Nancy Y. Villa

Sphingolipids Function as Downstream Effectors of a Fungal PAQR

The Izh2p protein from Saccharomyces cerevisiae belongs to the newly characterized progestin and adipoQ receptor (PAQR) superfamily of receptors whose mechanism of signal transduction is still unknown. Izh2p functions as a receptor for the plant PR-5 defensin osmotin and has pleiotropic effects on cellular biochemistry. One example of this pleiotropy is the Izh2p-dependent repression of FET3, a gene involved in iron-uptake. Although the physiological purpose of FET3 repression by Izh2p is a matter of speculation, it provides a reporter with which to probe the mechanism of signal transduction by this novel class of receptor. Receptors in the PAQR family share sequence similarity with enzymes involved in ceramide metabolism, which led to the hypothesis that sphingolipids are involved in Izh2p-dependent signaling. In this study, we demonstrate that drugs affecting sphingolipid metabolism, such as d-erythro-MAPP and myriocin, inhibit the effect of Izh2p on FET3. We also show that Izh2p causes an increase in steady-state levels of sphingoid base. Moreover, we show that Izh2p-independent increases in sphingoid bases recapitulate the effect of Izh2p on FET3. Finally, our data indicate that the Pkh1p and Pkh2p sphingoid base-sensing kinases are essential components of the Izh2p-dependent signaling pathway. In conclusion, our data indicate that Izh2p produces sphingoid bases and that these bioactive lipids probably function as the second messenger responsible for the effect of Izh2p on FET3.

Analysis of Vaccinia Virus−Host Protein−Protein Interactions: Validations of Yeast Two-Hybrid Screenings

Vaccinia virus, a large double-stranded DNA virus, is the prototype of the Orthopoxvirus genus, which includes several pathogenic poxviruses of humans, such as monkeypox virus and variola virus. Here, we report a comprehensive yeast two-hybrid (Y2H) screening for the protein−protein interactions between vaccinia and human proteins. A total of 109 novel vaccinia−human protein interactions were detected among 33 viral proteins. To validate subsets of those interactions, we constructed an ORFeome library of vaccinia virus strain WR using the Gateway plasmid cloning system. By co-expressing selected vaccinia and host proteins in a variety of expression systems, we found that at least 17 of the Y2H hits identified between vaccinia and human proteins can be verified by independent methods using GST pull-down assays, representing a 63% validation rate for the Y2H hits examined (17/27). Because the cloned ORFs are conveniently transferable from the entry vectors to various destination expression vectors, the vaccinia ORFeome library will be a useful resource for future high-throughput functional proteomic experiments.

Interplay between poxviruses and the cellular ubiquitin/ubiquitin-like pathways

Post‐translational polypeptide tagging by conjugation with ubiquitin and ubiquitin‐like (Ub/Ubl) molecules is a potent way to alter protein functions and/or sort specific protein targets to the proteasome for degradation. Many poxviruses interfere with the host Ub/Ubl system by encoding viral proteins that can usurp this pathway. Some of these include viral proteins of the membrane‐associated RING‐CH (MARCH) domain, p28/Really Interesting New Gene (RING) finger, ankyrin‐repeat/F‐box and Broad‐complex, Tramtrack and Bric‐a‐Brac (BTB)/Kelch subgroups of the E3 Ub ligase superfamily. Here we describe and discuss the various strategies used by poxviruses to target and subvert the host cell Ub/Ubl systems.

Antagonism of Human Adiponectin Receptors and Their Membrane Progesterone Receptor Paralogs by TNFα and a Ceramidase Inhibitor

The progestin and AdipoQ receptor (PAQR) family of proteins comprises three distinct structural classes, each with seemingly different agonist specificities. For example, Class I receptors, like the human adiponectin receptors (AdipoR1 and AdipoR2), sense proteins with a particular three-dimensional fold, while Class II receptors are nonclassical membrane receptors for the steroid hormone progesterone. Using a previously developed heterologous expression system to study PAQR receptor activity, we demonstrate that human PAQRs from all three classes are antagonized by both 1(S),2(R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, a ceramidase inhibitor, and TNFalpha, a homologue of adiponectin that functions antagonistically to both adiponectin and progesterone in human cells.

Poxvirus Proteomics and Virus-Host Protein Interactions

SUMMARY Studies of the functional proteins encoded by the poxvirus genome provide information about the composition of the virus as well as individual virus-virus protein and virus-host protein interactions, which provides insight into viral pathogenesis and drug discovery. Widely used proteomic techniques to identify and characterize specific protein-protein interactions include yeast two-hybrid studies and coimmunoprecipitations. Recently, various mass spectrometry techniques have been employed to identify viral protein components of larger complexes. These methods, combined with structural studies, can provide new information about the putative functions of viral proteins as well as insights into virus-host interaction dynamics. For viral proteins of unknown function, identification of either viral or host binding partners provides clues about their putative function. In this review, we discuss poxvirus proteomics, including the use of proteomic methodologies to identify viral components and virus-host protein interactions. High-throughput global protein expression studies using protein chip technology as well as new methods for validating putative protein-protein interactions are also discussed.

Myxoma and vaccinia viruses exploit different mechanisms to enter and infect human cancer cells

Myxoma (MYXV) and vaccinia (VACV) viruses have recently emerged as potential oncolytic agents that can infect and kill different human cancer cells. Although both are structurally similar, it is unknown whether the pathway(s) used by these poxviruses to enter and cause oncolysis in cancer cells are mechanistically similar. Here, we compared the entry of MYXV and VACV-WR into various human cancer cells and observed significant differences: 1--low-pH treatment accelerates fusion-mediated entry of VACV but not MYXV, 2--the tyrosine kinase inhibitor genistein inhibits entry of VACV, but not MYXV, 3--knockdown of PAK1 revealed that it is required for a late stage event downstream of MYXV entry into cancer cells, whereas PAK1 is required for VACV entry into the same target cells. These results suggest that VACV and MYXV exploit different mechanisms to enter into human cancer cells, thus providing some rationale for their divergent cancer cell tropisms.

Drug discovery targeting human 5-HT2C receptors: Residues S3.36 and Y7.43 impact ligand—binding pocket structure via hydrogen bond formation

Specific activation of serotonin (5-HT) 5-HT(2C) G protein-coupled receptors may be therapeutic for obesity and neuropsychiatric disorders. Mutagenesis coupled with computational and molecular modeling experiments based on the human β₂ adrenergic receptor structure was employed to delineate the interactions of different ligands at human 5-HT(2C) residues D3.32, S3.36 and Y7.43. No binding of the tertiary amine radioligand ([³H]-mesulergine) could be detected when the 5-HT(2C) D3.32 residue was mutated to alanine (D3.32A). The S3.36A point-mutation greatly reduced affinity of primary amine ligands, modestly reduced affinity of a secondary amine, and except for the 5-HT(2C)-specific agonist N(CH₃)₂-PAT, affinity of tertiary amines was unaffected. Molecular modeling results indicated that the primary amines form hydrogen bonds with the S3.36 residue, whereas, with the exception of N(CH₃)₂-PAT, tertiary amines do not interact considerably with this residue. The Y7.43A point-mutation greatly reduced affinity of 5-HT, yet reduced to a lesser extent the affinity of tryptamine that lacks the 5-hydroxy moiety present in 5-HT; modeling results indicated that the 5-HT 5-hydroxy moiety hydrogen bonds with Y7.43 at the 5-HT(2C) receptor. Additional modeling results showed that 5-HT induced a hydrogen bond between Y7.43 and D3.32. Finally, modeling results revealed two low-energy binding modes for 5-HT in the 5-HT(2C) binding pocket, supporting the concept that multiple agonist binding modes may stabilize different receptor active conformations to influence signaling. Ligand potencies for modulating WT and point-mutated 5-HT(2C) receptor-mediated phospholipase C activity were in accordance with the affinity data. Ligand efficacies, however, were altered considerably by the S3.36A mutation only.

Phylogenetic and Preliminary Phenotypic Analysis of Yeast PAQR Receptors: Potential Antifungal Targets

Proteins belonging to the Progestin and AdipoQ Receptor (PAQR) superfamily of membrane bound receptors are ubiquitously found in fungi. Nearly, all fungi possess two evolutionarily distinct paralogs of PAQR protein, which we have called the PQRA and PQRB subtypes. In the model fungus Saccharomyces cerevisiae, these subtypes are represented by the Izh2p and Izh3p proteins, respectively. S. cerevisiae also possesses two additional PQRA-type receptors called Izh1p and Izh4p that are restricted to other species within the “Saccharomyces complex”. Izh2p has been the subject of several recent investigations and is of particular interest because it regulates fungal growth in response to proteins produced by plants and, as such, represents a new paradigm for interspecies communication. We demonstrate that IZH2 and IZH3 gene dosage affects resistance to polyene antifungal drugs. Moreover, we provide additional evidence that Izh2p and Izh3p negatively regulate fungal filamentation. These data suggest that agonists of these receptors might make antifungal therapeutics, either by inhibiting fungal development or by sensitizing fungi to the toxic effects of current antifungal therapies. This is particularly relevant for pathogenic fungi such as Candida glabrata that are closely related to S. cerevisiae and contain the same complement of PAQR receptors.

Aromatic interactions impact ligand binding and function at serotonin 5-HT2C G protein-coupled receptors: Receptor homology modeling, ligand docking, and molecular dynamics results validated by experimental studies.

The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2 G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ∼75% transmembrane (TM) sequence identity. Agonists for 5-HT2C receptors are under development for psychoses; whereas, at 5-HT2A receptors, antipsychotic effects are associated with antagonists – in fact, 5-HT2A agonists can cause hallucinations and 5-HT2B agonists cause cardiotoxicity. It is known that 5-HT2A TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function; however, ligand interactions with these residues at the 5-HT2C receptor have not been reported. To predict and validate molecular determinants for 5-HT2C-specific activation, results from receptor homology modelling, ligand docking, and molecular dynamics simulation studies were compared with experimental results for ligand binding and function at wild type and W6.48A, F6.51A, and F6.52A point-mutated 5-HT2C receptors.

Dissecting the regulation of yeast genes by the osmotin receptor

The Izh2p protein from Saccharomyces cerevisiae is a receptor for the plant antifungal protein, osmotin. Since Izh2p is conserved in fungi, understanding its biochemical function could inspire novel strategies for the prevention of fungal growth. However, it has been difficult to determine the exact role of Izh2p because it has pleiotropic effects on cellular biochemistry. Herein, we demonstrate that Izh2p negatively regulates functionally divergent genes through a CCCTC promoter motif. Moreover, we show that Izh2p-dependent promoters containing this motif are regulated by the Nrg1p/Nrg2p and Msn2p/Msn4p transcription factors. The fact that Izh2p can regulate gene expression through this widely dispersed element presents a reasonable explanation of its pleiotropy. The involvement of Nrg1p/Nrgp2 in Izh2p-dependent gene regulation also suggests a role for this receptor in regulating fungal differentiation in response to stimuli produced by plants.