Cross-eyed stereo view of the context of the CBM of Couet <i>et al</i>[<b>6</b>] seen in the rat Gi1α protein (PDB code 1CIP; [<b>51</b>]).

<p>The β-hairpin structure of the motif is shown as a cartoon, coloured from blue to red, and the aromatic residues drawn as sticks (Phe189 is blue, Phe191 is cyan, Phe196 is yellow and Phe199 is red). The third strand of the three-stranded sheet to which the motif belongs is also shown in pink. The remainder of the protein is shown as lines and surface, the latter coloured green where contributed by side chains of the aromatic residues.</p

Evaluating Caveolin Interactions: Do Proteins Interact with the Caveolin Scaffolding Domain through a Widespread Aromatic Residue-Rich Motif?

<div><p>Caveolins are coat proteins of caveolae, small flask-shaped pits of the plasma membranes of most cells. Aside from roles in caveolae formation, caveolins recruit, retain and regulate many caveolae-associated signalling molecules. Caveolin-protein interactions are commonly considered to occur between a ∼20 amino acid region within caveolin, the caveolin scaffolding domain (CSD), and an aromatic-rich caveolin binding motif (CBM) on the binding partner (фXфXXXXф, фXXXXфXXф or фXфXXXXфXXф, where ф is an aromatic and X an unspecified amino acid). The CBM resembles a typical linear motif - a short, simple sequence independently evolved many times in different proteins for a specific function. Here we exploit recent improvements in bioinformatics tools and in our understanding of linear motifs to critically examine the role of CBMs in caveolin interactions. We find that sequences conforming to the CBM occur in 30% of human proteins, but find no evidence for their statistical enrichment in the caveolin interactome. Furthermore, sequence- and structure-based considerations suggest that CBMs do not have characteristics commonly associated with true interaction motifs. Analysis of the relative solvent accessible area of putative CBMs shows that the majority of their aromatic residues are buried within the protein and are thus unlikely to interact directly with caveolin, but may instead be important for protein structural stability. Together, these findings suggest that the canonical CBM may not be a common characteristic of caveolin-target interactions and that interfaces between caveolin and targets may be more structurally diverse than presently appreciated.</p> </div

Relative exposed surface area (RSA) of CBM aromatics.

<p>Relative exposed surface area (RSA) of CBM aromatics.</p

Predicted change in folding free energy (ΔΔG) resulting from alanine mutation.

<p>Predictions calculated using PoPMuSiC. ΔΔG values for proteins marked with * were determined from homology models (SWISS MODEL repository) and the PDB code given is that of the template (T) used for the model. Mutations predicted as significantly destabilising (ΔΔG>2.00 kcal/mol) in bold font.</p

SABLE estimates of relative exposed surface area (RSA) of CBM aromatics.

<p>Predictions are given in ranges spanning 10%. Buried residues (RSA<20%) are in bold font.</p

List of Cav-1-interacting molecules reported as containing CBM-like motifs.

<p>List of Cav-1-interacting molecules reported as containing CBM-like motifs.</p

DHCR24 activity of <i>B. mori</i> BGIBMGA005735 expressed in <i>S. cerevisiae</i>.

<p>Yeast homogenates containing expressed <i>B. mori</i> BGIBMGA005735 were added to assay mixtures containing desmosterol and various combinations of the cofactors NADPH and FAD. A yeast homogenate containing expressed pYES2 (vector control) was incubated with both cofactors as a negative control. Assays were incubated for 4 h at 37°C and products analysed by GC/MS. MS trace (total ion current) of positive reaction containing cholesterol and negative reaction containing only desmosterol are shown in Fig. 4a. Sterol peaks were calibrated using triplicate reactions containing 5α-cholestane as an internal standard and expressed as a percentage of desmosterol converted into cholesterol compared to the empty vector reaction (Fig 4b). The positions of elution of authentic cholesterol and desmosterol are shown by arrows (Fig. 4a).</p

Membrane association and complex formation of <i>B. mori</i> DHCR24.

<p>(a) Equal aliquots of midgut microsomes were washed with 10 volumes of 5 mM HEPES/NaOH, pH 7.5 on ice for 30 min before reisolating the microsomes and analysing by western blot using anti-DHCR24 antibody. The washes were as follows: (1) HEPES/NaOH, pH 7.5 only, (2) HEPES/NaOH, pH 7.5+200 mM KCl, (3) HEPES/NaOH, pH 7.5+500 mM KCl, (4) 0.1 M Na₂CO_3, pH 11.5. (b) Midgut microsomal protein was solubilised with 10% dodecyl maltoside and separated using native blue gel electrophoresis with native protein markers. This 1st dimension strip was then soaked in β-mercaptoethanol and SDS before running on a standard SDS PAGE gel and analysing by western blot using anti-DHCR24 antibody.</p

Western blot analysis of subcellular location and tissue distribution of <i>B. mori</i> DHCR24.

<p>(a) Samples of soluble supernatant (1) and microsomal (2) fractions, normalised for protein concentration were blotted with anti-DHCR24 antibodies. (b) Microsomes produced from various tissue homogenates normalised for protein concentration were probed with anti-DHCR24 antibodies: (1) foregut, (2) midgut, (3) hindgut, (4) testes, (5) ovary, (6) Malpighian tubules, (7) fat body, (8) head.</p

Pathway of transformation of sitosterol into cholesterol.

<p>Pathway of transformation of sitosterol into cholesterol.</p