Transcriptional response to stress in the dynamic chromatin environment of cycling and mitotic cells.

Heat shock factors (HSFs) are the master regulators of transcription under protein-damaging conditions, acting in an environment where the overall transcription is silenced. We determined the genomewide transcriptional program that is rapidly provoked by HSF1 and HSF2 under acute stress in human cells. Our results revealed the molecular mechanisms that maintain cellular homeostasis, including HSF1-driven induction of polyubiquitin genes, as well as HSF1- and HSF2-mediated expression patterns of cochaperones, transcriptional regulators, and signaling molecules. We characterized the genomewide transcriptional response to stress also in mitotic cells where the chromatin is tightly compacted. We found a radically limited binding and transactivating capacity of HSF1, leaving mitotic cells highly susceptible to proteotoxicity. In contrast, HSF2 occupied hundreds of loci in the mitotic cells and localized to the condensed chromatin also in meiosis. These results highlight the importance of the cell cycle phase in transcriptional responses and identify the specific mechanisms for HSF1 and HSF2 in transcriptional orchestration. Moreover, we propose that HSF2 is an epigenetic regulator directing transcription throughout cell cycle progression.

Cholesterol's interactions with serine phospholipids - a comparison of N-palmitoyl ceramide phosphoserine with dipalmitoyl phosphatidylserine.

In this study we have prepared ceramide phosphoserine (CerPS) and examined its sterol-interacting properties. CerPS is a hydrogen-bonding sphingolipid, but its head group differs from that found in sphingomyelin (SM). Based on diphenylhexatriene steady-state anisotropy measurements, we observed that fully hydrated N-palmitoyl CerPS had a gel-to-liquid crystalline phase transition temperature of about 51°C in 50mM sodium phosphate buffer (pH 7.4). This was close to the T(m) measured for 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) bilayers (T(m) 50.5°C). Based on cholestatrienol (CTL) quenching experiments in liquid disordered ternary bilayers (containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphcholine; POPC), cholesterol/CTL formed sterol-enriched ordered domains with CerPS. These had similar thermostability as the sterol domains formed with N-palmitoyl SM. Cholesterol failed to form sterol-enriched ordered domains with DPPS under comparable conditions. Based on the equilibrium partitioning of CTL, we observed that the affinity of sterol for bilayers containing POPC/CerPS/cholesterol (6:3:1 by mol) was much higher than the affinity measured for control fluid POPC/cholesterol (9:1 by mol) bilayers, but slightly less than seen for comparable PSM-containing bilayers. We conclude that the phosphoserine head group was less efficient than the phosphocholine head group in stabilizing sterol/sphingolipid interaction. However, hydrogen bonding apparently can overcome some of the negative effects of the phosphoserine head group, since CerPS interacted more favorably with cholesterol compared to DPPS.

Effects of sphingomyelin headgroup size on interactions with ceramide.

Sphingomyelins (SMs) and ceramides are known to interact favorably in bilayer membranes. Because ceramide lacks a headgroup that could shield its hydrophobic body from unfavorable interactions with water, accommodation of ceramide under the larger phosphocholine headgroup of SM could contribute to their favorable interactions. To elucidate the role of SM headgroup for SM/ceramide interactions, we explored the effects of reducing the size of the phosphocholine headgroup (removing one, two, or three methyls on the choline moiety, or the choline moiety itself). Using differential scanning calorimetry and fluorescence spectroscopy, we found that the size of the SM headgroup had no marked effect on the thermal stability of ordered domains formed by SM analog/palmitoyl ceramide (PCer) interactions. In more complex bilayers composed of a fluid glycerophospholipid, SM analog, and PCer, the thermal stability and molecular order of the laterally segregated gel domains were roughly identical despite variation in SM headgroup size. We suggest that that the association between PCer and SM analogs was stabilized by ceramide's aversion for disordered phospholipids, by interfacial hydrogen bonding between PCer and the SM analogs, and by attractive van der Waals' forces between saturated chains of PCer and SM analogs.

Structure-activity relationship of sphingomyelin analogs with sphingomyelinase from Bacillus cereus.

The aim of this study was to examine how structural properties of different sphingomyelin (SM) analogs affected their substrate properties with sphingomyelinase (SMase) from Bacillus cereus. Using molecular docking and dynamics simulations (for SMase-SM complex), we then attempted to explain the relationship between SM structure and enzyme activity. With both micellar and monolayer substrates, 3O-methylated SM was found not to be degraded by the SMase. 2N-methylated SM was a substrate, but was degraded at about half the rate of its 2NH-SM control. PhytoPSM was readily hydrolyzed by the enzyme. PSM lacking one methyl in the phosphocholine head group was a good substrate, but PSM lacking two or three methyls failed to act as substrates for SMase. Based on literature data, and our docking and MD simulations, we conclude that the 3O-methylated PSM fails to interact with Mg(2+) and Glu53 in the active site, thus preventing hydrolysis. Methylation of 2NH was not crucial for binding to the active site, but appeared to interfere with an induced fit activation of the SMase via interaction with Asp156. An OH on carbon 4 in the long-chain base of phytoPSM appeared not to interfere with the 3OH interacting with Mg(2+) and Glu53 in the active site, and thus did not interfere with catalysis. Removing two or three methyls from the PSM head group apparently increased the positive charge on the terminal N significantly, which most likely led to ionic interactions with Glu250 and Glu155 adjacent to the active site. This likely interaction could have misaligned the SM substrate and hindered proper catalysis.

Membrane properties of and cholesterol's interactions with a biologically relevant three-chain sphingomyelin: 3O-palmitoyl-N-palmitoyl-D-erythro-sphingomyelin.

Sphingomyelins (SMs) are order-imposing phospholipids in cell membranes which interact favorably with cholesterol. The hydrophobic part of SM constitutes a long-chain base with an amide-linked acyl chain, whereas the polar head group is phosphocholine. The long-chain base has a free hydroxyl group in position 3, which is an important donor/acceptor in hydrogen bonding. In newborn mammals, a SM in which a palmitic acid is esterified to the 3-OH has been reported. We have synthesized this SM analog (3O-P-PSM) and studied its properties in bilayer membranes, and also determined its interactions with cholesterol. Fully hydrated 3O-P-PSM bilayers underwent a gel-to-liquid crystalline phase transition at 55.5 °C (ΔH 8 kcal/mol), which is about 15 °C higher than the phase transition temperature of PSM. The 3O-P-PSM displayed rather poor miscibility with PSM in mixed bilayers, suggesting that the third acyl chain interfered significantly with lateral interactions. Bilayers made from 3O-P-PSM were much more resistant to detergent-induced solubilization than bilayers made from PSM. In binary bilayers, cholesterol was able to destabilize the gel phase, and order the fluid phase of 3O-P-PSM, in a concentration-dependent manner. Cholesterol was also able to form sterol-enriched ordered domains with 3O-P-PSM in fluid POPC bilayers. The interaction between cholesterol and 3O-P-PSM was not, however, as favorable as the interaction between cholesterol and PSM. It is unclear what physiological role 3O-P-PSM could play in newborn mammalian membranes. However, it is clear that 3O-P-PSM will form more highly ordered domains than PSM while still having a limited ability to interact with cholesterol.

N-cholesteryl sphingomyelin-A synthetic sphingolipid with unique membrane properties.

A sphingomyelin chimera in which the amide-linked acyl chain was replaced with cholesterol carbamate was prepared and its properties examined. The sphingomyelin/cholesterol chimera (N-cholesterol-D-erythro-sphingomyelin) was able to form unilamellar vesicles of defined size when extruded through 200nm pore size membranes. These N-cholesteryl sphingomyelin bilayers were resistant to solubilization by Triton X-100. When N-cholesteryl sphingomyelin was added to N-palmitoyl sphingomyelin (N-palmitoyl-d-erythro-sphingomyelin) bilayers, it increased acyl chain order as determined by 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy. N-cholesteryl sphingomyelin was, however, not as good an inducer of membrane order compared to cholesterol on a molar basis. Differential scanning calorimetry studies further showed that the miscibility of N-cholesteryl sphingomyelin with N-palmitoyl-d-erythro-sphingomyelin bilayers was non-ideal, and the effect of N-cholesteryl sphingomyelin on the N-palmitoyl-d-erythro-sphingomyelin gel-fluid transition enthalpy differed from that seen with cholesterol. Together with N-palmitoyl-d-erythro-sphingomyelin, the N-cholesteryl sphingomyelin chimera was able to form sterol-enriched ordered domains in a fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer. N-cholesteryl sphingomyelin in the absence of N-palmitoyl-d-erythro-sphingomyelin was unable to form such sterol-enriched ordered domains in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer. However, N-cholesteryl sphingomyelin markedly increased the affinity of cholestatrienol for N-cholesteryl sphingomyelin containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers, suggesting that N-cholesteryl sphingomyelin was able to somehow stabilize sterol interaction in fluid bilayers. Based on our results, we conclude that N-cholesteryl sphingomyelin behaved more like a cholesterol than a sphingolipid in fluid bilayer membranes. Because N-cholesteryl sphingomyelin increased bilayer order, conferred resistance against detergent solubilization, and is not degradable by phospholipases A(2), it could constitute a good lipocomplex matrix for drug delivery vehicles.