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K Docherty

Publications and source records attributed to K Docherty.

At least 55 records · Page 3Linked to original sources
PubMed1996

Processing of pro-islet amyloid polypeptide (proIAPP) by the prohormone convertase PC2.

Islet amyloid polypeptide (IAPP), 'amylin', is the component peptide of islet amyloid formed in Type 2 diabetes. IAPP is expressed in islet beta-cells and is derived from a larger precursor, proIAPP, by proteolysis. An in vitro translation/translocation system was used to separately examine processing of human proIAPP by the beta-cell endopeptidases PC2, PC3 or furin. ProIAPP was converted to mature IAPP by PC2 but there was little conversion by furin or PC3. These data are consistent with processing of proIAPP in beta-cell secretory granules. Abnormal cellular proteolysis associated with type 2 diabetes could contribute to IAPP amyloidosis.

Amino Acid Sequence↗
PubMed1995

A silencer and an adjacent positive element interact to modulate the activity of the human insulin promoter.

A negative regulatory element (NRE) is located between positions -279 and -261 relative to the transcription start site of the human insulin gene. The NRE contains at least three distinct overlapping binding sites for several nuclear proteins. These proteins may be distinguished by their interaction with mutant variants of the NRE. Mutagenesis of two of these protein-binding sites within an insulin gene fragment containing sequences from position -361 to position +112 attenuated the negative activity of the NRE, confirming the importance of these sites in the function of the NRE. When placed in isolation upstream of the herpes-simplex-virus thymidine-kinase promoter, the NRE exhibited stimulatory activity in non-beta (BHK) and beta (HIT) cells. The positive activity within the NRE was mapped to a sequence that resembled the binding site for the ubiquitous factor Oct-1. These results indicated that the negative activity of the NRE was dependent on its interaction with other regulatory sites within the insulin gene. Thus, the NRE exhibited negative activity in transfected HIT cells when placed upstream of an insulin gene fragment (positions -261 to +112). However, its activity was modulated in a positive manner by inclusion of additional sequences from position -279 to -341. This region contains the CT3 box that binds the homeodomain protein IUF1 (insulin upstream factor 1). The NRE resembled a silencer, being at least partly independent of precise location and orientation, and being able to operate upon a variety of promoters. The role of the NRE is unclear, although it may be involved in restricting expression of the insulin gene to cells of the islets of Langerhans.

Animals↗
PubMed1995

Identification and characterization of a functional retinoic acid/thyroid hormone-response element upstream of the human insulin gene enhancer.

A deletion analysis of the human insulin gene extending to 2 kb upstream of the transcription start site provided evidence of regulatory sequences located upstream of the insulin-linked polymorphic region (ILPR). Within this ILPR-distal region is a sequence (Ink, for insulin kilobase upstream) which contains three potential nuclear hormone-receptor half-sites, closely matching the consensus sequence AGGTCA. These sequences are arranged as a palindromic element with zero spacing over-lapping a direct repeat with 2 bp spacing. The Ink sequence was used in electrophoretic mobility-shift assays within nuclear extracts from COS-7 cells overexpressing the vitamin D, thyroid hormone or retinoic acid receptors, or from an insulin-expressing hamster cell line, HIT-T15. These studies suggest that the insulin-expressing cell line contains thyroid hormone and retinoic acid receptors at least, and that these receptors are able to recognize the Ink sequence. Three copies of the Ink sequence were placed upstream of the thymidine kinase promoter and firefly luciferase reporter gene. In COS-7 cells expressing the appropriate nuclear hormone receptor, this construct was responsive to both thyroid hormone (18-fold) and all-trans-retinoic acid (31-fold). In HIT-T15 cells the same construct responded to all-trans-retinoic acid, but not to thyroid hormone. Within the context of a 2 kb insulin gene fragment, the Ink sequence was shown to be activated by retinoic acid and by the retinoic acid receptor, but acted as a negative element in the presence of both retinoic acid and the retinoic acid receptor. Mutagenesis studies demonstrated that the palindromic sequence was important for the retinoic acid response, and for binding of complexes containing retinoic acid receptor. In human islets of Langerhans, retinoic acid was shown to stimulate insulin mRNA levels. These results demonstrate that a functional nuclear hormone-receptor-response element is located upstream of the human ILPR. As retinoic acid and thyroid hormone are frequently involved in developmental regulatory processes, it is possible that this element may be important in the process of islet cell differentiation.

Animals↗
PubMed1995

Differences between the catalytic properties of recombinant human PC2 and endogenous rat PC2.

Human prohormone convertase PC2 was expressed in Xenopus oocytes and its properties were compared with those of the Type-2 endopeptidase of rat insulin secretory granules, previously identified as PC2 [Bennett, Bailyes, Nielson, Guest, Rutherford, Arden and Hutton (1992) J. Biol. Chem. 267, 15229-15236]. Recombinant PC2 had the same substrate specificity as the Type-2 endopeptidase, cleaving at the CA-junction (Lys64, Arg65) of human des-31,32-proinsulin to generate insulin; little activity was found toward human des-64,65-proinsulin or proinsulin itself. Recombinant PC2 was maximally active in 5-7 mM Ca2+ (K0.5 = 1.6 mM) whereas the Type-2 endopeptidase was maximally active in 0.5-1 mM Ca2+ (K0.5 = 40 microM). Both enzymes had a pH optimum of 5.0-5.5 but the Type-2 endopeptidase was active over a wider pH range. Two molecular forms of recombinant PC2 (71 kDa and 68 kDa) were found, both had an intact C-terminus but differed by the presence of the propeptide. The endogenous PC2 comprised several overlapping forms (size range 64-68 kDa), approximately two-thirds of which lacked C-terminal immunoreactivity. Part of the size difference between recombinant and endogenous PC2 was attributable to differences in N-glycosylation. The different post-translational proteolytic modifications of recombinant and endogenous PC2 did not account for the different pH and Ca2+ sensitivities shown by the enzymes. A modulating effect of carbohydrate on enzyme activity could not be excluded.

Animals↗
PubMed1995

The insulin enhancer binding site 2 (IEB2; FAR) box of the insulin gene regulatory region binds at least three factors that can be distinguished by their DNA binding characteristics.

Located at approximately 230 bp upstream from the transcription start site, the insulin enhancer binding site 2 (IEB2) or FAR region of the insulin gene is one of several important sequences involved in regulating transcription of the gene. The present study was undertaken to characterize the transcription factors binding at the IEB2/FAR region of the rat insulin II gene and to compare these with factors known to bind to the equivalent sequence in the rat I and human insulin genes. An endocrine-enriched factor, EFD3, was identified, which bound to the sequence CAGGAG. A second factor (D4) was identified as the widely expressed factor USF (upstream stimulating factor), while a third factor (D5) remained largely uncharacterized. The binding affinities of these three factors differed in the three genes, suggesting that the role of the IEB2/FAR sequence may vary subtly between the rat insulin II, rat insulin I and human insulin genes.

Animals↗
PubMed1995

Differences in pH optima and calcium requirements for maturation of the prohormone convertases PC2 and PC3 indicates different intracellular locations for these events.

PC2 and PC3, which is also known as PC1, are subtilisin-like proteases that are involved in the intracellular processing of prohormones and proneuropeptides. Both enzymes are synthesized as propolypeptides that undergo proteolytic maturation within the secretory pathway. An in vitro translation/translocation system from Xenopus egg extracts was used to investigate mechanisms in the maturation of pro-PC3 and pro-PC2. Pro-PC3 underwent rapid (t1/2 < 10 min) processing of the 88-kDa propolypeptide at the sequence RSKR83 to generate the 80-kDa active form of the enzyme. This processing was blocked when the active site aspartate was changed to asparagine, suggesting that an autocatalytic mechanism was involved. In this system, processing of pro-PC3 was optimal between pH 7.0 and 8.0 and was not dependent on additional calcium. These results are consistent with pro-PC3 maturation occurring at an early stage in the secretory pathway, possibly within the endoplasmic reticulum, where the pH would be close to neutral and the calcium concentration less than that observed in later compartments. Processing of pro-PC2 in the Xenopus egg extract was much slower than that of pro-PC3 (t1/2 = 8 h). It exhibited a pH optimum of 5.5-6.0 and was dependent on calcium (K0.5 = 2-4 mM). The enzymatic properties of pro-PC2 processing were similar to that of the mature enzyme. Further studies using mutant pro-PC2 constructs suggested that cleavage of pro-PC2 was catalyzed by the mature 68-kDa PC2 molecule. The results were consistent with pro-PC2 maturation occurring within a late compartment of the secretory pathway that contains a high calcium concentration and low pH.

Amino Acid Sequence↗
PubMed1994

Glucose modulates the binding activity of the beta-cell transcription factor IUF1 in a phosphorylation-dependent manner.

In the human insulin gene, three regulatory sequences upstream of the transcription start site at -77 (the CT1 box), -210 (the CT2 box), and -315 (the CT3 box) bind a beta-cell-specific transcription factor, IUF1. Recent studies have mapped a glucose response element to a CT-like sequence in the rat insulin I gene. The present study was therefore undertaken to ascertain the role of IUF1 in glucose-stimulated insulin gene transcription. IUF1-binding activity was measured by electrophoretic mobility shift assay using the CT2 box as probe. When freshly isolated rat islets of Langerhans were incubated in medium containing low concentrations (3 mM) of glucose IUF1 activity fell to undetectable levels within 6 h. In high (20 mM) glucose IUF1 activity remained constant over a 24 h period. The loss of IUF1 activity was reversible. Thus when islets were incubated for 4 h in low glucose and transferred to high glucose, IUF1 levels recovered within 15 min. This effect was dependent on glucose metabolism as it was inhibited by mannoheptulose. Incubation of islets for 4 h in low concentrations of glucose supplemented with phosphatase inhibitors prevented the fall in IUF1 activity. No recovery in IUF1 activity was observed when islets were treated for 4 h with low glucose and then for a further 1 h with low glucose and dibutyryl cyclic AMP, or forskolin, or the phorbol ester phorbol 12-myristate 13-acetate. These results demonstrate that the IUF1-binding activity in islets of Langerhans is modulated by glucose in a phosphorylation-dependent manner, and that protein kinase A or protein kinase C are not involved. Finally, IUF1 was shown to be immunologically related to a recently cloned factor, IPF1, that binds to a CT-like sequence in the rat insulin I gene promoter.

Animals↗
PubMed1994

Calcium- and pH-dependent aggregation and membrane association of the precursor of the prohormone convertase PC2.

PC2 is a subtilisin-like protease which is thought to be involved in the processing of prohormones and proneuropeptides in neuroendocrine cells. The mature 68-kDa enzyme is generated by intracellular proteolytic processing of a 75-kDa pro-PC2 polypeptide. Neuroendocrine cells contain at least two secretory pathways: the regulated pathway whereby secreted products are concentrated, stored in granules, and released in response to external stimulation of the cell, and the constitutive pathway, whereby secretory and plasma membrane proteins are continuously transported to the cell surface without prior concentration or storage. An important step in the sorting of proteins into these pathways is thought to involve the aggregation of proteins destined for storage granules. To define the mechanisms in the intracellular sorting of PC2 to secretory vesicles, the present study was undertaken to investigate the aggregation and membrane association properties of precursor and mature forms of PC2. Using material expressed in microinjected Xenopus oocytes, it was demonstrated that the 75-kDa pro-PC2 polypeptide undergoes calcium- and acid pH-dependent aggregation. Calcium exhibited an effect on the aggregation of pro-PC2 at pH 7.0 and 6.5, whereas below 6.5 aggregation was independent of calcium. Association of pro-PC2 with membranes was observed at pH 5.5, but not at pH 7.0, 6.5, nor 6.0. The mature 68-kDa PC2 polypeptide remained soluble under conditions that caused aggregation and membrane association of the 75-kDa propolypeptide. Deletion of COOH-terminal sequences had no effect on the association of pro-PC2 with membranes, whereas a peptide corresponding to amino acids 57-85 of the propeptide was able to partially compete the membrane associated pro-PC2 away from the membranes.

Amino Acid Sequence↗
PubMed1994

Autocatalytic maturation of the prohormone convertase PC2.

PC2 is a member of the eukaryotic family of subtilisin-like proteases, which is thought to participate in the processing of prohormones and proneuropeptides in neuroendocrine cells. PC2 is synthesized as a 69-kDa prepropolypeptide. The NH2-terminal signal sequence is removed during segregation within the endoplasmic reticulum, where glycosylation occurs to generate a 75-kDa propolypeptide. A combination of site-directed mutagenesis and a cell-free translation/translocation system from Xenopus eggs was used to investigate the processing of the pro-PC2 precursor. The 75-kDa polypeptide underwent slow cleavage after the sequence Arg-Lys-Lys-Arg84 to generate a 68-kDa mature enzyme. Cleavage was blocked when the tetrabasic sequence was deleted (PC2M3) or when the active site Asp142 was changed to Asn (PC2M4). This latter observation suggested that cleavage of the 75-kDa propolypeptide to the mature 68-kDa enzyme was autocatalytic. Incubation of the PC2M4 mutant with the wild type PC2 precursor resulted in cleavage of both the wild type polypeptide and the catalytically inactive PC2M4 mutant. This indicates that cleavage could occur through an intermolecular reaction. The results also demonstrate that the novel Xenopus egg extract translation/translocation system represents a powerful cell-free method for studying proteolytic processing of propolypeptides.

Amino Acid Sequence↗
PubMed1994

Nutrient regulation of insulin gene expression.

The beta cell of the islets of Langerhans contributes along with other factors to glucose homeostasis by sensing changes in the plasma glucose concentrations and adjusting the rate of insulin production and release. Over short periods of time, insulin production is controlled principally through translation of pre-existing mRNA. Over longer periods, insulin mRNA levels are modulated through effects on the rate of transcription of the insulin gene, and also through changes in the rate of decay of insulin mRNA. These long-term effects may be important in allowing the beta cell to adapt to changes in diet or periods of fasting. Several mechanisms involved in the control of the rate of translation of insulin mRNA have been described. Effects of glucose metabolism on the turnover of insulin mRNA have yet to be characterized in detail. At the level of transcription, cis-acting DNA elements and trans-acting factors involved in the transient response of the insulin gene to changes in intracellular cAMP levels, or to signals generated as a result of glucose metabolism, have been identified.

Animals↗
PubMed1994

Synthesis and processing in vivo of the novel mouse thyrotropin beta-presubunit that contains an NH2-terminal extension sequence.

Expression of the single mouse TSH beta gene gives rise to multiple mRNAs, and we have previously shown that in vitro, one of these mRNAs gives rise to a novel TSH beta-presubunit due to initiation of translation at an in-frame start site unique to this mRNA which is up-stream of the normal start site. The novel presubunit contains a 17-amino acid NH2-terminal extension sequence compared to the normal presubunit. Although this extension sequence does not have the characteristics of a normal signal sequence, the novel TSH beta-presubunit was processed in vitro by microsomal membranes. In this study we have examined the translation product of this mRNA in intact cells and whether in vivo it gives rise to a processed secreted TSH beta-subunit that has an NH2-terminal sequence different from that of the established TSH beta-subunit. Firstly, mRNAs encoding alpha-presubunit and either the normal or novel TSH beta-presubunit were microinjected into Xenopus oocytes, and it was found that immunoprecipitable TSH dimer was secreted into the medium regardless of the mRNA used for TSH beta-subunit synthesis. However, less TSH was obtained when the TSH beta-subunit was derived from the extended TSH beta-presubunit. Secondly, when COS cells were transiently transfected with plasmids expressing alpha-presubunit and either the normal or novel TSH beta-presubunit, secreted TSH was obtained when the TSH beta-subunit was derived from either presubunit. TSH dimer was also obtained when the TSH beta-presubunit was derived from a mRNA encoding the extended presubunit in which the down-stream AUG had been eliminated by site-specific mutagenesis. This demonstrated that the up-stream translation start site was used in the intact cell and that secreted TSH beta-subunit was derived from the extended presubunit and not from normal presubunit resulting from translational readthrough to the down-stream AUG. When secreted TSH beta-subunits derived from the normal and extended TSH beta-presubunits were digested with endoproteinase LysC, the NH2-terminal fragments were similar in size, suggesting that the NH2-terminal extension had little if any effect on the site of cleavage by signal peptidase. Our data, therefore, demonstrate that the longer TSH beta-presubunit is synthesized in vivo and strongly suggest that it is processed in the intact cell to give a mature secreted TSH beta-subunit indistinguishable from that derived from the normal TSH beta-presubunit.

Amino Acid Sequence↗