Stimulation by endozepine of the side-chain cleavage of cholesterol in a reconstituted enzyme system

doi:10.1016/S0006-291X(05)81109-X

Biochemical and Biophysical Research Communications

Volume 180, Issue 2, 31 October 1991, Pages 609-614

https://doi.org/10.1016/S0006-291X(05)81109-X Get rights and content

Addition of endozepine in nanomolar concentrations to a system for side-chain cleavage reconstituted from highly purified P-450scc and electron carriers (adrenodoxin reductase and adrenodoxin) stimulates the conversion of cholesterol to pregnenolone (side-chain cleavage). This response is concentration and time-dependent and specific to the extent that a second steroidogenic P-450 located in the inner mitochondrial membrane (ie 11β-hydroxylase) was not stimulated by endozepine. Homogeneous endozepine prepared from bovine brain, the corresponding genetically engineered peptide and des(glu-ilu)-endozepine isolated from bovine adrenal cortex are all approximately equipotent in this system. Moreover, endozepine accelerates the rate of reduction of P-450scc by NADPH and the electron carriers. The results suggest that endozepine acts directly on P-450 and hence the rate of side-chain cleavage.

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Cited by (20)

Endozepines and their receptors: Structure, functions and pathophysiological significance
2020, Pharmacology and Therapeutics
Citation Excerpt :
As noted above (Section 2.6), DBI is highly expressed in adrenocortical and testicular Leydig cells (Bovolin et al., 1990; Costa & Guidotti, 1991; Duparc et al., 2003; Garnier et al., 1993; Papadopoulos, Berkovich et al., 1991; Yanagibashi et al., 1988, 1989). The effect of DBI, DBI(1–84), purified rat or bovine brain DBI, and rat testis and bovine testis DBI, on intramitochondrial cholesterol transport and steroidogenesis was confirmed in testicular Leydig and adrenal cortical cells (Boujrad, Hudson, & Papadopoulos, 1993; Brown & Hall, 1991; Garnier, Boujrad, Ogwuegbu, Hudson, & Papadopoulos, 1994). The naturally occurring processing fragments of DBI, TTN and ODN also increase steroid formation in Y-1 adrenocortical cells (Papadopoulos, Berkovich et al., 1991), in frog adrenal fragments (Lesouhaitier et al., 1998, 2000) and in human testicular explants (Duparc et al., 2003).
The existence of specific binding sites for benzodiazepines (BZs) in the brain has prompted the search for endogenous BZ receptor ligands designated by the generic term « endozepines ». This has led to the identification of an 86-amino acid polypeptide capable of displacing [³H]diazepam binding to brain membranes, thus called diazepam-binding inhibitor (DBI). It was subsequently found that the sequence of DBI is identical to that of a lipid carrier protein termed acyl-CoA-binding protein (ACBP). The primary structure of DBI/ACBP has been well preserved, suggesting that endozepines exert vital functions. The DBI/ACBP gene is expressed by astroglial cells in the central nervous system, and by various cell types in peripheral organs. Endoproteolytic cleavage of DBI/ACBP generates several bioactive peptides including a triakontatetraneuropeptide that acts as a selective ligand of peripheral BZ receptors/translocator protein, and an octadecaneuropeptide that activates a G protein-coupled receptor and behaves as an allosteric modulator of the GABA_AR. Although DBI/ACBP is devoid of a signal peptide, endozepines are released by astrocytes in a regulated manner. Consistent with the diversity and wide distribution of BZ-binding sites, endozepines appear to exert a large array of biological functions and pharmacological effects. Thus, intracerebroventricular administration of DBI or derived peptides induces proconflict and anxiety-like behaviors, and reduces food intake. Reciprocally, the expression of DBI/ACBP mRNA is regulated by stress and metabolic signals. In vitro, endozepines stimulate astrocyte proliferation and protect neurons and astrocytes from apoptotic cell death. Endozepines also regulate neurosteroid biosynthesis and neuropeptide expression, and promote neurogenesis. In peripheral organs, endozepines activate steroid hormone production, stimulate acyl chain ceramide synthesis and trigger pro-inflammatory cytokine secretion. The expression of the DBI/ACBP gene is enhanced in addiction/withdrawal animal models, in patients with neurodegenerative disorders and in various types of tumors. We review herein the current knowledge concerning the various actions of endozepines and discuss the physiopathological implications of these regulatory gliopeptides.
Neurosteroid biosynthesis: Enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides
2009, Frontiers in Neuroendocrinology
Citation Excerpt :
TTN also stimulates steroidogenesis in the Y-1 adrenocortical cell line [555,556] and the MA-10 and R2C Leydig cell lines [226,227,555,556]. TTN has been shown to increase Δ5P formation in isolated mitochondria [72] either by stimulating cholesterol transport into mitochondria [555,862] or by directly enhancing P450scc activity [98]. In frog, TTN stimulates corticosterone and aldosterone from perifused adrenal (interrenal) fragments [403,404,705].
Neuroactive steroids synthesized in neuronal tissue, referred to as neurosteroids, are implicated in proliferation, differentiation, activity and survival of nerve cells. Neurosteroids are also involved in the control of a number of behavioral, neuroendocrine and metabolic processes such as regulation of food intake, locomotor activity, sexual activity, aggressiveness, anxiety, depression, body temperature and blood pressure. In this article, we summarize the current knowledge regarding the existence, neuroanatomical distribution and biological activity of the enzymes responsible for the biosynthesis of neurosteroids in the brain of vertebrates, and we review the neuronal mechanisms that control the activity of these enzymes. The observation that the activity of key steroidogenic enzymes is finely tuned by various neurotransmitters and neuropeptides strongly suggests that some of the central effects of these neuromodulators may be mediated via the regulation of neurosteroid production.
Peripheral-type benzodiazepine receptor: Structure and function of a cholesterol-binding protein in steroid and bile acid biosynthesis
2003, Steroids
Cholesterol transport from the outer to the inner mitochondrial membrane is the rate-determining step in steroid and bile acid biosyntheses. Biochemical, pharmacological and molecular studies have demonstrated that the peripheral-type benzodiazepine receptor (PBR) is a five transmembrane domain mitochondrial protein involved in the regulation of cholesterol transport. PBR gene disruption in Leydig cells completely blocked cholesterol transport into mitochondria and steroid formation, while PBR expression in bacteria, devoid of endogenous PBR and cholesterol, induced cholesterol uptake and transport. Molecular modeling of PBR suggested that cholesterol might cross the membrane through the five helices of the receptor and that synthetic and endogenous ligands might bind to common sites in the cytoplasmic loops. A cholesterol recognition/interaction amino acid consensus (CRAC) sequence in the cytoplasmic carboxy-terminus of the PBR was identified by mutagenesis studies. In vitro reconstitution of PBR into proteoliposomes demonstrated that PBR binds both drug ligands and cholesterol with high affinity. In vivo polymeric forms of PBR were observed and polymer formation was reproduced in vitro, using recombinant PBR protein reconstituted into proteoliposomes, associated with an increase in drug ligand binding and reduction of cholesterol-binding capacity. This suggests that the various polymeric states of PBR might be part of a cycle mediating cholesterol uptake and release into the mitochondria, with PBR functioning as a cholesterol exchanger against steroid product(s) arising from cytochrome P450 action. Taking into account the widespread presence of PBR in many tissues, a more general role of PBR in intracellular cholesterol transport and compartmentalization might be considered.
Peripheral-type benzodiazepine receptors
1997, General Pharmacology
- 1.
  1. The pharmacological effects of benzodiazepines are mediated through a class of recognition sites associated with the γ-aminobutyric acid A receptor. A second class of benzodiazepine binding sites is found in virtually all mammalian peripheral tissues and is therefore called the peripheral type benzodiazepine receptor (PBR).
- 2.
  2. The first section of this review describes the tissue and subcellular distribution of the PBR in mammalian tissues and analyzes its many putative endogenous ligands.
- 3.
  3. The next section deals with the pharmacological, structural and molecular characterization of the PBR that has taken place in the past few years.
- 4.
  4. The final section describes the possible physiological role(s) of the PBR and identifies future work that would help deepen our understanding of the PBR and its function.
Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis
1997, Steroids
Steroidogenesis begins with the metabolism of cholesterol to pregneolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane and loading of P450scc with cholesterol. In previous in vitro studies, we demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR). We also showed that the polypeptide diazepam binding inhibitor (DBI), an endogenous PBR ligand, stimulates cholesterol transport and promotes loading of cholesterol to P450scc in vitro, and that its presence is vital for hCG-induced steroidogenesis by Leydig cells. Based on these data and the observations that i) the mitochondrial PBR binding and topography are regulated by hormones; ii) the 18-kDa PBR protein is functionally coupled to the mitochondrial contact site voltage-dependent anion channel protein; iii) the 18-kDa PBR protein is a channel for cholesterol, as shown by molecular modeling and in vitro reconstitution studies; iv) targeted disruption of the PBR gene in steroidogenic cells dramatically reduces the ability of the cells to transport cholesterol in the mitochondria and produce steroids; v) endocrine disruptors, with known anisteroidogenic effect, inhibit PBR ligand binding; and vi) in vivo reduction of adrenal PBR expression results in reduced circulating glucocorticoid levels, we conclude that PBR is an indispensable element of the steroidogenic machinery.
Role of the steroidogenic acute regulatory protein (StAR) in steroidogenesis
1996, Biochemical Pharmacology
The rate-limiting, hormone-regulated, enzymatic step in Steroidogenesis is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage enzyme system (CSCC), which is located on the matrix side of the inner mitochondrial membrane. However, it has long been observed that hydrophilic cholesterol-like substrates capable of traversing the mitochondrial membranes are cleaved to pregnenolone by the CSCC in the absence of any hormone stimulation. Therefore, the true regulated step in the acute response of steroidogenic cells to hormone stimulation is the delivery of cholesterol to the inner mitochondrial membrane and the CSCC. It has been known for greater than three decades that transfer of cholesterol requires de novo protein synthesis; however, prior to this time the regulatory protein(s) had yet to be identified conclusively. It is the purpose of this commentary to briefly review a number of the candidates that have been proposed as the acute regulatory protein. As such, we have summarized the available information that describes the roles of transcription, translation, and phosphorylation in this regulation, and have also reviewed the supporting cases that have been made for several of the proteins put forth as the acute regulator. We close with a comprehensive description of the Steroidogenic Acute Regulatory protein (StAR) that we and others have identified and characterized as a family of proteins that are synthesized and imported into the mitochondria in response to hormone stimulation, and for which strong evidence exists indicating that it is the long sought acute regulatory protein.

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Stimulation by endozepine of the side-chain cleavage of cholesterol in a reconstituted enzyme system

Biochem. Biophys. Acta.

Biochem. Biophys. Acta.

Hormonal proteins and peptides

Mol. Cell. Biochem.

Endocrinology.