RESUMEN
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. Then, using proteomic and transcriptomic experiments, RM-133 cytotoxicity was proven to be achieved via the endoplasmic reticulum (ER)-related apoptosis, which characterizes RM-133 as an endoplasmic reticulum stress aggravator (ERSA) anticancer drug. Furthermore, an shRNA-genome-wide screening has permitted to identify the steroidogenic acute regulator-related lipid transfer protein 5 (STARD5) as a major player in the RM-133 ER-related apoptosis mechanism, which was validated by an in vitro binding experiment. Altogether, the results presented herein suggest that RM-133 provokes a disturbance of cholesterol homeostasis via the implication of STARD5, which delivers an ERSA molecule to the ER. These results will be a springboard for RM-133 in its path toward clinical use.
Asunto(s)
Androstenos/farmacología , Antineoplásicos/farmacología , Proteínas Portadoras/metabolismo , Colesterol/metabolismo , Estrés del Retículo Endoplásmico , Proteínas Adaptadoras del Transporte Vesicular , Apoptosis/efectos de los fármacos , Proteínas Portadoras/genética , Línea Celular Tumoral , Homeostasis/efectos de los fármacos , HumanosRESUMEN
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. We found that the absence of the 7α-OH group and its epimerization increase the affinity of secondary bile acids for STARD5. According to NMR titration and molecular modeling, the affinity depends mainly on the number and positions of the steroid ring hydroxyl groups and to a lesser extent on the presence or type of bile acid side-chain conjugation. Primary and secondary bile acids have different binding modes and display different positioning within the STARD5 binding pocket. The relative STARD5 affinity for the different bile acids studied is: DCA>LCA>CDCA>GDCA>TDCA>CA>UDCA. TCA and GCA do not bind significantly to STARD5. The impact of the ligand chemical structure on the thermodynamics of binding is discussed. The discovery of these new ligands suggests that STARD5 is involved in the cellular response elicited by bile acids and offers many entry points to decipher its physiological role.
Asunto(s)
Ácidos y Sales Biliares/química , Proteínas Portadoras/química , Espectroscopía de Resonancia Magnética/métodos , Proteínas Adaptadoras del Transporte Vesicular , Humanos , Unión Proteica , TermodinámicaRESUMEN
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. The SAR by NMR [(1)H-(15)N heteronuclear single-quantum coherence (HSQC)] approach, complemented by circular dichroism (CD) and isothermal titration calorimetry (ITC), was used. Titration of STARD5 with cholic (CA) and chenodeoxycholic acid (CDCA), ligands of the farnesoid X receptor (FXR), leads to drastic perturbation of the (1)H-(15)N HSQC spectra and the identification of the residues in contact with those ligands. The most perturbed residues in presence of ligands are lining the internal cavity of the protein. Ka values of 1.8·10-(4) M(-1) and 6.3·10(4) M(-1) were measured for CA and CDCA, respectively. This is the first report of a START domain protein in complex with a sterol ligand. Our original findings indicate that STARD5 may be involved in the transport of bile acids rather than cholesterol.
Asunto(s)
Proteínas Portadoras/química , Ácido Quenodesoxicólico/química , Ácido Cólico/química , Proteínas Adaptadoras del Transporte Vesicular , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Clonación Molecular , Humanos , Ligandos , Espectroscopía de Resonancia Magnética/normas , Modelos Moleculares , Estabilidad Proteica , Estándares de Referencia , Relación Estructura-Actividad , TermodinámicaRESUMEN
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. The most intensively studied member of this subclass is the steroidogenic acute regulatory protein (StAR) or STARD1. While its role in steroid hormone production has been demonstrated, much less is understood about how its START domain specifically recognizes cholesterol and how it releases it to be transferred inside the mitochondria of steroidogenic cell of the gonads and adrenal cortex. A major obstacle that is slowing down progress in this area is the lack of knowledge of the 3D structures of the START domain of StAR in both its free and complexed forms. However, 3D models of the START domain of StAR and mechanisms of binding have been proposed. In addition biophysical studies aimed at validating the models and mechanism have been published. What's more, the crystal structures of the free forms of 3 START domains (STARD3, STARD4 and STARD5) known to specifically bind cholesterol have been elucidated so far. In this chapter, we will review and critically summarize existing data in order to provide the most current view and status of our understanding of the structure and reversible cholesterol binding mechanism of START domains.
Asunto(s)
Colesterol/metabolismo , Fosfoproteínas/fisiología , Proteínas Adaptadoras del Transporte Vesicular , Animales , Proteínas Portadoras/fisiología , Humanos , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana/fisiología , Modelos Químicos , Modelos Moleculares , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
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.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas/química , Conformación Proteica , Hiperplasia Suprarrenal Congénita/genética , Hiperplasia Suprarrenal Congénita/metabolismo , Humanos , Modelos Moleculares , MutaciónRESUMEN
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.
Asunto(s)
Colesterol/metabolismo , Simulación por Computador , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estructura Secundaria de Proteína , Reproducibilidad de los ResultadosRESUMEN
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.
Asunto(s)
Colesterol/metabolismo , Fosfoproteínas/metabolismo , Sitios de Unión/genética , Línea Celular Tumoral , Colesterol/química , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/aislamiento & purificación , Plásmidos , Unión Proteica/genética , Pliegue de Proteína , Estructura Secundaria de Proteína , Ensayo de Unión Radioligante , Temperatura , TermodinámicaRESUMEN
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.
Asunto(s)
Colesterol/metabolismo , Fosfoproteínas/metabolismo , Sitios de Unión , Colesterol/química , Clonación Molecular , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Fosfoproteínas/química , Conformación Proteica , Pliegue de Proteína , Temperatura , TermodinámicaRESUMEN
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 (Kb) and other parameters such as the enthalpy and Gibbs free energy of binding may be estimated from the changes in the stability curves ΔGu(T) of a protein in the presence of a ligand. Here we describe the application of two different procedures proposed by Layton and Hellinga et al. (Biochemistry 49:10831-10841, 2010) to evaluate the apparent Kb of testosterone to the START (StAR-related lipid transfer domain) domains.
Asunto(s)
Dicroismo Circular , Ligandos , Proteínas de Transporte de Membrana/metabolismo , Clonación Molecular , Expresión Génica , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/aislamiento & purificación , Unión Proteica , Proteínas Recombinantes , Análisis Espectral , TermodinámicaRESUMEN
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). In addition, two mutants of N174 genetically engineered by single amino acid substitutions (A104L and K164R) and one "consensus" (N174-CONS) chitosanase designed by multiple amino acid substitutions of N174 were analyzed. Chitosan used had a weight average molecular weight (Mw) of 220 kDa and was 85% deacetylated. Results showed a pH and concentration-dependent protective effect of chitosan in all the cases. However, the extent of thermal protection varied depending on chitosanases, suggesting that key amino acid residues contributed to resistance to heat denaturation. The transition temperatures (T(m)) of N174 were 54 degrees C and 69.5 degrees C in the absence and presence (6 g/l) of chitosan, respectively. T(m) were increased by 11.6 degrees C (N174-CONS), 13.8 degrees C (CSN-A104L), 15.6 degrees C (N174-K164R) and 25.2 degrees C (SCO) in the presence of chitosan (6 g/l). The thermal protective effect was attributed to an enzyme-ligand thermostabilization mechanism since it was not mimicked by the presence of anionic (carboxymethyl cellulose, heparin) or cationic (polyethylene imine) polymers, polyhydroxylated (glycerol, sorbitol) compounds or inorganic salts. Furthermore, the data from fluorometry experiments were in agreement with those obtained by analysis of reaction time-courses performed at 61 degrees C in which case CSN-A104L was rapidly inactivated whereas N174, N174-CONS and N174-K164R remained active over a reaction time of 90 min. This study presents evidence that (1) the fluorometric determination of T(m) in the presence of chitosan is a reliable technique for a rapid assessment of the thermal behavior of chitosanases, (2) it is applicable to structure-function studies of mutant chitosanases and, (3) it can be useful to provide an insight into the mechanism by which mutations can influence chitosanase stability.
Asunto(s)
Sustitución de Aminoácidos , Proteínas Bacterianas/química , Quitosano/química , Glicósido Hidrolasas/química , Pliegue de Proteína , Streptomyces coelicolor/enzimología , Proteínas Bacterianas/genética , Catálisis , Glicósido Hidrolasas/genética , Calor , Concentración de Iones de Hidrógeno , Desnaturalización Proteica/genética , Espectrometría de Fluorescencia , Streptomyces coelicolor/genética , Relación Estructura-ActividadRESUMEN
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., Lefebvre, A., 2001. Control of CYP11B2 gene expression through differential regulation of its promoter by atypical and conventional protein kinase C isoforms. J. Biol. Chem. 276 (11), 8021-8028; LeHoux, J.G., Lefebvre, A., 2006. Novel protein kinase C-epsilon inhibits human CYP11B2 gene expression through ERK1/2 signalling pathway and JunB. J. Mol. Endocrinol. 36 (1), 51-64]. The aim of the present work was to evaluate the physiological role of endogenous PKCepsilon and PKCalpha isoforms in the activation of p44/42 MAPK by AII. A 50% reduction of PKCepsilon protein by siRNA-PKCepsilon resulted in 35% inhibition of AII-induced p44/42 MAPK activation. Knockdown of PKCepsilon stimulated AII-induced CYP11B2 transcription indicating that the PKCepsilon is not involved in the activation of CYP11B2 gene expression by AII. Furthermore, knockdown of PKCalpha enhanced AII-stimulated CYP11B2 transcription without altering p44/42 MAPK indicating that inhibition of AII-stimulated CYP11B2 gene by PKCalpha does not involve the p44/42 MAPK signalling pathway. These results thus establish that physiologically, PKCepsilon and PKCalpha act through different signalling pathways to inhibit AII-stimulated CYP11B2 gene expression.
Asunto(s)
Angiotensina II/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Línea Celular Tumoral , Citocromo P-450 CYP11B2/genética , Humanos , Sistema de Señalización de MAP Quinasas , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C-epsilon/genética , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
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. Ang II activated nPKCepsilon and did not affect nPKCtheta, pinpointing PKCepsilon as the mediator of Ang II in p44/42 MAPK activation. Overexpression of wild-type ERK1 and ERK2 significantly reduced basal as well as Ang II-stimulated human -2023CYP11B2-CAT activity; conversely, the two dominant negative mutants increased them. Overexpression of constitutively active (ca) PKCsuppressed Ang II-induced -2023CYP11B2-CAT activity. Infection of H295R cells with adenoviruses (Adv) expressing caPKCepsilon activated endogenous MEK1/2 and p44/42 MAPK. Adv-caPKCepsilon inhibited Ang II-stimulated aldosterone synthase mRNA levels and this action was reversed by the MEK1 inhibitor, PD98059. Also, Ang II increased JunB protein levels and this effect was inhibited by PD98059 and Bis. Adv-caPKCepsilon enhanced JunB protein levels and PD98059 attenuated the increase. JunB overexpression abolished the Ang II-induced promoter activity within -138 bp of the 5'-flanking region of CYP11B2. Collectively, these results demonstrate that PKCepsilon inhibits CYP11B2 transcription through the p44/42 MAPK pathway and JunB in H295R cells.
Asunto(s)
Citocromo P-450 CYP11B2/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa C-epsilon/fisiología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Cloranfenicol O-Acetiltransferasa/genética , Cartilla de ADN , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Regiones Promotoras Genéticas , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , Transcripción Genética/efectos de los fármacosRESUMEN
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.
RESUMEN
START domain proteins are conserved α/ß 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. Here, we report the solution structure of STARD6 and the characterization of its backbone dynamics on multiple time-scales through (15)N spin-relaxation and amide exchange studies. We reveal for the first time the presence of concerted fluctuations in the Ω1 loop and the C-terminal helix on the microsecond-millisecond time-scale that allows for the opening of the binding site and ligand entry. We also report that STARD6 binds specifically testosterone. Our work represents a milestone for the study of ligand binding mechanism by other START domains and the elucidation of the biological function of STARD6.
Asunto(s)
Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Esteroides/química , Esteroides/metabolismo , Sitios de Unión , Humanos , Cinética , Ligandos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Unión Proteica , Conformación ProteicaRESUMEN
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. We thus investigated if computational analysis could explain the difference in activity profiles. Therefore, we inserted the four P450c17 substrates-pregnenolone, progesterone, and their 17alpha-hydroxylated forms-inside our hamster P450c17 model, which we derived from our human P450c17 model based on the crystal structure of P450BMP. We performed molecular dynamics (MD) simulations on the complexes and analyzed the resultant trajectories to identify amino acids that interact with substrates. Starting with substrates in two different orientations, we obtained two sets of binding trajectories in each case. The first set of trajectories reveal structural rearrangements that occur during binding, whereas the second set of trajectories reflects substrate orientations during catalysis. Our modeling suggests that three distinct steps are required for substrate selectivity and binding to the hamster P450c17: (1) recognition of the substrate at the putative substrate entrance, characterized by a pocket at the surface of the hamster P450c17 containing charged residues R96 and D116; (2) entry of the substrate into the active site, in an intermediate position directed by possible hydrogen bonding of the substrates with the heme D-ring propionate group, R96, R440, and T306; followed by (3) 90 degrees counterclockwise rotation of the substrates, positioning them in optimal position for reactivity, a process that may be directed by hydrogen bonding to the 110-112 region of the hamster P450c17. With some substrates, we obtained trajectories which suggest that major distortions in the I-helix and opening of the H-I loop occur during substrate binding. In conclusion, these modeling exercises provide insight to possible structural reorganizations that occur during substrate binding and suggest that amino acids that participate in three distinct steps of this process may all contribute to substrate binding and activity.
Asunto(s)
Esteroide 17-alfa-Hidroxilasa/química , 17-alfa-Hidroxipregnenolona/química , 17-alfa-Hidroxiprogesterona/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Simulación por Computador , Cricetinae , Isoenzimas/química , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Pregnenolona/química , Progesterona/química , Esteroide 17-alfa-Hidroxilasa/metabolismo , Especificidad por SustratoRESUMEN
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/ß-grip fold module of approximately 210 amino acids delimiting an internal cavity forming the binding site. However, the determinants that dictate ligand specificity and the mechanism of ligand entry and exit are ill-defined. Herein, we review and discuss the current knowledge on ligand specificity and binding mechanism of START domains. More specifically, we highlight that the conserved residues of STARD1, STARD3, STARD4, STARD5 and STARD6 START domains binding sterol play an important structural role for the global protein fold, whereas the residues forming the cavity that fits the shape of their respective ligand are divergent, suggesting their participation in ligand specificity. We also explore the potential binding of steroids to STARD6 in the context of ligand selectivity.
Asunto(s)
Colesterol/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Ácidos y Sales Biliares/metabolismo , Sitios de Unión , Humanos , Ligandos , Mamíferos/metabolismo , Proteínas de Transporte de Membrana/química , Datos de Secuencia MolecularRESUMEN
The specific development of the human fetal adrenal gland requires cell proliferation, migration, apoptosis, and zone-specific steroidogenic activity. The present work was designed to determine the physiological significance of the previously identified spatial distribution of extracellular matrix components in the fetal gland. Primary cultures of human fetal adrenal cells grown on collagen IV, laminin, or fibronectin revealed that cell morphology was affected by environmental cues. Matrices also modulated the profile of steroid secretion by the fetal cells. Collagen IV favored cortisol secretion after ACTH or angiotensin II stimulation and increased dehydroepiandrosterone production when the AT(2) receptor of angiotensin II was specifically stimulated. These effects were correlated by changes in the mRNA levels of 3beta-hydroxysteroid dehydrogenase and cytochrome P450C17. In contrast, fibronectin and laminin decreased cell responsiveness to ACTH in terms of cortisol secretion, but enhanced ACTH-stimulated androgen secretion. Finally, extracellular matrices were able to orchestrate cell behavior. Collagen IV and laminin enhanced cell proliferation, and fibronectin increased cell death. This study is the first to demonstrate that the nature of extracellular matrix coordinates specific steroidogenic pathways and cell turnover in the developing human fetal adrenal gland.
Asunto(s)
Glándulas Suprarrenales/embriología , Colágeno Tipo IV/fisiología , Fibronectinas/fisiología , Laminina/fisiología , 3-Hidroxiesteroide Deshidrogenasas/genética , Glándulas Suprarrenales/citología , Hormona Adrenocorticotrópica/farmacología , División Celular/efectos de los fármacos , División Celular/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Deshidroepiandrosterona/metabolismo , Sulfato de Deshidroepiandrosterona/metabolismo , Desarrollo Embrionario y Fetal/fisiología , Matriz Extracelular/fisiología , Feto/fisiología , Humanos , Hidrocortisona/metabolismo , ARN Mensajero/metabolismoRESUMEN
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. Northern blot analysis of total RNA from cells showed that the level of TIMP-1 mRNA increases sharply within 3h after ACTH stimulation. Since TIMP-1 inhibits some cell matrix metalloproteinases (MMPs) of the collagenase type, we examined the effect of ACTH on collagenase activity in bovine adrenocortical cells. Exposure of confluent cultures to ACTH for 24h showed dose-dependent inhibition of collagenase activity. Northern blot analysis of total RNA from rat adrenal zona fasciculata-reticularis and zona glomerulosa showed that in both of these zones TIMP-1 expression was induced within 12h after ACTH injection. Long-term (9 days) treatment with ACTH increased TIMP-1 mRNA levels nearly sixfold in zona fasciculata-reticularis. Overall, our results show that ACTH causes induction of TIMP-1 and suppression of collagenase activity, and suggest that ACTH may modulate the activities of MMPs and hence cell matrix remodeling.
Asunto(s)
Corteza Suprarrenal/efectos de los fármacos , Corteza Suprarrenal/metabolismo , Hormona Adrenocorticotrópica/farmacología , Inhibidores de la Metaloproteinasa de la Matriz , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Animales , Northern Blotting , Bovinos , Células Cultivadas , Clonación Molecular , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Biblioteca de Genes , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Distribución Tisular , Inhibidor Tisular de Metaloproteinasa-1/genéticaRESUMEN
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., unpublished). In this study, we are comparing the two-dimensional gel electrophoresis (2D-PAGE) characteristics of the WT StAR with those of the S194A and S55A/S56A/S194A-StAR mutants under control and (Bu)(2)-cAMP stimulation, using an anti-StAR antibody and an anti-phospho-(Ser/Thr) PKA substrate antibody. The 2D-PAGE migration pattern of the WT StAR analyzed by immunoblotting with the anti-StAR antibody revealed many StAR species with different pI and different molecular weights. In the (Bu)(2)-cAMP-WT preparations, except for three, all these StAR species were also recognized by the anti-phospho-(Ser/Thr) PKA substrate antibody; in contrast, less phosphorylated species were found in the non-stimulated WT preparations. The two-dimensional (2D) patterns of StAR revealed by the anti-StAR and the anti-phospho-(Ser/Thr) PKA substrate antibodies were modified for the S194A mutant and further modified for the S55A/S56A/S194A mutant. Whereas many species could still be detected by the anti-StAR antibody in the triple mutant S55A/S55A/S194A, none of these could be revealed by the anti-phospho-(Ser/Thr) PKA substrate antibody. Finally we found that, in addition to phosphorylation, the formation of different StAR species was also due to the hydrolysis of the molecule at its N-terminal and to a lesser degree at its C-terminal.
Asunto(s)
Corteza Suprarrenal/metabolismo , Mutación/genética , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Corteza Suprarrenal/citología , Corteza Suprarrenal/efectos de los fármacos , Animales , Bucladesina/farmacología , Células COS , Cricetinae , Electroforesis en Gel Bidimensional , Immunoblotting , Fosfoproteínas/genética , FosforilaciónRESUMEN
In order to understand the activity specificity of the hamster cytochrome P450 17 alpha-hydroxylase/17,20-lyase (P450c17), we have studied its structure/activity using three hamster P450c17 recombinant mutants (T202N/D240N/D407H). In transiently transfected COS-1 cells, the mutation T202N reduced 17 alpha-hydroxylation of pregnenolone and progesterone to 24 and 44% of wild type (WT), respectively, followed by reduced 17,20-cleavage to 71 and 67%, respectively. On the other hand, the mutation D240N decreased specifically 17,20-lyase activity to 61% of WT when incubated with pregnenolone while the mutation D407H only decreased 17 alpha-hydroxylation to 46% when incubated with progesterone.To comprehend the altered activity profiles of these hamster P450c17 mutants, we have elaborated a 3D model of the hamster P450c17 and compared it to our preceding model of the human P450c17. Analysis of the mutants with this model showed that, without direct contact to the substrates, these mutations transmit structural changes to the active site. By analogy, these results support the concept that any cellular changes modifying the external structure of P450c17, such as phosphorylation, could have influence on its active site and enzymatic activities.