Post-translational Processing Mechanisms Research Articles

Complex precursor structures of cytolytic cupiennins identified in spider venom gland transcriptomes

Analysis of spider venom gland transcriptomes focuses on the identification of possible neurotoxins, proteins and enzymes. Here, the first comprehensive transcriptome analysis of cupiennins, small linear cationic peptides, also known as cytolytic or antimicrobial peptides, is reported from the venom gland transcriptome of Cupiennius salei by 454- and Illumina 3000 sequencing. Four transcript families with complex precursor structures are responsible for the expression of 179 linear peptides. Within the transcript families, after an anionic propeptide, cationic linear peptides are separated by anionic linkers, which are transcript family specific. The C-terminus of the transcript families is characterized by a linear peptide or truncated linkers with unknown function. A new identified posttranslational processing mechanism explains the presence of the two-chain CsTx-16 family in the venom. The high diversity of linear peptides in the venom of a spider and this unique synthesis process is at least genus specific as verified with Cupiennius getazi.

Bacterial γ-glutamyltranspeptidases, physiological function, structure, catalytic mechanism and application.

γ-Glutamyltranspeptidase (GGT) has been widely used as a marker enzyme of hepatic and biliary diseases and relations between various diseases and its activity have been studied extensively. Nevertheless, several of its fundamental enzymatic characteristics had not been elucidated. We obtained homogeneous preparation of GGTs from bacteria, characterized them, and elucidated its physiological function that is common to mammalian cells, using GGT-deficient E. coli. Prior to GGT of all living organisms, we also identified catalytic nucleophile of E. coli GGT and revealed the post-translational processing mechanism for its maturation, and also its crystal structure was determined. The reaction intermediate was trapped and the structure-based reaction mechanism was presented. As for its application, using its transferase activity, we developed the enzymatic synthesis of various γ-glutamyl compounds that are promising in food, nutraceutical and medicinal industries. We found GGT of Bacillus subtilis is salt-tolerant and can be used as a glutaminase, which is important in food industry, to enhance umami of food, such as soy sauce and miso. We succeeded in converting bacterial GGT to glutaryl-7-aminocephalosporanic acid acylase, which is an important enzyme in cephem antibiotics production, by site-directed and random mutagenesis.

Post-translational cleavage pattern of Lupinus angustifolius γ-conglutin.

Protein post-translational modifications are a key element for the functional diversity of the proteome. The modifications generally refer to the addition of functional groups to certain proteins; however, proteolytic cleavage is also one of the relevant events during protein maturation. γ-Conglutin is a unique protein fraction present in lupin seeds that is marked by numerous unusual properties. This protein fraction undergoes very complex post-translational maturation. Unfortunately, the precise mechanism of γ-conglutin post-translational processing is not yet fully understood. Two independent methods were used to study γ-conglutin post-translational cleavage processing. Edman N-terminal sequencing indicates that the signal peptide is processed at Tyr34, while α- and β-subunit cleavage takes place between Ser295 and Ser296. High-resolution mass spectrometry revealed a great diversity of N-terminal sequences of γ-conglutin α-subunit. However, most abundant peptides also began from Tyr34. Mass spectrometric analyses additionally confirmed the subunit cleavage position between two serine residues. The results indicate that the proteolytic processing of γ-conglutin signal peptide is not precise. On the other hand, the post-translational cleavage between α- and β-subunits of γ-conglutin is very conserved. Interestingly, the results also indicate that proteolytic processing leading to the formation of two subunits of γ-conglutin is incomplete, leaving a certain amount of the protein in an uncut form. © 2018 Society of Chemical Industry.

The metabolic sensor Sirt1 and the hypothalamus: Interplay between peptide hormones and pro-hormone convertases

The last decade had witnessed a tremendous progress in our understanding of the causes of metabolic diseases including obesity. Among the contributing factors regulating energy balance are nutrient sensors such as sirtuins. Sirtuin1 (Sirt1), a NAD + - dependent deacetylase is affected by diet, environmental stress, and also plays a critical role in metabolic health by deacetylating proteins in many tissues, including liver, muscle, adipose tissue, heart, endothelium, and in the complexity of the hypothalamus. Because of its dependence on NAD+, Sirt1 also functions as a nutrient/redox sensor, and new novel data show a function of this enzyme in the maturation of hypothalamic peptide hormones controlling energy balance either through regulation of specific nuclear transcription factors or by regulating specific pro-hormone convertases (PCs) involved in the post-translational processing of pro-hormones. The post-translational processing mechanism of pro-hormones is critical in the pathogenesis of obesity as recently shown that metabolic and physiological triggers affect the biosynthesis and processing of many peptides hormones. Specific regulation of pro-hormone processing is likely another key step where final amounts of bioactive peptides can be tightly regulated. Different factors stimulate or inhibit pro-hormones biosynthesis in concert with an increase in the PCs involved in the maturation of bioactive hormones. Adding more complexity to the system, the new studies describe here suggest that Sirt1 could also regulate the fate of peptide hormone biosynthesis. The present review summarizes the recent progress in hypothalamic SIRT1 research with a particular emphasis on the tissue-specific control of neuropeptide hormone maturation. The series of studies done in mouse and rat models strongly advocate for the first time that a deacetylating enzyme could be a regulator in the maturation of peptide hormones and their processing enzymes. These discoveries are the culmination of the first in-depth understanding of the metabolic role of Sirt1 in the brain. It suggests that Sirt1 behaves differently in the brain than in organs such as the liver and pancreas, where the enzyme has been more commonly studied.

Post-translational modifications in mitochondria: protein signaling in the powerhouse.

There is an intimate interplay between cellular metabolism and the pathophysiology of disease. Mitochondria are essential to maintaining and regulating metabolic function of cells and organs. Mitochondrial dysfunction is implicated in diverse diseases, such as cardiovascular disease, diabetes and metabolic syndrome, neurodegeneration, cancer, and aging. Multiple reversible post-translational protein modifications are located in the mitochondria that are responsive to nutrient availability and redox conditions, and which can act in protein-protein interactions to modify diverse mitochondrial functions. Included in this are physiologic redox signaling via reactive oxygen and nitrogen species, phosphorylation, O-GlcNAcylation, acetylation, and succinylation, among others. With the advent of mass proteomic screening techniques, there has been a vast increase in the array of known mitochondrial post-translational modifications and their protein targets. The functional significance of these processes in disease etiology, and the pathologic response to their disruption, are still under investigation. However, many of these reversible modifications act as regulatory mechanisms in mitochondria and show promise for mitochondrial-targeted therapeutic strategies. This review addresses the current knowledge of post-translational processing and signaling mechanisms in mitochondria, and their implications in health and disease.

Isolation and characterization of Ts19 Fragment II, a new long-chain potassium channel toxin from Tityus serrulatus venom

Ts19 Fragment II (Ts19 Frag-II) was first isolated from the venom of the scorpion Tityus serrulatus (Ts). It is a protein presenting 49 amino acid residues, three disulfide bridges, Mr 5534Da and was classified as a new member of class (subfamily) 2 of the β-KTxs, the second one described for Ts scorpion. The β-KTx family is composed by two-domain peptides: N-terminal helical domain (NHD), with cytolytic activity, and a C-terminal CSαβ domain (CCD), with Kv blocking activity. The extensive electrophysiological screening (16 Kv channels and 5 Nav channels) showed that Ts19 Frag-II presents a specific and significant blocking effect on Kv1.2 (IC50 value of 544±32nM). However, no cytolytic activity was observed with this toxin. We conclude that the absence of 9 amino acid residues from the N-terminal sequence (compared to Ts19 Frag-I) is responsible for the absence of cytolytic activity. In order to prove this hypothesis, we synthesized the peptide with these 9 amino acid residues, called Ts19 Frag-III. As expected, Ts19 Frag-III showed to be cytolytic and did not block the Kv1.2 channel. The post-translational modifications of Ts19 and its fragments (I–III) are also discussed here. A mechanism of post-translational processing (post-splitting) is suggested to explain Ts19 fragments production. In addition to the discovery of this new toxin, this report provides further evidence for the existence of several compounds in the scorpion venom contributing to the diversity of the venom arsenal.

Free Glycine Accelerates the Autoproteolytic Activation of Human Asparaginase

Human asparaginase 3 (hASNase3), which belongs to the N-terminal nucleophile hydrolase superfamily, is synthesized as a single polypeptide that is devoid of asparaginase activity. Intramolecular autoproteolytic processing releases the amino group of Thr168, a moiety required for catalyzing asparagine hydrolysis. Recombinant hASNase3 purifies as the uncleaved, asparaginase-inactive form and undergoes self-cleavage to the active form at a very slow rate. Here, we show that the free amino acid glycine selectively acts to accelerate hASNase3 cleavage both in vitro and in human cells. Other small amino acids such as alanine, serine, or the substrate asparagine are not capable of promoting autoproteolysis. Crystal structures of hASNase3 in complex with glycine in the uncleaved and cleaved enzyme states reveal the mechanism of glycine-accelerated posttranslational processing and explain why no other amino acid can substitute for glycine.

Evolution of POMC: origin, phylogeny, posttranslational processing, and the melanocortins

The proopiomelanocortin (POMC) gene was most likely derived from an ancestral opioid-coding gene following the 1R chordate genome duplication event. During the radiation of the jawless fish, the POMC organization plan emerged multiple melanocortin sequences (α-MSH/ACTH and β-MSH) and a C-terminally extended opioid sequence (β-endorphin). Following the 2R genome duplication event, the γ-MSH sequence was gained. Among the jawed vertebrates, three distinct trends in the evolution of the POMC gene are apparent: the gain of the δ-MSH sequence (cartilaginous fish), the loss of the γ-MSH sequence (ray-finned fish), and the retention of the post 2R POMC organization plan (lobe-finned fish/tetrapods). POMC is synthesized in the pituitary gland and in neurons of the hypothalamus, where an array of posttranslational processing mechanisms, such as endoproteolytic cleavage and N-acetylation, generate distinct sets of end-products in these tissues. A striking feature of the melanocortin end-products is the rigorous conservation of the primary sequence of α-MSH and the first 25 amino acids of ACTH.

Identical Skin Toxins by Convergent Molecular Adaptation in Frogs

The Tree of Life is rife with adaptive convergences at all scales and biological levels of complexity. However, natural selection is not likely to result in the independent evolution of identical gene products. Here we report such a striking example of evolutionary convergence in the toxic skin secretions of two distantly related frog lineages. Caeruleins are important decapeptides in pharmacological and clinical research [1] and are commonly believed to represent a single evolutionary class of peptides [2-4]. Instead, our phylogenetic analyses combining transcriptome and genome data reveal that independently evolved precursor genes encode identical caeruleins in Xenopus and Litoria frogs. The former arose by duplication from the cholecystokinin (cck) gene, whereas the latter was derived from the gastrin gene. These hormone genes that are involved in many physiological processes diverged early in vertebrate evolution, after a segmental duplication during the Cambrian period. Besides implicating convergent mutations of the peptide-encoding sequence, recurrent caerulein origins entail parallel shifts of expression from the gut-brain axis to skin secretory glands. These results highlight extreme structural convergence in anciently diverged genes as an evolutionary mechanism through which recurrent adaptation is attained across large phylogenetic distances.

Analysis of pmpD Expression and PmpD Post-Translational Processing during the Life Cycle of Chlamydia trachomatis Serovars A, D, and L2

BackgroundThe polymorphic membrane protein D (PmpD) in Chlamydia is structurally similar to autotransporter proteins described in other bacteria and may be involved in cellular and humoral protective immunity against Chlamydia. The mechanism of PmpD post-translational processing and the role of its protein products in the pathogenesis of chlamydial infection have not been very well elucidated to date.Methodology/Principal FindingsHere we examined the expression and post-translational processing of the protein product of the pmpD gene during the life cycle of C. trachomatis serovars A, D, and L2. Each of these three serovars targets different human organs and tissues and encodes a different pmpD gene nucleotide sequence. Our quantitative real-time reverse transcription polymerase chain reaction results demonstrate that the pmpD gene is up-regulated at 12–24 hours after infection regardless of the Chlamydia serovar. This up-regulation is coincidental with the period of exponential growth and replication of reticulate bodies (RB) of Chlamydia and indicates a probable similarity in function of pmpD in serovars A, D, and L2 of Chlamydia. Using mass spectrometry analysis, we identified the protein products of post-translational processing of PmpD of C. trachomatis serovar L2 and propose a double pathway model for PmpD processing, with one cleavage site between the passenger and autotransporter domains and the other site in the middle of the passenger domain. Notably, when Chlamydia infected culture cells were subjected to low (28°C) temperature, PmpD post-translational processing and secretion was found to be uninhibited in the resulting persistent infection. In addition, confocal microscopy of cells infected with Chlamydia confirms our earlier hypothesis that PmpD is secreted outside Chlamydia and its secretion increases with growth of the chlamydial inclusion.Conclusion/SignificanceThe results of this current study involving multiple Chlamydia serovars support the general consensus that the pmpD gene is maximally expressed at mid infection and provide new information about PmpD as an autotransporter protein which is post-translationally processed and secreted outside Chlamydia during normal and low temperature induced persistent chlamydial infection.

Analyzing the evolution of β-endorphin post-translational processing events: Studies on reptiles

In many cartilaginous fishes, most ray-finned fishes, lungfishes, and amphibians, the post-translational processing of POMC includes the monobasic cleavage of β-endorphin to yield an opioid that is eight to ten amino acids in length. The amino acid motif within the β-endorphin sequence required for a monobasic cleavage event is -E-R-(S/G)-Q-. Mammals and birds lack this motif and as a result β-endorphin(1–8) is a not an end-product in either group. Since both mammals and birds were derived from ancestors with reptilian origins, an analysis of β-endorphin sequences from extant groups of reptiles should provide insights into the manner in which β-endorphin post-translational processing mechanisms have evolved in amniotes. To this end a POMC cDNA was cloned from the pituitary of the turtle, Chrysemys scripta. The β-endorphin sequence in this species was compared to other reptile β-endorphin sequences (i.e., Chinese soft shell turtle and gecko) and to known bird and mammal sequences. This analysis indicated that either the loss of the arginine residue at the cleavage site (the two turtle species, chick, and human) or a substitution at the glutamine position in the consensus sequence (gecko and ostrich) would account for the loss of the monobasic cleavage reaction in that species. Since amphibians are capable of performing the β-endorphin monobasic reaction, it would appear that the amino acid substitutions that eliminated this post-translational process event in reptilian-related tetrapods must have occurred in the ancestral amniotes.

Differential effects of fasting and leptin on proopiomelanocortin peptides in the arcuate nucleus and in the nucleus of the solitary tract

The alpha-melanocyte-stimulating hormone (alpha-MSH), derived from proopiomelanocortin (POMC), is generated by a posttranslational processing mechanism involving the prohormone convertases (PCs) PC1/3 and PC2. In the brain, alpha-MSH is produced in the arcuate nucleus (ARC) of the hypothalamus and in the nucleus of the solitary tract (NTS) of the medulla. This peptide is key in controlling energy balance, as judged by changes observed at transcriptional level. However, little information is available regarding the biosynthesis of the precursor POMC and the production of its processed peptides during feeding, fasting, and fasting plus leptin in the ARC compared with the NTS in conjunction with the PC activity. In this study we found that, in the ARC, pomc mRNA, POMC-derived peptides, and PC1/3 all decreased during fasting, and administration of leptin reversed these effects. In contrast, in the NTS, where there is a large amount of a 28.1-kDa peptide similar in size to POMC, the 28.1-kDa peptide and other POMC-derived peptides, including alpha-MSH, were further accumulated in fasting conditions, whereas pomc mRNA decreased. These changes were not reversed by leptin. We also observed that, during fasting, PC2 levels decreased in the NTS. These data suggest that, in the NTS, fasting induced changes in POMC biosynthesis, and processing is independent of leptin. These observations indicate that changes in energy status affect POMC in the brain in a tissue-specific manner. This represents a novel aspect in the regulation of energy balance and may have implications in the pathophysiology of obesity.

Human Polyserase-2, a Novel Enzyme with Three Tandem Serine Protease Domains in a Single Polypeptide Chain

We have cloned a human cDNA encoding a new serine protease that has been called polyserase-2 (polyserine protease-2) because it is the second identified human enzyme with several tandem serine protease domains in its amino acid sequence. The first serine protease domain contains all characteristic features of these enzymes, whereas the second and third domains lack one residue of the catalytic triad of serine proteases and are predicted to be catalytically inactive. This complex domain organization is also present in the sequences of mouse and rat polyserase-2 and resembles that of polyserase-1, which also contains three serine protease domains in its amino acid sequence. However, polyserase-2 lacks additional domains present in polyserase-1, including a type II transmembrane motif and a low-density lipoprotein receptor A module. Enzymatic analysis demonstrated that both full-length polyserase-2 and its first serine protease domain hydrolyzed synthetic peptides used for assaying serine proteases. Nevertheless, the activity of the isolated domain was greater than that of the entire protein, suggesting that the two catalytically inactive serine protease domains of polyserase-2 may modulate the activity of the first domain. Northern blot analysis showed that polyserase-2 is expressed in fetal kidney; adult skeletal muscle, liver, placenta, prostate, and heart; and tumor cell lines derived from lung and colon adenocarcinomas. Finally, analysis of post-translational processing mechanisms of polyserase-2 revealed that, contrary to those affecting to the membrane-bound polyserase-1, this novel polyprotein is a secreted enzyme whose three protease domains remain as an integral part of a single polypeptide chain.

Stepwise posttranslational processing of progrowth hormone-releasing hormone (proGHRH) polypeptide by furin and PC1.

Through a posttranslational processing mechanism, pro-growth hormone releasing hormone (proGHRH) gives rise to an amidated GHRH molecule, which in turn stimulates the synthesis and release of growth hormone. We have previously proposed a model for the biochemical processing of proGHRH [Nillni et al. (1999), Endocrinology 140, 5817-5827]. We demonstrated that the proGHRH peptide (10.5 kDa, 104 aa) is first processed to an 8.8 kDa intermediate form that is later cleaved to yield two products: the 5.2 kDa GHRH and the 3.6 kDa GHRH-RP. However, the proteolytic enzymes involved in this process are unknown. Therefore, in this study we determined which proconverting enzymes are involved in this process. We transfected different constructs in cell lines carrying different PC enzymes followed by analysis of the peptide products after metabolic labeling or Western blots. We found that in the absence of furin (LoVo cells) or CHO cells treated with BFA, only one moiety was observed, and that corresponds to the same electrophorectic mobility to the GHRH precursor. This finding strongly supports an initial role for furin in the processing of proGHRH. The results from transfections with preproGHRH alone or double or triple transfections with PC1 and PC2 in AtT-20, GH3, and GH4C1 cells indicated that PC1 is the primary enzyme involved in the generation of GHRH peptide from the 8.8 kDa intermediate form. We found that AtT-20 cells (high PC1, very low PC2) were able to generate GHRH. However, GH3 cells (high PC2, but not PC1) were able to process the 8.8 kDa peptide to GHRH only after the cotransfection with the PC1 enzyme. Transfections with preproGHRH-GFP and preproGHRH-V5 provided similar results in all the cell lines analyzed. These data support the hypothesis that proGHRH is initially cleave by furin at preproGHRH29-30, followed by a second cleavage at preproGHRH74 primarily by PC1 to generate GHRH and GHRH-RP peptides, respectively.

Site-specific proteolysis of the MALP-404 lipoprotein determines the release of a soluble selective lipoprotein-associated motif-containing fragment and alteration of the surface phenotype of Mycoplasma fermentans.

The mature MALP-404 surface lipoprotein of Mycoplasma fermentans comprises a membrane-anchored N-terminal lipid-modified region responsible for macrophage activation (P. F. Mühlradt, M. Kiess, H. Meyer, R. Süssmuth, and G. Jung, J. Exp. Med. 185:1951-1958, 1997) and an external hydrophilic region that contains the selective lipoprotein-associated (SLA) motif defining a family of lipoproteins from diverse but selective prokaryotes, including mycoplasmas (M. J. Calcutt, M. F. Kim, A. B. Karpas, P. F. Mühlradt, and K. S. Wise, Infect. Immun. 67:760-771, 1999). This family generally corresponds to a computationally defined group of orthologs containing the basic membrane protein (BMP) domain. Two discrete lipid-modified forms of the abundant MALP product which vary dramatically in ratio among isolates of M. fermentans occur on the mycoplasma surface: (i) MALP-404, the full-length mature product, and (ii) MALP-2, the Toll-like receptor 2-mediated macrophage-activating lipopeptide containing the N-terminal 14 residues of the mature lipoprotein. The role of posttranslational processing in the biogenesis of MALP-2 from the prototype MALP-404 SLA-containing lipoprotein was investigated. Detergent phase fractionation of cell-bound products and N-terminal sequencing of a newly discovered released fragment (RF) demonstrated that MALP-404 was subject to site-specific proteolysis between residues 14 and 15 of the mature lipoprotein, resulting in the cell-bound MALP-2 and soluble RF products. This previously unknown mechanism of posttranslational processing among mycoplasmas suggests that specific cleavage of some surface proteins may confer efficient "secretion" of extracellular products by these organisms, with concurrent changes in the surface phenotype. This newly identified form of variation may have significant implications for host adaptation by mycoplasmas, as well as other pathogens expressing lipoproteins of the SLA (BMP) family.

Vasopressin V2 receptor mRNA expression and cAMP accumulation in aging rat kidney.

The ability of the kidney to regulate water balance is impaired with age, although the secretion of vasopressin is maintained in senescent animals. This suggests that the cellular response to antidiuretic hormone is reduced in aging kidney. To test this hypothesis, the relationship between the expression of the vasopressin. V2 receptor mRNA and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was investigated in the medullary thick ascending limb of Henle's loop (MTAL) of adult and aging rats. Tubular suspensions of MTAL were prepared from 10- and 30-mo-old female WAG/Rij rats. The accumulation of cAMP for maximal concentration of vasopressin was 34% larger in adult than in old animals (9.5 +/- 0.5 pmol/4 min, n = 16, and 7.1 +/- 0.6 pmol/4 min, n = 12, respectively). The concentration of vasopressin corresponding to half-maximal stimulation was similar in the two groups (0.66 +/- 0.20 and 0.52 +/- 0.09 nmol, n = 5, in adult and old animals), indicating comparable sensitivity of the renal cells with age. The age-related impaired response to vasopressin of the V2 receptor was specific for females and was not observed in males. Direct stimulation of adenylyl cyclase by forskolin induced a comparable accumulation of cAMP in adult and senescent rats. The V2 receptor mRNA level in the MTAL was constant between 10 and 30 mo whether the animals were normally hydrated or dehydrated for 2 days. These data indicate that, in MTAL, the age-related impaired cAMP accumulation by vasopressin would be linked to a change either in the translation of V2 mRNA or in posttranslational processing mechanisms or in the coupling between the V2 receptor and adenylyl cyclase.

Molecular Cloning and Sequencing of Arylphorin-binding Protein in Protein Granules of the Sarcophaga Fat Body: IMPLICATION OF A POST-TRANSLATIONAL PROCESSING MECHANISM

Previously, we identified an arylphorin-binding protein of Sarcophaga peregrina (flesh fly) with a molecular mass of 120 kDa and suggested its participation in the selective uptake of arylphorin from the hemolymph into the pupal fat body at metamorphosis (Ueno, K., and Natori, S. (1984) J. Biol. Chem. 259, 12107-12111). This paper reports the isolation and sequencing of cDNA for the 120-kDa protein. This protein consists of 1146 amino acid residues. Immunoblotting and RNA blotting experiments revealed that this protein is present as two fragments of 76 kDa (695 residues) and 53 kDa (451 residues) in the larval fat body. When larvae pupate, the 120-kDa protein gene is further activated and the complete 120-kDa protein is synthesized without fragmentation. This suggests a novel mechanism for the production of the 120-kDa protein regulated by a proteinase depending upon the stage of development of Sarcophaga. All of these proteins were found to be localized in protein granules in the adipocytes.

Post-poly(Glu) cleavage and degradation modified by O-sulfated tyrosine: a novel post-translational processing mechanism.

Expression of bioactive peptides requires several modifications of the primary translation product. Gastrin, a vertebrate gut hormone, occurs in multiple forms, including a bioactive fragment of the predominant gastrin-17. Gastrin-17 is, however, without known cleavage sites. In order to identify the new site, we therefore isolated, from antral mucosa, fragments of gastrin-34 and -17 monitored by monospecific immunoassays. After three steps of reverse-phase chromatography, the short gastrins were identified as hepta-, hexa- and pentapeptide amides. By far the most abundant of these was tyrosine O-sulfated gastrin-6. The near complete sulfation contrasts with the larger gastrins, of which only half are sulfated. The longest N-terminal fragment of gastrin-34 was a hexadecapeptide without complementarity to the short gastrins. Instead, the predominant N-terminal fragment of gastrin-17 was the decapeptide complementary to gastrin-7. Therefore the novel processing site is the Glu10-Ala11 bond that follows a poly(Glu6-10) sequence. Moreover, gastrin-7 is apparently trimmed, with subsequent accumulation of sulfated gastrin-6. Consequently, O-sulfated tyrosine ensures production of a new hormone which stimulates gastric acid secretion as potently as gastrin-17.

Parathyroid Hormone-Related Protein as a Prohormone: Posttranslational Processing and Receptor Interactions*

Since the elucidation of the structures of the three human PRHrP isoforms in 1987, information has rapidly accured which indicates that the role of PTHrP in normal physiology will prove to be crucial as well as exceedingly complex. The importance of the role of PTHrP in normal physiology is underscored by its broad tissue expression, by its intense evolutionary conservation, by its extremely early expression after fertilization of the ovum, and by the lethal consequences of PTHrP gene disruption. The complexity of the role of PTHrP in normal physiology increases almost monthly. This complexity is reflected in the broad tissue distribution of the peptide, its complex transcriptional regulation and mRNA instability motifs, and its multiple transcripts and isoforms. It is now clear that additional complexity exists at the level of posttranslational processing. Expression of the PTHrP gene leads to the tissue-specific processing and secretion of an increasingly complex family of derivative peptides, each with its own repertoire of cognate receptors, signal transduction pathways, and physiological consequences. Further elucidation of the posttranslational processing pathways and mechanisms can be anticipated in the coming years, coupled with a corresponding elucidation of multiple PTHrP receptors, their specific signal transduction pathways, and their unique physiological roles. The role of PTHrP in causing HHM is now clearly established. Work in the coming decade will focus on the normal physiological roles played by PTHrP.

The Effects of Vitamin K1 and Warfarin on Prothrombin Expression in Human Hepatoblastoma (HepG2) Cells

Cultures of human hepatoblastoma (HepG2) cells were treated with vitamin K1 or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 micrograms vitamin K1 treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 micrograms/ml vitamin K1 treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with 35S-methionine and immunoprecipitation of 35S-labelled prothrombin demonstrated that, with vitamin K1 treatment (25 micrograms/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 micrograms/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K1 nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240-350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K1 and warfarin, in addition to effects on gamma-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.