Elena A. Shirokova

The prohibitin (PHB)-domain proteins are membrane proteins that regulate a variety of biological activities, including mechanosensation, osmotic homeostasis, and cell signaling, although the mechanism of this regulation is unknown. We have studied two members of this large protein family, MEC-2, which is needed for touch sensitivity in Caenorhabditis elegans, and Podocin, a protein involved in the function of the filtration barrier in the mammalian kidney, and find that both proteins bind cholesterol. This binding requires the PHB domain (including palmitoylation sites within it) and part of the N-terminally adjacent hydrophobic domain that attaches the proteins to the inner leaflet of the plasma membrane. By binding to MEC-2 and Podocin, cholesterol associates with ion-channel complexes to which these proteins bind: DEG/ENaC channels for MEC-2 and TRPC channels for Podocin. Both the MEC-2-dependent activation of mechanosensation and the Podocin-dependent activation of TRPC channels require cholesterol. Thus, MEC-2, Podocin, and probably many other PHB-domain proteins by binding to themselves, cholesterol, and target proteins regulate the formation and function of large protein-cholesterol supercomplexes in the plasma membrane.

Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein alphaolf and adenylyl cyclase type-III to the Ca2+ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of Galphaolf in odorant-specific signaling out of OR. The employment of the non-typical G protein alpha15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescence-imaging plate reader experiments, resulting in the de-orphaning of two new OR for the odorant (-)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (-)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of Galpha15 or Galphaolf. We finally established an EC50-ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR.

The human genome harbors 5 remnant genes coding for vomeronasal type-1 receptors, compared with 187 of such receptors in mice. In rodents, vomeronasal type-1 receptors are typically expressed in the vomeronasal organ. They are believed to be highly selective and sensitive pheromone detectors, as may be inferred from one receptor, V1rb2, responding to picomolar concentrations of the mouse pheromone 2-heptanone. Expression patterns, ligands, and signal transduction of human vomeronasal type-1 receptors have, however, remained largely obscure. Our aim was to deorphan and functionally characterize these 5 putative human pheromone receptors. Here, we report functional expression for all 5 receptors in HeLa/Olf cells. The recombinant, N-terminally tagged receptors expressed at the plasma membrane of HeLa/Olf cells and responded differentially to 19 of 140 odorants in a combinatorial way. C9-C10 aliphatic alcohols or aldehydes emerged as the best agonists at submicromolar concentrations above human odorant thresholds. Surprisingly, and in contrast to mouse V1rb2, all human vomeronasal type-1 receptors activated cAMP signaling via G protein alphaolf, when expressed in HeLa/Olf cells. While a biological function of human vomeronasal type-1 receptors is still elusive, our data show that their major functional characteristics are similar to those of odorant receptors.

IMPORTANCE OF THE FIELD: AIDS, a disease caused by human immunodeficiency virus, was called 'plague of the twentieth century'. 3'-Azido-3'-deoxythymidine (AZT), the first compound approved for the treatment of HIV, is still a mandatory component of treatment schemes. However, its toxicity stimulated a search for new agents. AREAS COVERED IN THIS REVIEW: This review presents the history and current state of the design of AZT prodrugs based on its phosphonate derivatives. WHAT THE READER WILL GAIN: Although every effort was made to include as many AZT structures bearing phosphonate residues and demonstrate the variety they offer, we also concentrated on the studies performed in our laboratory. Special attention was also paid to AZT 5'-H-phosphonate (phosphazide, Nikavir) approved in the Russian Federation as a drug for the prevention and treatment of HIV infection. TAKE HOME MESSAGE: The prodrug strategy applied to AZT phosphonate derivatives enriched chemistry, biology and medicine not only with new knowledge, methods and structures, but also with a new anti-HIV drug Nikavir. Currently, study of another phosphonate, AZT 5'-aminocarbonylphosphonate, is underway. Slow release of AZT following oral administration and penetration into cells, decreased toxicity and the lack of cumulative properties make the compounds of this group promising as extended-release forms of AZT.