Reversal of physiological deficits caused by diminished levels of peptidylglycine alpha-amidating monooxygenase by dietary copper.

Amidated peptides are critically involved in many physiological functions. Genetic deletion of peptidylglycine alpha-amidating monooxygenase (PAM), the only enzyme that can synthesize these peptides, is embryonically lethal. The goal of the present study was the identification of physiological functions impaired by haploinsufficiency of PAM. Regulation of the hypothalamic-pituitary-thyroid axis and body temperature, functions requiring contributions from multiple amidated peptides, were selected for evaluation. Based on serum T(4) and pituitary TSH-beta mRNA levels, mice heterozygous for PAM (PAM(+/-)) were euthyroid at baseline. Feedback within the hypothalamic-pituitary-thyroid axis was impaired in PAM(+/-) mice made hypothyroid using a low iodine/propylthiouracil diet. Despite their normal endocrine response to cold, PAM(+/-) mice were unable to maintain body temperature as well as wild-type littermates when kept in a 4 C environment. When provided with additional dietary copper, PAM(+/-) mice maintained body temperature as well as wild-type mice. Pharmacological activation of vasoconstriction or shivering also allowed PAM(+/-) mice to maintain body temperature. Cold-induced vasoconstriction was deficient in PAM(+/-) mice. This deficit was eliminated in PAM(+/-) mice receiving a diet with supplemental copper. These results suggest that dietary deficiency of copper, coupled with genetic deficits in PAM, could result in physiological deficits in humans.

Differential regulation of prohormone convertase 1/3, prohormone convertase 2 and phosphorylated cyclic-AMP-response element binding protein by short-term and long-term morphine treatment: implications for understanding the "switch" to opiate addiction.

Drug addiction is a state of altered brain reward and self-regulation mediated by both neurotransmitter and hormonal systems. Although an organism's internal system attempts to maintain homeostasis when challenged by exogenous opiates and other drugs of abuse, it eventually fails, resulting in the transition from drug use to drug abuse. We propose that the attempted maintenance of hormonal homeostasis is achieved, in part, through alterations in levels of processing enzymes that control the ratio of active hormone to pro-hormone. Two pro-hormone convertases, PC1/3 and PC2 are believed to be responsible for the activation of many neurohormones and expression of these enzymes is dependent on the presence of a cyclic-AMP response element (CRE) in their promoters. Therefore, we studied the effects of short-term (24-h) and long-term (7-day) morphine treatment on the expression of hypothalamic PC1/3 and PC2 and levels of phosphorylated cyclic-AMP-response element binding protein (P-CREB). While short-term morphine exposure down-regulated, long-term morphine exposure up-regulated P-CREB, PC1/3 and PC2 protein levels in the rat hypothalamus as determined by Western blot analysis. Quantitative immunofluorescence studies confirmed these regulatory actions of morphine in the paraventricular and dorsomedial nucleus of the hypothalamus. Specific radioimmunoassays demonstrated that the increase in PC1/3 and PC2 levels following long-term morphine led to increased TRH biosynthesis as evidence by increased TRH/5.4 kDa C-terminal proTRH-derived peptide ratios in the median eminence. Promoter activity experiments in rat somatomammotrope GH3 cells containing the mu-opioid receptor demonstrated that the CRE(s) in the promoter of PC1/3 and PC2 is required for morphine-induced regulation of PC1/3 and PC2. Our data suggest that the regulation of the prohormone processing system by morphine may lead to alterations in the levels of multiple bioactive hormones and may be a compensatory mechanism whereby the organism tries to restore its homeostatic hormonal milieu. The down-regulation of PC1/3, PC2 and P-CREB by short-term morphine and up-regulation by long-term morphine treatment may be a signal mediating the switch from drug use to drug abuse.

PreproTRH(178-199) and two novel peptides (pFQ7 and pSE14) derived from its processing, which are produced in the paraventricular nucleus of the rat hypothalamus, are regulated during suckling.

Suckling increases preproTRH messenger RNA in hypothalamic paraventricular neurons (PVN) and also markedly increases TRH release during the first period of lactation. Whether lactation alters preproTRH processing resulting in the generation of novel proTRH-derived peptides that may be involved in the regulation of PRL secretion lactation is not known. Therefore, in the present study we determine whether some other peptides derived from proTRH potentially contribute to lactation-induced PRL secretion. We have recently demonstrated that two members of the family of prohormone convertases PC1 and PC2 play a significant role in proTRH processing. PC1 is the major contributor in proTRH processing, whereas PC2 may have a specific role in cleaving TRH from its extended forms. In this study, we used a recombinant vaccinia virus system to coexpress rat preproTRH complementary DNA with PC1, PC2, and the neuropeptide 7B2 in GH4C1 cells (somatomammothophs, rat). We found that two novel peptides, preproTRH(178-184) (pFQ(7)), and preproTRH(186-199) (pSE(14)), were formed after the cleavage of their precursor preproTRH(178-199) (pFE(22)) by only PC2. Their formation was confirmed by microsequence analysis. Anatomical analyses revealed that these peptides are also found in the rat PVN. In addition, we found that pFE(22), pSE(14) and pFQ(7) produced a dose-dependent release of PRL from primary cultures of pituitary cells compared with one of the well studied secretagogues of PRL, TRH. To establish whether these peptides might play a role in vivo in the regulation of PRL release, we took rat litters on postnatal day 4, separated the pups from their mothers for 6 h, and then reunited the pups and mothers for 45 min. At the end of this period, the mothers were killed, acidic extracts of microdissected PVN were prepared and subjected to SDS-PAGE, followed by slicing and analysis by pFE(22) RIA. Forty-five minutes of suckling induced a marked 6-fold increase in serum levels of PRL. In addition, PVN levels of pFE(22) and pSE(14) increased approximately 5-fold during the same period in the acutely suckling females. Lactating animals that were separated from their litters and never reunited with their pups had low levels of PRL, and pFE(22) and pSE(14). These data provide the first evidence for alterations in proTRH processing in the PVN during lactation and suggest that the products of this altered processing may play a physiological role in the regulation of PRL secretion.

Transcriptional regulation of the thyrotropin-releasing hormone gene by leptin and melanocortin signaling.

Starvation causes a rapid reduction in thyroid hormone levels in rodents. This adaptive response is caused by a reduction in thyrotropin-releasing hormone (TRH) expression that can be reversed by the administration of leptin. Here we examined hypothalamic signaling pathways engaged by leptin to upregulate TRH gene expression. As assessed by leptin-induced expression of suppressor of cytokine signaling-3 (SOCS-3) in fasted rats, TRH neurons in the paraventricular nucleus are activated directly by leptin. To a greater degree, they also contain melanocortin-4 receptors (MC4Rs), implying that leptin can act directly or indirectly by increasing the production of the MC4R ligand, alpha-melanocyte stimulating hormone (alpha-MSH), to regulate TRH expression. We further demonstrate that both pathways converge on the TRH promoter. The melanocortin system activates the TRH promoter through the phosphorylation and DNA binding of the cAMP response element binding protein (CREB), and leptin signaling directly regulates the TRH promoter through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). Indeed, a novel Stat-response element in the TRH promoter is necessary for leptin's effect. Thus, the TRH promoter is an ideal target for further characterizing the integration of transcriptional pathways through which leptin acts.

Leptin regulates prothyrotropin-releasing hormone biosynthesis. Evidence for direct and indirect pathways.

The hypothalamic-pituitary-thyroid axis is down-regulated during starvation, and falling levels of leptin are a critical signal for this adaptation, acting to suppress preprothyrotropin-releasing hormone (prepro-TRH) mRNA expression in the paraventricular nucleus of the hypothalamus. This study addresses the mechanism for this regulation, using primary cultures of fetal rat hypothalamic neurons as a model system. Leptin dose-dependently stimulated a 10-fold increase in pro-TRH biosynthesis, with a maximum response at 10 nm. TRH release was quantified using immunoprecipitation, followed by isoelectric focusing gel electrophoresis and specific TRH radioimmunoassay. Leptin stimulated TRH release by 7-fold. Immunocytochemistry revealed that a substantial population of cells expressed TRH or leptin receptors and that 8-13% of those expressing leptin receptors coexpressed TRH. Leptin produced a 5-fold induction of luciferase activity in CV-1 cells transfected with a TRH promoter and the long form of the leptin receptor cDNA. Although the above data are consistent with a direct ability of leptin to promote TRH biosynthesis through actions on TRH neurons, addition of alpha-melanocyte-stimulating hormone produced a 3.5-fold increase in TRH biosynthesis and release, whereas neuropeptide Y treatment suppressed pro-TRH biosynthesis approximately 3-fold. Furthermore, the melanocortin-4 receptor antagonist SHU9119 partially inhibited leptin-stimulated TRH release from the neuronal culture. Consequently, our data suggest that leptin regulates the TRH neurons through both direct and indirect pathways.

Posttranslational processing of progrowth hormone-releasing hormone.

The prepro-GH-releasing hormone (prepro-GHRH; 12.3 kDa) precursor, like other neuropeptide precursors, undergoes proteolytic cleavage to give rise to mature GHRH, which is the primary stimulatory regulator of pituitary GH secretion. In this study we present the first model of in vitro pro-GHRH processing. Using pulse-chase analysis, we demonstrate that at least five peptide forms in addition to GHRH are produced. The pro-GHRH (after removal of its signal peptide, 10.5 kDa) is first processed to an 8.8-kDa intermediate form that is cleaved to yield two products: the 5.2-kDa GHRH and GHRH-related peptide (GHRH-RP; 3.6 kDa). GHRH-RP is a recently described peptide derived from proteolytic processing of pro-GHRH that activates stem cell factor, a factor known to be essential for hemopoiesis, spermatogenesis, and melanocyte function. Further cleavage results in a 3.5-kDa GHRH and a 2.2-kDa product of GHRH-RP. Like GHRH, there is GHRH-RP immunostaining in hypothalamic neurons in the median eminence as detected by immunohistochemistry and immunoelectron microscopy. Based on deduced amino acid sequences of the pro-GHRH processing products, several peptides were synthesized and tested for their ability to stimulate the cAMP second messenger system. GHRH, GHRH-RP, and one of these peptides [prepro-GHRH-(75-92)-NH2] all significantly stimulated the PKA pathway. This work delineates a new model of pro-GHRH processing and demonstrates that novel peptides derived from this processing may have biological action.

Neuroregulation of ProTRH biosynthesis and processing.

This review presents an overview of the current knowledge on proTRH biosynthesis, its processing, its tissue distribution, and the role of known processing enzymes in proTRH maturation. The neuroendocrine regulation of TRH biosynthesis, the biological actions of its products, and the signal transduction and catabolic pathways used by those products are also reviewed. The widespread expression of proTRH, PC1, and PC2 rnRNAs in hypophysiotropic and extrahypophysiotropic areas of the brain, with their overlapping distribution in many areas, indicates the striking versatility provided by tissue-specific processing in generating quantitative and qualitative differences in nonTRH peptide products as well as TRH. Evidence is presented suggesting that differential processing for proTRH at the intracellular level is physiologically relevant. It is clear that control over the diverse range of proTRH-derived peptides within a specific cell is accomplished most from the regulation at the posttranslational level rather than the translational or transcriptional levels. Several examples supporting this hypothesis are presented in this review. A better understanding of proTRH-derived peptides role represents an exciting new frontier in proTRH research. These connecting sequences in between TRH molecules to form the precursor protein may function as structural or targeting elements that guide the folding and sorting of proTRH and its larger intermediates so that subsequent processing and secretion are properly regulated. The particular anatomical distribution of the proTRH end products, as well as regulation of their levels by neuroendocrine or pharmacological manipulations, supports a unique potential biologic role for these peptides.

Glucocorticoids modulate the biosynthesis and processing of prothyrotropin releasing-hormone (proTRH).

The thyrotropin- (TRH) releasing hormone precursor (26 kDa) undergoes proteolytic cleavage at either of two sites, generating N-terminal 15 kDa/9.5 kDa or C-terminal 16.5/10 kDa intermediate forms that are processed further to yield five copies of TRH-Gly and seven non-TRH peptides. Glucocorticoids (Gcc) have been shown to enhance TRH gene expression in three different cell systems in vitro, an effect that occurs, at least in part, through transcriptional activation. Although this implies that an increase of TRH prohormone biosynthesis would take place, this had not been demonstrated as yet. We report here that the synthetic glucocorticoid dexamethasone (Dex) substantially elevated the de novo biosynthesis of the intact 26-kDa TRH prohormone and its intermediate products of processing in cultured anterior pituitary cells, an observation that is consistent with an overall upregulation of both the biosynthesis and degradation of the TRH precursor. We reasoned that Gcc may act not only at the transcriptional, but also at the translational/posttranslational level. To address this question we chose a different cell system, AtT20 cells transfected with a cDNA encoding preproTRH. Since TRH gene expression in these cells is driven by the CMV-IE promoter and not by an endogenous "physiological" promoter, these cells provide an ideal model to study selectively the effects of Gcc on the translation and posttranslational processing of proTRH without interference from a direct transcriptional activation of the TRH gene. Dex caused a significant 75.7% increase in newly synthesized 26-kDa TRH prohormone, suggesting that the glucocorticoid raised the translation rate. We then demonstrated that Dex treatment accelerated TRH precursor processing. Of interest, processing of the N- vs the C-terminal intermediate was influenced differentially by the glucocorticoid. Although the N-terminal intermediate product of processing accumulated, the C-terminal intermediate was degraded more rapidly. Consistent with these observations was the finding that the intracellular accumulation of the N-terminally derived peptide preproTRH 25-50 was enhanced, but levels of the C-terminally derived peptide preproTRH208-255 were reduced. Accumulation of TRH itself, whose five copies are N- and C-terminally derived, was also enhanced. We conclude that Gcc induce changes in the biosynthesis and processing of proTRH by increasing the translation rate and by differentially influencing the processing of N- vs C-terminal intermediates of the precursor molecule. These effects of Gcc at the translational and posttranslational levels result in an increase in TRH production accompanied by differential effects on the accumulation of N- and C-terminal non-TRH peptides.

Processing of prothyrotropin-releasing hormone by the family of prohormone convertases.

The post-translational processing of prothyrotropin-releasing hormone (pro-TRH25-255) has been extensively studied in our laboratory, and the processing pathway to mature TRH has been elucidated. We have also demonstrated that recombinant PC1 and PC2 process partially purified pro-TRH to cryptic peptides in vitro and that pro-TRH and PC1 mRNAs are coexpressed in primary cultures of hypothalamic neurons. To further define the role of each convertase, and particularly PC1 and PC2, in pro-TRH processing, recombinant vaccinia viruses were used to coexpress the prohormone convertases PC1, PC2, PACE4, PC5-B, furin, or control dynorphin together with rat prepro-TRH in constitutively secreting LoVo cells or in the regulated endocrine GH4C1 cell line. Radioimmunoassays from LoVo-derived secreted products indicated that furin cleaves the precursor to generate both N- and C-terminal intermediates. PC1, PC2, and PACE4 only produced N-terminal intermediates, but less efficiently than furin. In GH4C1 cells, PC1, PC2, furin, PC5-B, and PACE4 produced both N-terminal and C-terminal forms. Significantly, TRH-Gly and TRH were mostly produced by PC1, PC2, and furin. Utilizing gel electrophoresis to further analyze the cleavage specificities of PC1 and PC2, we found that PC1 seems primarily responsible for cleavage to both intermediates and mature TRH, since it generated all products at significantly higher levels than PC2. The addition of 7B2 to the coinfection did not augment the ability of PC2 to cleave pro-TRH to either N- or C-terminal forms.

Identification of the thyrotropin-releasing hormone precursor, its processing products, and its coexpression with convertase 1 in primary cultures of hypothalamic neurons: anatomic distribution of PC1 and PC2.

The processing of pro-TRH, has been extensively studied in our laboratory using a corticotropic cell line, AtT20, transfected with the pro-TRH gene. We have also demonstrated that the convertases PC1 and PC2 process pro-TRH to cryptic peptides in vitro. However, although these processing pathways have been well characterized in vitro, little is known about the processing and subcellular distribution of pro-TRH and its derived peptides in hypothalamic neurons, an endogenous source of pro-TRH and PC enzymes. In this study we used multiple approaches to identify, both biochemically and anatomically, the presence and localization of pro-TRH (26 kDa) and its processing products. We also investigated the presence of PC1 and PC2 enzymes and the coexpression of pro-TRH and PC1 messenger RNAs. Identification of the TRH precursor was demonstrated by 1) Western blot analysis of cellular extracts, 2) immunoprecipitation of radiolabeled pro-TRH followed by analysis on acrylamide gel electrophoresis, 3) fluorescence immunocytochemistry, and 4) immunoelectron microscopy. The presence of the convertases PC1 and PC2 was determined by Western blot analysis of cellular extracts and fluorescence immunocytochemistry. The coexpression of pro-TRH with PC1 was shown by double in situ hybridization. Our findings support three main conclusions. First, this primary culture system of hypothalamic neurons is suitable for characterizing pro-TRH processing as well as identifying the anatomical location of its processing products. Second, prohormome processing takes place during axonal transport after removal of the signal peptide in the endoplasmic reticulum, and subsequent cleavages of the prohormone occur as intermediate peptides move down the axon toward the nerve terminal. This coupled transport-processing phenomenon may provide the necessary mechanism to ensure flexibility in differential processing of specific protein sequences that are determined by the secretory needs of cells. It appears that certain intermediate peptides differ in their subcompartmental distribution, suggesting the possibility of a differential processing and maturation of pro-TRH-derived peptides. Thirdly, the 87-kDa form of PC 1 may initiate the processing of pro-TRH at the Golgi complex level, which then continues to be processed by PC1 and PC2 in later stages of the secretory pathway.

Intracellular sites of prothyrotropin-releasing hormone processing.

Post-translational processing of proteins plays a key role in regulating their subcellular localization, enzymatic activity, and protein-protein interactions by such diverse mechanisms as phosphorylation, glycosylation, and proteolytic cleavage. The prothyrotropin-releasing hormone (pro-TRH) precursor (26 kDa) undergoes proteolytic cleavage at either of two sites, generating a 15/10-kDa or a 9.5/16.5-kDa N/C-terminal pair of intermediates. Using transfected AtT20 cells encoding a prepro-TRH cDNA, we have previously reported that this initial set of cleavages occurs prior to entry into the secretory granules (Nillni, E. A., Sevarino, K. A., and Jackson, I. M. D. (1993) Endocrinology 132, 1271-1277). In this study, we set out to identify the subcellular compartment where this initial cleavage takes place as well as to determine the sites of processing of the intermediates produced. Our strategy was to block the transport of pro-TRH or its intermediates from one subcellular compartment to the next and to assay for the accumulation of intermediates, presumably because their processing occurs in a post-blockade compartment. Radiolabeling experiments in AtT20 cells in the presence of the drug brefeldin A, which blocks transport from the endoplasmic reticulum to the Golgi complex, led to an accumulation of the 26-kDa precursor, suggesting a post-endoplasmic reticulum site of processing. When Golgi complex-to-secretory granule transport was blocked at 20 degrees C, the processing of the 26-kDa precursor was not affected, suggesting a Golgi complex site of processing. At this temperature, the 15-kDa N-terminal intermediate accumulated, suggesting a post-Golgi complex processing site, while the 16.5-kDa C-terminal intermediate was processed in the Golgi complex to produce a 5.4-kDa peptide.

Opiate withdrawal increases ProTRH gene expression in the ventrolateral column of the midbrain periaqueductal gray.

The midbrain periaqueductal gray matter (PAG) has a critical role in the modulation of behavioral and autonomic manifestations of the opiate withdrawal syndrome. We report a nearly 5-fold increase in proTRH gene expression in neurons of the ventrolateral column of the PAG following naltrexone precipitated morphine withdrawal. The accumulation of immunoreactive proTRH-derived peptides, but not the mature TRH tripeptide was concomitantly observed in these cells. These findings indicate that proTRH-derived peptides synthesized in neurons of the ventrolateral PAG may function as modifiers of opiate withdrawal responses.

Pro-thyrotropin-releasing hormone processing by recombinant PC1.

Pro-thyrotropin-releasing hormone (proTRH) is the precursor to thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26-kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [3H]leucine-labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26-kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well-established antibodies, we propose that the initial cleavage gave rise to the formation of a 15-kDa N-terminal peptide (preproTRH25-152 or pre-proTRH25-158) and a 10-kDa C-terminal peptide (pre-proTRH154-255 or preproTRH160-255). Some initial cleavage occurred after amino acid 108 to generate a 16.5-kDa C-terminal peptide. The 15-kDa N-terminal intermediate was further processed to a 6-kDa peptide (prepro-TRH25-76 or preproTRH25-82) and a 3.8-kDa peptide (preproTRH83-108), whereas the 10-kDa C-terminal intermediate was processed to a 5.4-kDa peptide (prepro-TRH206-255). The optimal pH for these cleavages was 5.5. ZnCl2, EDTA, EGTA, and the omission of Ca2+ inhibited the formation of pYE27 (preproTRH25-50), one of the proTRH N-terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.

Processing of prothyrotropin-releasing hormone (Pro-TRH) by bovine intermediate lobe secretory vesicle membrane PC1 and PC2 enzymes.

TRH is synthesized from a larger 26-kilodalton (kDa) prohormone (pro-TRH). Rat pro-TRH contains five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven other cryptic peptides. Each of the five TRH progenitor sequences is flanked by pairs of basic amino acids. We used a bovine intermediate lobe secretory vesicle membrane preparation, which contains the prohormone convertases (PCs) PC1 and PC2, to study the in vitro processing of pro-TRH. Pro-TRH was radiolabeled using [3H]Leu in AtT20 cells transfected with prepro-TRH complementary DNA, and the labeled 26-kDa pro-TRH was isolated from the cell extract by preparative sodium dodecyl sulfate-gel electrophoresis. Incubation of [3H]pro-TRH with the intermediate lobe secretory vesicle membrane preparation was followed by immunoprecipitation with antibodies specific for various regions of the pro-TRH sequence, and the immunoprecipitates were analyzed by sodium dodecyl sulfate-gel electrophoresis. Immunoprecipitation of the reaction mixture with anti-pCC10 antibody (an antibody that recognizes the intact precursor and amino-terminal intermediate products of processing) showed a time-dependent appearance of a 15-kDa and a 6-kDa peptide and, at times, a 3.8-kDa peptide with diminution of the 26-kDa substrate. Immunoprecipitation of the incubate with the C-terminal-directed antibody, pYE17 (an antibody that recognizes the intact precursor and C-terminal intermediate products of processing), showed the generation of 16.5-, 10-, and 5.4-kDa products in a time-dependent manner, with disappearance of the substrate. Western blot analysis demonstrated that the secretory vesicle membrane preparation contains PC1 and PC2. Immunodepletion studies with antiserum specific for PC1 or PC2 demonstrated that PC1 and PC2 can process pro-TRH to these intermediate products. An initial site of cleavage appeared to be either at the 152-153 or the 158-159 pair of basic residues to yield a 15-kDa N-terminal fragment that was then processed to the 6-kDa [TRH-(25-74)] and 3.8-kDa [TRH-(83-112)] forms. The 10-kDa C-terminal peptide generated by this cleavage was then processed to a 5.4-kDa peptide [TRH-(208-255)]. Alternatively, an initial cleavage at the 107-108 or the 112-113 bonds was also observed, yielding a 16.5-kDa C-terminal product that was further processed to the 5.4-kDa peptide. The pH profile for the appearance of both C- and N-terminal products showed a bimodal distribution, with optima at both 5.5 and 7.5. The cleavage of pro-TRH was enhanced by Ca2+ and partially inhibited by Zn2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Processing of proTRH to its intermediate products occurs before the packing into secretory granules of transfected AtT20 cells.

The intracellular compartments where posttranslational processing of proTRH takes place have not been identified. Using AtT20 cells transfected with a complementary DNA for preproTRH, we have used purified antibodies that recognize the intact precursor, intermediate and end products of processing to identify the subcellular compartments in which cleavage occur. Further, pulse-chase experiments followed by subcellular fractionation were undertaken to determine the order of processing of proTRH during its transport to the secretory granules. Cells were homogenized by nitrogen cavitation and subjected to a centrifugation of 1.065 mg/ml density gradient of Percoll to separate secretory granules (SG) from rough endoplasmic reticulum (RER)/Golgi apparatus. The purity of the SG and RER fractions was assessed by assays of marker enzymes for mitochondria, RER, Golgi, and cytoplasm. ProTRH derived cryptic peptides and TRH in each fraction were determined by RIA. Golgi and SG fractions were subjected to polyacrylamide gel electrophoresis followed by extraction and RIA. Using the anti-pCC10 antiserum which recognizes intact (26 kd) as well as partially processed prohormone, the RER/Golgi fraction contained 0.3 pmol intact ProTRH and 0.2 pmol each 15 and 6 kilodalton (kDa) fragments; the SG contained the 15 kDa moiety (0.2 pmol) along with a 6 kDa (0.4 pmol) material but not the 26 kDa ProTRH. The SG were also enriched by 0.21 pmol pYE27 (PreproTRH 25-50), 0.23 pmol pFT (PreproTRH 53-74), 0.31 pmol pEH24 (PreProTRH 86-106), and 0.5 pmol TRH. None of these were present in the RER/Golgi. Pulse-chase studies also showed that the intact proTRH (26 kDa) precursor was only present in the RER/Gg fraction along with two of its N-terminal intermediate processing products, a 15 k mol wt peptide and a 6 k mol wt peptide, and two of its C-terminal processing products, a 16.5 k mol wt and a 9.6 k mol wt peptides. In addition, fully processed peptides as well as TRH were only detected in the neurosecretory granules. These observations suggest that after the initial conversion of proTRH in the RER/Golgi fraction, the peptides are delivered to the granules where processing to TRH and cryptic peptides takes place. Supporting this, our pulse-chase studies unequivocally showed that, pEH24, an end product of proTRH processing, was only produced in secretory granules. Thus, initial cleavage of the TRH precursor may be required for packing and sorting of the end products to occur.

Identification of the thyrotropin-releasing hormone-prohormone and its posttranslational processing in a transfected AtT20 tumoral cell line.

By using an AtT20 cell line transfected with complementary DNA for preproTRH, we have identified the proTRH polyeptide precursor [26 kilodaltons (kDa)] and shown that this molecule gives rise to the proTRH derived sequences as determined by pulse-chase and trypsinization studies. The predicted proTRH precursor composed of 231 amino acids with 5 copies of a TRH progenitor sequence (Gln-His-Pro-Gly) and 7 other cryptic peptides was demonstrated by: 1) Western Blot analysis of an AtT20 cell extract with anti-pCC10 antibodies (an antibody that recognizes the intact prohormone as well as some intermediate products of processing); 2) Immunoprecipitation of the radiolabelled 26 kDa protein with anti-pCC10 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis; 3) Gel filtration chromatography of the radiolabeled 26 kDa extracted from SDS-PAGE. 4) RIA with anti-pCC10 antiserum against peptides extracted from adult rat hypothalamus and olfactory lobe after SDS-PAGE. 5) Trypsinization of the proTRH precursor which generated the proTRH cryptic peptides preproTRH25-50 (pYE27) and preproTRH53-74 (pFT22). These moieties were also produced during trypsinization of intermediate products of processing. By means of pulse-chase studies, the 26 kDa polypeptide was shown to be the biosynthetic precursor to all the proTRH derived cryptic peptides. Cleavage at two positions in the center of the molecule (Lys107-Arg108 and Lys152-Arg153) generated two moieties of 16.5 and 15 kDa. The 15 kDa N-terminal fragment is later cleaved to a 6 kDa peptide that includes the proTRH derived peptides, pYE27, pFT22, and pEH24. The carboxy-terminal 16.5 kDa fragment of the prohormone is processed to a 9.6 kDa fragment which contains the proTRH derived peptide pST10 (preproTRH160-169) and a fragment of 5.4 kDa that may be the C-terminal peptide preproTRH208-255 recognized by antisera pAC12 and pYE17. In further processing, the 9.6 kDa molecule is cleaved to produce a 5.4 kDa peptide from either sequences 115-169 or 160-199.

Membrane potential of erythrocytic stages of Plasmodium chabaudi free of the host cell membrane.

Free parasites were isolated from Plasmodium chabaudi-infected rat erythrocytes by N2-cavitation and purified on Percoll gradients. The membrane potential of the free parasites determined from the transmembrane distribution of the lipophilic cation, tetraphenylphosphonium, was -93 +/- 10 mV for late stage parasites and -90 +/- 3 mV for ring forms. Studies with intact infected erythrocytes demonstrated that the membrane potential of ring forms was much smaller compared to late trophozoites and schizonts and thus the present findings with free parasites suggest that host cell cytoplasmic factors may determine the magnitude of the parasite membrane potential. Both extracellular pH and [Na+] were found to modify the membrane potential of free parasites. Electrogenic protonophores, the H+-ATPase inhibitor dicyclohexylcarbodiimide and orthovanadate collapsed the potential of free parasites. Ouabain (or its membrane permeant derivative, strophanthidin), and oligomycin were without effect. These inhibitor studies suggest that an electrogenic H+-ATPase similar to that found in yeast generates in part the membrane potential of malaria parasites. Using weak acid distribution or a pH sensitive fluorescent dye, it was demonstrated that free parasites maintain an alkaline intracellular pH at extracellular pH greater than 6.5. The pH gradient was partially collapsed by orthovanadate or dicyclohexylcarbodiimide and by substitution of Na+ for K+ in the suspending buffer. The H+-ATPase and K+:H+ exchange may therefore both contribute to regulation of intracellular pH in Plasmodium.

Extracellular development of Plasmodium knowlesi erythrocytic stages in an artificial intracellular medium.

The development of erythrocytic stages of Plasmodium knowlesi separated from their host cells has been determined in terms of the capacity of the isolated organisms to carry out the synthesis and secretion of proteins. P. knowlesi trophozoites and schizonts were released from host cells by nitrogen decompression and cultivated in a medium consisting of 20 mM Na+; 120 mM K+; 1 mM Mg2+; no Ca2+; 100 mM Cl-; 20 mM HCO3-; 5 mM Hepes [pH 6.73], glucose, vitamins, amino acids and 10% fetal calf serum. The yield was about 97% intact parasites, judging by their ability to maintain a membrane potential, and these parasites had more than 80% the capacity of infected cells for nuclear replication and macromolecule biosynthesis. Pulse and pulse-chase labeling studies with [35S]methionine show that parasite-synthesized proteins with Mr 160 000, 140 000, 100 000 and 58 000 are exported from the parasite in soluble form. Proteins with Mr 140 000, 100 000, 58 000-60 000, 40 000 were recovered in a particulate fraction isolated from the parasite culture fluid. An Mr 62 000 protein synthesized in large amounts by isolated parasites during the last 2h of the developmental cycle, could not be detected in infected erythrocytes, and a minor early Mr 74 000 protein becomes prominent in free parasites but not infected cells toward the end of the developmental cycle. Parasite-synthesized proteins with Mr 230 000, 160 000, 140 000, 62 000, 58 000 and 45 000 were labeled by incubation with radioactive N-acetylglucosamine during short term incubation in vitro. About 80% of label incorporation occurred via N-glycosylation supported by dolichol derived from the blood, and about 20% via glycolytic intermediates.

Characterization of Plasmodium berghei antigens inducing blast transformation in immune rat lymphocytes.

Rat erythrocytes, infected with Plasmodium berghei, were disrupted by freezing and thawing or parasites were released by mechanical breaking of the agglutinated erythrocytes. The released soluble antigens, inducing blast transformation in non-adherent immune rat spleen cells, were fractionated by a sequence of salting out, ion exchange chromatography and gel filtration. At the final step a purification factor of 83.7-fold was achieved. This fraction contained three components discernible by SDS-PAGE of molecular weights 56, 66 and 72 kD respectively. The free parasites, also induced blast transformation in immune rat spleen cells. After disruption by freeze-thawing the majority of the active antigens was found in the sediment after centrifugation at 17,300 g.