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.

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.

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.

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.