Rabih Darwiche

Lamins as mediators of oxidative stress

The nuclear lamina defines both structural and functional properties of the eukaryotic cell nucleus. Mutations in the LMNA gene, encoding A-type lamins, lead to a broad spectrum of diseases termed laminopathies. While different hypotheses have been postulated to explain disease development, there is still no unified view on the mechanistic basis of laminopathies. Recent observations indicate that laminopathies are often accompanied by altered levels of reactive oxygen species and a higher susceptibility to oxidative stress at the cellular level. In this review, we highlight the role of reactive oxygen species for cell function and disease development in the context of laminopathies and present a framework of non-exclusive mechanisms to explain the reciprocal interactions between a dysfunctional lamina and altered redox homeostasis.

Schistosoma mansoni venom allergen-like protein 4 (SmVAL4) is a novel lipid-binding SCP/TAPS protein that lacks the prototypical CAP motifs

The first structure of an S. mansoni venom allergen-like protein is presented.

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

&NA; 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. Significance Statement Pathogenesis‐related proteins 1 (PR‐1) are extracellular antimicrobial proteins whose expression is induced by pathogens, but their biochemical function was elusive. Here we provide genetic and biochemical evidence that PR‐1 proteins are sterol‐binding proteins that sequester sterols from pathogens and thereby inhibit their growth.

The caveolin-binding motif of the pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is required for in vivo export of cholesteryl acetate

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in different physiological processes. Their molecular mode of action, however, is poorly understood. Saccharomyces cerevisiae expresses three members of this superfamily, pathogen-related yeast (Pry)1, -2, and -3. We have recently shown that Pry function is required for the secretion of cholesteryl acetate and that Pry proteins bind cholesterol and cholesteryl acetate, suggesting that CAP superfamily members may generally act to bind sterols or related small hydrophobic compounds. Here, we analyzed the mode of sterol binding by Pry1. Computational modeling indicates that ligand binding could occur through displacement of a relatively poorly conserved flexible loop, which in some CAP family members displays homology to the caveolin-binding motif. Point mutations within this motif abrogated export of cholesteryl acetate but did not affect binding of cholesterol. Mutations of residues located outside the caveolin-binding motif, or mutations in highly conserved putative catalytic residues had no effect on export of cholesteryl acetate or on lipid binding. These results indicate that the caveolin-binding motif of Pry1, and possibly of other CAP family members, is crucial for selective lipid binding and that lipid binding may occur through displacement of the loop containing this motif.

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.

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 Mg2+ 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 Mg2+-dependent sterol binding by Pry1.

Crystal structure of MpPR-1i, a SCP/TAPS protein from Moniliophthora perniciosa , the fungus that causes witches’ broom disease of cacao

The pathogenic fungi Moniliophthora perniciosa causes Witches’ Broom Disease (WBD) of cacao. The structure of MpPR-1i, a protein expressed by M. perniciosa when it infects cacao, are presented. This is the first reported de novo structure determined by single-wavelength anomalous dispersion phasing upon soaking with selenourea. Each monomer has flexible loop regions linking the core alpha-beta-alpha sandwich topology that comprise ~50% of the structure, making it difficult to generate an accurate homology model of the protein. MpPR-1i is monomeric in solution but is packed as a high ~70% solvent content, crystallographic heptamer. The greatest conformational flexibility between monomers is found in loops exposed to the solvent channel that connect the two longest strands. MpPR-1i lacks the conserved CAP tetrad and is incapable of binding divalent cations. MpPR-1i has the ability to bind lipids, which may have roles in its infection of cacao. These lipids likely bind in the palmitate binding cavity as observed in tablysin-15, since MpPR-1i binds palmitate with comparable affinity as tablysin-15. Further studies are required to clarify the possible roles and underlying mechanisms of neutral lipid binding, as well as their effects on the pathogenesis of M. perniciosa so as to develop new interventions for WBD.

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.

Cholesterol-Binding by the Yeast CAP Family Member Pry1 Requires the Presence of an Aliphatic Side Chain on Cholesterol

Pathogen-related yeast protein 1 (Pry1) is a Saccharomyces cerevisiae member of the CAP/SCP/TAPS superfamily. Although, CAP proteins have been proposed to be implicated in a number of physiological processes, such as pathogen virulence, sperm maturation andfertilization, host-pathogen interactions and defense mechanisms, the molecular mode of action of these proteins is poorly understood. CAP proteins are mostly secreted and they are stable in the extracellular space over a wide a range of conditions. All members of this superfamily contain a common CAP domain of approximately 150 amino acids, which adopts a unique α-β-α sandwich fold. We have previously shown that the yeast CAP family members act as sterol-binding and -export proteins in vivo and that the Pry proteins bind cholesterol and cholesteryl acetate in vitro. The conserved CAP domain of Pry1 is necessary and sufficient for sterol binding. Based on these observations, it is conceivable that CAP proteins exert their biological function through a common mechanism, such as binding and sequestration of sterols or related small hydrophobic compounds. Here we analyze the ligand specificity of Pry1 in more detail and show that the presence of the aliphatic isooctane side chain of the sterol but not the 3-hydroxyl group is important for binding to Pry1.

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

Graphical abstract