A systematic study of yeast sterol biosynthetic protein-protein interactions using the split-ubiquitin system.
ABSTRACT Sterol biosynthesis occurs in the ER and most sterol biosynthetic enzymes have transmembrane domains. However, due to difficulties in characterizing membrane protein-protein interactions, the nature of the sterol biosynthetic complex as well as in vivo interactions between various enzymes have not been described. We employed a split-ubiquitin membrane protein yeast two-hybrid system to characterize interactions between sterol biosynthetic proteins. Fourteen bait constructs were co-transformed into a reporter yeast strain with 14 prey constructs representing all sterol enzymatic reactions beginning with the synthesis of squalene. Our results not only confirmed several previous interactions, but also allowed us to identify novel interactions. Based on these results, ergosterol biosynthetic enzymes display specific protein-protein interactions forming a functional complex we designate, the ergosome. In this complex, Erg11p, Erg25p, Erg27p, and Erg28p appear to form a core center that can interact with other enzymes in the pathway. Also Erg24p and Erg2p, two enzymes that are sensitive to morpholine antifungals, appear to interact with one another; however, the profile of protein interaction partners appears to be unique. Erg2p and Erg3p, two enzymes catalyzing sequential reactions also appear to have different interaction partners. Our results provide a working model as to how sterol biosynthetic enzymes are topologically organized not only in yeast but in plant and animal systems that share many of these biosynthetic reactions.
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ABSTRACT: Bitopic integral membrane proteins with a single transmembrane helix play diverse roles in catalysis, cell signaling, and morphogenesis. Complete monospanning protein structures are needed to show how interaction between the transmembrane helix and catalytic domain might influence association with the membrane and function. We report crystal structures of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase, a membrane monospanning cytochrome P450 of the CYP51 family that catalyzes the first postcyclization step in ergosterol biosynthesis and is inhibited by triazole drugs. The structures reveal a well-ordered N-terminal amphipathic helix preceding a putative transmembrane helix that would constrain the catalytic domain orientation to lie partly in the lipid bilayer. The structures locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding drug resistance.Proceedings of the National Academy of Sciences 02/2014; DOI:10.1073/pnas.1324245111 · 9.81 Impact Factor
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ABSTRACT: Currently available drugs for Chagas' disease are limited by toxicity and low efficacy in the chronic stage. Posaconazole, the most advanced new anti-chagasic drug candidate, did not fully confirm its initial potential in a Phase II clinical trial for chronic Chagas' disease. Given that posaconazole is highly active against Trypanosoma cruzi in vitro, and was very well tolerated in clinical trials, it should not be abandoned. Rather, a combination therapy may provide a highly promising outlook. Systems-scale approaches facilitate the hunt for a combination partner for posaconazole, which acts by blocking sterol biosynthesis. Mounting evidence suggests the functional interactions between sterols and sphingolipids in vivo. Here, we propose combining sterol and sphingolipid biosynthesis inhibitors to advance drug development in Chagas' disease. Copyright © 2014 Elsevier Ltd. All rights reserved.Trends in Parasitology 12/2014; 31(2). DOI:10.1016/j.pt.2014.11.004 · 6.22 Impact Factor
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ABSTRACT: Cholesterol is essential to human health, and its levels are tightly regulated by a balance of synthesis, uptake and efflux. Cholesterol synthesis requires the actions of more than twenty enzymes to reach the final product, through two alternate pathways. Here we describe a physical and functional interaction between the two terminal enzymes. 24-dehydrocholesterol reductase (DHCR24) and 7-dehydrocholesterol reductase (DHCR7) co-immunoprecipitate, and when the DHCR24 gene is knocked down by siRNA, DHCR7 activity is also ablated. Conversely, overexpression of DHCR24 enhances DHCR7 activity, but only when a functional form of DHCR24 is used. DHCR7 is important for both cholesterol and vitamin D synthesis, and we have identified a novel layer of regulation, whereby its activity is controlled by DHCR24. This suggests the existence of a cholesterol ″metabolon″, where enzymes from the same metabolic pathway interact with each other, to provide a substrate channeling benefit. We predict that other enzymes in cholesterol synthesis may similarly interact, and this should be explored in future studies. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.The Journal of Lipid Research 01/2015; 56(4). DOI:10.1194/jlr.M056986 · 4.73 Impact Factor