Pheno-RNA, a method to associate genes with a specific phenotype, identifies genes linked to cellular transformation.

Cellular transformation is associated with dramatic changes in gene expression, but it is difficult to determine which regulated genes are oncogenically relevant. Here we describe Pheno-RNA, a general approach to identifying candidate genes associated with a specific phenotype. Specifically, we generate a "phenotypic series" by treating a nontransformed breast cell line with a wide variety of molecules that induce cellular transformation to various extents. By performing transcriptional profiling across this phenotypic series, the expression profile of every gene can be correlated with the strength of the transformed phenotype. We identify ∼200 genes whose expression profiles are very highly correlated with the transformation phenotype, strongly suggesting their importance in transformation. Within biological categories linked to cancer, some genes show high correlations with the transformed phenotype, but others do not. Many genes whose expression profiles are highly correlated with transformation have never been associated with cancer, suggesting the involvement of heretofore unknown genes in cancer.

Necator americanus Ancylostoma Secreted Protein-2 (Na-ASP-2) Binds an Ascaroside (ascr#3) in Its Fatty Acid Binding Site.

During their infective stages, hookworms release excretory-secretory (E-S) products, small molecules, and proteins to help evade and suppress the host's immune system. Small molecules found in E-S products of mammalian hookworms include nematode derived metabolites like ascarosides, which are composed of the sugar ascarylose linked to a fatty acid side chain. The most abundant proteins found in hookworm E-S products are members of the protein family known as Ancylostoma secreted protein (ASP). In this study, two ascarosides and their fatty acid moieties were synthesized and tested for in vitro binding to Na-ASP-2 using both a ligand competition assay and microscale thermophoresis. The fatty acid moieties of both ascarosides tested and ascr#3, an ascaroside found in rat hookworm E-S products, bind to Na-ASP-2's palmitate binding cavity. These molecules were confirmed to bind to the palmitate but not the sterol binding sites. An ascaroside, oscr#10, which is not found in hookworm E-S products, does not bind to Na-ASP-2. More studies are required to determine the structural basis of ascarosides binding by Na-ASP-2 and to understand the physiological significance of these observations.

Localization and functional characterization of the pathogenesis-related proteins Rbe1p and Rbt4p in Candida albicans.

Members of the Cysteine-rich secretory protein, Antigen 5 and Pathogenesis-related 1 (CAP) protein superfamily are important virulence factors in fungi but remain poorly characterized on molecular level. Here, we investigate the cellular localization and molecular function of Rbe1p and Rbt4p, two CAP family members from the human pathogen Candida albicans. We unexpectedly found that Rbe1p localizes to budding sites of yeast cells in a disulfide bond-dependent manner. Furthermore, we show that Rbe1p and Rbt4p bind free cholesterol in vitro and export cholesteryl acetate in vivo. These findings suggest a previously undescribed role for Rbe1p in cell wall-associated processes and a possible connection between the virulence attributes of fungal CAP proteins and sterol binding.

Heligmosomoides polygyrus Venom Allergen-like Protein-4 (HpVAL-4) is a sterol binding protein.

Heligmosomoides polygyrus bakeri is a model parasitic hookworm used to study animal and human helminth diseases. During infection, the parasite releases excretory/secretory products that modulate the immune system of the host. The most abundant protein family in excretory/secretory products comprises the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. There are >30 secreted Heligmosomoides polygyrus VAL proteins (HpVALs) and these proteins are characterised by having either one or two 15 kDa CAP (cysteine-rich secretory protein (CRISP)/antigen 5/pathogenesis related-1) domains. The first known HpVAL structure, HpVAL-4, refined to 1.9 Šis reported. HpVAL-4 was produced as a homogeneously glycosylated protein in leaves of Nicotiana benthamiana infiltrated with recombinant plasmids, making this plant expression platform amenable for the production of biological products. The overall topology of HpVAL-4 is a three layered αßα sandwich between a short N-terminal loop and a C-terminal cysteine rich extension. The C-terminal cysteine rich extension has two strands stabilized by two disulfide bonds and superposes well with the previously reported extension from the human hookworm Necator americanus Ancylostoma secreted protein-2 (Na-ASP-2). The N-terminal loop is connected to alpha helix 2 via a disulfide bond previously observed in Na-ASP-2. HpVAL-4 has a central cavity that is more similar to the N-terminal CAP domain of the two CAP Na-ASP-1 from Necator americanus. Unlike Na-ASP-2, mammalian CRISP, and the C-terminal CAP domain of Na-ASP-1, the large central cavity of HpVAL-4 lacks the two histidines required to coordinate divalent cations. HpVAL-4 has both palmitate-binding and sterol-binding cavities and is able to complement the in vivo sterol export phenotype of yeast mutants lacking their endogenous CAP proteins. More studies are required to determine endogenous binding partners of HpVAL-4 and unravel the possible impact of sterol binding on immune-modulatory functions.

Crystal structure of Brugia malayi venom allergen-like protein-1 (BmVAL-1), a vaccine candidate for lymphatic filariasis.

Brugia malayi is a causative agent of lymphatic filariasis, a major tropical disease. The infective L3 parasite stage releases immunomodulatory proteins including the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. BmVAL-1 is a major target of host immunity with >90% of infected B. malayi microfilaraemic cases being seropositive for antibodies to BmVAL-1. This study is part of ongoing efforts to characterize the structures and functions of important B. malayi proteins. Recombinant BmVAL-1 was produced using a plant expression system, crystallized and the structure was solved by molecular replacement and refined to 2.1 Å, revealing the characteristic alpha/beta/alpha sandwich topology of eukaryotic SCP/TAPS proteins. The protein has more than 45% loop regions and these flexible loops connect the helices and strands, which are longer than predicted based on other parasite SCP/TAPS protein structures. The large central cavity of BmVAL-1 is a prototypical CRISP cavity with two histidines required to bind divalent cations. The caveolin-binding motif (CBM) that mediates sterol binding in SCP/TAPS proteins is large and open in BmVAL-1 and is N-glycosylated. N-glycosylation of the CBM does not affect the ability of BmVAL-1 to bind sterol in vitro. BmVAL-1 complements the in vivo sterol export phenotype of yeast mutants lacking their endogenous SCP/TAPS proteins. The in vitro sterol-binding affinity of BmVAL-1 is comparable with Pry1, a yeast sterol transporting SCP/TAPS protein. Sterol binding of BmVAL-1 is dependent on divalent cations. BmVAL-1 also has a large open palmitate-binding cavity, which binds palmitate comparably to tablysin-15, a lipid-binding SCP/TAPS protein. The central cavity, CBM and palmitate-binding cavity of BmVAL-1 are interconnected within the monomer with channels that can serve as pathways for water molecules, cations and small molecules.

A Ligand-Binding Assay to Measure the Affinity and Specificity of Sterol-Binding Proteins In Vitro.

Sterols are major constituents of the plasma membrane of eukaryotic cells and serve as a precursor for several classes of signaling molecules, including steroids and hydroxy sterols. They maintain the functionality and permeability barrier of the plasma membrane through lipid-lipid and lipid-protein interactions. The S. cerevisiae pathogen-related yeast proteins 1, 2, and 3 (Pry) belong to a large protein superfamily known as CAP/SCP/TAPS. Members of this superfamily have been implicated in a wide variety of processes, including immune defense in mammals and plants, pathogen virulence, sperm maturation and fertilization, venom toxicity, and prostate and brain cancer. Pry proteins bind and export sterols in vivo and the purified Pry1 protein binds sterols and related small hydrophobic compounds in vitro. Here we describe a method to determine lipid binding of a purified protein in vitro.

The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein.

Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites.

The sterol-binding activity of PATHOGENESIS-RELATED PROTEIN 1 reveals the mode of action of an antimicrobial protein.

Pathogenesis-related proteins played a pioneering role 50 years ago in the discovery of plant innate immunity as a set of proteins that accumulated upon pathogen challenge. The most abundant of these proteins, PATHOGENESIS-RELATED 1 (PR-1) encodes a small antimicrobial protein that has become, as a marker of plant immune signaling, one of the most referred to plant proteins. The biochemical activity and mode of action of PR-1 proteins has remained elusive, however. Here, we provide genetic and biochemical evidence for the capacity of PR-1 proteins to bind sterols, and demonstrate that the inhibitory effect on pathogen growth is caused by the sequestration of sterol from pathogens. In support of our findings, sterol-auxotroph pathogens such as the oomycete Phytophthora are particularly sensitive to PR-1, whereas sterol-prototroph fungal pathogens become highly sensitive only when sterol biosynthesis is compromised. Our results are in line with previous findings showing that plants with enhanced PR-1 expression are particularly well protected against oomycete pathogens.

Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein.

The production, crystal structure, and functional characterization of the C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of pathogen-related yeast protein-1 (Pry1) from Saccharomyces cerevisiae is presented. The CAP domain of Pry1 (Pry1CAP) is functional in vivo as its expression restores cholesterol export to yeast mutants lacking endogenous Pry1 and Pry2. Recombinant Pry1CAP forms dimers in solution, is sufficient for in vitro cholesterol binding, and has comparable binding properties as full-length Pry1. Two crystal structures of Pry1CAP are reported, one with Mg(2+) coordinated to the conserved CAP tetrad (His208, Glu215, Glu233 and His250) in spacegroup I41 and the other without divalent cations in spacegroup P6122. The latter structure contains four 1,4-dioxane molecules from the crystallization solution, one of which sits in the cholesterol binding site. Both structures reveal that the divalent cation and cholesterol binding sites are connected upon dimerization, providing a structural basis for the observed Mg(2+)-dependent sterol binding by Pry1.