassinosteroid (BR) treatment, and a 1939-bp full-length cDNA, OsCKI1, was isolated and found to encode a putative 463-aa protein. RT-PCR and Northern blot analysis indicated that OsCKI1 was constitutively expressed in various rice tissues and upregulated by treatments with BR and abscisic acid (ABA). Enzymatic assay of recombinant OsCKI1 proteins expressed in Escherichia coli showed that the protein was capable of phosphorylating casein. The physiological roles of OsCKI1 were studied through antisense transgenic approaches, and homozygous transgenic plants showed abnormal root development, including fewer lateral and adventitious roots, and shortened primary roots as a result of reduced cell elongation. Treatment of wild-type plants with CKI-7, a specific inhibitor of CKI, also confirmed these functions of OsCKI1. Interestingly, in transgenic and CKI-7-treated plants, exogenously supplied IAA could restore normal root development, and measurement of free IAA content in CKI-deficient primary and adventitious roots revealed altered auxin content, indicating that OsCKI1 is involved in auxin metabolism or that it may affect auxin levels. Transgenic plants were less sensitive than control plants to ABA or BR treatment during germination, suggesting that OsCKI1 may be involved in various hormone-signaling pathways. OsCKI1-GFP fusion studies revealed the localization of OsCKI1 to the nucleus, suggesting a possible involvement in regulation of gene expression. In OsCKI1-deficient plants, differential gene expression was investigated using cDNA chip technology, and results indicated that genes related to signal transduction and hormone metabolism were indeed with altered expression.     

Regulation of gonadotropin-releasing hormone (GnRH) gene expression in hypothalamic neuronal cells

Summary 1. Gonadotropin-releasing hormone (GnRH) is the hypothalamic releasing factor that controls pituitary gonadotropin subunit gene expression and indirectly gametogenesis and steroidogenesis from the gonad, which results in reproductive competence.2. GnRH is synthesized in only about 1000 neurons in the hypothalamus and released in an episodic fashion down the median eminence to regulate gonadotropin biosynthesis.3. Although much is known about the secretory dynamics of GnRH release, little is known about the pretranslational control of GnRH biosynthesis due to lack of appropriate model systems. The recent availability of immortalized neuronal cell lines that produce GnRH allows investigators for the first time to begin to dissect the factors that directly regulate GnRH gene expression.4. This article reviews the current state of knowledge concerning the mechanisms that direct tissue-specific and peptide hormone control of GnRH biosynthesis.     

Molecular cloning and functional characterization of the canine parathyroid hormone/parathyroid hormone related peptide receptor (PTH1)

Parathyroid hormone (PTH) is a major mediator of calcium and phosphate metabolism through its interactions with receptors in kidney and bone. PTH binds with high affinity to PTH1 and PTH2, members of the superfamily of G protein-coupled receptors. In order to clone the canine PTH1 receptor, a canine kidney cDNA library was screened using the human PTH1 receptor cDNA and two clones were further characterized. The longest clone was 2177 bp and contained a single open reading frame of 1785 bp, potentially encoding a protein of 595 amino acids with a predicted molecular weight of 66.4 kD. This open reading frame exhibits >91% identity to the human PTH1 receptor cDNA and >95% identity when the putative canine and human protein sequences are compared. Competition binding following transfection of the canine PTH1 receptor into CHO cells demonstrated specific displacement of ¹²⁵I-human PTH 1-34 by canine PTH 1-34, human PTH 1-34, and canine/human parathyroid hormone related peptide (PTHrP) 1-34. Treatment of canine PTH1 receptor transfected cells, but not mock transfected cells, with these ligands also resulted in increased levels of intracellular cAMP. In contrast, the non-related aldosterone secretion inhibiting factor 1-35 neither bound nor activated the canine PTH1 receptor. Northern blot analysis revealed high levels of PTH1 receptor mRNA in the kidney, with much lower, but detectable, levels in aorta, heart, lung, prostate, testis, and skeletal muscle. Together, these data indicate that we have cloned the canine PTH1 receptor and that it is very similar, both in sequence and in functional characteristics, to the other known PTH1 receptors.     

Human parathyroid hormone as a secretory peptide in milk of transgenic mice

In a transgenic mouse model we have targeted the expression of recombinant human parathyroid hormone (hPTH) to the mammary gland yielding hPTH as a secretory, soluble peptide in milk. A 2.5 kb upstream regulatory sequence of the murine whey acidic protein (WAP) directed the expression of the hPTH cDNA in a fusion gene construct (WAPPTHSV2) containing the SV40 small t-antigen intron and polyadenylation site in the 3′ end. Established lines of transgenic mice secreted hPTH to milk in concentrations up to 415 ng/ml. Recombinant hPTH recovered from the milk was purified by HPLC and shown to be identical to hPTH standard as analyzed by SDS-PAGE followed by immunoblotting. Expression of the WAPPTHSV2 was limited to the mammary gland as analyzed by polymerase chain reaction (PCR) and Southern blot of reversed transcribed mRNA from different tissues. hPTH is an important bone anabolic hormone and may be a potentially important pharmaceutical for treatment of demineralization disorders such as osteoporosis. We present the transgenic animal as a possible production system for hPTH. © 1995 Wiley-Liss, Inc.     

Effects of adipokinetic hormone and its related peptide on maintaining hemolymph carbohydrate and lipid levels in the two-spotted cricket,Gryllus bimaculatus

Adipokinetic hormone (AKH) regulates energy homeostasis in insects by mobilizing lipid and carbohydrate from the fat body. Here, using RNA sequencing data, we identified cDNAs encoding AKH (GbAKH) and its highly homologous hormone AKH/corazonin-related peptide (GbACP) in the corpora cardiaca of the two-spotted cricket, Gryllus bimaculatus. RT-PCR revealed that GbAKH and GbACP are predominantly expressed in the corpora cardiaca and corpora allata, respectively. Phylogenetic analysis confirmed that the identified GbAKH and GbACP belong to the clades containing other AKHs and ACPs, respectively. Injection of synthetic GbAKH and GbACP elevated hemolymph carbohydrate and lipid levels and reduced food intake significantly. In contrast, knockdown of GbAKH and GbACP by RNA interference increased the food intake, although hemolymph lipid level was not altered. Collectively, this study provides evidence that ACP regulates hemolymph carbohydrate and lipid levels in cricket, possibly collaborative contribution with AKH to the maintenance of energy homeostasis.     

Targeting the insulin receptor with hormone and peptide dimers

Insulin is a key hormone involved in the regulation of overall energetic homeostasis of the organism. The dimeric character of the receptor for insulin evokes ideas about its activation or inhibition with peptide dimers that could either trigger or block the structural transition of the insulin receptor, leading to its activation. Herewith, we present the chemical engineering and biological characterization of several series of insulin dimers or dimers of specific peptides that should be able to bind receptors for insulin or insulin growth factor 1. The hormones or peptides in the dimers were interconnected with different linkers, consisting of triazole moieties and 3, 6, 8, 11, or 23 polyethylene glycol units. The prepared dimers were weaker in binding to insulin receptors than human insulin. However, some of the insulin dimers showed preferential binding specificity toward the isoform A of the insulin receptor, and the insulin dimers also stimulated the insulin receptor more strongly than would be consistent with their binding affinities. Our results suggest that designing insulin dimers may be a promising strategy for modulating the ability of the hormone to activate the receptor or to alter its specificity toward insulin receptor isoforms.     

Regulation of hormone metabolism in Arabidopsis seeds: phytochrome regulation of abscisic acid metabolism and abscisic acid regulation of gibberellin metabolism

In a wide range of plant species, seed germination is regulated antagonistically by two plant hormones, abscisic acid (ABA) and gibberellin (GA). In the present study, we have revealed that ABA metabolism (both biosynthesis and inactivation) was phytochrome-regulated in an opposite fashion to GA metabolism during photoreversible seed germination in Arabidopsis. Endogenous ABA levels were decreased by irradiation with a red (R) light pulse in dark-imbibed seeds pre-treated with a far-red (FR) light pulse, and the reduction in ABA levels in response to R light was inhibited in a phytochrome B (PHYB)-deficient mutant. Expression of an ABA biosynthesis gene, AtNCED6, and the inactivation gene, CYP707A2, was regulated in a photoreversible manner, suggesting a key role for the genes in PHYB-mediated regulation of ABA metabolism. Abscisic acid-deficient mutants such as nced6-1, aba2-2 and aao3-4 exhibited an enhanced ability to germinate relative to wild type when imbibed in the dark after irradiation with an FR light pulse. In addition, the ability to synthesize GA was improved in the aba2-2 mutant compared with wild type during dark-imbibition after an FR light pulse. Activation of GA biosynthesis in the aba2-2 mutant was also observed during seed development. These data indicate that ABA is involved in the suppression of GA biosynthesis in both imbibed and developing seeds. Spatial expression patterns of the AtABA2 and AAO3 genes, responsible for last two steps of ABA biosynthesis, were distinct from that of the GA biosynthesis gene, AtGA3ox2, in both imbibed and developing seeds, suggesting that biosynthesis of ABA and GA in seeds occurs in different cell types.     

Demonstration and characterization of hippocampal cholinergic neurostimulating peptide (HCNP) processing enzyme activity in rat hippocampus

Hippocampal cholinergic neurostimulating peptide (HCNP) stimulates cholinergic activity of cultured medial septal nuclei explants. It consists of eleven amino acids that are located at the N-terminal region of its precursor protein. This report concerns the demonstration and characterization of an HCNP processing enzyme that cleaves the bioactive undecapeptide from the precursor. The enzyme was purified from the hippocampus of young Wistar rats. A synthetic deacetylated peptide (peptide_1–26) consisting of the N-terminal 26 amino acids of the HCNP precursor protein served as substrate. The product of the enzyme reaction was identified and quantitated by HPLC using deacetylated HCNP as standard. The amount of undecapeptide generated was directly proportional to the time of incubation of the enzyme reaction mixture. From molecular sieving chromatography it was estimated that the molecular mass of the enzyme is close to 68 kDa. The HCNP processing enzyme has a pH optimum of 6.0 and a K_m of 0.50 mM for peptide_1–26. Preincubation at 56°C causes rapid inactivation of the HCNP processing activity. Enzyme activity is enhanced by EDTA and 1,4-dithiothreitol, and inhibited by antipain, chymostatin and E-64. These findings suggest that the enzyme probably has a thiol group in its active site. This novel enzyme of the hippocampus may represent a valuable tool for further studies on the general protein metabolism in the central nervous system, as well as for elucidating the neurochemical aspects of neurodegenerative disorders.     

抗坏血酸、表皮生长因子和促卵泡素对绵羊卵巢皮质体外培养的影响

本研究旨在评估抗坏血酸(VC)、表皮生长因子(EGF)、促卵泡素(FSH)对绵羊原始卵泡体外培养的影响以及它们之间的相互关系。实验按照2×2×2因子试验设计分为8组,分别为:MEM(对照组),MEM+VC(50μg/mL),MEM+EGF(100ng/mL),MEM+FSH(50ng/mL),MEM+VC+EGF,MEM+VC+FSH,MEM+EGF+FSH,MEM+VC+EGF+FSH。在培养0(未培养对照组)、2、6、12d后,对培养的卵巢皮质薄片进行组织学和增殖细胞核抗原(PCNA)检测以及透射电镜(TEM)观察。结果表明,与未培养组(发育卵泡比例15.4%±1.9%,正常卵泡比例88.2%±4.6%)比较,所有培养组中发育卵泡比例显著增加(P0.05),正常卵泡比例下降(P0.05)。培养12d后,与对照组(卵泡直径(34.5±3.3)μm,卵泡存活比例(38.9%±3.9%))比较,MEM+VC+FSH和MEM+EGF+FSH组中卵泡直径(分别为(39.7±3.4)μm和(42.5±5.1)μm)和卵泡存活比例(分别为58.5%±4.3%和59.3%±3.7%)都显著提高(P0.05);各处理组中,培养12d后,MEM+VC+EGF组中发育卵泡比例(49.3%±3.2%)和卵泡直径((32.3±2.3)μm)最低,颗粒细胞PCNA阳性卵泡比例(26.4%±1.2%)也最少,而MEM+VC+EGF+FSH组中卵泡存活率(59.7%±6.1%)和卵泡直径((42.5±5.1)μm)都显著增加(P0.05),颗粒细胞PCNA(43.5%±4.1%,P0.05)表达增加。电镜结果表明,VC+EGF+FSH组能够维持与正常卵泡类似的超微结构,而在MEM和MEM+VC+EGF组却显示不同程度的退化特征。本研究结果提示在培养中联合添加VC与EGF抑制卵泡的发育和生长,而联合添加VC、EGF和FSH可能是促进绵羊原始卵泡体外激活和生长,维持卵泡存活以及结构完整的最有效的处理手段之一。     

植物激素对微管和纤维素微纤丝排向的调节

回顾了微管和纤维素微纤丝在细胞骨架构成和延展中的作用;综述了植物激素在微管和纤维素微纤丝排向中的调节功能,并对细胞扩大和伸长的机制进行了探讨。     

Effects of growth hormone-releasing factor, somatostatin and dopamine on growth hormone and prolactin secretion from cultured ovine pituitary cells

A synthetic form of human pancreatic growth hormone releasing factor (GRF-44-NH2) was shown to be a potent stimulator of growth hormone (GH) secretion and cellular cyclic AMP levels in cultured sheep pituitary cells. A small dose-dependent stimulation of prolactin secretion was also observed. Somatostatin (0.5 microM) completely blocked the maximal GRF (1 nM)-stimulated secretion without a significant effect on cyclic AMP levels. Dopamine (0.1 microM) inhibited the GRF-elevated GH secretion by 50% and lowered cyclic AMP levels by 30%. Dopamine (0.1 microM) inhibition of basal prolactin secretion was not affected by GRF (1 nM). The data support the hypothesis that cyclic AMP is involved in the action of GRF but suggest that somatostatin can inhibit GRF-induced secretion of GH independently of cyclic AMP.     

Production of a biologically active novel goldfish growth hormone in Escherichia coli

Goldfish pituitary contains two types of growth hormones. One with five cysteine residues (type-I) similar to other Cyprinid GHs, and the other with four Cys residues (type-II) similar to those of other fish and tertapod species. Recombinant goldfish type II GH (gfGH-II) was produced in Escherichia coli using the pRSETB expression vector. The gfGH-II was produced fused to a leader sequence, which sequestered into inclusion bodies after expression. The inclusion bodies were solubilized using sodium hydroxide and the fusion protein purified by chelating affinity chromatography. Subsequently, gfGH-II was cleaved and analyzed by Western blotting, using a specific antiserum. For comparison we also produced recombinant common carp GH (cGH) which has 95% similarity to gfGH-II, and tested their growth promoting activity in goldfish. Both forms of GH significantly increased the growth rate of goldfish (P<0.05), although cGH was found to have a somewhat higher potency than gfGH-II.