Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment.

Proline is established as a potent breaker of both alpha-helical and beta-sheet structures in soluble (globular) proteins. Thus, the frequent occurrence of the Pro residue in the putative transmembrane helices of integral membrane proteins, particularly transport proteins, presents a structural dilemma. We propose that this phenomenon results from the fact that the structural propensity of a given amino acid may be altered to conform to changes imposed by molecular environment. To test this hypothesis on proline, we synthesized model peptides of generic sequence H2N-(Ser-LyS)2-Ala- Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH (Z = Ala and/or Pro). Peptide conformations were analyzed by circular dichroism spectroscopy in aqueous buffer, SDS, lysophosphatidylglycerol micelles, and organic solvents (methanol, trifluoroethanol, and 2-propanol). The helical propensity of Pro was found to be greatly enhanced in the membrane-mimetic environments of both lipid micelles and organic solvents. Proline was found to stabilize the alpha-helical conformation relative to Ala at elevated temperatures in 2-propanol, an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media. Parallel studies in deoxycholate micelles of the temperature-induced conformational transitions of the single-spanning membrane bacteriophage IKe major coat protein, in which the Pro-containing wild type was compared with Pro30 --> Ala mutant, Pro was found to protect the helix, but disrupt the beta-sheet structure as effectively as it does to model peptides in water. The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels. The overall data provide a rationale for predicting and understanding the structural consequences when Pro occurs in the context of a membrane.

Sphingolipid synthesis as a target for chemotherapy against malaria parasites.

The human malaria parasite Plasmodium falciparum contains sphingomyelin synthase in its Golgi apparatus and in a network of tubovesicular membranes in the cytoplasm of the infected erythrocyte. Palmitoyl and decanoyl analogues of 1-phenyl-2-acylamino-3-morpholino-1-propanol inhibit the enzyme activity in infected erythrocytes. An average of 35% of the activity is extremely sensitive to these drugs and undergoes a rapid, linear decrease at drug concentrations of 0.05-1 microM. The remaining 65% suffers a slower linear inhibition at drug concentrations ranging from 25 to 500 microM. Evidence is presented that inhibition of the sensitive fraction alone selectively disrupts the appearance of the interconnected tubular network in the host cell cytoplasm, without blocking secretory development at the parasite plasma membrane or in organelles within the parasite, such as the Golgi and the digestive food vacuole. This inhibition also blocks parasite proliferation in culture, indicating that the sensitive sphingomyelin synthase activity as well as the tubovesicular network may provide rational targets for drugs against malaria.

Electrophile and antioxidant regulation of enzymes that detoxify carcinogens.

Detoxication (phase 2) enzymes, such as glutathione S-transferases (GSTs), NAD(P)H:(quinone-acceptor) oxidoreductase (QR), and UDP-glucuronsyltransferase, are induced in animal cells exposed to a variety of electrophilic compounds and phenolic antioxidants. Induction protects against the toxic and neoplastic effects of carcinogens and is mediated by activation of upstream electrophile-responsive/antioxidant-responsive elements (EpRE/ARE). The mechanism of activation of these enhancers was analyzed by transient gene expression of growth hormone reporter constructs containing a 41-bp region derived from the mouse GST Ya gene 5'-upstream region that contains the EpRE/ARE element and of constructs in which this element was replaced with either one or two consensus phorbol 12-tetradecanoate 13-acetate (TPA)-responsive elements (TREs). When these three constructs were compared in Hep G2 (human) and Hepa 1c1c7 (murine) hepatoma cells, the wild-type sequence was highly activated by diverse inducers, including tert-butylhydroquinone, Michael reaction acceptors, 1,2-dithiole-3-thione, sulforaphane,2,3-dimercapto-1-propanol, HgCl2, sodium arsenite, and phenylarsine oxide. In contrast, constructs with consensus TRE sites were not induced significantly. TPA in combination with these compounds led to additive or synergistic inductions of the EpRE/ARE construct, but induction of the TRE construct was similar to that induced by TPA alone. Transfection of the EpRE/ARE reporter construct into F9 cells, which lack endogenous TRE-binding proteins, produced large inductions by the same compounds, which also induced QR activity in these cells. We conclude that activation of the EpRE/ARE by electrophile and antioxidant inducers is mediated by EpRE/ARE-specific proteins.