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N Masson

Publications and source records attributed to N Masson.

16 recordsLinked to original sources
PubMed2001

C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.

HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.

2,2'-Dipyridyl↗
PubMed2001

Independent function of two destruction domains in hypoxia-inducible factor-alpha chains activated by prolyl hydroxylation.

Oxygen-dependent proteolytic destruction of hypoxia-inducible factor-alpha (HIF-alpha) subunits plays a central role in regulating transcriptional responses to hypoxia. Recent studies have defined a key function for the von Hippel-Lindau tumour suppressor E3 ubiquitin ligase (VHLE3) in this process, and have defined an interaction with HIF-1 alpha that is regulated by prolyl hydroxylation. Here we show that two independent regions within the HIF-alpha oxygen-dependent degradation domain (ODDD) are targeted for ubiquitylation by VHLE3 in a manner dependent upon prolyl hydroxylation. In a series of in vitro and in vivo assays, we demonstrate the independent and non-redundant operation of each site in regulation of the HIF system. Both sites contain a common core motif, but differ both in overall sequence and in the conditions under which they bind to the VHLE3 ligase complex. The definition of two independent destruction domains implicates a more complex system of pVHL-HIF-alpha interactions, but reinforces the role of prolyl hydroxylation as an oxygen-dependent destruction signal.

Amino Acid Motifs↗
PubMed2000

Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein.

The von Hippel-Lindau tumor suppressor protein (pVHL) has emerged as a key factor in cellular responses to oxygen availability, being required for the oxygen-dependent proteolysis of alpha subunits of hypoxia inducible factor-1 (HIF). Mutations in VHL cause a hereditary cancer syndrome associated with dysregulated angiogenesis, and up-regulation of hypoxia inducible genes. Here we investigate the mechanisms underlying these processes and show that extracts from VHL-deficient renal carcinoma cells have a defect in HIF-alpha ubiquitylation activity which is complemented by exogenous pVHL. This defect was specific for HIF-alpha among a range of substrates tested. Furthermore, HIF-alpha subunits were the only pVHL-associated proteasomal substrates identified by comparison of metabolically labeled anti-pVHL immunoprecipitates from proteosomally inhibited cells and normal cells. Analysis of pVHL/HIF-alpha interactions defined short sequences of conserved residues within the internal transactivation domains of HIF-alpha molecules sufficient for recognition by pVHL. In contrast, while full-length pVHL and the p19 variant interact with HIF-alpha, the association was abrogated by further N-terminal and C-terminal truncations. The interaction was also disrupted by tumor-associated mutations in the beta-domain of pVHL and loss of interaction was associated with defective HIF-alpha ubiquitylation and regulation, defining a mechanism by which these mutations generate a constitutively hypoxic pattern of gene expression promoting angiogenesis. The findings indicate that pVHL regulates HIF-alpha proteolysis by acting as the recognition component of a ubiquitin ligase complex, and support a model in which its beta domain interacts with short recognition sequences in HIF-alpha subunits.

Animals↗
PubMed1998

The T-cell oncogenic protein HOX11 activates Aldh1 expression in NIH 3T3 cells but represses its expression in mouse spleen development.

Hox11 is a homeobox gene essential for spleen formation in mice, since atrophy of the anlage of a developing spleen occurs in early embryonic development in Hox11 null mice. HOX11 is also expressed in a subset of T-cell acute leukemias after specific chromosomal translocations. Since the protein has a homeodomain and can activate transcription, it probably exerts at least some of its effects in vivo by regulation of target genes. Representational difference analysis has been used to isolate cDNA clones corresponding to mRNA species activated following stable expression of HOX11 in NIH 3T3 cells. The gene encoding the retinoic acid-synthesizing enzyme aldehyde dehydrogenase 1 (Aldh1), initially called Hdg-1, was found to be ectopically activated by HOX11 in this system. Study of Aldh1 gene expression during spleen development showed that the presence of Aldh1 mRNA inversely correlated with Hox11. Hox11 null mouse embryos have elevated Aldh1 mRNA in spleen primordia prior to atrophy, while Aldh1 seems to be repressed by Hox11 during organogenesis of the spleens of wild-type mice. This result suggests that expression of Aldh1 protein is negatively regulated by Hox11 and that abnormal expression of Aldh1 in Hox11 null mice may cause loss of splenic precursor cells by aberrant retinoic acid metabolism.

3T3 Cells↗
PubMed1998

Optimal activation of an endogenous gene by HOX11 requires the NH2-terminal 50 amino acids.

The HOX11 homeobox gene was first identified through studies of the t(7;10) and t(10;14) chromosomal translocations of acute T-cell leukemia. In addition, analysis of Hox11-/- mice has demonstrated a critical role for this gene in murine spleen development. A possible mode of in vivo function for the HOX11 protein in these two situations is regulation of target genes following DNA binding via the homeodomain, but little is known about how HOX11 regulates transcription in vivo. By performing transcriptional studies in yeast and mammalian one-hybrid systems, a modular transcriptional transactivation region at the NH2 terminus of HOX11 has been functionally dissected from other parts of the protein. This NH2-terminal region includes the previously identified short conserved Hep motif, which itself activates transcription in one-hybrid assays. The importance of the NH2-terminal region for the function of HOX11 in vivo was assayed by activating a HOX11-dependent gene in NIH 3T3 cells. Activation of this gene was found to be dependent upon an intact homeodomain in HOX11, but maximal activation was obtained only when the NH2-terminal 50 amino acids of HOX11 was present, showing that this region of HOX11 is important for in vivo transcriptional control of a chromosomal target gene.

3T3 Cells↗
PubMed1997

A novel mechanism for isolated central hypothyroidism: inactivating mutations in the thyrotropin-releasing hormone receptor gene.

Isolated central hypothyroidism, characterized by insufficient TSH secretion resulting in low levels of thyroid hormones, is a rare disorder. We report a boy in whom isolated central hypothyroidism was diagnosed at 9 yr of age. Complete absence of TSH and PRL responses to TRH led us to speculate that he had an inactivating mutation of the TRH receptor gene. The patients' genomic DNA was isolated, and the entire coding region of the TRH receptor was amplified by the PCR and sequenced directly. Confirmation of the mutations and haplotyping of the family was performed using restriction enzymes. The biological activity of the wild-type and mutated TRH receptors was verified by evaluating the binding of labeled TRH and stimulation by TRH of total inositol phosphate accumulation in transfected HEK-293 and COS-1 cells. The patient was found to be a compound heterozygote, having inherited a different mutated allele from each of the parents; both mutations were in the 5'-part of the gene. Mutated receptors were unable to bind TRH and to activate total inositol phosphate accumulation. Our report is the first description of naturally occurring inactivating mutations of a G protein-coupled receptor linked to the phospholipase C second messenger pathway. The prevalence and phenotypic spectrum of TRH receptor mutations in isolated central hypothyroidism remain to be established.

Cell Line↗
PubMed1996

The chemistry of pseudomonic acid. 17. Dual-action C-1 oxazole derivatives of pseudomonic acid having an extended spectrum of antibacterial activity.

A series of C-1 oxazole isosteres of pseudomonic acid A (mupirocin) bearing a nitroheterocycle have been synthesized, and significant differences in both spectrum of activity and potency were found between these derivatives and mupirocin. Additionally, the antibacterial potency of two members of this class of compounds against mupirocin-resistant staphylococci could not be accounted for solely by inhibition of the target enzyme isoleucyl-tRNA synthetase (IRS), indicating an additional mode of action. The most potent compound, the nitrofuran 3f (SB 205952), was the most electron affinic derivative prepared and was transformed by NAD(P)H-dependent bacterial reductases at a rate similar to that for nitrofurantoin. The second mode of action of this compound may therefore arise from its reduction to a species with cellular targets other than IRS. In in vivo studies, 3f was shown to be a very effective agent by both the subcutaneous and oral routes of administration.

Anti-Bacterial Agents↗
PubMed1993

In vitro phosphorylation studies of a conserved region of the transcription factor ATF1.

A large family of mammalian transcription factors including multiple variants of CREB, CREM and ATF1 have been implicated in signal transduction by cAMP and other cellular pathways. Although the roles of some members of the family have been characterised the function of ATF1 is poorly understood. We have identified one or more key serine residues that are required for a phosphorylation-induced conformational change in ATF1. The critical serines map to a putative transcriptional activation domain of ATF1 and affect the stability of ATF1 DNA-binding. Intriguingly phosphorylation is modulated by ATF1 homodimerization and by ATF1 binding to DNA. One of the key serine residues required for ATF1 phosphorylation is not conserved in CREB and CREM suggesting that it is likely to determine some specialised function of ATF1.

Activating Transcription Factors↗
PubMed1993

Identification of proteins that interact with CREB during differentiation of F9 embryonal carcinoma cells.

The mammalian transcription factor CREB is thought to activate cAMP-inducible genes in a variety of differentiated cell types and is probably involved in other signalling pathways. Undifferentiated F9 embryonal carcinoma (UF9) cells are refractory to cAMP and become cAMP-responsive following differentiation to endoderm like cells. It has been proposed that UF9 cells contain a negative regulator(s) of the cAMP-response that might act through direct interaction with CREB. We have used a protein blotting assay and 32P-labelled CREB to probe for CREB-binding proteins in nuclear extracts from F9 cells and to examine their abundance during differentiation. We find that ATF1 (a protein that is highly homologous to CREB) and a novel polypeptide(s) of approximately 100 kDa (CBP100) are the major CREB-binding proteins in extracts from UF9 cells. As expected ATF1 is detected due to leucine zipper-dependent heterodimerisation with CREB. In contrast CBP100 interacts with CREB independently of the leucine zipper. The total amount of ATF1 and the amount of ATF1 that is complexed with CREB are substantially reduced following differentiation. In addition, ATF1 mRNA levels are lower in differentiated F9 cells indicating that a pretranslational mechanism contributes to the decreased ATF1 protein levels observed. CBP100 levels are also reduced or CBP100 is modified upon differentiation. We discuss the potential roles of ATF1 and CBP100 in regulating CREB activity during differentiation of F9 embryonal carcinoma cells.

Amino Acid Sequence↗
PubMed1992

Cyclic AMP response element-binding protein and the catalytic subunit of protein kinase A are present in F9 embryonal carcinoma cells but are unable to activate the somatostatin promoter.

The cyclic AMP (cAMP) response elements (CREs) of the somatostatin and vasoactive intestinal peptide (VIP) promoters contain binding sites for CRE-binding protein (CREB) that are essential for cAMP-regulated transcription. Using F9 embryonal carcinoma cells, we show that the somatostatin and VIP promoters exhibit a differentiation-dependent cAMP response, demonstrating that these promoters are regulated by transcription factors that become active during differentiation. Lack of cAMP responsiveness of the somatostatin promoter in undifferentiated cells is not due to the absence of known positive-acting factors (the catalytic subunit of protein kinase A [cPKA] and CREB) or a general inhibition of protein kinase A activity. Since overexpression of exogenous cPKA and CREB is sufficient to activate the somatostatin promoter in undifferentiated cells, these findings suggest that a negative factor(s) represses endogenous cPKA and CREB. In contrast to their effects on somatostatin, exogenous CREB and cPKA do not activate the VIP promoter. Thus, despite coregulation during differentiation and the ability to bind CREB, the somatostatin and VIP promoters are not coordinately activated by CREB in undifferentiated F9 cells.

Base Sequence↗
PubMed1990

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.

Activating Transcription Factors↗
PubMed1982

ECT use for a patient with malignant hyperthermia.

The authors used dantrolene as a prophylactic during ECT with a depressed patient susceptible to malignant hyperthermia. Succinylcholine and tricyclic and MAO-inhibiting antidepressants can precipitate malignant hyperthermia and were thus avoided.

Adult↗
PubMedDate not supplied

Inhibiting albumin glycation ameliorates diabetic nephropathy in the db/db mouse.

Albumin modified by Amadori glucose adducts stimulates the expression of extracellular matrix proteins by glomerular mesangial and endothelial cells, and has been mechanistically linked to the pathogenesis of diabetic nephropathy. To test the hypothesis that inhibiting the formation of glycated albumin might beneficially influence the development of kidney disease in diabetes, we treated diabetic db/db mice for 12 weeks with a low-molecular-weight compound (EXO-226) that impedes the condensation of free glucose with lysine epsilon-amino groups in albumin. Administration of EXO-226 (3 mg/kg) twice daily by gavage normalized the plasma concentration of glycated albumin within days after initiation of treatment and maintained glycated albumin within the normal range throughout the study, despite persistent and severe hyperglycemia. Urine albumin excretion, which was markedly increased at the start of the study (age 12 weeks), was significantly reduced in treated diabetic animals compared with their untreated diabetic littermates. The fall in creatinine clearance that was observed in untreated diabetic animals was prevented in diabetic littermates that received treatment. Compared with the nondiabetic controls, the amount of glomerular mesangial matrix was threefold greater in untreated diabetic mice; in contrast, the mesangial matrix fraction was only 1. 5 times that of nondiabetic controls in the treated diabetic animals, representing a reduction in mesangial matrix accumulation of more than 50%. EXO-226 also reduced the overexpression of mRNA encoding for alpha1 (IV) collagen in renal cortex of db/db mice. We conclude that normalization of plasma glycated albumin concentrations with the glycation inhibitor EXO-226 ameliorates the glomerular structural and functional abnormalities associated with diabetic nephropathy in the db/db mouse.

Albuminuria↗