Preparation of isotopically labelled benzophenone-containing lipid analogues

Wang, Pingzhen; Spencer, Thomas A.

doi:10.1002/jlcr.996

Cited by 3 publications

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“…Effect of SUV lipid composition on the α-helical content of proSCP-2 and SCP-2 N-terminal peptides upon membrane interaction. Circular dichroism spectra were obtained as described in Experimental Procedures: (A and B) peptide [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Pro, (C and D) peptide Pro, (E and F) peptide Colocalization of Cy5-SCP-2 and Cy5-proSCP-2 with a plasma membrane marker in L cells. L cells were incubated in the presence of Cy5-SCP-2 (A-D) or Cy5-proSCP-2 (E-H) and the plasma membrane marker, AlexaFluor 488-cholera toxin B (AF488-CTB), and examined by laser scanning confocal microscopy (LSCM) as described in Experimental Procedures.…”

Section: Discussionmentioning

confidence: 99%

“…Free cholesterol benzophenone (FCBP) was synthesized by T. A. Spencer (Department of Chemistry, Dartmouth College) (19). Radiolabelled free cholesterol benzophenone ([ 3 H]FCBP) was prepared for our laboratory by Moravek Biochemicals (Brea, CA) utilizing a procedure (20) that incorporates 3 H at position C-3 of the cholesterol moiety. Cholesterol, oleic acid, and oleoyl-CoA were from Sigma (St. Louis, MO).…”

Section: Experimental Procedures Materialsmentioning

confidence: 99%

“…FIGURE 7: Effect of SUV lipid composition on the R-helical content of proSCP-2 and SCP-2 N-terminal peptides upon membrane interaction.Circular dichroism spectra were obtained as described in Experimental Procedures: (A and B) peptide[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Pro, (C and D) peptide Pro, (E and F) peptide Pro, and (G and H) peptide 1-24 SCP-2. Symbols refer to the peptide(15 µM) spectra obtained in the presence of (b) 10 mM potassium phosphate (pH 7.4), (O) 1 mM SUV1 (65:35:0 POPC/cholesterol/DOPS), (1) 1 mM SUV2 (55:35:10 POPC/cholesterol/ DOPS), (3) 1 mM SUV3 (40:50:10 POPC/cholesterol/DOPS), and (9) 1 mM SUV4 (35:35:30 POPC/cholesterol/DOPS).…”

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Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence

et al. 2008

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Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2's affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts/caveolae (AF488-CTB), 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488-antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting.Keywords sterol carrier protein-2; presequence; cholesterol; peroxin; peroxisome Sterol carrier protein-2 (SCP-2) 1 is a ubiquitous, soluble protein present at highest level in tissues involved in cholesterol uptake (intestine), oxidation (liver, steroidogenic cells), and/or elimination (liver) (reviewed in ref 1). SCP-2 exhibits high (nanomolar K d values) affinity for

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Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence

et al. 2008

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“…1D), 25-NBD-cholesterol (not shown), etc. (16,(30)(31)(32)(33)(34)(35)(36)(37)(38). Although there is considerable discussion regarding the solubility and orientation of these synthetic fluorescent sterol probes in membrane bilayers, especially 22-NBD-cholesterol and 25-NBD-cholesterol, at low concentrations some are thought to mimic the distribution and orientation of cholesterol (16,(35)(36)(37) (64,(69)(70)(71).…”

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Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

McIntosh

Atshaves

Huang

et al. 2008

Lipids

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Cholesterol itself has very few structural/chemical features suitable for real-time imaging in living cells. Thus, the advent of dehydroergosterol [ergosta-5,7,9(11),22-tetraen-3β-ol, DHE] the fluorescent sterol most structurally and functionally similar to cholesterol to date, has proven to be a major asset for real-time probing/elucidating the sterol environment and intracellular sterol trafficking in living organisms. DHE is a naturally-occurring, fluorescent sterol analog that faithfully mimics many of the properties of cholesterol. Because these properties are very sensitive to sterol structure and degradation, such studies require the use of extremely pure (>98%) quantities of fluorescent sterol. DHE is readily bound by cholesterol-binding proteins, is incorporated into lipoproteins (from the diet of animals or by exchange in vitro), and for real-time imaging studies is easily incorporated into cultured cells where it co-distributes with endogenous sterol. Incorporation from an ethanolic stock solution to cell culture media is effective, but this process forms an aqueous dispersion of dehydroergosterol crystals which can result in endocytic cellular uptake and distribution into lysosomes which is problematic in imaging DHE at the plasma membrane of living cells. In contrast, monomeric DHE can be incorporated from unilamellar vesicles by exchange/fusion with the plasma membrane or from DHE-methyl-β-cyclodextrin (DHE-MβCD) complexes by exchange with the plasma membrane. Both of the latter techniques can deliver large quantities of monomeric dehydroergosterol with significant distribution into the plasma membrane. The properties and behavior of DHE in protein-binding, lipoproteins, model membranes, biological membranes, lipid rafts/caveolae, and real-time imaging in living cells indicate that this naturally-occurring fluorescent sterol is a useful mimic for probing the properties of cholesterol in these systems. Keywordsdehydroergosterol; cholesterol; ergosterol; fluorescent sterols; microscopy Historical PerspectiveIn order to resolve the dynamics and factors governing cholesterol trafficking within cells, it is important to recognize that the cholesterol molecule has very few polar constituents (Fig. 1A). Consequently, cholesterol is poorly soluble in aqueous environments such as cytosol as evidenced by critical micellar concentrations of cholesterol and other sterols being very low, *To whom correspondence should be addressed: Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 862-1433, FAX: (979) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript near 20-30 nM (1-5). The physiological impact of cholesterol's low aqueous solubility is that spontaneous cholesterol desorption from the cytofacial leaflet of the plasma membrane or lysosomal membrane into the cytosol for transfer to intracellular sites for esterification, oxidation, or secretion is extremely slow (t 1/2 3 hours-days) (6-9). Therefore, it is important to resolve factors...

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