Post-translational Processing Mechanisms Research Articles

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.

The Molecular Evolution of Neuropeptides: Prospects for the '90s (Part 2 of 2)

Three distinct opioid precursors have been identified in the central nervous system of mammals: proopiomelanocortin (POMC), proenkephalin, and prodynorphin. These precursors are derived from separate genes, synthesized in distinct neurons, and yield unique sets of opioid end products. This review will discuss the general mechanisms involved in the biosynthesis of neuropeptide precursors and consider the roles of posttranscriptional and posttranslational processing mechanisms in the generation of multiple sets of end products from a single gene. In addition, techniques that can be used for isolating and characterizing neuropeptide genes, mRNAs, and end products will be reviewed. These introductory comments will serve as the framework for a discussion of the phylogeny of the opioid precursors in the major groups of non-mammalian vertebrates.

The Molecular Evolution of Neuropeptides: Prospects for the '90s

Three distinct opioid precursors have been identified in the central nervous system of mammals: proopiomelanocortin (POMC), proenkephalin, and prodynorphin. These precursors are derived from separate genes, synthesized in distinct neurons, and yield unique sets of opioid end products. This review will discuss the general mechanisms involved in the biosynthesis of neuropeptide precursors and consider the roles of posttranscriptional and posttranslational processing mechanisms in the generation of multiple sets of end products from a single gene. In addition, techniques that can be used for isolating and characterizing neuropeptide genes, mRNAs, and end products will be reviewed. These introductory comments will serve as the framework for a discussion of the phylogeny of the opioid precursors in the major groups of non-mammalian vertebrates.

Purification and characterization of two distinct forms of rat adrenal cytochrome P450 11β: Functional and structural aspects

It is generally accepted that the last three steps of aldosterone biosynthesis are catalyzed by a single enzyme, i.e., cytochrome P450 11β (P450XIB). We have previously reported that rat adrenal mitochondria may be capable of producing two forms of P450 11β which differ in molecular weight (49 and 51 kDa). In the present study we describe the purification, the enzymatic activities, and some structural properties of these two proteins. Using zona fasciculata mitochondria, the 51-kDa protein was purified to electrophoretic homogeneity by means of octyl-Sepharose chromatography. In a reconstituted system the protein catalyzed 18- and 11β-hydroxylation of deoxycorticosterone, but exhibited no 18-hydroxylation or 18-hydroxydehydrogenation of corticosterone. The 49-kDa protein was isolated from zona glomerulosa mitochondria of rats kept on a low-sodium, high-potassium regimen. Using octyl-Sepharose chromatography, it could be separated from the 51-kDa protein. A reconstituted eluate fraction, containing the 49-kDa protein, converted deoxycorticosterone not only to 18-OH-deoxycorticosterone and corticosterone, but also to 18-OH-corticosterone and aldosterone. These findings indicate that the rat adrenal cortex is capable of producing two distinct forms of active cytochrome P450 11β. A structural relationship of the 49- and 51-kDa proteins was indicated by experiments involving limited proteolysis. Thus, digestion with α-chymotrypsin and V8-protease yielded very similar peptide maps for both proteins. During potassium repletion of potassium-deficient rats, the disappearance of the active 51-kDa protein coincided with the appearance of the 49-kDa protein. These results are suggestive of a post-translational processing mechanism converting the 51-kDa protein into the smaller 49-kDa form. However, the 49-kDa protein might also be encoded by a distinct gene, regulated separately depending on the physiological conditions.

A single 12.5-kilobase androgen-regulated mRNA encoding multiple proline-rich polypeptides in the ventral prostate of the rat.

Synthetic 32P-labeled oligonucleotides have been used to identify the prostatic proline-rich polypeptide (PRP) mRNA which has partially been characterized. The 14-mer d(G-G-T-T-C-T-G-C-A-T-A-A-T-G) complementary to the coding sequence for His-Tyr-Ala-Glu-Pro, a sequence element occurring in all 38-residue PRP variants, hybridizes specifically with a 12.5-kilobase mRNA which is clearly androgen-controlled. This oligonucleotide was used as an efficient primer for the construction of a PRP-specific lambda gt10 cDNA library. The nucleotide sequence of the inserts from several recombinant clones has been determined. This structural analysis revealed a PRP mRNA encoding a large precursor containing a number of tandemly repeated units. Each repeat codes for a sequence of 100 amino acids in which the highly conserved PRP sequence is embedded. From this polyprotein the large number of PRP variants must be generated by a post-translational processing mechanism which is still unknown. The high degree of conservation of both nucleotide and amino acid sequence in the entire unit also indicates that the PRP gene(s) likely evolved by multiplication of a 300-base pair ancestral DNA sequence. This has resulted in a noninterrupted repetitive DNA coding segment which is detected at the genomic level.

Development of gastrin synthesis and posttranslational processing mechanisms in rats.

To examine the ontogeny of gastrin synthesis and posttranslational processing mechanisms, we utilized region-specific antisera to determine the factors that regulate the contents of gastrin and its precursors in the stomachs of developing rats. Gastrin content increased gradually from birth to a peak at 5 wk of age, after which there was a slight decrease to levels observed in mature animals. Unlike gastrin, glycine-extended precursors of gastrin (G-Gly) increased abruptly in the first day of life but thereafter increased at a steady state. Dexamethasone treatment and early weaning (at 11 days of age) rapidly increased gastrin content, whereas G-Gly content was decreased. On weaning animals to adult rat chow abruptly at an age (18 days) when the corticosteroid surge associated with the weaning process had already occurred, evidence for increases in both gastrin and G-Gly content were observed 7 days later. In contrast, administration of dexamethasone to 18-day-old rats enhanced gastrin content to a greater extent than G-Gly. These results suggest that the natural surge in corticosteroids associated with weaning may enhance gastrin amidation activity, whereas the concomitant dietary changes may exert a stimulatory effect of gastrin synthesis.

Synthesis and expression of MHC class II molecules in the absence of attached invariant chains by recombinant-interferon-gamma-activated bone-marrow-derived macrophages.

Pure populations of in vitro propagated bone marrow-derived macrophages are constitutively Ia negative. Co-culturing of these cells with recombinant interferon-gamma (rIFN-gamma) resulted in the appearance of high amounts of Ia antigens at the cell surface of essentially all cells. The continuous presence of the stimulus was a prerequisite for sustained Ia expression because removal of the stimulus resulted in rapid decline of surface Ia. Two-dimensional (2D) gel analysis (1D isoelectric focusing, 2D sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of class II molecules synthesized by rIFN-gamma-stimulated bone marrow macrophages (BMM phi) revealed that, in contrast to class II complexes hitherto described, BMM phi-derived I-A and I-E subregion-encoded subunits are synthesized without invariant chains. The invariant chain-deficient alpha,beta heterodimers are expressed at the cell surface in high proportions demonstrating that their correct assembly and transport to the cell surface is accomplished in the absence of invariant chains. The lack of invariant chains appears not to be due to a failure of rIFN-gamma to induce transcription of the gamma-chain gene because rIFN-gamma-induced, in contrast to uninduced, BMM phi accumulate high levels of invariant chain-specific transcripts as evidenced by Northern blot analysis. These findings suggest that translation of gamma-chain-specific mRNA is blocked in BMM phi for as yet unknown reasons. Alternatively, newly synthesized gamma chains might have escaped their regular intracellular maturation pathway as a result of unidentified modifications mediated by altered post-translational processing mechanisms.

β-Endorphin is present in active and inactive forms in rat gastric antrum

Lipotropin, β-endorphin and a series of peptides related to β-endorphin were extracted from rat antrum and resolved by gel filtration, ion exchange chromatography and high pressure liquid chromatography; the concentrations of the peptides were determined by radioimmunoassay. The major peptide with β-endorphin immunoreactivity present in the antrum was lipotropin but it was accompanied by substantial quantities of β-endorphin in its biologically active form; in addition there were minor quantities of a number of inactive β-endorphin related peptides. The experiments demonstrate that in rat antrum gastrin can be accompanied by both active and inactive forms of β-endorphin. The implications of post-translational processing mechanisms common to gastrin and β-endorphin are discussed.

Precursors of Vasopressin and Oxytocin

Publisher Summary It is now known that in addition to transcription and translation processes, various signal sequences and post-translational processing events are also involved in determining the type of peptides that are secreted by the cell. This chapter describes unequivocal criteria for the identification of precursors of peptides with regard to both vasopressin and oxytocin. These criteria include (1) demonstration in classical pulse-chase experiments in intact cellular systems that a larger form of the peptide is first synthesized and subsequently decreases in radioactivity as the radioactive peptide is formed, (2) demonstration by in vitro translation experiments that a larger precursor form of the peptide is synthesized, and (3) utilizing modem recombinant DNA techniques to clone cyclic DNA (cDNA) obtained from a purified messenger RNA (mRNA) template using reverse transcriptase. A variety of biosynthesis studies conducted in vivo and in vitro have shown that the vasopressin precursor is a 19–23,000 molecular weight glycoprotein, which contains vasopressin at its N-terminus, neurophysin in the middle, and a 39 amino acid glycopeptide at the C-terminus. While, the 15–16,000 molecular weight oxytocin precursor is not glycosylated. The vasopressin precursor has been completely sequenced by recombinant DNA methods, thereby providing insights into the nature of the glycopeptide as well as post-translational processing mechanisms. However, many questions remain to be answered such as the nature of the post-translational processing enzymes and mechanisms that fashion the final peptide products or the mechanisms underlying the transcriptional and translational regulation of the precursors during functional activity.