Proteasomal degradation of N-acetyltransferase 1 is prevented by acetylation of the active site cysteine - A mechanism for the slow acetylator phenotype and substrate-dependent down-regulation

Many drugs and chemicals found in the environment are either detoxified by N-acetyltransferase 1 (NAT1, EC 2.3.1.5) and eliminated from the body or bioactivated to metabolites that have the potential to cause toxicity and/or cancer. NAT1 activity in the body is regulated by genetic polymorphisms as well as environmental factors such as substrate-dependent down-regulation and oxidative stress. Here we report the molecular mechanism for the low protein expression from mutant NAT1 alleles that gives rise to the slow acetylator phenotype and show that a similar process accounts for enzyme down-regulation by NAT1 substrates. NAT1 allozymes NAT1 14, NAT1 15, NAT1 17, and NAT1 22 are devoid of enzyme activity and have short intracellular half-lives (similar to4 h) compared with wild-type NAT1 4 and the active allozyme NAT1 24. The inactive allozymes are unable to be acetylated by cofactor, resulting in ubiquitination and rapid degradation by the 26 S proteasome. This was confirmed by site-directed mutagenesis of the active site cysteine 68. The NAT1 substrate p-aminobenzoic acid induced ubiquitination of the usually stable NAT1 4, leading to its rapid degradation. From this study, we conclude that NAT1 exists in the cell in either a stable acetylated state or an unstable non-acetylated state and that mutations in the NAT1 gene that prevent protein acetylation produce a slow acetylator phenotype

Identification of a minimal promoter sequence for the human N-acetyltransferase Type I gene that binds AP-1 (activator protein 1) and YY-1 (Yin and Yang 1).

Human N -acetyltransferase Type I (NAT1) catalyses the acetylation of many aromatic amine and hydrazine compounds and it has been implicated in the catabolism of folic acid. The enzyme is widely expressed in the body, although there are considerable differences in the level of activity between tissues. A search of the mRNA databases revealed the presence of several NAT1 transcripts in human tissue that appear to be derived from different promoters. Because little is known about NAT1 gene regulation, the present study was undertaken to characterize one of the putative promoter sequences of the NAT1 gene located just upstream of the coding region. We show with reverse-transcriptase PCR that mRNA transcribed from this promoter (Promoter I) is present in a variety of human cell-lines, but not in quiescent peripheral blood mononuclear cells. Using deletion mutant constructs, we identified a 20 bp sequence located 245 bases upstream of the translation start site which was sufficient for basal NAT1 expression. It comprised an AP-1 (activator protein 1)-binding site, flanked on either side by a TCATT motif. Mutational analysis showed that the AP-1 site and the 3' TCATT sequence were necessary for gene expression, whereas the 5' TCATT appeared to attenuate promoter activity. Electromobility shift assays revealed two specific bands made up by complexes of c-Fos/Fra, c-Jun, YY-1 (Yin and Yang 1) and possibly Oct-1. PMA treatment enhanced expression from the NAT1 promoter via the AP-1-binding site. Furthermore, in peripheral blood mononuclear cells, PMA increased endogenous NAT1 activity and induced mRNA expression from Promoter I, suggesting that it is functional in vivo

14-3-3 proteins in calcium-sensing receptor signalling

The Calcium-sensing receptor (CaR) belongs to Family C of G protein-coupled receptors. The receptor’s main role is to maintain calcium homeostasis. In addition, the CaR plays an important functional role in processes such as cell proliferation and apoptosis. These processes are mediated by various intracellular signalling pathways and the CaR tail is believed to play a major role in receptor signal transduction. To provide insight into mechanisms that control CaR signalling, recent yeast two-hybrid (Y2H) studies in our laboratory, using the CaR tail as bait, identified a number of interacting proteins including two isoforms of 14-3-3 (theta and zeta). 14-3-3 proteins are ubiquitously expressed and highly conserved, and are emerging as a group of multifunctional adapter proteins with a recognised role as chaperones. 14-3-3 proteins bind to numerous partner proteins and have a preference for targets containing phosphorylated motifs. Y2H deletion mapping studies performed in our laboratory have delineated the interaction region for 14-3-3 theta to residues 865-923 in the CaR tail. This region contains a consensus 14-3-3 binding motif that includes serine 895, a known protein kinase C substrate. The in vivo interaction between the CaR and 14-3-3 theta has been demonstrated by co-immunoprecipitation in mammalian cells. In addition, a direct in vitro interaction has been confirmed between 14-3-3 theta and the CaR tail using pulldown assays. An immediate aim of our studies is to establish the significance of the consensus 14-3-3 interaction motif in CaR signalling

Genomic organization of human arylamine \u3cem\u3eN\u3c/em\u3e-acetyltransferase Type I reveals alternative promoters that generate different 5´-UTR splice variants with altered translational activities

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NAT1 (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NAT1 is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5´ non-coding region of NAT1. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NAT1 expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9–11-fold in a luciferase reporter assay, while the other isoforms were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NAT1 transcripts may contribute to the variation in NAT1 activity in vivo

The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling

A yeast two-hybrid screen performed to identify binding partners of the CaR (calcium-sensing receptor) intracellular tail identified the adaptor protein 14-3-3? as a novel binding partner that bound to the proximal membrane region important for CaR expression and signalling. The 14-3-3? protein directly interacted with the CaR tail in pull-down studies and FLAG-tagged CaR co-immunoprecipitated with EGFP (enhanced green fluorescent protein)-tagged 14-3-3? when co-expressed in HEK (human embryonic kidney)-293 or COS-1 cells. The interaction between the CaR and 14-3-3? did not require a putative binding site in the membrane-proximal region of the CaR tail and was independent of PKC (protein kinase C) phosphorylation. Confocal microscopy demonstrated co-localization of the CaR and EGFP-14-3-3? in the ER (endoplasmic reticulum) of HEK-293 cells that stably expressed the CaR (HEK-293/CaR cells), but 14-3-3? overexpression had no effect on membrane expression of the CaR. Overexpression of 14-3-3? in HEK-293/CaR cells attenuated CaR-mediated Rho signalling, but had no effect on ERK (extracellular-signal-regulated kinase) 1/2 signalling. Another isoform identified from the library, 14-3-3?, exhibited similar behaviour to that of 14-3-3? with respect to CaR tail binding, cellular co-localization and impact on receptor-mediated signalling. However, unlike 14-3-3?, this isoform, when overexpressed, significantly reduced CaR plasma membrane expression. Results indicate that 14-3-3 proteins mediate CaR-dependent Rho signalling and may modulate the plasma membrane expression of the CaR. © The Authors Journal compilation © 2012 Biochemical Society

The role of 14-3-3 proteins in calcium-sensing receptor-mediated Rho signalling

The calcium-sensing receptor (CaR) is pivotal in maintaining calcium homeostasis, but also regulates a number of other cellular processes. To achieve this, the CaR activates a number of different cell signalling pathways including Rho-dependent serum-response element (SRE) activation, which is enhanced by the partner binding protein, filamin. To provide further insight into mechanisms controlling CaR signalling, a yeast two-hybrid screen was performed using the CaR intracellular tail as bait. A number of interacting proteins were identified including the 14-3-3 isoforms theta and zeta. 14-3-3 proteins are chaperones which bind to numerous partner proteins, influencing a multitude of cellular processes, including cell signalling. Yeast two-hybrid mapping studies delineated the interaction site for both 14-3-3 isoforms to residues 865-922 on the CaR tail, and co-immunoprecipitation studies confirmed CaR/14-3-3 theta and zeta in vivo interaction in mammalian cells. To investigate the possible role of 14-3-3 theta and zeta in CaR-mediated Rho signalling, HEK-293 cells stably expressing the CaR (HEK-293/CaR) were transfected with either 14-3-3 theta or zeta and an SRE luciferase reporter, which allowed for the measurement of SRE activation. Results demonstrated that over-expression of both 14-3-3 theta and zeta inhibited CaR-mediated SRE activation in these cells. This effect was not seen in CaR-transfected M2 cells unable to express filamin but was not restored in CaR-transfected M2 cells stably expressing filamin. We propose a mechanism whereby 14-3-3 theta and zeta either competitively bind with or sequester filamin leading to the inhibition of CaR-mediated SRE activation in HEK-293/CaR cells

The 14-3-3 isoforms theta and zeta bind the calcium-sensing receptor and modulate receptor signalling through the Rho pathway, but not the ERK1/2 pathway

The calcium-sensing receptor (CaR) maintains calcium homeostasis, but also influences processes such as cell proliferation, differentiation and apoptosis through activation of signalling pathways such as Rho and ERK1/2. To provide insight into mechanisms controlling CaR signalling, a yeast two-hybrid (Y2H) screen was performed using the CaR intracellular tail as bait. Several interacting proteins were identified including the 14-3-3 isoforms theta and zeta. 14-3-3 proteins are chaperones which bind to numerous partner proteins, preferring targets containing phosphorylated motifs. They influence a multitude of cellular processes, including cell signalling. Co-immunoprecipitation (co-IP) studies have confirmed CaR-14-3-3 in vivo interaction for both 14-3-3 theta and zeta. Confocal microscopy experiments have demonstrated that both 14-3-3 isoforms co-localise with the CaR most likely in the endoplasmic reticulum. Y2H deletion mapping delineated the interaction site for 14-3-3 zeta to residues 965-980 in the CaR tail, which overlaps the filamin interaction domain. By contrast, the 14-3-3 theta interaction site is confined to residues 865-923. A 14-3-3 consensus binding motif, Rx1-2Sx2-3S, exists within this region of the CaR tail where serine 895 is putatively phosphorylated. Site-directed mutagenesis and co-IP assays have shown that the serine 895 is not primarily responsible for mediating CaR-14-3-3 theta interaction. Further studies aim to establish the significance of the entire consensus motif for CaR-14-3-3 theta interaction. To determine the role of 14-3-3 proteins in CaR-mediated ERK1/2 activity, HEK-293 cells stably expressing the CaR were transfected with 14-3-3 theta or zeta, stimulated with extracellular calcium and analysed for ERK1/2 phosphorylation by Western blot analysis. Neither 14-3-3 isoform modulated ERK1/2 activity through the CaR. To determine the possible role of 14-3-3 theta or zeta in CaR-mediated Rho signalling, HEK-293 cells stably expressing the CaR were transfected with 14-3-3 theta or zeta and a luciferase reporter gene which allowed for the measurement of luciferase activity through Rho-dependent stimulation of serum-response element transcription. Results demonstrated that both 14-3-3 theta and zeta activated Rho signalling. The enhancement of CaR-mediated Rho signalling by 14-3-3 isoforms suggests a general role for 14-3-3 proteins as chaperones in the regulation of Rho signalling, and possibly other CaR-mediated signalling pathways

14-3-3 protein isoforms interact differentially with the calcium-sensing receptor intracellular tail

The main role of the calcium-sensing receptor (CaR) is to maintain calcium homeostasis but the receptor also participates in other functions such as cell proliferation and apoptosis mediated by various intracellular signalling pathways. To provide insight into mechanisms that control CaR signalling, yeast two-hybrid (Y2H) studies in our laboratory, using the CaR tail as bait, identified a number of interacting proteins including the 14-3-3 isoforms zeta and theta. 14-3-3 proteins are ubiquitously expressed and highly conserved, and are emerging as a group of multifunctional adapter proteins with a recognised role as chaperones. 14-3-3 proteins bind to numerous partner proteins having a preference for phosphorylated targets. Y2H deletion mapping studies have delineated the interaction region for 14-3-3 zeta to residues 923-1078 in the CaR tail. By contrast, the 14-3-3 theta interaction site is confined to residues 865-923 in the CaR tail. This region of 14-3-3 theta interaction contains a consensus 14-3-3 binding motif that includes a phosphorylated serine, residue 895. In vivo interaction between the CaR and 14-3-3 theta has been demonstrated by co-immunoprecipitation in mammalian cells. In addition, a direct in vitro interaction has been confirmed between 14-3-3 theta and the CaR tail using pulldown assays. Site-directed mutagenesis and subsequent co-immunoprecipitation experiments have shown that the phosphoserine residue (S895) in the identified consensus motif in the CaR tail is not primarily responsible for mediating CaR and 14-3-3 theta interaction. Differential binding of the 14-3-3 isoforms to the CaR tail points to differences in the way these isoforms might influence CaR-mediated signalling

The importance of transgene and cell type on the regeneration of adult retinal ganglion cell axons within reconstituted bridging grafts

When grafted onto the cut optic nerve, chimeric peripheral nerve (PN) sheaths reconstituted with adult Schwann cells (SCs) support the regeneration of adult rat retinal ganglion cell (RGC) axons. Regrowth can be further enhanced by using PN containing SCs transduced ex vivo with lentiviral (LV) vectors encoding a secretable form of ciliary neurotrophic factor (CNTF). To determine whether other neurotrophic factors or different cell types also enhance RGC regrowth in this bridging model, we tested the effectiveness of (1) adult SCs transduced with brain-derived neurotrophic factor (BDNF) or glial cell line-derived neurotrophic factor (GDNF), and (2) fibroblasts (FBs) genetically modified to express CNTF. SCs transduced with LV-BDNF and LV-GDNF secreted measurable and bioactive amounts of each of these proteins, but reconstituted grafts containing LV-BDNF or LV-GDNF transduced SCs did not enhance RGC survival or axonal regrowth. LV-BDNF modified grafts did, however, contain many pan-neurofilament immunolabeled axons, many of which were also immunoreactive for calcitonin gene-related peptide (CGRP) and were presumably of peripheral sensory origin. Nor-adrenergic and cholinergic axons were also seen in these grafts. There were far fewer axons in LV-GDNF engineered grafts. Reconstituted PN sheaths containing FBs that had been modified to express CNTF did not promote RGC viability or regeneration, and PN reconstituted with a mixed population of SCs and CNTF expressing FBs were less effective than SCs alone. These data show that both the type of neurotrophic factor and the cell types that express these factors are crucial elements when designing bridging substrates to promote long-distance regeneration in the injured CNS

14-3-3 proteins in calcium-sensing receptor cell signalling

The calcium-sensing receptor (CaR) maintains calcium homeostasis, but also influences processes such as cell proliferation, differentiation and apoptosis through activation of signalling pathways such as Rho and ERK1/2. To provide insight into mechanisms controlling CaR signalling, a yeast two-hybrid (Y2H) screen was performed using the CaR intracellular tail as bait. Several interacting proteins were identified including the 14-3-3 isoforms theta and zeta. 14-3-3 proteins are chaperones which bind to numerous partner proteins, preferring targets containing phosphorylated motifs. They influence a multitude of cellular processes, including ERK1/2 signalling. Co-immunoprecipitation (co-IP) and pulldown assays have confirmed CaR-14-3-3 in vivo and in vitro interaction, respectively, for 14-3-3 theta. Confocal microscopy has demonstrated that both 14-3-3 isoforms co-localise with the CaR in the endoplasmic reticulum. Y2H deletion mapping delineated the interaction site for 14-3-3 zeta to residues 965-980 in the CaR tail. By contrast, the 14-3-3 theta interaction site is confined to residues 865-923. A 14-3-3 consensus binding motif, Rx1-2Sx2-3S, exists within this region of the CaR tail where serine 895 is putatively phosphorylated. Site-directed mutagenesis and co-IP assays have shown that the serine 895 is not primarily responsible for mediating CaR-14-3-3 theta interaction. Further studies aim to establish the significance of the entire consensus motif for CaR-14-3-3 theta interaction. To determine the role of 14-3-3 proteins in CaR-mediated ERK1/2 activity, HEK-293 cells stably expressing the CaR were transfected with 14-3-3 theta or zeta, stimulated with extracellular calcium and analysed for ERK1/2 activity by Western blot analysis. Neither 14-3-3 isoform modulated ERK1/2 activity through the CaR. In addition, the possible role of 14-3-3 theta and zeta in CaR-mediated modulation of Rho-dependent stimulation of serum-response element transcription was examined in HEK293 cells using a luciferase assay. Preliminary results suggest that both 14-3-3 theta and zeta inhibit Rho signalling to a similar extent. Differential binding of the two 14-3-3 isoforms to the CaR tail initially suggested differences in the way these isoforms may influence CaR-mediated signalling but experiments to date have not exposed such differences. The functional significance of the CaR-14-3-3 interaction will be further examined with other CaR-mediated signalling pathways