RESUMO
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. Point mutations or truncations in the StAR gene produce a partial to non-functional protein that hinders the StAR-induced delivery of cholesterol to the mitochondria during an acute hormonal stimulation of steroidogenic cells. So far, homology modeling, structure-based thermodynamics and biophysical studies have allowed us to propose the existence of an open state of StAR where the C-terminal alpha-helix 4 undergoes partial unfolding. This may act as a gating mechanism to the cholesterol binding site. Once bound, cholesterol leads to the stabilization and the refolding of alpha-helix 4, and eventually to the interaction with an import complex at the surface of the mitochondria. Though the current homology models have proven useful in understanding StAR function, only the full determination of the 3D structure of the apo- and holo-states will further validate this two-state model. In this context, we have used solution-state nuclear magnetic resonance (NMR) and obtained high-resolution (1)H-(15)N-HSQC spectra of StAR in its apo- and holo-states at physiological pH. Both spectra displayed well-dispersed resonances. However, key differences are observed on the spectra which indicate that both states have stable but slightly different tertiary structures. In conjunction with the binding/activity assays and biophysical methods, this original NMR data constitutes another structural step into the validation of the two-state model and the three-dimensional structure of StAR.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Fosfoproteínas/química , Conformação Proteica , Hiperplasia Suprarrenal Congênita/genética , Hiperplasia Suprarrenal Congênita/metabolismo , Humanos , Modelos Moleculares , MutaçãoRESUMO
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. In this study, we present a structural in silico analysis of StAR. Molecular dynamics simulations showed that point mutations of Phe(267), Leu(271) or Leu(275) at the alpha-helix 4 increased the gyration radius (more flexibility) of the protein's structure, whereas the salt bridge double mutant E169M/R188M showed a decrease in flexibility (more compactness). Also, in the latter case, an interaction between Met(169) and Phe(267) disrupted the hydrophobic cavity, rendering it impervious to ligand binding. These obtained results are in agreement with previous in vitro experiments, and provide further validation of the two-state binding mode of action.
Assuntos
Colesterol/metabolismo , Simulação por Computador , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estrutura Secundária de Proteína , Reprodutibilidade dos TestesRESUMO
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. A detailed understanding of the mechanism of the specific binding of free cholesterol by StAR would be a critical asset in understanding the molecular origin of this disease. Previous studies have led to the proposal that the C-terminal alpha-helix 4 of StAR was undergoing a folding/unfolding transition. This transition is thought to gate the cholesterol-binding site. Moreover, a conserved salt bridge (Glu169-Arg188) in the cholesterol-binding site is also proposed to be critical to the binding process. Interestingly, some of the documented clinical mutations occur at this salt bridge (E169G, E169K and R188C) and in the C-terminal alpha-helix 4 (L275P). In the present study, using rationalized mutagenesis, activity assays, CD, thermodynamic studies and molecular modelling, we characterized the alpha-helix 4 mutations L271N and L275P, as well as the salt bridge double mutant E169M/R188M. The results provide experimental validation for the gating mechanism of the cholesterol-binding site by the C-terminal alpha-helix and the importance of the salt bridge in the binding mechanism. Altogether, our results offer a molecular framework for understanding the impact of clinical mutations on the reduction of the binding affinity of StAR for free cholesterol.
Assuntos
Colesterol/metabolismo , Fosfoproteínas/metabolismo , Sítios de Ligação/genética , Linhagem Celular Tumoral , Colesterol/química , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Plasmídeos , Ligação Proteica/genética , Dobramento de Proteína , Estrutura Secundária de Proteína , Ensaio Radioligante , Temperatura , TermodinâmicaRESUMO
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. In the bound form, cholesterol interacts with hydrophobic side-chains located in the C-terminal alpha-helix 4, thereby favouring the folding of this helix. To verify this model experimentally, we have characterized the in vitro activity, overall structure, thermodynamic stability and cholesterol-binding affinity of StAR lacking the N-terminal 62 amino acid residues (termed N-62 StAR). This mature form is biologically active and has a well-defined tertiary structure. Addition of cholesterol to N-62 StAR led to an increase in the alpha-helical content and T degrees (melting temperature), indicating the formation of a stable complex. However, the mutation F267Q, which is located in the C-terminal helix interface lining the cholesterol-binding site, reduced the biological activity of StAR. Furthermore, the cholesterol-induced thermodynamic stability and the binding capacity of StAR were significantly diminished in the F267Q mutant. Titration of StAR with cholesterol yielded a 1:1 complex with an apparent K(D) of 3 x 10(-8). These results support our model and indicate that StAR can readily bind to cholesterol with an apparent affinity that commensurates with monomeric cholesterol solubility in water. The proper function of the C-terminal alpha-helix is essential for the binding process.
Assuntos
Colesterol/metabolismo , Fosfoproteínas/metabolismo , Sítios de Ligação , Colesterol/química , Clonagem Molecular , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Fosfoproteínas/química , Conformação Proteica , Dobramento de Proteína , Temperatura , TermodinâmicaRESUMO
Objective: 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. Design: Published data in the literature and a detailed 38-year follow-up of two sibling LCAH patients. Molecular structure and modeling of the STARD1 L275P mutation. Setting: University hospital. Patients: Patient A (46,XY female phenotype) and patient B (46,XX female) with LCAH bearing the L275P mutation in STARD1. Interventions: Since early-age diagnosis, both patients underwent corticoid replacement therapy. Patient A received estrogen therapy at pubertal age. Clomiphene therapy was given to Patient B to induce ovulation. Pregnancies were protected with progesterone administration. Main Outcome Measures: Clinical and molecular assessment of adrenal and gonadal functions. Results: Both patients have classic manifestations of corticosteroid deficiency observed in LCAH. Time of onset and severity were different. Patient A developed into a female phenotype due to early and severe damage of Leydig cells. Patient B started a progressive pubertal development, menarche and regular non-ovulatory cycle. She was able to have successful pregnancies. Conclusions: Understanding the molecular structure and function of STARD1 in all steroidogenic tissues is the key for comprehending the heterogeneous clinical manifestations of LCAH, and the development of an appropriate strategy for the induction of ovulation and protecting pregnancies in this disease.
RESUMO
4-aminopyridine (4AP) is a general blocker of voltage-dependent K+ channels. This pyridine derivative has also been shown to inhibit T cell proliferation, to modulate immune responses and to alleviate some of the symptoms associated with neurological disorders such as multiple sclerosis, myasthenia gravis and Alzheimer's disease. 4AP triggers a Ca2+ response in lymphocytes, astrocytes, neurons and muscle cells but little is known about the regulation of the 4AP response in these cells. We report that 4AP induced a non-capacitative transplasma membrane influx of Ca2+ in Jurkat T lymphocytes. The influx of Ca2+ was not affected by activation or inhibition of protein kinase A (PKA). In contrast, activation of protein kinase C (PKC) by phorbol myristyl acetate (PMA), mezerein or 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited the influx of Ca2+ triggered by 4AP. The inhibitory effect of PKC could be prevented by prior exposure of the cells to the PKC inhibitor GF 109203X. Under these conditions, mezerein and OAG no longer inhibited the 4AP-dependent Ca2+ response. Inhibition of serine and threonine protein phosphatases PP1 and PP2A by treating the cells with calyculin A (CalA) reduced the Ca2+ response to 4AP. Okadaic acid (OA) had no effect, suggesting an involvement of PP1. A combination of CalA and OAG (or PMA) abolished the influx of Ca2+ induced by 4AP, adding further evidence to the importance of protein phosphorylation in the modulation of the 4AP response. Our data suggest that the transplasma membrane influx of Ca2+ triggered by 4AP in Jurkat T cells can be modulated by the opposite actions of PKC and protein serine and threonine phosphatase(s).
Assuntos
4-Aminopiridina/farmacologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Células Jurkat/efeitos dos fármacos , Proteína Quinase C/fisiologia , 4-Aminopiridina/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Inibidores Enzimáticos/farmacologia , Fura-2 , Humanos , Células Jurkat/metabolismo , Toxinas Marinhas , Oxazóis/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Proteína Quinase C/farmacologiaRESUMO
Collagen type IV (CnIV) and fibronectin (Fn) were used as ligands to study the distribution of alpha(2)beta(1) and alpha(4)beta(1) integrins in low-density, detergent-resistant microdomains (DRM) of Jurkat lymphocytes. CnIV-coated microspheres induced (optical trapping) the redistribution of GM(1)-associated fluorescence from the cell periphery to the area of contact. This was not observed in cells treated with beta-methyl cyclodextrin (MCD). Fn- or bovine serum albumin-coated microspheres did not modify the peripheral distribution of fluorescence. These observations were confirmed by confocal microscopy. Western blot analysis of cells exposed to surfaces coated with CnIV revealed that the alpha(2)-subunit was initially present at low levels in DRM, became strongly associated after 40 min, and returned to basal levels after 75 min. Fn induced a slight recruitment of the beta(1)-integrin alpha(4)-subunit in DRM after 5 and 10 min, followed by a return to basal levels. Neither CnIV nor Fn triggered significant changes in the distribution of the beta(1)-subunit in DRM. Fn- and CnIV-coated microspheres or surfaces coated with these ligands triggered a MCD-sensitive mobilization of Ca(2)(+). MCD did not alter the state of the Ca(2)(+) reserves. The differential distributions of the alpha(2)beta(1) and alpha(4)beta(1) integrins in DRM may provide one additional step in the regulation of outside-in signaling involving these integrins.
Assuntos
Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Integrina alfa2beta1/metabolismo , Integrina alfa4beta1/metabolismo , Microdomínios da Membrana/metabolismo , Cálcio/metabolismo , Fluorescência , Gangliosídeo G(M1)/metabolismo , Humanos , Células Jurkat , Microesferas , Fatores de TempoRESUMO
Proper folding is a crucial step for the trafficking of proteins through the secretory pathway. We hypothesized that the secretory granules of endocrine cells provide optimal folding conditions of prohormone precursors for cleavage. Here, using circular dichroism and in vitro processing on purified prourotensin II (ProUII), we show that the precursor undergoes pH- and Ca(2+)-dependent conformational and stability changes. ProUII has a stable tertiary structure at pH 5.5 in presence of Ca(2+) and is correctly cleaved in these conditions by prohormone convertases. Taken together, our results support the notion that precursors may need to be optimally folded in the lumen of secretory granules for their processing.
Assuntos
Cálcio/farmacologia , Dobramento de Proteína/efeitos dos fármacos , Urotensinas/química , Animais , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Conformação Proteica/efeitos dos fármacos , Precursores de Proteínas/química , Estabilidade Proteica/efeitos dos fármacos , Vesículas Secretórias/metabolismoRESUMO
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. OBJECTIVE: We describe the first cases of pregnancy in a LCAH female patient bearing the L275P mutation in the StAR gene. DESIGN: We studied the gonadal function, pubertal development, and apply the appropriate hormonal therapy to support pregnancies. PATIENT: A 46,xx patient of French Canadian descent was diagnosed with LCAH at the age of 4.5 months. Substitution therapy with glucocorticoids and mineralocorticoids led to normal growth and development. Progressive pubertal development started at the age of 11 7/12 yr. Menarche occurred at 14 2/12 yr with normal regular menstruations thereafter but without ovulation. RESULTS: Clomiphene stimulation induced the first pregnancy at 25 4/12 yr of age. Spontaneous abortion occurred after 6 wk gestation. The second pregnancy (with clomiphene stimulation) was induced at the age of 26 yr. Progesterone (Prog) therapy was added at the 17th day of the cycle to protect pregnancy. Vaginal delivery of dichorionic-diamniotic twin pregnancy occurred at 30 wk gestation (two normal weight male babies). Two years later, again under clomiphene stimulation, she underwent another successful singleton pregnancy and delivered a normal weight female baby at 36 wk. The pregnancies were almost uncomplicated. CONCLUSION: Despite the dysfunctional StAR, pregnancy is possible under the proper therapeutic strategy.