Emily Westover - Academia.edu (original) (raw)

Papers by Emily Westover

Steroids, Feb 1, 2003

We report the first synthesis of the unnatural enantiomer of desmosterol (ent-desmosterol). The s... more We report the first synthesis of the unnatural enantiomer of desmosterol (ent-desmosterol). The sterol nucleus was constructed enantiospecifically, followed by stepwise addition of the side chain. Beginning with ent-androst-4-ene-3,17-dione, ent-desmosterol was synthesized in 13 steps and 20% yield. Protected ent-desmosterol was subjected to catalytic deuteration to afford ent-deuterocholesterol. Ent-desmosterol and ent-deuterocholesterol will be used to study the importance of sterol absolute configuration for sterol-lipid interactions in biophysical studies and in biological systems.

Journal of Biological Chemistry, Nov 1, 2001

The Journal of Membrane Biology, Dec 1, 2004

Cholesterol plays a variety of significant roles in biological systems. However, the mechanisms b... more Cholesterol plays a variety of significant roles in biological systems. However, the mechanisms by which cholesterol functions remain largely unclear. The enantiomer of cholesterol (ent-cholesterol)--which has identical physical properties, but opposite three-dimensional configuration compared to cholesterol--is a unique tool that can be used to better understand the mechanisms of cholesterol function. We review the literature pertaining to ent-cholesterol, focusing in particular on its use in biological studies.

Journal of Biological Chemistry, Sep 1, 2006

Steroids, Jun 1, 2006

25-Hydroxycholesterol (25-HC) appears to play a role in several important biological processes, i... more 25-Hydroxycholesterol (25-HC) appears to play a role in several important biological processes, including regulating cellular cholesterol levels and promoting apoptosis. However, in most cases the mechanisms by which 25-HC elicits its biological effects are not known. Insights into mechanisms of 25-HC action can be gained by studying the activity of its enantiomer (ent-25-HC). ent-25-HC is physically and chemically identical to 25-HC; however, 25-HC and ent-25-HC can be distinguished in chiral environments, like a protein binding site. In order to probe the mechanisms of 25-HC action, we have synthesized the enantiomer of 25-HC (ent-25-HC).

FEBS Letters, Sep 24, 2003

Membrane cholesterol is essential to the activity of at least two structurally unrelated families... more Membrane cholesterol is essential to the activity of at least two structurally unrelated families of bacterial poreforming toxins, represented by streptolysin O (SLO) and Vibrio cholerae cytolysin (VCC), respectively. Here, we report that SLO and VCC di¡er sharply in their interaction with liposome membranes containing enantiomeric cholesterol (ent-cholesterol). VCC had very low activity with ent-cholesterol, which is in line with a stereospeci¢c mode of interaction of this toxin with cholesterol. In contrast, SLO was only slightly less active with ent-cholesterol than with cholesterol, suggesting a rather limited degree of structural speci¢city in the toxin^cholesterol interaction.

Biochemical Journal, Oct 10, 2005

Tankyrase 1 is a PARP [poly(ADP-ribose) polymerase] that localizes to multiple subcellular sites,... more Tankyrase 1 is a PARP [poly(ADP-ribose) polymerase] that localizes to multiple subcellular sites, including telomeres and mitotic centrosomes. Previous studies demonstrated that cells deficient in tankyrase 1 suffered a block in resolution of sister telomeres and arrested in early anaphase [Dynek and Smith (2004) Science 304, 97-100]. This phenotype was dependent on the catalytic PARP activity of tankyrase 1. To identify critical acceptors of PARsylation [poly(ADP-ribosyl)ation] by tankyrase 1 in mitosis, tankyrase 1 immunoprecipitates were analysed for associated PARsylated proteins. We identified NuMA (nuclear mitotic apparatus protein) as a major acceptor of poly(ADP-ribose) from tankyrase 1 in mitosis. We showed by immunofluorescence and immunoprecipitation that association between tankyrase 1 and NuMA increases dramatically at the onset of mitosis, concomitant with PARsylation of NuMA. Knockdown of tankyrase 1 by siRNA (small interfering RNA) eliminates PARsylation of NuMA in mitosis, confirming tankyrase 1 as the PARP responsible for this modification. However, even in the absence of tankyrase 1 and PARsylation, NuMA localizes to spindle poles. By contrast, siRNA knockdown of NuMA results in complete loss of tankyrase 1 from spindle poles. We discuss our result in terms of a model where PARsylation of NuMA by tankyrase 1 in mitosis could play a role in sister telomere separation and/or mitotic progression.

Journal of Lipid Research, Nov 1, 2006

To probe the pathway and specificity of cholesterol absorption, the synthetic enantiomer of chole... more To probe the pathway and specificity of cholesterol absorption, the synthetic enantiomer of cholesterol (ent-cholesterol) and cholesterol were labeled with deuterium, gavaged into hamsters, and measured by negative ion mass spectrometry. Initial uptake of both tracers into the intestinal mucosa at 30 min was similar but cholesterol was temporarily retained there, whereas mucosal entcholesterol declined rapidly with concomitantly increased enrichment in both the systemic circulation and the gut lumen. In a 3 day fecal recovery study, ent-cholesterol was quantitatively recovered in the stool, whereas cholesterol absorption was 53.2%. ent-Cholesterol given by intracardiac injection was selectively secreted into bile, and the ratio of ent-cholesterol to cholesterol tracers in the gut lumen increased down the length of the small bowel, with the largest value being found in stool. ent-Cholesterol is efficiently taken up by the intestinal mucosa and undergoes transient enterohepatic recirculation, but it is quantitatively eliminated over 3 days as a result of selective secretion into bile and selective enrichment within the lumen of the intestine. These findings suggest that cholesterol absorption is structurally specific and likely to be mediated by enantiospecific cellular proteins.

Steroids, 2006

Steroids, 2003

The Journal of Membrane Biology, 2004

The Journal of Lipid Research, 2006

The Journal of Lipid Research, 2010

Phosphatidylinositol 4-phosphate (PI4P) is generated by phosphorylation of phosphatidylinositol (... more Phosphatidylinositol 4-phosphate (PI4P) is generated by phosphorylation of phosphatidylinositol (PI) on the 4-position by phosphatidylinositol 4-kinases (1). In mammalian cells (2, 3), PI4P synthesis is predominately accounted for by the type II phosphatidylinositol 4-kinase II ␣ isoform (PI4KII ␣). Our most recent work has shown that loss of PI4KII ␣ activity in mice leads to late-onset neurodegeneration (2). In addition, Li et al. (4) have demonstrated that PI4KII ␣ is overexpressed in a wide range of common cancers where it has a key role in promoting angiogenesis. However, despite its emerging importance in disease, little is known about endogenous factors that regulate PI4KII ␣ , and there are currently no pharmacological reagents available to modulate its activity in cells. We previously demonstrated that PI4KII ␣ activity is sensitive to membrane cholesterol levels (5). Given the signifi cant role of the enzyme in major pathologies, we sought to investigate the biochemical and biophysical mechanisms that underlie sterol-sensitive PI4P synthesis. One possible mechanism through which cholesterol could affect PI4KII ␣ is by modulating its membrane mobility (6-10). Lateral diffusion of a membrane-associated protein determines its interaction dynamics with other membrane components (reviewed in Ref. 6), and in the case of an enzyme such as PI4KII ␣ , this is likely to be important for the kinetics of product formation. However, it is important to point out that nothing is known Abstract Type II phosphatidylinositol 4-kinase II ␣ (PI4KII ␣) is the dominant phosphatidylinositol kinase activity measured in mammalian cells and has important functions in intracellular vesicular traffi cking. Recently PI4KII ␣ has been shown to have important roles in neuronal survival and tumorigenesis. This study focuses on the relationship between membrane cholesterol levels, phosphatidylinositol 4-phosphate (PI4P) synthesis, and PI4KII ␣ mobility. Enzyme kinetic measurements, sterol substitution studies, and membrane fragmentation analyses all revealed that cholesterol regulates PI4KII ␣ activity indirectly through effects on membrane structure. In particular, we found that cholesterol levels determined the distribution of PI4KII ␣ to biophysically distinct membrane domains. Imaging studies on cells expressing enhanced green fl uorescent protein (eGFP)tagged PI4KII ␣ demonstrated that cholesterol depletion resulted in morphological changes to the juxtanuclear membrane pool of the enzyme. Lateral membrane diffusion of eGFP-PI4KII ␣ was assessed by fl uorescence recovery after photobleaching (FRAP) experiments, which revealed the existence of both mobile and immobile pools of the enzyme. Sterol depletion decreased the size of the mobile pool of PI4KII ␣. Further measurements revealed that the reduction in the mobile fraction of PI4KII ␣ correlated with a loss of trans-Golgi network (TGN) membrane connectivity. We conclude that cholesterol modulates PI4P synthesis through effects on membrane organization and enzyme diffusion.-Minogue, S.

Journal of Biological Chemistry, 2008

Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory e... more Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory element-binding protein maturation and promoting degradation of hydroxymethylglutaryl-CoA reductase. To examine whether oxysterol-membrane interactions contribute to the regulation of cellular cholesterol homeostasis, we synthesized the enantiomer of 25-hydroxycholesterol. Using this unique oxysterol probe, we provide evidence that oxysterol regulation of cholesterol homeostatic responses is not mediated by enantiospecific oxysterol-protein interactions. We show that side chain oxysterols, but not steroid ringmodified oxysterols, exhibit membrane expansion behavior in phospholipid monolayers and bilayers in vitro. This behavior is non-enantiospecific and is abrogated by increasing the saturation of phospholipid acyl chain constituents. Moreover, we extend these findings into cultured cells by showing that exposure to saturated fatty acids at concentrations that lead to endoplasmic reticulum membrane phospholipid remodeling inhibits oxysterol activity. These studies implicate oxysterol-membrane interactions in acute regulation of sterol homeostatic responses and provide new insights into the mechanism through which oxysterols regulate cellular cholesterol balance.

Journal of Biological Chemistry, 2004

Journal of Biological Chemistry, 2001

Journal of Biological Chemistry, 2006

Journal of Biological Chemistry, 2003

In A431 cells, depletion of cholesterol with methyl-␤cyclodextrin induced an increase in both bas... more In A431 cells, depletion of cholesterol with methyl-␤cyclodextrin induced an increase in both basal and epidermal growth factor (EGF)-stimulated EGF receptor phosphorylation. This increase in phosphorylation was site-specific, with significant increases occurring at Tyr 845 , Tyr 992 , and Tyr 1173 , but only minor changes at Tyr 1045 and Tyr 1068. The elevated level of receptor phosphorylation was associated with an increase in the intrinsic kinase activity of the EGF receptor kinase, possibly as a result of the cyclodextrin-induced enhancement of the phosphorylation of Tyr 845 , a site in the kinase activation loop known to be phosphorylated by pp60 src. Cholesterol and its enantiomer (ent-cholesterol) were used to investigate the molecular basis for the modulation of EGF receptor function by cholesterol. Natural cholesterol (nat-cholesterol) was oxidized substantially more rapidly than entcholesterol by cholesterol oxidase, a protein that contains a specific binding site for the sterol. By contrast, the ability of nat-and ent-cholesterol to interact with sphingomyelins and phosphatidylcholine and to induce lipid condensation in a monolayer system was the same. These data suggest that, whereas cholesterol-protein interactions may be sensitive to the absolute configuration of the sterol, sterol-lipid interactions are not. nat-and ent-cholesterol were tested for their ability to physically reconstitute lipid rafts following depletion of cholesterol. nat-and ent-cholesterol reversed to the same extent the enhanced phosphorylation of the EGF receptor that occurred following removal of cholesterol. Furthermore, the enantiomers showed similar abilities to reconstitute lipid rafts in cyclodextrin-treated cells. These data suggest that cholesterol most likely affects EGF receptor function because of its physical effects on membrane properties, not through direct enantioselective interactions with the receptor.

FEBS Letters, 2003

Biochemical Journal, 2005

ACAT1 (acyl-CoA:cholesterol acyltransferase 1) is thought to have two distinct sterol-binding sit... more ACAT1 (acyl-CoA:cholesterol acyltransferase 1) is thought to have two distinct sterol-binding sites: a substrate-binding site and an allosteric-activator site. In the present work, we investigated the structural features of various sterols as substrates and/or activators in vitro. The results show that without cholesterol, the plant sterol sitosterol is a poor substrate for ACAT. In the presence of cholesterol, ACAT1-mediated esterification of sitosterol is highly activated while ACAT2-mediated esterification of sitosterol is only moderately activated. For ACAT1, we show that the stereochemistry of the 3-hydroxy group at steroid ring A is a critical structural feature for a sterol to serve as a substrate, but less critical for activation. Additionally, enantiomeric cholesterol, which has the same biophysical properties as cholesterol in membranes, fails to activate ACAT1. Thus ACAT1 activation by cholesterol is the result of stereo-specific interactions between cholesterol and ACAT1...