Missense mutations identified in patients manifesting with non-classic StAR deficiency.

<p>*Mutation on partner allele is given in brackets (p.).</p><p>**Loss of function mutation manifesting clinically at birth with signs of classic StAR deficiency. Data given for comparison.</p><p>***<i>In vitro</i> activity (% of WT) is assessed by pregnenolone production (immunoassay) in COS cells transfected with expression vectors for wild-type or mutant StAR and F2 (the fusion protein P450 side-chain cleavage/adrenodoxin/adrenodoxin reductase). Note that data derive from different laboratories employing similar assay systems.</p

Genetic analysis of the StAR gene.

<p>Upper panel, scheme of the identified mutations at the nucleotide (c.DNA) and protein (p.) level. Lower panel, family tree with individual electropherograms showing the novel StAR mutations.</p

Model of StAR protein showing reported, non-classic StAR mutations.

<p>Mutations manifesting clinically with NCLAH and having >10% WT StAR activity in a direct functional assay (V187M, R188C, R192C, G221S) are depicted in red. Mutations A218V, M225T, F267S, L275P, for which clinical and functional results are conflicting, are given in blue. StAR loss-of-function mutation L260P is given in black for comparison.</p

Exit of cholesterol from StAR as studied by steered MD simulations.

<p>StAR protein is shown as a ribbons model colored in a rainbow gradient from the amino terminus in blue to carboxy terminus in red. The A174–V179 loop that was observed to move and make way for the exit of cholesterol is shown in grey. Major amino acids involved in interaction of cholesterol with StAR and formation of cholesterol binding pocket are shown as stick models. The exit route of cholesterol observed during simulation is shown as a solid model in red. In case of S221-StAR a delay in exit of cholesterol was observed, potentially due to altered binding pattern caused by shift in H220 side chain and additional interactions with R188.</p

A closeup of the cholesterol binding pocket of StAR.

<p>After docking of cholesterol to both WT (A) and S221-StAR (B), we calculated potential residues interacting with cholesterol molecule during docking as well as exit of cholesterol from the binding cavity. In case of S221-StAR a loss of interaction with H220 was observed due to a shift in the H220 side chain. Cholesterol is shown as a ball and stick model in magenta. Amino acids interacting with cholesterol are shown as stick models.</p

Functional testing of the identified StAR mutations <i>in vitro.</i>

<p>COS-1 cells were transiently transfected with expression vectors for the side chain cleavage system (F2) and wild-type (WT) or mutant StAR. A) Protein expression was assessed by Western blot. B) The ability to produce pregnenolone (Preg) was measured in the cell supernatants after 5–600 min using a commercially available ELISA assay. Results are given as mean ± SD. StAR independent Preg production of the cell system was assessed by using empty vector/F2 and adding 22(R)-hydroxycholesterol (22R OH-Chol) to the cell medium.</p

Secondary structural and amino acid conservation of human StAR.

<p>(A) A secondary structure prediction was performed to start the model building of human StAR. Locations of amino acids variants of StAR found in UniProt database and pubmed are indicated. (B) A partial sequencing alignment of human StAR amino acid sequence with a range of StAR proteins from other species found in the UniProt database. Overall, StAR amino acid sequence is very well conserved across species with only occasional difference showing up across whole alignment. All major residues at cholesterol binding pocket, E169, R188, R192 and T223 are conserved in all species studied in this analysis, while H220 was found to be replaced with an asparagine in Chicken and Zebrafish, potentially conserving the core structural elements.</p