ReviewToward the NMR structure of StAR
Section snippets
Steroidogenesis
The first step in steroidogenesis is the conversion of precursor cholesterol to pregnenolone by the action of the cytochrome P450 side chain cleavage enzyme (P450scc, product of the CYP11A1 gene) present at the inner mitochonrial membrane (IMM) (Brownie et al., 1972, Privalle et al., 1983). For a long time, the activity of P450scc was thought to be the rate-limiting step in steroidogenesis. However, it was found that cycloheximide blocked the translocation of cholesterol from the outer
The START domain
StAR has an N-terminal mitochondrial import sequence (Arakane et al., 1998, Wang et al., 1998), and a steroidogenic acute regulatory protein-related lipid transfer (START) domain that plays a role in the binding and transfer of cholesterol (Tsujishita and Hurley, 2000, Petrescu et al., 2001). The START domain is part of a superfamily of proteins found in prokaryotes, animals and plants (Iyer et al., 2001, Schrick et al., 2004, Alpy and Tomasetto, 2005). Proteins with a START domain possess a
StAR mutations in LCAH
More than 30 mutations in the StAR gene lead to non-functional proteins that cause lipoid congenital adrenal hyperplasia (LCAH) (Bose et al., 1996, Bose et al., 2000). Mutated StAR lacks the ability to bind free cholesterol and to transfer it to the OMM. During an acute hormonal stimulation, this causes an accumulation of cholesterol in the cytoplasm and an engorgement of the cell with lipid droplets, thereby impairing steroid synthesis in LCAH patients. According to the molecular models of
Role of α-helix 4
To understand the precise mechanism of StAR and its role in steroidogenesis, it is necessary to elucidate its structure as well as its interaction with cholesterol. In a previous study, we characterized the thermodynamics of N-62 StAR (StAR without its mitochondrial import sequence) and determined that cholesterol bound the protein with a 1:1 ratio with an apparent KD of 3 × 10−8 (Roostaee et al., 2008). These results are in agreement with the current structural model illustrating the cholesterol
The clinical mutation L275P and the role of the salt bridge
In another series of experiments (Roostaee et al., in press), the role of StAR's salt bridge and α-helix 4 in the binding mechanism of free cholesterol was investigated with more detail using structure-based mutagenesis. The hydrophobic cluster formed by α-helix 4 in relation to the bound cholesterol (Fig. 2) was studied. For instance, the clinical mutation L275P had a lesser α-helicity, it retained only a fraction of the cholesterol binding and had less steroidogenic activity in comparison to
NMR spectroscopy of StAR
Thus far, the structural validation of the two-state model for StAR action relied on biochemical and thermodynamic analyses. However, this methodology is limited to macroscopic parameters, and a structural approach at the atomic level is required to confirm the homology models of the free and bound states. Crystallization of StAR was previously attempted (Tsujishita and Hurley, 2000), but due to aggregation problems the task did not succeed. And so, we focused our efforts to study StAR by
Cholesterol release/delivery mechanism
StAR acts at the OMM (Bose et al., 2002) and has an increased capacity to bind synthetic membranes at very low (artificial) pH (Christensen et al., 2001). It was hypothesized that the acidic polar head groups of membrane phospholipids would cause StAR to become a molten globule which in turn becomes the active form of the protein (Bose et al., 1999). Acidic pH is a particularly effective method for artificially generating molten globule states for structural studies (Kumar et al., 1995,
Conclusion
In conclusion, the homology modeling, structure-based mutagenesis and biophysical studies have provided us with a theoretical framework for understanding the mechanism of StAR action in steroidogenesis and to rationalize the impact of mutations regarding the accumulation of cholesterol in the clinical cases of LCAH. Thus far biochemical, cellular, structural and thermodynamic studies have brought a substantial experimental validation of our two-state model. However, to gain deeper insights into
Acknowledgment
This work was supported by a grant to Jean-Guy LeHoux from the Canadian Institute of Health Research, MT-10983.
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2020, Saudi Journal of Biological SciencesCitation Excerpt :It has been observed that patients with PCOS have an irregularity in androgen biosynthesis, which results in hyperandrogenemia (Overbeek and Lambalk, 2009; Kahsar-Miller et al., 2001). It is considered as an initial step in androgen biosynthesis to transport cholesterol via the StAR protein from outer to the inner mitochondrial membrane (Barbar, LeHoux and Lavigne, 2009; Miller, 2007). The central nervous system, gonads, adrenal glands, and placenta are regions for synthesizing steroid hormones.
Structural basis of sterol binding and transport by a yeast StARkin domain
2018, Journal of Biological ChemistryCitation Excerpt :Nevertheless, complete and substantial shifts in the positions of a large number of resonances indicate that the oxysterol is bound quantitatively and that binding is accompanied by a conformational rearrangement of the protein (see enlargement of a portion of the spectrum in Fig. 3). Similar changes have been observed in the 2D NMR spectra of other StART domain proteins upon ligand binding (21–23). Thus, the NMR results confirm that Lam4S2 is well folded and able to bind to 25-hydroxycholesterol and that it maintains its overall structure and topology upon ligand binding.
Solution structure of human steroidogenic acute regulatory protein STARD1 studied by small-angle X-ray scattering
2017, Biochemical and Biophysical Research CommunicationsCitation Excerpt :This indicated that STARD1 may be a very conformationally labile protein undergoing significant rearrangements for cholesterol binding and transfer. MD simulations and studies on engineered STARD1 mutants suggested that cholesterol binding involves unfolding of the C-terminal α4-helix of STARD1 which may serve as a lid for the steroid binding pocket [17,19–22]. Alternative hypothesis considered the so-called “clam-shell” like mechanism implying even more significant movements of a START domain halves to open the lipid-binding cavity [23].
A single cell level measurement of StAR expression and activity in adrenal cells
2017, Molecular and Cellular EndocrinologyCitation Excerpt :Mutations, which cause the human disease, concentrate in the cholesterol binding domain rather than the NTD (Sahakitrungruang et al., 2010). However, the R182 mutation retains full cholesterol exchange activity but does not stimulate activity at CYP11A1 (Baker et al., 2005; Barbar et al., 2009). The StAR activity under hormonal control is mediated by phosphorylation at S194 by cAMP and protein kinase A (PKA) in fasciculata cells, and by Ca–dependent kinases in glomerulosa cells (Dyson et al., 2009; Elliott et al., 1993).
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