Thyroid hormone receptor-associated proteins and general positive cofactors mediate thyroid hormone receptor function in the absence of the TATA box-binding protein-associated factors of TFIID.
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Coactivators previously implicated in ligand-dependent activation functions by thyroid hormone receptor (TR) include p300 and CREB-binding protein (CBP), the steroid receptor coactivator-1 (SRC-1)-related family of proteins, and the multicomponent TR-associated protein (TRAP) complex. Here we show that two positive cofactors (PC2 and PC4) derived from the upstream stimulatory activity (USA) cofactor fraction act synergistically to mediate thyroid hormone (T3)-dependent activation either by TR or by a TR-TRAP complex in an in vitro system reconstituted with purified factors and DNA templates. Significantly, the TRAP-mediated enhancement of activation by TR does not require the TATA box-binding protein-associated factors of TFIID. Furthermore, neither the pleiotropic coactivators CBP and p300 nor members of the SRC-1 family were detected in either the TR-TRAP complex or the other components of the in vitro assay system. These results show that activation by TR at the level of naked DNA templates is enhanced by cooperative functions of the TRAP coactivators and the general coactivators PC2 and PC4, and they further indicate a potential functional redundancy between TRAPs and TATA box-binding protein-associated factors in TFIID. In conjunction with earlier studies on other nuclear receptor-interacting cofactors, the present study also suggests a multistep pathway, involving distinct sets of cofactors, for activation of hormone responsive genes. (+info)
The neuroendocrine protein 7B2 is required for peptide hormone processing in vivo and provides a novel mechanism for pituitary Cushing's disease.
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The neuroendocrine protein 7B2 has been implicated in activation of prohormone convertase 2 (PC2), an important neuroendocrine precursor processing endoprotease. To test this hypothesis, we created a null mutation in 7B2 employing a novel transposon-facilitated technique and compared the phenotypes of 7B2 and PC2 nulls. 7B2 null mice have no demonstrable PC2 activity, are deficient in processing islet hormones, and display hypoglycemia, hyperproinsulinemia, and hypoglucagonemia. In contrast to the PC2 null phenotype, these mice show markedly elevated circulating ACTH and corticosterone levels, with adrenocortical expansion. They die before 9 weeks of severe Cushing's syndrome arising from pituitary intermediate lobe ACTH hypersecretion. We conclude that 7B2 is indeed required for activation of PC2 in vivo but has additional important functions in regulating pituitary hormone secretion. (+info)
The differential processing of proenkephalin A in mouse and human breast tumour cell lines.
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We have carried out an investigation into the processing of the enkephalin-like immunoreactivity reported in breast tissue using two human breast tumour cell lines and a mouse tumour cell line. A 46 kDa form of proenkephalin (PE) has been observed in the cell lysates of two human breast tumour cell lines (MCF-7, ZR-75-1) and the mouse androgen-responsive Shionogi breast carcinoma cell line (SC115). PE processing in the cell lysates of these cells was assessed by a specific met-enkephalin RIA. The basal levels of processed PE in the MCF-7, ZR-75-1 and SC115 cell lysates were 30, 30 and 76% respectively. The processing enzymes PC1 and PC2, which have been implicated in the differential processing of PE, were detected by immunoblot analysis in these cells. PC1 was found within the cell extracts of all three cell lines. PC2 was only observed in the SC115 cell line, which may account for the higher percentage of processed PE measured. The cDNA of PC2 has been transfected into ZR-75-1 cells and this was accompanied by an increase in the level of processed PE from 30 to 76%. These breast tumour cell lines may provide a useful insight into the function of enkephalin-containing peptides in breast cancer. (+info)
Long-term elevation of free fatty acids leads to delayed processing of proinsulin and prohormone convertases 2 and 3 in the pancreatic beta-cell line MIN6.
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To explore the role of chronically elevated free fatty acids (FFAs) in the pathogenesis of the hyperproinsulinemia of type 2 diabetes, we have investigated the effect of FFAs on proinsulin processing and prohormone convertases PC2 and PC1/PC3 in MIN6 cells cultured in Dulbecco's modified Eagle's medium with or without 0.5 mmol/l FFA mixture (palmitic acid:oleic acid = 1:2). After 7 days of culture, the percent of proinsulin in FFA-exposed cells was increased (25.9 +/-0.3% intracellular and 75.4 +/- 1.2% in medium vs. 13.5 +/-0.2 and 56.2 +/- 4.1%, respectively, in control cells). The biosynthesis and secretion of proinsulin and insulin were analyzed by comparing the incorporation of [3H]Leu and [35S]Met. In pulse-chase studies, proinsulin-to-insulin conversion was inhibited, and proinsulin in the medium was increased by 50% after 3 h of chase, while insulin secretion was decreased by 50% after FFA exposure. Levels of cellular PC2 and PC3 analyzed by Western blotting were decreased by 23 and 15%, respectively. However, PC2, PC3, proinsulin, and 7B2 mRNA levels were not altered by FFA exposure. To test for an effect on the biosynthesis of PC2, PC3, proinsulin, and 7B2, a protein required for PC2 activation, MIN6 cells were labeled with [35S]Met for 10-15 min, followed by a prolonged chase. Most proPC2 was converted after 6 h of chase in control cells, but conversion was incomplete even after 6 h of chase in FFA-exposed MIN6 cells. Media from chase incubations showed that FFA-exposed cells secreted more proPC2 than controls. Similar inhibitory effects were noted on the processing of proPC3, proinsulin, and 7B2. In conclusion, prolonged exposure of beta-cells to FFAs may affect the biosynthesis and posttranslational processing of proinsulin, PC2, PC3, and 7B2, and thereby contribute to the hyperproinsulinemia of type 2 diabetes. The mechanism of inhibition of secretory granule processing by FFAs may be through changes in Ca2+ concentration, the pH in the secretory granules, and/or other factors that may influence the activation and function of the convertases. (+info)
Exposure of human islets to cytokines can result in disproportionately elevated proinsulin release.
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Infiltration of immunocytes into pancreatic islets precedes loss of beta cells in type 1 diabetes. It is conceivable that local release of cytokines affects the function of beta cells before their apoptosis. This study examines whether the elevated proinsulin levels that have been described in prediabetes can result from exposure of beta cells to cytokines. Human beta-cell preparations were cultured for 48 or 72 hours with or without IL-1beta, TNF-alpha, or IFN-gamma, alone or in combination. None of these conditions were cytotoxic, nor did they reduce insulin biosynthetic activity. Single cytokines did not alter medium or cellular content in insulin or proinsulin. Cytokine combinations, in particular IL-1beta plus IFN-gamma, disproportionately elevated medium proinsulin levels. This effect expresses an altered functional state of the beta cells characterized by preserved proinsulin synthesis, a slower hormone conversion, and an increased ratio of cellular proinsulin over insulin content. The delay in proinsulin conversion can be attributed to lower expression of PC1 and PC2 convertases. It is concluded that disproportionately elevated proinsulin levels in pre-type 1 diabetic patients might result from exposure of their beta cells to cytokines released from infiltrating immunocytes. This hormonal alteration expresses an altered functional state of the beta cells that can occur independently of beta-cell death. (+info)
A 36-residue peptide contains all of the information required for 7B2-mediated activation of prohormone convertase 2.
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The prohormone convertases (PCs) are serine proteinases responsible for the processing of secretory protein precursors. PC2 is the only member of this family whose activation requires intracellular interaction with a helper protein, the neuroendocrine protein 7B2. In order to gain a better understanding of the mechanism of proPC2 activation, we have characterized the structural determinants of 7B2 required for proPC2 activation. We had already identified a proline-rich binding determinant in the 21-kDa domain, the portion of 7B2 responsible for proPC2 activation. We have now investigated the function of the weakly conserved amino-terminal portion of 21-kDa 7B2 by sequential deletions. Mutant proteins were analyzed in four assays: binding to proPC2, facilitation of proPC2 maturation, and activation of proPC2 in vivo and in vitro. We found that the amino-terminal half of 7B2 is not involved in proPC2 activation, and we identified an active 36-residue peptide that contains the previously characterized proline-rich sequence as well as an alpha-helix and the only disulfide bond of 7B2. Mutation of the alpha-helix and of the cysteines demonstrated that these determinants are absolutely required for PC2 activation. Thus, the 186-residue full-length 7B2 rat protein can be functionally reduced to an internal segment of only 36 residues. (+info)
A role for amontillado, the Drosophila homolog of the neuropeptide precursor processing protease PC2, in triggering hatching behavior.
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Accurate proteolytic processing of neuropeptide and peptide hormone precursors by members of the kexin/furin family of proteases is key to determining both the identities and activities of signaling peptides. Here we identify amontillado (amon), the Drosophila melanogaster homolog of the mammalian neuropeptide processing protease PC2, and show that in contrast to vertebrate PC2, amontillado expression undergoes extensive regulation in the nervous system during development. In situ hybridization reveals that expression of amontillado is restricted to the final stages of embryogenesis when it is found in anterior sensory structures and in only 168 cells in the brain and ventral nerve cord. After larvae hatch from their egg shells, the sensory structures and most cells in the CNS turn off or substantially reduce amontillado expression, suggesting that amontillado plays a specific role late in embryogenesis. Larvae lacking the chromosomal region containing amontillado show no gross anatomical defects and respond to touch. However, such larvae show a greatly reduced frequency of a hatching behavior of wild-type Drosophila in which larvae swing their heads, scraping through the eggshell with their mouth hooks. Ubiquitous expression of amontillado can restore near wild-type levels of this behavior, whereas expression of amontillado with an alanine substitution for the catalytic histidine cannot. These results suggest that amontillado expression is regulated as part of a programmed modulation of neural signaling that controls hatching behavior by producing specific neuropeptides in particular neurons at an appropriate developmental time. (+info)
Activation and routing of membrane-tethered prohormone convertases 1 and 2.
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Many peptide hormones and neuropeptides are processed by members of the subtilisin-like family of prohormone convertases (PCs), which are either soluble or integral membrane proteins. PC1 and PC2 are soluble PCs that are primarily localized to large dense core vesicles in neurons and endocrine cells. We examined whether PC1 and PC2 were active when expressed as membrane-tethered proteins, and how tethering to membranes alters the biosynthesis, enzymatic activity, and intracellular routing of these PCs. PC1 and PC2 chimeras were constructed using the transmembrane domain and cytoplasmic domain of the amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The membrane-tethered PCs were rerouted from large dense core vesicles to the Golgi region. In addition, the chimeras were transiently expressed at the cell surface and rapidly internalized to the Golgi region in a fashion similar to PAM. Membrane-tethered PC1 and PC2 exhibited changes in pro-domain maturation rates, N-glycosylation, and in the pH and calcium optima required for maximal enzymatic activity against a fluorogenic substrate. In addition, the PC chimeras efficiently cleaved endogenous pro-opiomelanocortin to the correct bioactive peptides. The PAM transmembrane domain/cytoplasmic domain also prevented stimulated secretion of pro-opiomelanocortin products in AtT-20 cells. (+info)