Dimerization-driven interaction of hepatitis C virus core protein with NS3 helicase.

Hepatitis C virus (HCV) infects over 130 million people causing a worldwide epidemic of liver cirrhosis and hepatocellular-carcinoma. Because current HCV treatments are only partially effective, molecular mechanisms involved in HCV propagation are actively being pursued as possible drug targets. Here, we report on a new macromolecular interaction between the HCV capsid core protein and the helicase portion of HCV non-structural protein 3 (NS3h), confirmed by four different biochemical methods. The protease portion of NS3 is not required. Interaction between the two proteins could be disrupted by two types of specific inhibitors of core dimerization, the small molecule SL201 and core106, a C-terminally truncated core protein. Cross-linking experiments suggest that the physical interaction with NS3h is probably driven by core oligomerization. Moreover, SL201 blocks the production of infectious virus, but not the production of a subgenomic HCV replicon by hepatoma cells. Time-of-addition experiments confirm that SL201 has no effect on entry of the virus. These data underline the essential role of core as a key organizer of HCV particle assembly, confirm the importance of oligomerization, reveal the interaction with viral helicase and support a new molecular understanding of the formation of the viral particle at the level of the lipid droplets, before its migration to the site of release and budding.

Peptide inhibitors of hepatitis C virus core oligomerization and virus production.

Hepatitis C virus (HCV) nucleocapsid assembly requires dimerization of the core protein, an essential step in the formation of the virus particle. We developed a novel quantitative assay for monitoring this protein-protein interaction, with the goal of identifying inhibitors of core dimerization that might block HCV production in infected Huh-7.5 hepatoma cells. Two core-derived, 18-residue peptides were found that inhibited the dimerization of a fragment of core comprising residues 1-106 (core106) by 68 and 63%, respectively. A third, related 15-residue peptide displayed 50% inhibition, with an IC50 of 21.9 microM. This peptide was shown, by fluorescence polarization, to bind directly to core106 with a Kd of 1.9 microM and was displaced by the unlabelled peptide with an IC50 of 18.7 microM. When measured by surface plasmon resonance, the same peptide bound core169 with a Kd of 7.2 microM. When added to HCV-infected cells, each of the three peptides blocked release, but not replication, of infectious virus. When measured by real-time RT-PCR, the RNA levels were reduced by 7-fold. The 15-residue peptide had no effect on HIV propagation. Such inhibitors may constitute useful tools to investigate the role of core dimerization in the virus cycle.

G-protein-coupled receptor signalling through protein networks.

This short review provides a broad, and therefore necessarily incomplete and personal, overview of G-protein-coupled receptors, which are often targets for a wide range of important drugs: I will discuss successively their structure, function and interactions with associated proteins. Examples will be drawn from work done over the last 30 years by scientists that worked at different times in my laboratories, mainly in the field of beta-adrenoceptors, muscarinic acetylcholine, melatonin and angiotensin receptors.

Structural organization and expression of human MTUS1, a candidate 8p22 tumor suppressor gene encoding a family of angiotensin II AT2 receptor-interacting proteins, ATIP.

The Mitochondrial Tumor suppressor 1 (MTUS1) gene is a newly identified candidate tumor suppressor gene at chromosomal position 8p22. We report here that MTUS1 encodes a family of proteins whose leader member (ATIP1) was previously isolated in our laboratory as a novel interacting partner of the angiotensin II AT2 receptor involved in growth inhibition (Nouet, JBC 279: 28989-97, 2004). The MTUS1 gene contains 17 coding exons distributed over 112 kb of genomic DNA. Alternative exon usage generates three major transcripts (ATIP1, ATIP3 and ATIP4), each showing different tissue distribution. ATIP polypeptides are identical in their carboxy-terminal region carrying four coiled-coil domains. In their amino-terminal portion, ATIP polypeptides exhibit distinct motifs for localisation in the cytosol, nucleus or cell membrane, suggesting that MTUS1 gene products may be involved in a variety of intracellular functions in an AT2-dependent and independent manner. ATIP1 is ubiquitous and highly expressed in the brain. ATIP3 is the major transcript in tissues (prostate, bladder, breast, ovary, colon) corresponding to cancer types with frequent loss of heterozygosity at 8p22. Interestingly, ATIP4 is a brain-specific transcript highly abundant in the cerebellum and fetal brain. High evolutionary conservation of ATIP amino-acid sequences suggests important biological roles for this new family of proteins in tumor suppression and/or brain function.

Mutation analysis of the 8p22 candidate tumor suppressor gene ATIP/MTUS1 in hepatocellular carcinoma.

A high frequency of allelic loss affecting chromosome 8p and a minimal region of deletion at p21-22 have been previously reported in hepatocellular carcinoma (HCC), suggesting that at least one tumor suppressor gene is present in this region. In this study, we assessed whether the angiotensin II AT2 receptor interacting protein (ATIP)/mitochondrial tumor suppressor gene (MTUS1), a gene newly identified at position 8p22, may be a candidate tumor suppressor gene mutated in HCC. We searched for alterations in the 17 coding exons of ATIP/MTUS1 by means of denaturating high-performance liquid chromatography and sequencing, in 51 HCC tumors and 58 cell lines for which loss of heterozygosity status was known. Five major nucleotide substitutions were identified, all located in exons used by the ATIP3 transcript which is the only ATIP transcript variant expressed in liver. These nucleotide variations result in amino-acid substitution or deletion of conserved structural motifs (nuclear localisation signal, polyproline motif, leucine zipper) and also affect exonic splicing enhancer motifs and physiological splice sites, suggesting potential deleterious effects on ATIP3 function and/or expression.

New series of N-substituted phenyl ketone oxime ethers: synthesis and bovine beta3-adrenergic agonistic activities.

A series of ten novel phenyl ketone oxime ethers substituted on the terminal nitrogen by either 1,3 benzodioxole, alkyl, aralkyl or aryl moiety were synthesized and tested for their activity at bovine beta3-adrenoceptors. The best compound, which was the benzodioxole dicarboxylate derivative, showed potent beta3-adrenergic agonistic activities in Chinese hamster ovary cells expressing the bovine beta3-adrenoceptors with Kact and Ki values better than compound CL 316,243 used as reference (14 +/- 6 nM and 203 +/- 71 nM, respectively). In this series three compounds showed an antagonistic activity. Structure-activity relationships in these ketone oxime ethers are discussed.

Serum leptin activity in obese and lean patients.

Blood levels of the satiety hormone leptin are directly correlated to fat stores in obese and lean people. Therefore, leptin resistance is the logical explanation for the phenomenon of common obesity. However, the important question of whether or not the intrinsic leptin activity could differ between obese and lean people has not been examined before. In the present study, serum leptin activity was measured by an in vitro assay of leptin signaling in a modified culture of HEK-293 cells. The system is based on activation of a luciferase reporter gene through a leptin receptor-dependent activation of the signal transducer and activator of transcription (STAT3). Serum samples from 20 obese and 20 non-obese individuals with leptin levels ranging from 3 to 75 ng/ml, as determined by radioimmunoassay (RIA), were used. A high correlation was observed for each serum sample between leptin RIA values and leptin activity in the bioassay. The results indicate that obesity in the 20 obese patients among the 40 individuals examined cannot be accounted for by alterations in leptin activity in our assay. The assay system provides a tool to screen for possible rare cases exhibiting alteration in leptin activity either due to a change in leptin itself or through interaction with other serum factors.

The iodocyanopindolol and SM-11044 binding protein belongs to the TM9SF multispanning membrane protein superfamily.

SM-11044 is the only beta-adrenergic agonist that inhibits guinea pig eosinophil chemotaxis and induces relaxation of depolarized rat colon tonus. We have previously reported the purification of a 34 kDa photoaffinity-labeled SM-11044 binding protein (SMBP) from rat colon that may mediate the biological effects of the ligand and that differs from all known monoamine receptors (Sugasawa et al., J. Biol. Chem. 272 (1997) 21244). The present report describes partial amino acid sequence of rat SMBP and molecular cloning of corresponding human SMBP (hSMBP) cDNA. This cDNA encodes a 588 amino acid residue polypeptide comprising a signal peptide, a long hydrophilic amino-terminal region, and a highly hydrophobic C-terminal portion organized into nine putative transmembrane domains. The sequence and structure of hSMBP shows homology to members of a new transmembrane protein 9 superfamily (TM9SF). Comparison of hSMBP with related protein sequences from yeast, plant and human revealed two subgroups within TM9SF. The members of these groups differ in length and have characteristic amino acid sequence motifs in their amino-terminal portion. Northern blot analysis revealed two major SMBP mRNAs, at 3.4 and 3.8 kb, that were present in all the human tissues examined. Western blot experiments detected SMBP as a 70 kDa protein that may be further cleaved into an active 34 kDa N-terminal polypeptide. Stable Chinese Hamster Ovary cell transfectants expressing hSMBP cDNA displayed specific binding of [(125)I]iodocyanopindolol that was displaced by SM-11044 in a dose-dependent manner. Thus, SMBP is the first member of TM9SF with functional ligand binding properties, suggesting that some of these integral membrane proteins may function as channels, small molecule transporters or receptors.

Functional expression of MT2 (Mel1b) melatonin receptors in human PAZ6 adipocytes.

Several reports have demonstrated that the pineal hormone, melatonin, plays an important role in body mass regulation in mammals. To date, however, the target tissues and relevant biochemical mechanisms involved remain uncharacterized. As adipose tissue is the principal site of energy storage in the body, we investigated whether melatonin could also act on this tissue. Semiquantitative RT-PCR analysis revealed the expression of MT1 and MT2 melatonin receptor mRNAs in the human brown adipose cell line, PAZ6, as well as in human brown and white adipose tissue. Binding analysis with 2-[(125)I]iodomelatonin ((125)I-Mel) revealed the presence of a single, high affinity binding site in PAZ6 adipocytes with a binding capacity of 7.46 +/- 1.58 fmol/mg protein and a K(d) of 457 +/- 5 pM. Both melatonin and the MT2 receptor-selective antagonist, 4-phenyl-2-propionamidotetraline, competed with 2-[(125)I]iodomelatonin binding, with respective K(i) values of 3 x 10(-11) and 1.5 x 10(-11) M. Functional expression of melatonin receptors in PAZ6 adipocytes was indicated by the melatonin-induced, dose-dependent inhibition of forskolin-stimulated cAMP levels and basal cGMP levels with IC(50) values of 2 x 10(-9) and 3 x 10(-10) M, respectively. Modulation of the cGMP pathway by melatonin further supports functional expression of MT2 receptors, as this pathway was shown to be specific for that subtype in humans. In addition, long-term melatonin treatment of PAZ6 adipocytes was found to decrease the expression of the glucose transporter Glut4 and glucose uptake, an important parameter of adipocyte metabolism. These results suggest that melatonin may act directly at MT2 receptors on human brown adipocytes to regulate adipocyte physiology.

Synthesis and bovine beta 3-adrenergic agonistic activities of a novel series of aryloxypropanolamines.

We synthesized a novel series of 21 aryloxypropanolamine compounds characterized by N-alkyl, aralkyl, and aryl substituents. The compounds showed potent beta 3-adrenergic agonistic activities in Chinese hamster ovary cells expressing the bovine beta 3-adrenoceptors with Kact and Ki values of 4.2 +/- 3.0 nM and 459 +/- 169 nM respectively, for the ligand with the best compromise between potency and affinity. Structure-activity relationships are discussed.

A common polymorphism in the promoter of UCP2 is associated with decreased risk of obesity in middle-aged humans.

Obesity is the most common nutritional disorder in Western society. Uncoupling protein-2 (UCP2) is a recently identified member of the mitochondrial transporter superfamily that is expressed in many tissues, including adipose tissue. Like its close relatives UCP1 and UCP3, UCP2 uncouples proton entry in the mitochondrial matrix from ATP synthesis and is therefore a candidate gene for obesity. We show here that a common G/A polymorphism in the UCP2 promoter region is associated with enhanced adipose tissue mRNA expression in vivo and results in increased transcription of a reporter gene in the human adipocyte cell line PAZ-6. In analyzing 340 obese and 256 never-obese middle-aged subjects, we found a modest but significant reduction in obesity prevalence associated with the less-common allele. We confirmed this association in a population-based sample of 791 middle-aged subjects from the same geographic area. Despite its modest effect, but because of its high frequency (approximately 63%), the more-common risk allele conferred a relatively large population-attributable risk accounting for 15% of the obesity in the population studied.

Effect of dexamethasone on adipocyte differentiation markers and tumour necrosis factor-alpha expression in human PAZ6 cells.

AIMS/HYPOTHESIS: Adipose tissue-derived tumour necrosis factor-alpha (TNF-alpha) has been implicated in the insulin resistance observed in animal models of obesity. Moreover, TNF-alpha has inhibitory effects on adipocyte differentiation. Glucocorticoids play important roles in the regulation of insulin sensitivity and adipose tissue distribution. We therefore studied the effect of dexamethasone on TNF-alpha expression and adipocyte differentiation in human PAZ6 cells. METHODS: The expression of TNF-alpha and adipocyte differentiation markers was assessed by reverse-transcription polymerase chain reaction in PAZ6 cells. RESULTS: In cells cultured for 15 days in the presence of dexamethasone, adipocyte differentiation marker expression was higher and TNF-alpha expression was lower than in cells cultured in the absence of dexamethasone. The presence of dexamethasone was necessary during the whole period of differentiation because removal of dexamethasone during the second week resulted in poorly differentiated adipocytes that express higher levels of TNF-alpha. Dexamethasone also reduced TNF-alpha expression during early stages of differentiation. The use of a TNF-alpha-neutralising antibody showed, however, that endogenously-produced TNF-alpha did not play an important part in the control of PAZ6 cell differentiation. During early stages of adipocyte differentiation, dexamethasone induced the expression of the transcription factors PPAR gamma (peroxisome proliferator activated receptor gamma) and C/EBP alpha (CCAAT/enhancer binding protein alpha) while inhibiting the expression of the inhibitor of DNA binding Id2. CONCLUSION/INTERPRETATION: The effect of dexamethasone on human adipocyte differentiation is not mediated by reduction of TNF-alpha expression but more likely by regulation of the expression of nuclear factors such as PPAR gamma, CEBP alpha and Id2.

Genetic variation in the stress protein hsp70-2 gene is highly associated with obesity.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) expression is increased in adipose tissue of both rodent models of obesity and obese humans. It has therefore been considered as a candidate gene for obesity. Several studies have indeed shown statistical evidence of linkage between obesity and the chromosomal region encompassing the TNF-alpha gene, suggesting that TNF-alpha and/or a nearby gene (eg hsp70 gene) is involved in the onset and progression of weight gain. We designed a case-controlled study to investigate the potential association of polymorphism of the TNF-alpha and that of a stress protein (hsp70-2) with obesity. METHODS: We used the polymerase chain reaction and restriction enzyme digestion to characterize the variation of the TNF-alpha promoter region and that of the hsp70-2 gene in 343 unrelated Tunisian patients with obesity and 174 healthy control subjects. RESULTS: Analysis of the -308 TNF-alpha polymorphism in patients with obesity and in control subjects did not reveal an association between TNF-alpha alleles and obesity. In contrast, polymorphism analysis of the hsp70-2 gene in patients with obesity demonstrated highly significant differences in genotypic distribution of this bi-allelic locus compared to the control subject group. Homozygosity for one hsp70-2 allele was highly associated with obesity (r2=7.12; P<10(-6)). CONCLUSION: Tunisian persons carrying the P2/P2 genotype of the hsp70-2 gene may have an increased risk of obesity.

Chemokines control fat accumulation and leptin secretion by cultured human adipocytes.

In addition to their role in inflammation, cytokines like TNFalpha have been reported to regulate the adipose tissue function suggesting a role for these soluble mediators in metabolism. However, it is not known whether adipocytes have the capacity to secrete chemokines, a group of low molecular weight inflammatory mediators that control leukocyte migration into tissues. Here we show that primary cultures of human preadipocytes constitutively produce three chemokines, interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1), while their level of expression is low in mature adipocytes. Upon TNFalpha treatment, the expression of all the three chemokines is upregulated in adipocytes differentiated in vitro. In addition, we describe the presence of seven different chemokine receptors, mainly in mature adipocytes, both in vitro and in human fat tissue sections. Prolonged stimulation of cultured human adipocytes with exogenous chemokines leads to a decrease in lipid content in association with the downregulation of PPARgamma mRNA expression. Moreover, chemokines positively control the secretion of leptin, a hormone that regulates appetite, by a post-transcriptional mechanism. These findings reveal a new role for chemokines in the regulation of adipose tissue and suggest a novel therapeutic basis for the treatment of obesity, diabetes and cachexia.

Functional proteomics to exploit genome sequences.

The sequencing of various genomes has inaugurated a new stage in the understanding of normal and pathological cell function through the analysis of the role of proteins. Proteins, after all, that intervene in the different molecular mechanisms of life, during growth, reproduction, and in the interaction between cells, thus making it possible to describe the biology of integrated systems. In this article, we briefly describe the various stages in the progression of our knowledge, from the genome to the "functional" proteome. Emphasis is placed on a global approach to the protein-protein interactions used to describe the cellular "interactome".

The uncoupling protein-3 gene is transcribed from tissue-specific promoters in humans but not in rodents.

Uncoupling protein-3 (UCP3), a mitochondrial membrane transporter, is a candidate effector of thermogenesis. Even though mice with targeted disruption of the UCP3 gene are not obese, indirect evidence suggests that this protein contributes to the control of energy expenditure in humans. We therefore characterized the human UCP3 gene and compared it with its rodent homologues with respect to tissue-specific expression and regulatory regions. Like rodent UCP3, human UCP3 was expressed in skeletal muscle and brown adipose tissue (BAT). The short mRNA isoform, UCP3(S), which is absent in rodents, was relatively more abundant in human skeletal muscle in comparison to human BAT. Two tissue-specific transcription start sites for each skeletal muscle and BAT were delineated for human UCP3. Tissue-specific transcript initiation was maintained in both tissues and cultured cells over a wide range of expression levels. In contrast, rodent transcripts were initiated at the same site in BAT and muscle tissue. Comparison of human and rodent promoters indicated a rapid phylogenetic evolution suggesting functional diversification. The transcription from tissue-specific promoters in humans is a novel finding that may provide the basis for therapeutic interventions aimed at regulating energy expenditure in a tissue-specific fashion.

Functional trans-inactivation of insulin receptor kinase by growth-inhibitory angiotensin II AT2 receptor.

The present study demonstrates negative intracellular cross-talk between angiotensin II type 2 (AT2) and insulin receptors. AT2 receptor stimulation leads to inhibition of insulin-induced extracellular signal-regulated protein kinase (ERK2) activity and cell proliferation in transfected Chinese hamster ovary (CHO-hAT2) cells. We show that AT2 receptor interferes at the initial step of insulin signaling cascade, by impairing tyrosine phosphorylation of the insulin receptor (IR) beta-chain. AT2-mediated inhibition of IR phosphorylation is insensitive to pertussis toxin and is also detected in neuroblastoma N1E-115 and pancreatic acinar AR42J cells that express endogenous receptors. We present evidence that AT2 receptor inhibits the autophosphorylating tyrosine kinase activity of IR, with no significant effect on insulin binding properties. AT2-mediated inactivation of IR does not mainly involve tyrosine dephosphorylation by vanadate-sensitive tyrosine phosphatases nor serine/threonine phosphorylation by protein kinase C. As a consequence of IR inactivation, AT2 receptor inhibits tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and signal-regulatory protein (SIRPalpha1) and prevents subsequent association of both IRS-1 and SIRPalpha1 with Src homology 2 (SH2)-containing tyrosine phosphatase SHP-2. Our results thus demonstrate functional trans-inactivation of IR kinase by G protein-coupled AT2 receptor, illustrating a novel mode of negative communication between two families of membrane receptors.

B219/OB-R 5'-UTR and leptin receptor gene-related protein gene expression in mouse brain and placenta: tissue-specific leptin receptor promoter activity.

Leptin receptor (OB-R) splice variants either encode proteins with different 3' cytoplasmic domains or have different 5' untranslated regions (UTR), indicative of dual promoters. The B219/OB-R promoter transcribes only OB-R transcripts, whereas the OB-R/GRP promoter initiates transcription of both OB-R and another protein of unknown function, called the leptin receptor gene-related protein (OB-RGRP). We compared expression of B219/OB-R 5'-UTR and OB-RGRP mRNAs by in situ hybridization. We thus assessed, by inference, the contributions of the two promoters to the leptin receptor transcript pool, in murine brain or in placenta, a tissue with abundant leptin receptor mRNA. Expression of B219/OB-R 5'-UTR mRNA (and thus by inference B219/OB-R promoter activity) in brain was similar in both distribution and relative intensity to OB-R mRNA. OB-RGRP mRNA (and thus by inference OB-R/GRP promoter activity) was widely distributed in murine brain, with elevated expression in the hypothalamic regions that express the leptin receptor mRNA, including the paraventricular nucleus. B219/OB-R 5'-UTR mRNA, but not OB-RGRP mRNA, was upregulated in hypothalamus of obese ob/ob mice. In placenta, B219/OB-R 5'-UTR mRNA was restricted to the maternal interface, and transcription of both long and short leptin receptor splice variants in the main body of the tissue thus proceeds via the OB-R/GRP promoter, strongly indicative of tissue-specific promoter usage.

Substituting selenocysteine for catalytic cysteine 41 enhances enzymatic activity of plant phospholipid hydroperoxide glutathione peroxidase expressed in Escherichia coli.

The citrus phospholipid hydroperoxide glutathione peroxidase (cit-PHGPx) was the first plant peroxidase demonstrated to exhibit PHGPx-specific enzymatic activity, although it was 500-fold weaker than that of the pig heart analog. This relatively low activity is accounted for the catalytic residue of cit-PHGPx, which was found to be cysteine and not the rare selenocysteine (Sec) present in animal enzymes. Sec incorporation into proteins is encoded by a UGA codon, usually a STOP codon, which, in prokaryotes, is suppressed by an adjacent downstream mRNA stem-loop structure, the Sec insertion sequence (SECIS). By performing appropriate nucleotide substitutions into the gene encoding cit-PHGPx, we introduced bacterial-type SECIS elements that afforded the substitution of the catalytic Cys(41) by Sec, as established by mass spectrometry, while preserving the functional integrity of the peroxidase. The recombinant enzyme, whose synthesis is selenium-dependent, displayed a 4-fold enhanced peroxidase activity as compared with the Cys-containing analog, thus confirming the higher catalytic power of Sec compared with Cys in cit-PHGPx active site. The study led also to refinement of the minimal sequence requirements of the bacterial-type SECIS, and, for the first time, to the heterologous expression in Escherichia coli of a eukaryotic selenoprotein containing a SECIS in its open reading frame.

Beta(beta)3-adrenergic receptors in human pancreatic islet and duodenal somatostatin neuroendocrine cells.

BACKGROUND: We previously localized beta3-adrenergic receptors immunohistochemically in human gastrointestinal smooth muscle and incidently found a population of human pancreatic islet cells and duodenal epithelial neuroendocrine cells that also expressed beta3-adrenergic receptors. AIM: To identify the nature of the islet and duodenal cells that stained positive for beta3-adrenergic receptors. METHODS: Paraffin sections of human pancreas and duodenum and Chinese hamster ovary cells transfected with the human beta3-adrenergic receptor were immuno-stained for beta3-adrenergic receptors using an affinity-purified rabbit polyclonal antibody (anti-P12) raised against a 15 amino acid sequence (P12) of the human receptor. Immunohistochemical staining for the receptor was carried out in the presence and absence of P12 peptide and both somatostatin 14 and 18 peptides. beta3-adrenergic receptor-stained sections were also double-immunostained with anti-insulin, -glucagon, -somatostatin and -pancreatic polypeptide antibodies. RESULTS: A subpopulation of human pancreatic islet cells and duodenal epithelial cells expressed positive cytoplasmic beta3-adrenergic receptor immunostaining. Using distribution and double-staining techniques, these cells were found to be somatostatin-positive D cells but not A or B cells. The positive staining of D cells with anti-P12 antibody was inhibited by prior incubation of the antibody with P12 peptide but not somatostatin-14 or -28 peptides. Pancreatic vascular smooth muscle and duodenal vascular and non-vascular smooth muscle also stained with anti-P12 antibody. Transfected Chinese hamster ovary cells showed positive membrane staining. CONCLUSION: We have identified a population of neuroendocrine cells in the human pancreas and duodenum that express beta3-adrenergic receptors. These cells appear to be somatostatin D cells.