Biomedical subjects

Y Bourbonnais

Publications and source records attributed to Y Bourbonnais.

7 recordsLinked to original sources

PubMed1992

The pro-region of the Kex2 endoprotease of Saccharomyces cerevisiae is removed by self-processing.

We have produced in the baculovirus/insect cells expression system a soluble secreted form of the Saccharomyces cerevisiae Kex2 endoprotease. This secreted enzyme was purified and its NH2-terminal sequence determined. The NH2-terminal sequence started at residue Leu109 of the sequence deduced from the KEX2 gene nucleotide sequence, showing that the Kex2 enzyme is produced as a proenzyme. Residue Leu109 is preceded by a pair of basic amino acid residues (Lys107-Arg108) which is a potential processing site for the Kex2 endopeptidase. Furthermore, expression of an inactive form of this truncated enzyme resulted in the production of a protein with a higher molecular weight. These observations suggest that the pro-region of Kex2 endoprotease is removed by a self-processing event.

Amino Acid Sequence↗

PubMed1991

Heterologous expression of peptide hormone precursors in the yeast Saccharomyces cerevisiae. Evidence for a novel prohormone endoprotease with specificity for monobasic amino acids.

The peptide somatostatin (SRIF) exists as two different molecular species. In addition to the most common form, which is a 14-residue peptide, there is also a 14-amino acid amino-terminally extended form of the tetradecapeptide, SRIF-28. Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which, upon cleavage, generate either SRIF-14 or -28, respectively. In mammals a single prepro-SRIF molecule undergoes tissue-specific processing to generate the mature hormone whereas in some species of fish separate genes encode two distinct but homologous precursors prepro-SRIF-I and -II that give rise to SRIF-14 and -28, respectively. To investigate the molecular basis for differential processing of the prohormones we introduce their cDNAs into yeast cells (Saccharomyces cerevisiae). The signal peptides of both precursors were poorly recognized by the yeast endoplasmic reticulum translocation apparatus, consequently only low levels of SRIF peptides were synthesized. To circumvent this problem a chimeric precursor consisting of the alpha-factor signal peptide plus 30 residues of the proregion was fused to pro-SRIF-II. This fusion protein was efficiently transported through the yeast secretory pathway and processed to SRIF-28 exclusively, which is identical to the processing of the native precursor in pancreatic islet D-cells. Most significantly, cleavage of the precursor to SRIF-28 was independent of the Kex 2 endoprotease since processing occurred efficiently in a kex 2 mutant strain. We conclude that in addition to the Kex 2 protease, yeast possess a distinct prohormone converting enzyme with specificity toward monobasic processing sites.

Amino Acid Sequence↗

PubMed1991

Prohormone processing by yeast proteases.

Investigations of the precursors of alpha-pheromone and killer toxin in the yeast Saccharomyces cerevisiae have defined the genes coding (KEX1 and KEX2) for the proteases which are responsible for their processing. In addition to processing at pairs of basic residues it is evident that yeast can also process at monobasic sites. We present data on the Kex1p and Kex2p enzymes, their cellular localization, and their post-translational modification. In addition initial characterisation of the monobasic specific protease and the isolation of mutants defective in this activity are presented. The use of the yeast system as a model for the processing of mammalian prohormones is discussed.

Amino Acid Sequence↗

PubMed1988

Secretion of somatostatin by Saccharomyces cerevisiae. Correct proteolytic processing of pro-alpha-factor-somatostatin hybrids requires the products of the KEX2 and STE13 genes.

Somatostatin is a 14-amino-acid peptide hormone that is proteolytically excised from its precursor, prosomatostatin, by the action of a paired-basic-specific protease. Yeast (Saccharomyces cerevisiae Mat alpha) synthesizes an analogous peptide hormone precursor, pro-alpha-factor, which is proteolytically processed by at least two separate proteases, the products of the KEX2 and STE13 genes, to generate the mature bioactive peptide. Expression in yeast of recombinant DNAs encoding hybrids between the proregion of alpha-factor and somatostatin results in proteolytic processing of the chimeric precursors and secretion of mature somatostatin. To determine if the chimeras were processed by the same enzymes that cleave endogenous pro-alpha-factor, the hybrid DNAs were introduced into kex2 and ste13 mutants, and the secreted proteins were analyzed. Expression of the pro-alpha-factor-somatostatin hybrids in kex2 mutant yeast resulted in secretion of a high molecular weight hyperglycosylated precursor. No mature somatostatin was secreted, and there was no proteolytic cleavage at the Lys-Arg processing site. Similarly, in ste13 yeast, only somatostatin molecules containing the (Glu-Ala)3 spacer peptide at the amino terminus were secreted. Our results demonstrate that in yeast processing mutants, the behavior of the chimeric precursors with respect to proteolytic processing was exactly as that of endogenous pro-alpha-factor. We conclude that the same enzymes that generate mature alpha-factor proteolytically process hybrid precursors. This suggests that structural domains of the proregion rather than the mature peptide are recognized by the processing proteases.

Amino Acid Sequence↗

PubMed1986

Posttranslational modifications of proopiomelanocortin in rat intermediate lobe cells.

Proopiomelanocortin (POMC), the common precursor to beta-endorphin and alpha-melanocyte-stimulating hormone synthesized in rat intermediate lobe cells, exhibits both charge and size heterogeneity on two-dimensional gels. Pulse-labeling and pulse-chase studies revealed that this heterogeneity is due to co- and post-translational modifications of a single common polypeptide. Short 5-min-pulse incubation with [3H]phenylalanine allowed the preferential labeling of two major forms characterized by an identical isoelectric point (8.2), but slightly different apparent molecular weights (MW = 34,000 and 36,000). These peptides could be labeled with [3H]mannose and the analysis of their tryptic fragments by high-pressure liquid chromatography revealed that they correspond to polypeptides bearing one or two N-linked carbohydrate side chains. Accumulation of more acidic forms was observed during subsequent chase incubations in the absence of phenylalanine. These acidic forms were shown to incorporate sulfate and (or) phosphate groups. Sulfation and phosphorylation occurred on POMC within 5 min after its synthesis and were concomitant with the processing of the N-linked carbohydrates from the high mannose to the complex structure. Finally, partial digestion of the phosphorylated and nonphosphorylated analogs of POMC with either Staphylococcus aureus (V8 strain) protease or chymotrypsin suggests that the presence of a phosphate group may alter POMC sensitivity to exogenously added proteases.

Alkaline Phosphatase↗

PubMed1985

Post-translational incorporation of [35S]sulfate into oligosaccharide side chains of pro-opiomelanocortin in rat intermediate lobe cells.

Pro-opiomelanocortin (POMC), the common precursor to beta-endorphin and alpha-melanocyte-stimulating hormone in rat neurointermediate lobe cells, exhibits both charge and size heterogeneity on two-dimensional gel electrophoretograms. Short term [3H]phenylalanine pulse-labeling, and pulse-chase studies, revealed that this heterogeneity is acquired either co-translationally, through the addition of mannose-rich oligosaccharide chains to the nascent protein, or post-translationally, probably during the period of oligosaccharide processing from the high mannose to the complex forms. In this process, radioactive sulfate is incorporated into different glycoprotein variants of POMC. In the presence of tunicamycin, an inhibitor of the N-glycosylation process, [35S]sulfate incorporation does not occur in any of the major variant forms of POMC, thereby preventing the appearance of the most acidic forms on two-dimensional gels. POMC tryptic fragments were separated by high-pressure liquid chromatography. Sulfate incorporation occurred in only two peptides that were also labeled with [3H]glucosamine. Extensive alkaline digestion of these peptides in the presence of sodium borohydride released the sulfate-containing moieties which were separated from free amino acids by gel filtration. Sulfate bearing moieties could also be released by almond emulsin peptide:N-glycosidase digestion. All these results unambiguously show that sulfate moieties preferentially enter asparagine-linked carbohydrate side chains and not amino acid residues of the POMC polypeptide. It is also likely that differential sulfation, conferring unequal amounts of negative charge upon various glycoprotein variants of POMC, is responsible for much of the charge heterogeneity displayed by the prohormone.

Amino Acid Sequence↗

PubMedDate not supplied

Characterization of sulfated forms of the pro-opiomelanocortin amino-terminal glycopeptide in rat intermediate lobe cells.

Explants of rat neurointermediate lobes were incubated in the presence of radioactive amino acids, sugars or sulfate and the labeled proteins were separated by two-dimensional gel electrophoresis. A double series of acidic peptides (Mr = 16,000-21,500) were identified as variant forms of the amino-terminal glycopeptide of pro-opiomelanocortin (N-POMC). The series of peptides with the higher molecular weights (Mr = 18,000-21,500) contain a tryptic fragment (tentatively identified as the tryptic peptide of the "joining peptide": sequence 77 to 93 of rat POMC) which is absent from the forms of the lower molecular weight series (Mr = 16,000 to 18,000). Pulse-chase studies further showed that the high molecular weight forms of N-POMC could be post-translationally cleaved albeit slowly into the species of Mr = 16,000-18,000 which constitute, at least in part, the final maturation products of the N-terminal region of the precursor molecule. All the variant forms of the N-POMC glycopeptide could be labeled with [35S]sulfate. Our results strongly suggest that most of the sulfate groups are attached to N-linked oligosaccharide side chains of N-POMC. We therefore propose that one of the final maturation products of the N-terminal portion of POMC in rat intermediate lobes is a sulfated glycopeptide (Mr = 16,000-18,000) composed of the 1-74 sequence of rat POMC.

Amino Acid Sequence↗