Determination of Ligand Binding Affinity and Specificity of Purified START Domains by Thermal Shift Assays Using Circular Dichroism.

The use of direct calorimetric methods such as isothermal titration calorimetry for measuring the affinity and specificity of protein-ligand interactions requires large amounts of proteins and ligands. When material is scarce and/or in the absence of calorimeters, thermal Shift Assays (TSA) using Circular Dichroism (CD) or other spectroscopic methods offers an alternative and quantitative method for the determination of apparent or indirect thermodynamical parameters describing the affinity of ligands for proteins. Indeed, the binding constants of ligands (K) and other parameters such as the enthalpy and Gibbs free energy of binding may be estimated from the changes in the stability curves ΔG(T) of a protein in the presence of a ligand.

Implication of STARD5 and cholesterol homeostasis disturbance in the endoplasmic reticulum stress-related response induced by pro-apoptotic aminosteroid RM-133.

The aminosteroid derivative RM-133 is an effective anticancer molecule for which proof of concept has been achieved in several mouse xenograph models (HL-60, MCF-7, PANC-1 and OVCAR-3). To promote this new family of molecules toward a clinical phase 1 trial, the mechanism of action governing the anticancer properties of the representative candidate RM-133 needs to be characterized. In vitro experiments were first used to determine that RM-133 causes apoptosis in cancer cells.

Thirty-Eight-Year Follow-Up of Two Sibling Lipoid Congenital Adrenal Hyperplasia Patients Due to Homozygous Steroidogenic Acute Regulatory (STARD1) Protein Mutation. Molecular Structure and Modeling of the STARD1 L275P Mutation.

Review the impact of StAR (STARD1) mutations on steroidogenesis and fertility in LCAH patients. Examine the endocrine mechanisms underlying the pathology of the disorder and the appropriate therapy for promoting fertility and pregnancies. Published data in the literature and a detailed 38-year follow-up of two sibling LCAH patients.

STARD6 on steroids: solution structure, multiple timescale backbone dynamics and ligand binding mechanism.

START domain proteins are conserved α/β helix-grip fold that play a role in the non-vesicular and intracellular transport of lipids and sterols. The mechanism and conformational changes permitting the entry of the ligand into their buried binding sites is not well understood. Moreover, their functions and the identification of cognate ligands is still an active area of research.

The binding site specificity of STARD4 subfamily: Breaking the cholesterol paradigm.

Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain proteins display diverse expression patterns and cellular localisations. They bind a large variety of lipids and sterols and are involved in lipid metabolism, lipid transfer and cell signalling. The START domain tertiary structure is an α-helix/β-grip fold module of approximately 210 amino acids delimiting an internal cavity forming the binding site.

Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated bile acids to STARD5.

STARD5 is a member of the STARD4 sub-family of START domain containing proteins specialized in the non-vesicular transport of lipids and sterols. We recently reported that STARD5 binds primary bile acids. Herein, we report on the biophysical and structural characterization of the binding of secondary and conjugated bile acids by STARD5 at physiological concentrations.

STARD5 specific ligand binding: comparison with STARD1 and STARD4 subfamilies.

We present herein a review of our recent results on the characterization of the binding sites of STARD1, STARD5 and STARD6 using NMR and other biophysical techniques. Whereas STARD1 and STARD6 bind cholesterol, no cholesterol binding could be detected for STARD5. However, titration of STARD5 with cholic acid and chenodeoxycholic acid led to specific binding.

StAR-related lipid transfer domain protein 5 binds primary bile acids.

Steroidogenic acute regulatory-related lipid transfer (START) domain proteins are involved in the nonvesicular intracellular transport of lipids and sterols. The STARD1 (STARD1 and STARD3) and STARD4 subfamilies (STARD4-6) have an internal cavity large enough to accommodate sterols. To provide a deeper understanding on the structural biology of this domain, the binding of sterols to STARD5, a member of the STARD4 subfamily, was monitored.

(1)H, (13)C, and (15)N backbone chemical shift assignments of StAR-related lipid transfer domain protein 5 (STARD5).

Steroidogenic acute regulatory (StAR)-related lipid transfer proteins possess a START (steroidogenic acute regulatory-related lipid transfer) domain. START domains are conserved protein modules involved in the non-vesicular intracellular transport of lipids and cholesterol in mammals. Fifteen mammalian proteins, divided in five subfamilies, are reported to possess a START domain.

Mammalian StAR-related lipid transfer (START) domains with specificity for cholesterol: structural conservation and mechanism of reversible binding.

The StAR-related lipid transfer (START) domain is an evolutionary conserved protein module of approximately 210 amino acids. There are 15 mammalian proteins that possess a START domain. Whereas the functions and specific ligands are being elucidated, 5 of them have already been shown to bind specifically cholesterol.

Gonadal function, first cases of pregnancy, and child delivery in a woman with lipoid congenital adrenal hyperplasia.

Context: Mutations in the steroidogenic acute regulatory protein (StAR) gene often cause lipoid congenital adrenal hyperplasia (LCAH). In this disorder an impairment of steroid synthesis leads to adrenal and gonadal insufficiencies with a particular female genital phenotype in both human karyotypes. Pregnancy in LCAH has not been yet reported.

Toward the NMR structure of StAR.

The steroidogenic acute regulatory (StAR) protein plays a crucial role in steroidogenesis, as it accelerates the transport of cholesterol to the inner mitochondrial membrane where the cytochrome P450scc enzyme is located. Mutations in the StAR gene can lead to lipoid congenital adrenal hyperplasia (LCAH), a disease that is fatal if not treated with hormone replacement therapy. Solving the structure of StAR is an important aspect of understanding LCAH.

Validation of the mechanism of cholesterol binding by StAR using short molecular dynamics simulations.

We previously proposed an original two-state cholesterol binding mechanism by StAR, in which the C-terminal alpha-helix of StAR gates the access of cholesterol to its binding site cavity. This cavity, which can accommodate one cholesterol molecule, was proposed to promote the reversible unfolding of the C-terminal alpha-helix and allow for the entry and dissociation of cholesterol. In our molecular model of the cholesterol-StAR complex, the hydrophobic moiety of cholesterol interacts with hydrophobic amino acid side-chains located in the C-terminal alpha-helix and at the bottom of the cavity.

The mechanism of specific binding of free cholesterol by the steroidogenic acute regulatory protein: evidence for a role of the C-terminal alpha-helix in the gating of the binding site.

Steroidogenesis depends on the delivery of free cholesterol to the inner mitochondrial membrane by StAR (steroidogenic acute regulatory protein). Mutations in the StAR gene leads to proteins with limited cholesterol-binding capacity. This gives rise to the accumulation of cytoplasmic cholesterol, a deficit in steroid hormone production and to the medical condition of lipoid congenital adrenal hyperplasia.

Cholesterol binding is a prerequisite for the activity of the steroidogenic acute regulatory protein (StAR).

Steroidogenesis depends on the delivery of cholesterol from the outer to the inner mitochondrial membrane by StAR (steroidogenic acute regulatory protein). However, the mechanism by which StAR binds to cholesterol and its importance in cholesterol transport are under debate. According to our proposed molecular model, StAR possesses a hydrophobic cavity, which can accommodate one cholesterol molecule.

Quantitative fluorometric analysis of the protective effect of chitosan on thermal unfolding of catalytically active native and genetically-engineered chitosanases.

We have taken advantage of the intrinsic fluorescence properties of chitosanases to rapidly and quantitatively evaluate the protective effect of chitosan against thermal denaturation of chitosanases. The studies were done using wild type chitosanases N174 produced by Streptomyces sp. N174 and SCO produced by Streptomyces coelicolor A3(2).

Angiotensin II activates p44/42 MAP kinase partly through PKCepsilon in H295R cells.

Using pharmaceutical and overexpression approaches we have previously reported that in H295R cells, (a) angiotensin II (AII) activates PKCepsilon, PKCalpha and p44/42 MAPK pathway, (b) PKCepsilon, PKCalpha and p44/42 MAPK overexpression inhibits AII-induced CYP11B2 gene transcription and (c) overexpression of PKCepsilon inhibits CYP11B2 gene transcription through p44/42 MAPK activation [LeHoux, J.G., Dupuis, G.

Novel protein kinase C-epsilon inhibits human CYP11B2 gene expression through ERK1/2 signalling pathway and JunB.

We previously reported that H295R cells co-express three diacylglycerol (DAG)-dependent protein kinase Cs (PKCs), namely conventional (c) PKCalpha and novel (n) PKCepsilon and PKCtheta. The aim of the present work was to evaluate the implication of DAG-dependent PKCs in the activation of p44/42 MAP kinase (MAPK) by angiotensin II (Ang II) and to define the role of this pathway towards CYP11B2 regulation in H295R cells. The PKC inhibitor bisindolylmaleimide 1 (Bis) inhibited Ang II-induced p44/42 MAPK phosphorylation whereas the cPKC inhibitor Gö6976 failed to do so, thus ruling out the participation of PKCalpha.

Family of two patients with congenital lipoid adrenal hyperplasia due to StAR mutation.

We are reporting the case of two sisters born to nonrelated French Canadian parents. Patient A is of female phenotype with 46,xy, and patient B with 46,xx. The children had severe manifestations of mineralocorticoid deficiency at the age of 11 and 4.

On the control of the hCYP11B2 gene expressing cytochrome P450 aldosterone synthase.

We have previously reported that the protein kinase C ligand 12-O-tetradecanoyphorbol-13-acetate (TPA) inhibited the angiotensin II (AII) stimulated CYP11B2 gene expression in the adrenocortical H295R cell line. Here we report that TPA increased the level of phospho-p44/42 MAPK but AII did not. The MEK1 inhibitor PD98059 was found to increase the level of aldosterone synthase mRNA and the activity of a human CYP11B2(-2023 bp)-promoter construct.

Phosphorylation and function of the hamster adrenal steroidogenic acute regulatory protein (StAR).

In order to study the effect of phosphorylation on the function of the steroidogenic acute regulatory protein (StAR), 10 putative phosphorylation sites were mutated in the hamster StAR. In pcDNA3.1-StAR transfected COS-1 cells, decreases in basal activity were found for the mutants S55A, S185A and S194A.

Phosphorylation of the hamster adrenal steroidogenic acute regulatory protein as analyzed by two-dimensional polyacrylamide gel electrophoreses.

Post-translational modifications such as phosphorylation and specific proteolysis affect the steroidogenic acute regulatory protein (StAR) activity. We have found that in pcDNA3.1-StAR-transfected COS-1 cells, StAR was phosphorylated on S55, S56 and S194 (Fleury et al.

ACTH induces TIMP-1 expression and inhibits collagenase in adrenal cortex cells.

To identify genes that are induced by corticotropin (ACTH) in adrenal cortex cells, we carried out a differential hybridization screening of adrenal cortex cDNA libraries. Some of the clones we identified represented tissue inhibitor of metalloproteinase 1 (TIMP-1) mRNA. We examined ACTH dependence of the expression of TIMP-1 in vitro in cultured bovine adrenocortical cells, and in ACTH-treated rats.

Adrenocorticotropin regulation of steroidogenic acute regulatory protein.

We studied the effect of the adrenocorticotropin hormone (ACTH) on the expression of the steroidogenic acute regulatory protein (StAR) in vivo in rat and hamster adrenals and also in transfection experiments using COS-1 cells. In vivo, ACTH increased the level of StAR mRNA within 30-60 minutes and also increased the quantity of StAR, but with a 2-3-hour delay. ACTH induced the formation of many acidic StAR species as analyzed by two-dimensional gel electrophoresis and immunoblotting.

Molecular dynamics of substrate complexes with hamster cytochrome P450c17 (CYP17): mechanistic approach to understanding substrate binding and activities.

The cytochrome P450c17 isoforms from various animal species have different substrate selectivity, especially for 17,20-lyase activity. In particular, the human P450c17 selectively produces dehydroepiandrosterone with little androstenedione (AD). Hamster P450c17, on the other hand, produces both of these steroids at comparable rates.