Bruno Cardinaud

Suppression of microRNA-silencing pathway by HIV-1 during virus replication.

MicroRNAs (miRNAs) are single-stranded noncoding RNAs of 19 to 25 nucleotides that function as gene regulators and as a host cell defense against both RNA and DNA viruses. We provide evidence for a physiological role of the miRNA-silencing machinery in controlling HIV-1 replication. Type III RNAses Dicer and Drosha, responsible for miRNA processing, inhibited virus replication both in peripheral blood mononuclear cells from HIV-1–infected donors and in latently infected cells. In turn, HIV-1 actively suppressed the expression of the polycistronic miRNA cluster miR-17/92. This suppression was found to be required for efficient viral replication and was dependent on the histone acetyltransferase Tat cofactor PCAF. Our results highlight the involvement of the miRNA-silencing pathway in HIV-1 replication and latency.

An evolutionary view of drug-receptor interaction : the bioamine receptor family

The large molecular diversity of receptors and their subtypes means that the pharmacologist is faced with many puzzling characterization questions. First, the molecular diversity of the receptors is deciphered only in part by a pharmacological approach, which precludes a satisfactory receptor classification based solely on pharmacological characteristics. Second, the physiological counterpart of the numerous subtypes of receptors specifically activated by single endogenous ligands remains unclear. Here, Philippe Vernier and colleagues use the example of the bioamine G protein-coupled receptors to show that many of the apparent inconsistencies that emerge from pharmacological and molecular characterizations of receptors can be better understood if the evolutionary history of the receptors is taken into account.

Early Emergence of Three Dopamine D1 Receptor Subtypes in Vertebrates MOLECULAR PHYLOGENETIC, PHARMACOLOGICAL, AND FUNCTIONAL CRITERIA DEFINING D1A, D1B, AND D1C R/ECEPTORS IN EUROPEAN EELANGUILLA ANGUILLA

The existence of dopamine D1C and D1D receptors in Xenopus and chicken, respectively, challenged the established duality (D1A and D1B) of the dopamine D1 receptor class in vertebrates. To ascertain the molecular diversity of this gene family in early diverging vertebrates, we isolated four receptor-encoding sequences from the European eel Anguilla anguilla. Molecular phylogeny assigned two receptor sequences (D1A1 and D1A2) to the D1A subtype, and a third receptor to the D1B subtype. Additional sequence was orthologous to the Xenopus D1C receptor and to several other previously unclassified fish D1-like receptors. When expressed in COS-7 cells, eel D1A and D1B receptors display affinity profiles for dopaminergic ligands similar to those of other known vertebrate homologues. The D1C receptor exhibits pharmacological characteristics virtually identical to its Xenopus homologue. Functionally, while all eel D1 receptors stimulate adenylate cyclase, the eel D1B receptor exhibits greater constitutive activity than either D1A or D1C receptors. Semiquantitative reverse transcription-polymerase chain reaction reveals the differential distribution of D1A1, D1A2, D1B, and D1C receptor mRNA within the hypothalamic-pituitary axis of the eel brain. Taken together, these data suggest that the D1A, D1B, and D1C receptors arose prior to the evolutionary divergence of fish and tetrapods and exhibit molecular, pharmacological, and functional attributes that unambiguously allow for their classification as distinct D1 receptor subtypes in the vertebrate phylum.

Evolution and Origin of the Diversity of Dopamine Receptors in Vertebrates

Publisher Summary In this chapter a comparative approach of the dopamine 1 (D1)-receptor class has been undertaken to analyze in detail the dopamine-receptor multiplicity of D1-like receptors in vertebrates by cloning the corresponding genes in most of the main groups of vertebrates. The aim of the chapter is to identify the events that led to the emergence of a “new” D1-receptor gene in the evolution of vertebrates. This phylogenetical approach provides conclusive evidence for the existence of two other subtypes of D1 receptors, one named D1C, found in all the jawed vertebrates except mammals, and one subtype termed D1D, only to be found in birds. Therefore, the D1-receptor class comprises three to four subtypes in “higher” vertebrates, virtually indentical to other catecholamine receptor families, indicating that the gene duplications at the origin of this receptor multiplicity occurred before or concomitantly with the appearance of Chondrychtians (cartilaginous fish). As all of the known bioamine receptor subtypes are expressed in the central nervous system, it is proposed that the bioamine and dopamine D1-receptor diversification accompanies in some respect the genetic mechanisms leading to the encephalization of the vertebrate nervous system. Acquisition and changes in the expression pattern of bioamine receptors is likely to have been the major factor of duplicated gene conservation in vertebrates.

Comparative analysis of melanin-concentrating hormone structure and activity in fishes and mammals.

A comparative analysis of the structure of the melanin-concentrating hormone (MCH) precursor reveals that this sequence has been subjected to a higher selection pressure in mammals than in teleosts, suggesting that the structural constraints have not been the same throughout the vertebrate lineage. In contrast, the MCH peptide sequence has been very well conserved in all species. A sensitive and reproducible eel skin assay was developed and allowed us to define the structural features needed for a full MCH bioactivity. It was shown that the minimal structure carrying the critical residues was the same in fishes and in mammals. A pharmacological approach confirmed that MCH receptor activation decreased the cAMP levels in the fish skin, but this effect appeared to be independent from a Galphai protein. We propose that one of the intracellular signaling pathways of the MCH receptor in fish skin is the activation of one or several cellular phosphodiesterases.

Characterization of MCH-gene-overprinted-polypeptide-immunoreactive material in hypothalamus reveals an inhibitory role of pro-somatostatin1−64 on somatostatin secretion

The melanin‐concentrating hormone (MCH) gene encodes two proteins, pro‐MCH and MCH‐gene‐overprinted polypeptide (MGOP), produced through alternative splicing of the primary transcript. Our initial purpose was to characterize the MGOP‐immunoreactive material. First, MGOP mRNA was clearly found in rat and mouse hypothalami but Western blot analysis failed to unambiguously identify MGOP in protein extracts. Immunohistochemical experiments with wild‐type and MCH gene‐null mice demonstrated genuine expression of MGOP confined to the MCH‐containing neurons in the lateral hypothalamus area and the presence of an ‘MGOP‐like’ antigen in periventricular nucleus and arcuate nucleus neurons and their area of projection. This suggested a colocalization in somatostatin (SRIF) hypophysiotropic neurons. Further characterization, using SRIF gene‐null mice and Western blot analysis with recombinant proteins, revealed that the MGOP‐like product was pro‐SRIF1−64. The role of pro‐SRIF1−64 on fetal hypothalamic neurons was evaluated and a strong tonic inhibitory effect on SRIF secretion was found. These results (i) indicate that MGOP expression is restricted to the MCH neurons in the lateral hypothalamus and that MGOP‐like immunoreactivity outside this system corresponds to pro‐SRIF1−64, and (ii) provide the first evidence for a negative feedback regulation by pro‐SRIF1−64 on SRIF secretion, suggesting new mechanisms by which the pro‐region of a neuropeptide precursor may control the regulated secretion of a neuropeptide derived from the same precursor.

SVK14 cells express an MCH binding site different from the MCH1 or MCH2 receptor

Melanin-concentrating hormone (MCH) is a cyclic peptide, mainly involved in the regulation of skin pigmentation in teleosts and feeding behavior in mammals. The human keratinocyte SVK14 cell line has been previously shown to express binding sites for the MCH analog [125I]-[Phe13,3-iodo-Tyr19]MCH. We report here that: (1) this binding site similarly recognized [125I]-[3-iodo-Tyr13]MCH; (2) its pharmacological profile clearly differed from those observed at the two human MCH receptor subtypes, MCH1-R and MCH2-R; (3) MCH did not induce any effect on second messenger systems (including cAMP, calcium, and MAP kinase signaling pathways), and (4) no mRNAs corresponding to the MCH receptors were found. In conclusion, the binding site characterized in the SVK14 cell line is distinct from the MCH1 and MCH2 receptors and deserves therefore further investigation.

The classification of bioamine receptors. How helpful are molecular phylogenies

Monoamine receptors coupled to G proteins constitute a gene family (i.e., genes encoding proteins with similar structure and functions) which exhibits a large molecular diversity. The concept of gene family implies that the origin of its multiple members is primarily gene duplications. These genes derived from a common ancestor gene by successive steps of duplications followed by mutations and selection by physiological constraints in animal species and exhibit ancestor-descent relationships. Two kind of diversification mechanisms should be considered. Firstly, gene duplications spreading in large populations yield receptor subtypes (paralogous genes), and secondly, speciation events multiply species populations which evolve on their own and homologous receptors in these species (orthologous receptors) genetically diverge to the same extent than the species themselves. The pharmacological differences between receptors reflect in part this evolutionary diversification, but pharmacology alone cannot unravel the nature of receptor homologies. “Natural” classifications based on phylogenetic methods use sequences of genes and proteins and provide clues to the genetic events which generated molecular diversity. They easily distinguish between paralogous receptors (true subtypes) and orthologous receptors (species homologues), and help to homogenize receptor nomenclature. To c l a m the diversification of bioamine receptor family close to the origin of vertebrates we have cloned a dozen of dopamine receptors in “lower” vertebrates (lampreys, hagfish, European eel) and used various phylogenetical methods to classify all the known sequences of vertebrate bioamine receptors (FIG. 1). Important results are: i) Classes, subtypes, species homologues and polymorphic variants of the receptors are clearly individualized on the structural basis. ii) Carefully controlled tree topologies suggest that the main classes of receptors were generated before the divergence of vertebrates from invertebrates (Dl, D2, al, a2, b, SHTlA, 5HT2, etc.), and that the diversification of these receptor classes into subtypes (Dla, Dlb, Dlc, a2a, a2b, a2c, etc.) was contemporary to vertebrate emergence and is likely to depend on two steps of genome tetraploidization. However, the use of molecular phylogeny methods has also severe limitations: the rate of receptor evolution is highly irregular in this protein family (but proportionally similar for all the domains of the receptors). iii) “Saturation” of sequence similarity occurs at the basis of the tree and the relationships of the main classes of bioamine

Ligand Binding Profile and Effects of Melanin-Concentrating Hormone on Fish and Mammalian Skin Cells

and a 19-amino-acid peptide in mammals. 2 In teleost fish, MCH is produced by hypothalamic neurons, and it acts as a potent paling factor either by direct action on melanocytes, or by inhibition of α -MSH release at the level of the pituitary gland (for a review see Ref. 3). In mammals, MCH synthesis is confined to the lateral hypothalamus and zona incerta, whereas MCH fibers are widely distributed in the central nervous system. 4 MCH is involved as a neuromediator/neuromodulator in a broad array of functions in the brain (see Refs. 3 and 5 for reviews). In the periphery, MCH acts as a paracrine/autocrine inducer of water and electrolyte secretion in the gut. It may also regulate spermatogenesis in rodent and human testis (see Ref. 5 for a review).

Subtype-specific regulation of dopamine D1 receptors in vertebrates cells

Annexins constimte a multigene family of proteins broadly expressed, both phylogenetically and within cells and tissues. They arc characterized by theJr ability to bii calcium and anionic phospholipids and by a conserved 70-aminoacid domain, repeated either four or eight times. These proteins have been implicated in multiple aspects of cell biology including regulation of membrane trafficking, transmcmbrane channel activity, inhibition of phospholipase A2, inhibition of coagulation, transduction of mitogenic signal ad SCtKhmcnt of cchnaKrix inKcractions. Howcvcr e~acf bidlogical functions of the annexins are not yet known. (1) i&-&e-de G&of%kpe tdoldcula~re. CNRS. 91190 Giisur Yvette