Showing papers on "Protein maturation published in 1983"

Synthetic peptide immunogens as vaccines.

Abstract: Synthetic peptide immunogens have been shown to elicit antibodies that can react with full-length proteins containing that peptide. Such antibodies are directed against a specific region of the protein chosen in advance by the investigator and so have a predetermined specificity. In basic research, these antibodies are useful in identifying the protein product of an open reading frame, localizing the gene product to particular cells or subcellular organelles, identifying the enzymatic function of a protein product, following the fate of particular regions of a product through protein maturation processes, analyzing the expression of exons following DNA rearrangements and RNA splicing, and purifying the protein by immunoaffinity chromatography techniques. In medicine, such antibodies may provide reagents for passive vaccination, antitoxin therapy, and targeted immunotherapy of neoplasia. The peptides themselves may be used as synthetic vaccines. The immediate future of the synthetic peptide immunogen in medicine is clear--the promise demonstrated in the laboratory must be reduced to safe application in the hospital. Two barriers to this are the selection of precisely the best peptide and the selection of the proper adjuvant. Currently, a brute force approach is utilized to find the best peptide for eliciting the desired antibodies. This is clearly a problem when the pathogenic organism is assayable only in man. Possibly, by combining studies on the antigenicity of the pathogenic organism with an analysis of naturally occurring variants that alter its immunogenicity, peptide selection will be made easier. Also, since the adjuvants and carriers used in the laboratory are in general too harsh for widespread use in humans and animals, much work needs to be done to find suitable adjuvants and carriers. Nonetheless, now that the major conceptual hurdle to synthetic peptide vaccines has been cleared (that is, it is not necessary to reproduce conformation exactly), it should be relatively straightforward to solve the remaining problems.

Monensin blocks the maturation of receptors for insulin and somatomedin C: identification of receptor precursors.

Abstract: Cultured human lymphoid (IM-9) cells were labeled with [35S]methionine in the presence and absence of monensin, a carboxylic ionophore that inhibits post-translational protein maturation. Labeled receptors for insulin and somatomedin C were immunoprecipitated with antibodies specific for each receptor. Monensin inhibits the biosynthesis of mature α and β subunits of both receptors and leads to the accumulation of immunoreactive polypeptides with molecular weights of 180,000. These 180,000 molecular weight polypeptides exist as disulfide-linked dimers and may be biosynthetic precursors of both α and β subunits. In the presence of monensin, small amounts of immunoreactive polypeptides with molecular weights 115,000 and 89,000 also are produced. These may be abnormally processed forms of the α and β subunits lacking residues normally added during terminal glycosylation. In cells treated with monensin, the polypeptides of molecular weights 180,000 and 115,000 can be affinity-labeled with 125I-labeled insulin. These labeled polypeptides are immunoprecipitated by antibodies specific for insulin receptors but not by antibodies specific for somatomedin-C receptors. This indicates that the putative precursors for insulin and somatomedin-C receptors are distinct polypeptides, although they have similar molecular weights and similar modes of processing. A possible structural relationship between the precursors for these receptors and the type II insulin-like growth factor receptor is discussed.

Genetic analysis of the folding pathway for the tail spike protein of phage P22

Abstract: Temperature-sensitive mutations in the gene encoding the trimeric tail spike protein of phage P22 interfere with protein maturation at 39 degrees C. We show here that temperature-sensitive mutations at many sites block the folding pathway prior to accumulation of the partially folded protrimer intermediate. Temperature-shift experiments indicate that at least some of the mutants accumulate an earlier intermediate in the folding pathway. Immunoprecipitation experiments suggest that the conformation of the isolated temperature-sensitive polypeptide chains is closer to that of the unfolded chain than to that of the mature spike formed at permissive temperature. The sites of these mutations probably represent amino acid sequences that play key roles during the folding of the tail spike polypeptide chain but are not important in the mature protein.

Two classes of mutant mammary tumor virus-infected HTC cell with defects in glucocorticoid-regulated gene expression.

Abstract: We have isolated mutant derivatives of M1.54 (a mammary tumor virus [MTV]-infected rat hepatoma [HTC] cell line containing multiple integrated proviruses) that fail to express hormone-inducible cell surface viral glycoproteins. In wild-type M1.54, the synthetic glucocorticoid dexamethasone selectively stimulates the rate of synthesis of MTV RNA. In addition, dexamethasone is essential for posttranslational maturation of three of the four cell surface viral glycoproteins processed from the MTV glycosylated precursor polyprotein; the fourth mature species is produced constitutively. Two mutant phenotypes are described; each contains glucocorticoid receptors that are indistinguishable from the wild-type receptor with respect to hormone affinity, intracellular concentration, nuclear translocation efficiency, DNA-cellulose chromatography, and sedimentation rate. In one class, represented by the mutant line CR1, dexamethasone fails to stimulate the low basal rate of MTV gene transcription; surprisingly, hormonal regulation of tyrosine aminotransferase activity is also defective in CR1, whereas several other cellular responses to dexamethasone are normal. In the second class of mutants, represented by CR4, dexamethasone stimulates synthesis of MTV transcripts indistinguishable from those produced in M1.54, but only the constitutive cell surface viral glycoprotein is expressed. Thus, these mutants define two distinct and novel aspects of glucocorticoid regulated gene expression in HTC cells: CR4 contains a defect in a hormone inducible protein maturation pathway that acts on specific viral (and presumably cellular) precursor polypeptides, whereas the lesion in CR1 appears to affect the expression of a subset of the gene products normally under glucocorticoid control in M1.54.