The new enzymology of precursor processing endoproteases.
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Animals↗
Biomedical subjects
D F Steiner
Publications and source records attributed to D F Steiner.
Conservation of the prohormone convertase gene family in metazoa: analysis of cDNAs encoding a PC3-like protein from hydra.
A subclass of proteolytic enzymes that correctly cleave precursor proteins at paired basic residues and are structurally related to the bacterial subtilisins has recently been identified. In yeast, a single membrane-bound proteolytic processing enzyme encoded by the kex2 gene has been found, whereas in higher vertebrates cDNAs encoding four distinct enzymes (PC2, PC3, furin, and PACE 4) have been identified. Like kex2, furin (also known as PACE) contains a hydrophobic transmembrane domain, but PC2, PC3, and PACE 4 lack this feature. All five enzymes exhibit striking similarities in their catalytic domains, and this suggests that they have arisen from a common ancestral subtilisin-like gene. We report here the identification of cDNAs encoding a protein that is similar in structure to PC3 from a simple metazoan, Hydra vulgaris (formerly Hydra attenuata). cDNAs encoding two isoforms of this PC3-like enzyme were obtained that differ only in their carboxyl-terminal sequences, probably due to alternative splicing of a common pre-mRNA. Neither form contains a transmembrane domain. Predicted amino acid sequence comparisons revealed that the hydra PC3-like enzyme is 55.4% and 56.7% identical in the catalytic domain to mouse PC3 and human furin, respectively. RNA blot analyses revealed that the PC3-like RNA is expressed predominantly in the hydra body column and not in the head region, although the hydra head contains a high density of nerve cells, which synthesize a variety of neuropeptides. For this reason, we suspect that another proprotein cleavage enzyme isoform may be expressed in head nerve cells. The isolation of a PC3-like cDNA from hydra is consistent with the presence of neuroendocrine cells and indicates that the PC/furin gene family has been well conserved in all metazoa. A simplified nomenclature for the group of mammalian processing proteases is proposed.
Amino Acid Sequence↗
A member of the eukaryotic subtilisin family (PC3) has the enzymic properties of the type 1 proinsulin-converting endopeptidase.
PC3, a mammalian homologue of the yeast subtilisin-like proteinase Kex2, was expressed in Xenopus oocytes and its activity was characterized. PC3 cleaved human proinsulin at one of the two dibasic sites (KTRR32 but not LQKR65). The specificity, inhibitor profile, pH optimum (5.5) and Ca(2+)-dependence (K0.5 = 2.5-3 mM) paralleled those of the insulin-granule type 1 endopeptidase activity, suggesting a role for PC3 in the conversion of prohormones.
Animals↗
Identification and analysis of the gene encoding human PC2, a prohormone convertase expressed in neuroendocrine tissues.
In recent studies we have identified PC2 and PC3, members of a family of serine proteases that are related structurally to subtilisin, and have provided evidence that these are involved in the tissue-specific processing of prohormones and neuropeptides. PC2 is expressed at high levels in the islets of Langerhans, where it participates in the processing of proinsulin to insulin (S.P.S. and D.F.S., unpublished data). To evaluate the regulated expression of the human PC2 (hPC2) gene we have analyzed its structure and characterized its promoter. A map of the gene was constructed by using 11 clones isolated from two human genomic DNA libraries. The gene spans greater than 130 kilobase pairs and consists of 12 exons. Comparison with the structure of the gene encoding human furin, another member of this superfamily, revealed a high degree of conservation of exon-intron junctions. The hPC2 gene was localized to chromosome 20, band p11.2. The 5' flanking region of the hPC2 gene is very G+C-rich and contains six potential Sp1 binding sites but no TATA or CAAT box. Expression of chloramphenicol acetyltransferase reporter fusions containing the putative promoter region was observed to occur in beta TC-3 mouse insulinoma cells but not in HepG2 human hepatoma cells, consistent with the known tissue-specific pattern of expression of the hPC2 gene. Analysis of the level of chloramphenicol acetyltransferase activity with several deletion mutants identified the region from -1100 to -539 from the translation start site as essential for hPC2 promoter activity.
Amino Acid Sequence↗
Cellular and molecular biology of the beta cell.
Considerable progress has been made in our understanding of islet-cell function and its relationship to regulation of whole body glucose metabolism. At the genetic level, the regulatory regions in islet-specific genes are being characterised. Transcription factors that interact with these regions have been cloned and these will be instructive in elucidating how islet-specific genes are regulated during development and regeneration. Identification of the enzymes responsible for proteolytic conversion of proinsulin to insulin represents a major advance in understanding prohormone processing. Cleavage of proinsulin is mediated by at least two prohormone convertases (PC3/PC1 and PC2). Their activity is regulated by an acidic gradient between the Golgi and secretory granules and by calcium ions. It is not yet clear how insulin or the PC's are specifically diverted into the regulated secretory pathway. Regulation at this step may be defective in some diabetic patients resulting in relatively elevated circulating proinsulin levels. Specific features of GLUT 2 and glucokinase (GK), proteins that regulate Beta-cell glucose transport and phosphorylation, indicate that these may be key components of the glucose sensor. GLUT 2 is necessary to reconstitute glucose-sensitive insulin secretion in pituitary tumour cells expressing a proinsulin cDNA. Furthermore, the expression of GLUT 2 in Beta cells, but not in hepatocytes, is decreased in diabetes mellitus. However, under normal circumstances GK is probably rate limiting for Beta-cell glucose utilisation. Thus, it is likely that both GLUT 2 and GK determine the set point for glucose-stimulated insulin secretion. Elucidation of distal effectors that regulate insulin secretion is also crucial to our understanding of Beta-cell function.(ABSTRACT TRUNCATED AT 250 WORDS)
Amyloid↗
Molecular biology of islet amyloid polypeptide.
We investigated the relationship between non-insulin-dependent diabetes mellitus (NIDDM) and islet amyloid polypeptide (IAPP) gene by restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-direct sequencing analysis. Endonuclease BglII and/or PvuII RFLP analysis revealed no positive correlation of IAPP gene with NIDDM. In PCR-direct sequencing of 25 NIDDM patients, no nucleotide sequence differences were found. These data do not support the view that IAPP plays an important role in the pathogenesis of NIDDM. cDNAs encoding cat, rat, mouse, guinea pig and degu IAPP precursors were also cloned, and comparison of these predicted amino acid sequences clarified the species difference, especially between amyloid-forming and non-amyloid-forming species. Amino acid residues 25-28 of mature IAPP might be responsible for their amyloidogeneity. The alternative splicing transcripts of guinea pig IAPP gene were identified by using PCR. If these types of transcripts are translated, N-terminal mutated IAPP might be produced and act as an antagonist. The signal peptide cleavage site of rat IAPP precursor was also identified by an in vitro translation and processing system.
Amino Acid Sequence↗
Effects of islet amyloid polypeptide (IAPP) on insulin biosynthesis or secretion in rat islets and mouse beta TC3 cells. Biosynthesis of IAPP in mouse beta TC3 cells.
Effects of rat islet amyloid polypeptide (IAPP) on insulin biosynthesis and secretion were examined in isolated rat islets and mouse beta TC3 cells. Culture of islets or mouse beta TC3 cells for 24 h in the presence of 10(-6) M IAPP and 5.5 mM glucose had no effect on insulin mRNA levels. The rates of proinsulin biosynthesis were not altered in islets incubated in 10(-4)-10(-9) M IAPP. In beta TC3 cells, proinsulin biosynthesis was stimulated by glucose, though no effects of IAPP were shown. Addition of 10(-5) M IAPP to islets incubated in 11 mM glucose decreased the fractional insulin secretion rates; however, the secretion of insulin from beta TC3 cells was not affected by 10(-5) M IAPP. On the other hand, mouse beta TC3 cells expressed the elevated level of IAPP mRNA. Metabolic labeling of beta TC3 cells revealed the synthesis of both proIAPP and mature IAPP. In pulse chase experiments, proIAPP was processed to IAPP in a manner similar to proinsulin. These data indicate that IAPP is a possible polypeptide hormone synthesized in pancreatic beta cells though it is unlikely that IAPP is a physiologically relevant modulator of insulin biosynthesis or secretion.
Amyloid↗
Structure and evolution of insulin and insulin-like growth factors in chordates.
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Amino Acid Sequence↗
The biosynthesis and processing of neuroendocrine peptides: identification of proprotein convertases involved in intravesicular processing.
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Amino Acid Sequence↗
Altered glucose regulation of insulin biosynthesis in insulinoma cells: mouse beta TC3 cells secrete insulin-related peptides predominantly via a constitutive pathway.
The effects of glucose on insulin gene expression and proinsulin biosynthesis, processing, and secretion were studied in mouse beta TC3 cells, an established insulinoma cell line derived from transgenic mice carrying a hybrid insulin promotor-simian virus-40 tumor antigen. The level of insulin mRNA was maintained at high levels by culture in 11 mM glucose, but essentially disappeared after 48 h of culture without glucose. The rate of insulin biosynthesis in beta TC3 cells was also dependent on glucose concentration over periods of 24 or 48 h (but not during 3 h) of stimulation. Insulin biosynthesis decreased about 50% after 24 h and about 85% after 48 h of incubation without glucose. When beta TC3 cells were incubated without glucose for 48 h, the rate of conversion of proinsulin to insulin was decreased compared to that at 11 mM glucose. Insulin secretion was sustained by medium glucose and also exhibited a much lower threshold for maximal stimulation; 2-deoxyglucose uptake decreased about 50% after 48 h of incubation without glucose. Studies on the secretion of newly synthesized proinsulin/insulin revealed that up to 80% of the total cellular pool of labeled proinsulin was released during a 60-min chase compared to only 10% of labeled insulin. The release of immunoreactive insulin (IRI) during the chase period was stimulated by forskolin and phorbol-12-myristate-13-acetate 1.6- and 10-fold, respectively. However, the release of newly synthesized proinsulin was insensitive to these secretagogues. It is concluded that 1) as in normal islets, glucose influences the steady state levels of proinsulin mRNA in beta TC3 cells; 2) the rate of proinsulin biosynthesis reflects only the level of insulin mRNA; translational control is absent; 3) cellular conversion of proinsulin to insulin is up-regulated by glucose as in normal rat islets; 4) newly synthesized proinsulin is released predominantly via a constitutive, rather than a regulated pathway, in contrast to normal beta-cells.
Actins↗
Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes.
The biosynthesis and post-translational maturation of PC2, a neuroendocrine-specific Kex2-like endoprotease, following expression in Xenopus oocytes is described. The initial translation product was a 75-kDa membrane-associated protein which was released from the oocytes as a glycosylated 71-kDa protein. During extended chase periods, the extracellular 71-kDa protein was converted to a mature 68-kDa product. A deletion mutant lacking a putative COOH-terminal amphipathic helix was still membrane-associated, suggesting that this domain was not essential for attachment of PC2 to membranes. Two putative proregion cleavage site mutants were also constructed. Conversion of the 75-kDa peptide to the 71-kDa peptide involved cleavage at the sequence Lys-Arg-Arg-Arg (amino acids 78-81), since mutation of this sequence to Lys-Val-Arg-Leu resulted in the secretion of the 75-kDa peptide. Extracellular conversion of the 71-kDa peptide to the 68-kDa peptide involved cleavage at the sequence Arg-Lys-Lys-Arg (amino acids 106-109), since deletion of this tetrabasic sequence resulted in secretion of the 71-kDa peptide without further conversion to the 68-kDa form. Finally, a mutation which changed a catalytically important Asp to Asn did not affect processing of proPC2. These results may be relevant to our understanding of mechanisms in the intracellular sorting and maturation of proPC2 in neuroendocrine cells.
Amino Acid Sequence↗
Biosynthesis of islet amyloid polypeptide. Elevated expression in mouse beta TC3 cells.
Islet amyloid polypeptide (IAPP) messenger RNA levels, biosynthesis, processing, and secretion were studied in cultured mouse beta TC3 insulinoma cells. Northern blot analysis revealed that the size of IAPP mRNA (0.9 kb) in beta TC3 cells was the same as that in normal mouse islets; IAPP mRNA was approximately 60% of the level of insulin mRNA in beta TC3 cells. However, the ratio of synthesis of insulin to IAPP was approximately 6:1, suggesting that IAPP mRNA is not translated efficiently in these cells. Metabolic labeling of beta TC3 cells with [3H]leucine revealed the synthesis of both a precursor form of IAPP (pro-IAPP) of apparent Mr 7400 and a mature form (IAPP) of apparent Mr 3900. In pulse-chase experiments, pro-IAPP could be shown to be processed to IAPP in a manner similar to proinsulin. The t1/2 for conversion of pro-IAPP to IAPP was about 25 min, faster than the t1/2 for proinsulin to insulin of 70 min. A significant proportion of newly synthesized IAPP and insulin precursors were secreted via a constitutive pathway from beta TC3 cells. Possible effects of dexamethasone and forskolin on IAPP mRNA levels and biosynthesis were examined but no effects were observed. In conclusion, the IAPP gene is strongly expressed in beta TC3 cells leading to the biosynthesis, proteolytic processing, and secretion of IAPP, a putative islet hormone.
Amyloid↗
Characterization of PC2, a mammalian Kex2 homologue, following expression of the cDNA in microinjected Xenopus oocytes.
A human insulinoma cDNA (PC2) that encodes a protein homologous to the Kex2/subtilisin-like proteinases has recently been described [1990, J. Biol. Chem. 265, 2997-3000]. In order to characterise the associated proteinase activity, mRNA encoding PC2 was synthesised in vitro and microinjected into Xenopus oocytes. The proteinase activity released into the media from oocytes microinjected with PC2 mRNA was assayed using small peptide fluorogenic substrates. Boc.Gln.Arg.Arg aminomethyl coumarin was hydrolysed in a Ca(2+)-dependent manner, but substrate analogues bearing a single basic aminoacid were not. The substrate specificity, inhibitor profile, and pH optimum of 5.5 were compatible with an involvement of PC2 in prohormone processing in mammalian cells.
Animals↗
Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes.
Two mammalian gene products, PC2 and PC3, have been proposed as candidate neuroendocrine-precursor processing enzymes based on the structural similarity of their catalytic domains to that of the yeast precursor-processing endoprotease Kex2. In this report we demonstrate that these two proteases can cleave proopiomelanocortin (POMC) in the secretory pathway of mammalian cells. Similarly to pituitary corticotrophs, PC3 expressed in processing-deficient BSC-40 cells cleaved native mouse POMC at the -Lys-Arg- sites flanking corticotropin. The -Lys-Arg- within beta-lipotropin was less efficiently cleaved to release beta-endorphin. Expression of PC2 together with PC3 resulted in efficient conversion of beta-lipotropin, as occurs in pituitary melanotrophs. Furthermore, coexpression of PC2 together with mouse POMC in bovine adrenomedullary chromaffin cells resulted in conversion of beta-lipotropin to gamma-lipotropin and beta-endorphin in the regulated secretory pathway. Finally, the processing selectivities of PC3 and PC2 expressed together in BSC-40 cells were determined by using a series of mutant mouse POMCs containing all possible pairs of basic residues at certain sites. The observed pattern of cleavage site selectivities mimicked that of the endogenous endoproteases of the insulinoma and bovine adrenomedullary chromaffin cells, suggesting that PC2 and PC3 may represent important core endoproteases in the catalysis of prohormone processing in many neuroendocrine cell types.
Adrenocorticotropic Hormone↗
Evolution of the insulin gene superfamily. Sequence of a preproinsulin-like growth factor cDNA from the Atlantic hagfish.
Complementary DNAs encoding a preproinsulin-like growth factor (prepro-IGF) have been cloned from a primitive vertebrate species, the Atlantic hagfish, by using a DNA amplification strategy based on the polymerase chain reaction. A composite sequence containing a 414-nucleotide open reading frame encoding 138 amino acids and 164 nucleotides in the 3'-untranslated region was obtained. The deduced partial sequence of hagfish prepro-IGF reveals that it is organized like the mammalian prepro-IGFs with an unusually large (greater than 39-amino acid) signal peptide (initiator methionine residue is missing), 29-amino acid B, 15-amino acid C, 21-amino acid A, 10-amino acid D, and 26-amino acid E domains. All the invariant residues necessary to form the correct tertiary fold of an insulin-like molecule have been conserved in hagfish IGF. Sequence comparisons revealed that the A and B domains of hagfish IGF are equally similar to those of human IGF-I (35 out of 50 amino acids) or IGF-II (37 out of 53 amino acids). In contrast, the similarity between hagfish and mammalian pro-IGFs in the C, D, and E domains is relatively low. Northern blot analysis of RNA isolated from hagfish brain, heart, liver, skeletal muscle, and islet organ, however, indicated that hagfish IGF, like mammalian IGF-I, is expressed predominantly in the liver as a 4.2-kilobase transcript. DNA blot analysis revealed that hagfish IGF is a single copy gene. The predicted sequence of hagfish prepro-IGF thus demonstrates that the divergence of the IGF and insulin genes occurred prior to the separation of the Agnatha and that the organization and tertiary structure of IGF have been well maintained throughout 550 million years of vertebrate evolution.
Amino Acid Sequence↗
Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans.
PC2 and furin are two recently identified members of a class of mammalian proteins homologous to the yeast precursor processing protease kex2 and the bacterial subtillisins. We have used the polymerase chain reaction to identify and clone a cDNA (PC3) from the mouse AtT20 anterior pituitary cell line that represents an additional member of this growing family of mammalian proteases. PC3 encodes a 753-residue protein that begins with a signal peptide and contains a 292-residue domain closely related to the catalytic modules of PC2, furin, and kex2. Within this region 58%, 65%, and 50% of the amino acids of PC3 are identical to those of the aligned PC2, furin, and kex2 sequences, respectively, and the catalytically important Asp, His, and Ser residues are all conserved. On Northern blots, PC3 hybridizes to two transcripts of 3 and 5 kilobases. Tissue distribution studies indicate that both PC2 and PC3 are expressed in a variety of neuroendocrine tissues, including pancreatic islets and brain, but are not expressed in liver, kidney, skeletal muscle, and spleen. The high degree of similarity of PC3, PC2, and furin suggests that they are all members of a superfamily of mammalian proteases that are involved in the processing of prohormones and/or other protein precursors. In contrast to furin, PC3, like PC2, lacks a hydrophobic transmembrane anchor, but it has a potential C-terminal amphipathic helical segment similar to the putative membrane anchor of carboxypeptidase H. These and other differences suggest that these proteins carry out compartmentalized proteolysis within cells, such as processing within regulated versus constitutive secretory pathways.
Adrenal Gland Neoplasms↗
Sequence and functional expression in Xenopus oocytes of a human insulinoma and islet potassium channel.
Regulation of insulin secretion involves the coordinated control of ion channels in the beta-cell membrane. We have isolated and characterized cDNA and genomic clones encoding a voltage-dependent K+ channel isoform expressed in human islets and in a human insulinoma. This K+ channel isoform, designated hPCN1, with a deduced amino acid sequence of 613 residues (Mr = 67,097), is related to the Shaker family of Drosophila K+ channels. hPCN1 is homologous to two other human K+ channel isoforms we have isolated, hPCN2 and hPCN3, with 55% and 65% amino acid sequence identity, respectively. The electrophysiological characteristics of hPCN1 were determined after microinjection of synthetic RNA into Xenopus oocytes. Two-microelectrode voltage-clamp recordings of oocytes injected with hPCN1 RNA revealed a voltage-dependent outward K+ current that inactivated slowly with time. Outward currents were inhibited by 4-aminopyridine with a Ki less than 0.10 mM and were relatively insensitive to tetraethylammonium ion or Ba2+. A delayed rectifier K+ channel such as hPCN1 could restore the resting membrane potential of beta cells after depolarization and thereby contribute to the regulation of insulin secretion.
Amino Acid Sequence↗
Sequences of islet amyloid polypeptide precursors of an Old World monkey, the pig-tailed macaque (Macaca nemestrina), and the dog (Canis familiaris).
The 37-amino acid islet amyloid polypeptide represents the major protein component present in islet amyloid deposits. Although the presence of islet amyloid is a characteristic pathological feature of the islets of humans, monkeys and cats with Type 2 (non-insulin-dependent) diabetes mellitus, it is not found in the islets of diabetic rats, mice or dogs. To further explore the molecular basis for these species differences in amyloid deposition we have used a polymerase chain reaction based method to clone cDNAs encoding the monkey (Macaca nemestrina) and dog (Canis familiaris) islet amyloid polypeptide precursors. The predicted amino acid sequence of the monkey precursor is 96% identical to that of the human protein; differences include one replacement in the signal peptide and three in the islet amyloid polypeptide domain. The sequence of the dog precursor is most closely related to that of the cat protein (85% identity); the sequences of dog and cat islet amyloid polypeptide differ only at two positions and are identical in the region of amino acids 20-29, the region thought to be primarily responsible for amyloidogenesis. Thus, amino acid residues in addition to those at positions 20-29 may facilitate the aggregation of islet amyloid polypeptide. The presence of amyloid deposits in some dog pancreatic endocrine tumours suggests that the dog protein can be amyloidogenic, perhaps due to elevated expression of islet amyloid polypeptide by the tumours relative to normal islets.
Amino Acid Sequence↗