The human peripheral renzodiazepine receptor gene: Cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia

doi:10.1016/S0888-7543(05)80367-2

Genomics

Volume 18, Issue 3, December 1993, Pages 643-650

https://doi.org/10.1016/S0888-7543(05)80367-2 Get rights and content

Abstract

The mitochondrial benzodiazepine receptor (mBzR)appears to be a key factor in the flow of cholesterol into mitochondia to permit the initiation of steroid hormone synthesis. The mBzR consists of three components; the 18-kDa component on the outer mitochondrial membrane appears to contain the benzodiazepine binding site, and is hence often termed the peripheral benzodiazepine receptor (PBR). Using a cloned human PBR cDNA as probe, we have cloned the human PBR gene. The 13-kb gene is divided into four exons, with exon 1 encoding only a short 5′ untranslated segment. The 5′ flanking DNA lacks TATA and CAAT boxes but contains a cluster of SP-1 binding sites, typical of “housekeeping” genes. The encoded PBR mRNA is alternately spliced into two forms: “authentic” PBR mRNA retains all four exons, while a short form termed PBRS lacks exon 2. While PBR-S contains a 102-codon open reading frame with a typical initiator sequence, the reading frame differs from that of PBR, so that the encoded protein is unrelated to PBR. RT-PCR and RNase protection experiments confirm that both PBR and PBR-S are expressed in all tissues examined and that expression PBR-S is about 10 times the level of PBR. Expression of PBR cDNA in pCMV5 vectors transfected into COS-1 cells resulted in increased binding of [³H]PK11195, but expression of PBR-S did not. It has been speculated that patients with congenital lipoid adrenal hyperplasia, who cannot make any steroids, might have a genetic lesion in mBzR. RT-PCR analysis of testicular RNA from such a patient, sequencing of the cDNA, and blotting analysis of genomic DNA all indicate that the gene and mRNA for the PBR component of mBzR are normal in this disease.

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Translocator protein (18 kDa) (Tspo), formerly known as peripheral benzodiazepine receptor is a highly conserved transmembrane protein primarily located in the outer mitochondrial membrane. In the central nervous system (CNS), especially in glia cells, Tspo is upregulated upon inflammation. Consequently, Tspo was used as a tool for diagnostic in vivo imaging of neuroinflammation in the brain and as a potential therapeutic target. Several synthetic Tspo ligands have been explored as immunomodulatory and neuroprotective therapy approaches. Although the function of Tspo and how its ligands exert these beneficial effects is not fully clear, it became a research topic of interest, especially in ocular diseases in the past few years. This review summarizes state-of-the-art knowledge of Tspo expression and its proposed functions in different cells of the retina including microglia, retinal pigment epithelium and Müller cells. Tspo is involved in cytokine signaling, oxidative stress and reactive oxygen species production, calcium signaling, neurosteroid synthesis, energy metabolism, and cholesterol efflux. We also highlight recent developments in preclinical models targeting Tspo and summarize the relevance of Tspo biology for ocular and retinal diseases. We conclude that glial upregulation of Tspo in different ocular pathologies and the use of Tspo ligands as promising therapeutic approaches in preclinical studies underline the importance of Tspo as a potential disease-modifying protein.
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Steroidogenesis begins with internalization of low-density lipoprotein particles and subsequent intracellular processing of cholesterol. Disorders in these steps include adrenoleukodystrophy, Wolman disease, and Niemann–Pick type C disease, which may present as adrenal insufficiency. Cholesterol delivery to the inner mitochondrial membrane is regulated by the steroidogenic acute regulatory protein, StAR, and cholesterol is converted to pregnenolone within mitochondria by the cholesterol side-chain cleavage enzyme, P450scc. Severe StAR mutations cause classic congenital lipoid adrenal hyperplasia (CAH), characterized by adrenal insufficiency and 46,XY disorders of sexual development (DSD). The lipoid CAH phenotype, including spontaneous puberty in 46,XX females, is explained by a two-hit model. StAR mutations that retain partial function cause milder nonclassic disease characterized by glucocorticoid deficiency, with lesser disorders of mineralocorticoid and sex steroid synthesis. Rare P450scc mutations are clinically and hormonally indistinguishable from lipoid CAH, and may also present as milder nonclassic disease. Adrenal imaging may distinguish these but is not 100% reliable, necessitating DNA sequencing.
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Although these new PET tracers show improved SNR, there is a limitation that there is variability in TSPO binding potential (BP) among individuals due to a single nucleotide polymorphism in the TSPO gene54. The human TSPO gene is located on chromosome 22q13.3, consists of four exons, and encodes 169 amino acids55. Recent studies discovered a single-nucleotide polymorphism (rs6971) in exon 4 of the human TSPO gene that results in a nonconservative alanine to threonine substitution, influencing the TSPO protein's ligand binding affinity56.
The 18 kDa translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is predominately localized to the outer mitochondrial membrane in steroidogenic cells. Brain TSPO expression is relatively low under physiological conditions, but is upregulated in response to glial cell activation. As the primary index of neuroinflammation, TSPO is implicated in the pathogenesis and progression of numerous neuropsychiatric disorders and neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), multiple sclerosis (MS), major depressive disorder (MDD) and obsessive compulsive disorder (OCD). In this context, numerous TSPO-targeted positron emission tomography (PET) tracers have been developed. Among them, several radioligands have advanced to clinical research studies. In this review, we will overview the recent development of TSPO PET tracers, focusing on the radioligand design, radioisotope labeling, pharmacokinetics, and PET imaging evaluation. Additionally, we will consider current limitations, as well as translational potential for future application of TSPO radiopharmaceuticals. This review aims to not only present the challenges in current TSPO PET imaging, but to also provide a new perspective on TSPO targeted PET tracer discovery efforts. Addressing these challenges will facilitate the translation of TSPO in clinical studies of neuroinflammation associated with central nervous system diseases.
Subcellular distribution of the 18 kDa translocator protein and transcript variant PBR-S in human cells
2017, Gene
Citation Excerpt :
During the analysis of human PBR/TSPO for construction of the fusion constructs, we noted the presence of a transcript variant. Lin et al. (1993) cloned the human PBR/TSPO gene and determined that the full-length PBR/TSPO mRNA consists of four exons while a shorter isoform, termed PBR-S, lacks exon 2. The PBR-S mRNA contains a 102-codon open reading frame that differs to PBR/TSPO, meaning that the deduced PBR-S protein (also known as putative peripheral benzodiazepine receptor-related protein) is completely distinct from PBR/TSPO (Lin et al., 1993).
Despite continued interest in the 18 kDa translocator protein (PBR/TSPO) as a biomarker and a therapeutic target for a range of diseases, its functional role, such as in the steroid synthesis pathway and energy metabolism has either become contentious or remains to be described more precisely.
The PBR/TSPO gene consists of four exons, while a shorter isoform termed PBR-S lacks exon 2. The PBR-S 102-codon open reading frame differs to that of PBR/TSPO, resulting in a protein that is completely unrelated to PBR/TSPO. To our knowledge, PBR-S protein has never been described and has no known or proposed function.
To obtain possible clues on the role of this uncharacterised protein, we compared the subcellular distribution of PBR-S to that of PBR/TSPO. By expressing fluorescently tagged PBR/TSPO, we confirmed that full-length PBR/TSPO co-localises with mitochondria in HeLa, HEK-293, MDA-MB-231, BJ and U87-MG human cell lines.
Unlike the strictly mitochondrial localisation of PBR/TSPO, PBR-S has a punctate distribution throughout the cytosol that co-localises with lysosomes in HeLa, HEK-293, MDA-MB-231, BJ and U87-MG cells.
In summary, within the cell lines examined we confirm mitochondria rather than occasionally reported other localisations, such as the cell nucleus, to be the only site where PBR/TSPO resides. Due to the lack of any shared protein sequences and the different subcellular locations, we suggest that the previously uncharacterised PBR-S protein variant of the PBR/TSPO gene is likely to serve a different yet to be discovered function compared to PBR/TSPO.
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2017, Journal of Steroid Biochemistry and Molecular Biology
Steroidogenesis begins with cellular internalization of low-density lipoprotein particles and subsequent intracellular processing of cholesterol. Disorders in these steps include Adrenoleukodystrophy, Wolman Disease and its milder variant Cholesterol Ester Storage Disease, and Niemann-Pick Type C Disease, all of which may present with adrenal insufficiency. The means by which cholesterol is directed to steroidogenic mitochondria remains incompletely understood. Once cholesterol reaches the outer mitochondrial membrane, its delivery to the inner mitochondrial membrane is regulated by the steroidogenic acute regulatory protein (StAR). Severe StAR mutations cause classic congenital lipoid adrenal hyperplasia, characterized by lipid accumulation in the adrenal, adrenal insufficiency, and disordered sexual development in 46,XY individuals. The lipoid CAH phenotype, including spontaneous puberty in 46,XX females, is explained by a two-hit model. StAR mutations that retain partial function cause a milder, non-classic disease characterized by glucocorticoid deficiency, with lesser disorders of mineralocorticoid and sex steroid synthesis. Once inside the mitochondria, cholesterol is converted to pregnenolone by the cholesterol side-chain cleavage enzyme, P450scc, encoded by the CYP11A1 gene. Rare patients with mutations of P450scc are clinically and hormonally indistinguishable from those with lipoid CAH, and may also present as milder non-classic disease. Patients with P450scc defects do not have the massive adrenal hyperplasia that characterizes lipoid CAH, but adrenal imaging may occasionally fail to distinguish these, necessitating DNA sequencing.
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The human peripheral renzodiazepine receptor gene: Cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia

Abstract

J. Biol. Chem.

Gene

J. Biochem.

J. Biol. Chem.

J. Biochem. Chem.

J. Steroid Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Identification of des-(Gly-Ile)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: Stimulation of cholesterol deliveryis mediated by the peripheral benzodiazepine receptor

The human “peripheral-type” benzodiazepine receptor: Regional mapping of the gene and characterization of the receptor expressed from cDNA

DNA Cell Biol.

Studies on the role of protein synthesis in the regulation of corticosterone production by adrenocorticotropic hormone in vivo

Construction and function of fusion enzymes of the human cytochrome P450scc system

DNA Cell Biol.

A rapid and simple one-step method for isolation of poly(A)+ RNA from cells in monolayer

Endocrinology

Congenital adrenal hyperplasia due to deficient cholesterol side-chain cleavage activity (20, 22 desmolase) in a patient treated for 18 years

Clin. Endocrinol.

The influence of exogenous free cholesterol on steroid synthesis in cultured cells

Endocrinology

A rapid and simple one-step method for isolation of poly(A)⁺ RNA from cells in monolayer