scholarly journals De novo disruption of promoter and exon 1 of STAR gene reveals essential role for gonadal development

2017 ◽  
Vol 2017 ◽  
Author(s):  
Anil Piya ◽  
Jasmeet Kaur ◽  
Alan M Rice ◽  
Himangshu S Bose
Keyword(s):  
Protein Import ◽  
De Novo ◽  
Regulatory Protein ◽  
Mitochondrial Protein ◽  
Protein Translation ◽  
Gonadal Development ◽  
Cholesterol Transport ◽  
List Type ◽  
Star Protein ◽  
Exon 1

Summary Cholesterol transport into the mitochondria is required for synthesis of the first steroid, pregnenolone. Cholesterol is transported by the steroidogenic acute regulatory protein (STAR), which acts at the outer mitochondrial membrane prior to its import. Mutations in the STAR protein result in lipoid congenital adrenal hyperplasia (CAH). Although the STAR protein consists of seven exons, biochemical analysis in nonsteroidogenic COS-1 cells showed that the first two were not essential for pregnenolone synthesis. Here, we present a patient with ambiguous genitalia, salt-lossing crisis within two weeks after birth and low cortisol levels. Sequence analysis of the STAR, including the exon–intron boundaries, showed the complete deletion of exon 1 as well as more than 50 nucleotides upstream of STAR promoter. Mitochondrial protein import with the translated protein through synthesis cassette of the mutant STAR lacking exon 1 showed protein translation, but it is less likely to have synthesized without a promoter in our patient. Thus, a full-length STAR gene is necessary for physiological mitochondrial cholesterol transport in vivo. Learning points: STAR exon 1 deletion caused lipoid CAH. Exon 1 substitution does not affect biochemical activity. StAR promoter is responsible for gonadal development.

scholarly journals Function and crystal structure of the dimeric P-loop ATPase CFD1 coordinating an exposed [4Fe-4S] cluster for transfer to apoproteins

2018 ◽  
Vol 115 (39) ◽  
pp. E9085-E9094 ◽  
Author(s):  
Oliver Stehling ◽  
Jae-Hun Jeoung ◽  
Sven A. Freibert ◽  
Viktoria D. Paul ◽  
Sebastian Bänfer ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Iron Homeostasis ◽  
De Novo ◽  
Regulatory Protein ◽  
Protein Translation ◽  
Cluster Assembly ◽  
Cellular Iron ◽  
Chaetomium Thermophilum ◽  
Cellular Iron Homeostasis ◽  
S Proteins

Maturation of iron-sulfur (Fe-S) proteins in eukaryotes requires complex machineries in mitochondria and cytosol. Initially, Fe-S clusters are assembled on dedicated scaffold proteins and then are trafficked to target apoproteins. Within the cytosolic Fe-S protein assembly (CIA) machinery, the conserved P-loop nucleoside triphosphatase Nbp35 performs a scaffold function. In yeast, Nbp35 cooperates with the related Cfd1, which is evolutionary less conserved and is absent in plants. Here, we investigated the potential scaffold function of human CFD1 (NUBP2) in CFD1-depleted HeLa cells by measuring Fe-S enzyme activities or 55Fe incorporation into Fe-S target proteins. We show that CFD1, in complex with NBP35 (NUBP1), performs a crucial role in the maturation of all tested cytosolic and nuclear Fe-S proteins, including essential ones involved in protein translation and DNA maintenance. CFD1 also matures iron regulatory protein 1 and thus is critical for cellular iron homeostasis. To better understand the scaffold function of CFD1-NBP35, we resolved the crystal structure of Chaetomium thermophilum holo-Cfd1 (ctCfd1) at 2.6-Å resolution as a model Cfd1 protein. Importantly, two ctCfd1 monomers coordinate a bridging [4Fe-4S] cluster via two conserved cysteine residues. The surface-exposed topology of the cluster is ideally suited for both de novo assembly and facile transfer to Fe-S apoproteins mediated by other CIA factors. ctCfd1 specifically interacted with ATP, which presumably associates with a pocket near the Cfd1 dimer interface formed by the conserved Walker motif. In contrast, ctNbp35 preferentially bound GTP, implying differential regulation of the two fungal scaffold components during Fe-S cluster assembly and/or release.

scholarly journals The Steroidogenic Acute Regulatory Protein Is Expressed in Steroidogenic Cells of the Day-Old Brain

Endocrinology ◽  
2004 ◽  
Vol 145 (10) ◽  
pp. 4775-4780 ◽  
Author(s):  
Steven R. King ◽  
Stephen D. Ginsberg ◽  
Tomohiro Ishii ◽  
Roy G. Smith ◽  
Keith L. Parker ◽  
...  
Keyword(s):  
De Novo ◽  
Metastatic Lymph Node ◽  
Regulatory Protein ◽  
Cell Types ◽  
Adult Brain ◽  
Peripheral Tissues ◽  
Cytochrome P450scc ◽  
Star Protein ◽  
Steroidogenic Acute Regulatory ◽  
The Brain

Abstract Although recent research has focused on the fundamental role(s) of steroids synthesized de novo in the brain on development, the mechanism by which production of these neurosteroids is regulated remains unclear. Steroid production in peripheral tissues is acutely regulated by the steroidogenic acute regulatory (StAR) protein, which mediates the rate-limiting step in steroid biosynthesis: the intramitochondrial delivery of cholesterol to cytochrome P450scc for conversion to steroid. We recently demonstrated that StAR is present in discrete cell types in the adult brain, suggesting that neurosteroid production is mediated by StAR. Nevertheless, little is known regarding the presence of StAR in the developing brain. In the present study, the presence of StAR and for the first time, its homolog, the putative cholesterol transport protein metastatic lymph node 64 (MLN64), were defined in the neonatal mouse brain using immunocytochemical techniques. Both StAR and MLN64 were found to be present in the brain with staining patterns characteristic to each protein, indicating the authenticity of StAR and MLN64 immunoreactivity. Furthermore, we found MLN64 to be expressed in the adult brain as well, apparently at higher levels than StAR. Importantly, StAR protein is present in cells that also express P450scc. These data suggest that, as with the adult, neurosteroid production during development occurs through a StAR-mediated pathway.

scholarly journals Mice harboring a SCA28 patient mutation in AFG3L2 develop late-onset ataxia associated with enhanced mitochondrial proteotoxicity.

2018 ◽  
Author(s):  
Cecilia Mancini ◽  
Eriola Hoxha ◽  
Luisa Iommarini ◽  
Alessandro Brussino ◽  
Uwe Richter ◽  
...  
Keyword(s):  
Network Formation ◽  
De Novo ◽  
Neurodegenerative Disorder ◽  
Late Onset ◽  
Mitochondrial Protein ◽  
Atp Synthesis ◽  
Protein Translation ◽  
Mutant Mice ◽  
Mitochondrial Morphology ◽  
Missense Mutations

Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but signs of ataxia were detectable by beam test at 18 months. Cerebellar pathology was negative; electrophysiological analysis showed increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls, although not statistically significant. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was also altered, in line with greatly reduced expression of Opa1 fusogenic protein L-isoforms. The mitochondrial alterations observed in MEFs were also detected in cerebella of 18-month-old heterozygous mutants, suggesting they may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.

scholarly journals A Hypoxia Sensitive to Hypoxia Resistant Transformation in Long-Lived Germline Mutants

2021 ◽  
Author(s):  
Marc Ryan Van Gilst ◽  
C Hemphill ◽  
E Pylarinou-Sinclair ◽  
O Itani ◽  
B Scott ◽  
...  
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Resistance Mechanism ◽  
Protein Translation ◽  
Germline Development ◽  
Mitochondrial Protein Import ◽  
C Elegans ◽  
Physiological Properties ◽  
Rnaseq Data ◽  
Somatic Tissues

Signals from the germline play a significant role in determining longevity in numerous animal models. In C. elegans , ablation of the germline leads to long life span and various other types of stress resistance. It has been reported that mutations that block oogenesis or an upstream step in germline development confer strong resistance to hypoxia. We report here that the hypoxia resistance of sterile mutants is dependent on developmental stage and age. In just a 12-hour period, sterile animals transform from hypoxia sensitive L4 larvae into highly hypoxia resistant adults. Since this transformation occurs in animals with no germline, the physiological programs that determine hypoxia sensitivity must occur independently of germline signals and instead rely on developmental signals from somatic tissues. Furthermore, we found two distinct mechanisms of hypoxia resistance in long-lived germline deficient animals. First, a DAF-16/FoxO independent mechanism that occurs in all hypoxia resistant sterile adults and, second, a DAF-16/FoxO dependent mechanism that confers an added layer of resistance, or “super-resistance”, to animals with no germline as they age past day 1 of adulthood. RNAseq data showed that nearly all genes involved in both cytosolic and mitochondrial protein translation, as well as in mitochondrial protein import, are repressed in germline deficient adults and further repressed as they age. The hypoxia super-resistance of aging germline deficient animals was suppressed by dual mutation of ncl-1 and larp-1 , two regulators of nucleolar biology and protein translation, demonstrating that the hypoxia super-resistance mechanism involves reduced protein translation. These studies provide novel insight into a profound physiological transformation that takes place in germline mutants during development, showing that some of the unique physiological properties of these long-lived animals are dependent on developmental repression of genes involved in protein translation, which operate independently of germline signals.

scholarly journals GnRH agonist treatment decreases progesterone synthesis, luteal peripheral benzodiazepine receptor mRNA, ligand binding and steroidogenic acute regulatory protein expression during pregnancy

1999 ◽  
Vol 22 (1) ◽  
pp. 45-54 ◽  
Author(s):  
R Sridaran ◽  
GH Philip ◽  
H Li ◽  
M Culty ◽  
Z Liu ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Molecular Mechanisms ◽  
Radioligand Binding ◽  
Regulatory Protein ◽  
Benzodiazepine Receptor ◽  
Steroidogenic Acute Regulatory Protein ◽  
Cholesterol Transport ◽  
Pregnant Rat ◽  
Star Protein ◽  
Steroidogenic Acute Regulatory

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) suppresses luteal steroidogenesis in the pregnant rat. We further demonstrated that the peripheral-type benzodiazepine receptor (PBR) and the steroidogenic acute regulatory protein (StAR) play key roles in cholesterol transport leading to steroidogenesis. The purpose of this study was to understand the cellular and molecular mechanisms involved in the suppression of luteal steroidogenesis leading to a fall in serum progesterone levels in GnRH-Ag-treated rats during early pregnancy. Pregnant rats were treated individually starting on day 8 of pregnancy with 5 microgram/day GnRH-Ag using an osmotic minipump. Sham-operated control rats received no treatment. At 0, 4, 8 and 24 h after initiation of the treatment, rats were killed and corpora lutea (CL) were removed for PBR mRNA, protein and radioligand binding analyses, immunoblot 1-D gel analysis of StAR, P450 scc and 3beta-hydroxysteroid dehydrogenase as well as 2-D gel analysis of StAR. The treatment decreased the luteal PBR mRNA expression at all time periods starting at 4 h compared with that in corresponding sham controls. GnRH-Ag also reduced, in the CL, the PBR protein/ligand binding, the StAR protein and P450 scc protein and its activity as early as 8 h after the treatment and they remained low compared with those in corresponding sham controls. The data from 2-D gel studies suggest that the majority of the decrease in StAR protein appears to be in the phosphorylated forms of StAR. Thus, we have demonstrated, for the first time, the presence of PBR and StAR in the pregnant rat CL and that the coordinated suppression of these proteins involved in the mitochondrial cholesterol transport along with P450 scc by GnRH-Ag leads to reduced ovarian steroidogenesis.

scholarly journals De novo mutations in TOMM70, a receptor of the mitochondrial import translocase, cause neurological impairment

2020 ◽  
Vol 29 (9) ◽  
pp. 1568-1579 ◽  
Author(s):  
Debdeep Dutta ◽  
Lauren C Briere ◽  
Oguz Kanca ◽  
Paul C Marcogliese ◽  
Melissa A Walker ◽  
...  
Keyword(s):  
White Matter ◽  
Protein Import ◽  
De Novo ◽  
Mitochondrial Protein ◽  
Neurological Impairment ◽  
Loss Of Function ◽  
De Novo Mutations ◽  
Coding Region ◽  
Mitochondrial Protein Import ◽  
Mitochondrial Proteome

Abstract The translocase of outer mitochondrial membrane (TOMM) complex is the entry gate for virtually all mitochondrial proteins and is essential to build the mitochondrial proteome. TOMM70 is a receptor that assists mainly in mitochondrial protein import. Here, we report two individuals with de novo variants in the C-terminal region of TOMM70. While both individuals exhibited shared symptoms including hypotonia, hyper-reflexia, ataxia, dystonia and significant white matter abnormalities, there were differences between the two individuals, most prominently the age of symptom onset. Both individuals were undiagnosed despite extensive genetics workups. Individual 1 was found to have a p.Thr607Ile variant while Individual 2 was found to have a p.Ile554Phe variant in TOMM70. To functionally assess both TOMM70 variants, we replaced the Drosophila Tom70 coding region with a Kozak-mini-GAL4 transgene using CRISPR-Cas9. Homozygous mutant animals die as pupae, but lethality is rescued by the mini-GAL4-driven expression of human UAS-TOMM70 cDNA. Both modeled variants lead to significantly less rescue indicating that they are loss-of-function alleles. Similarly, RNAi-mediated knockdown of Tom70 in the developing eye causes roughening and synaptic transmission defect, common findings in neurodegenerative and mitochondrial disorders. These phenotypes were rescued by the reference, but not the variants, of TOMM70. Altogether, our data indicate that de novo loss-of-function variants in TOMM70 result in variable white matter disease and neurological phenotypes in affected individuals.

scholarly journals Post-Transcriptional Control of Coenzyme Q Biosynthesis Revealed by Transomic Analysis of the RNA-Binding Protein Puf3p

2017 ◽  
Author(s):  
Christopher P. Lapointe ◽  
Jonathan A. Stefely ◽  
Adam Jochem ◽  
Paul D. Hutchins ◽  
Gary M. Wilson ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Protein Import ◽  
Rna Binding ◽  
Protein Complexes ◽  
Binding Protein ◽  
Coenzyme Q ◽  
Mitochondrial Protein ◽  
Rna Binding Protein ◽  
List Type

SUMMARYCoenzyme Q (CoQ) is a redox active lipid required for mitochondrial oxidative phosphorylation (OxPhos). How CoQ biosynthesis is coordinated with the biogenesis of OxPhos protein complexes is unclear. Here, we show that the Saccharomyces cerevisiae RNA-binding protein (RBP) Puf3p directly regulates CoQ biosynthesis. To establish the mechanism for this regulation, we employed a transomic strategy to identify mRNAs that not only bind Puf3p, but also are regulated by Puf3p in vivo. The CoQ biosynthesis enzyme Coq5p is a critical Put3p target: Puf3p regulates the level of Coq5p and prevents its toxicity, thereby enabling efficient CoQ production. In parallel, Puf3p represses a specific set of proteins involved in mitochondrial protein import, translation, and OxPhos complex assembly — pathways essential to prime mitochondrial biogenesis. Our data reveal a mechanism for post-transcriptionally coordinating CoQ production with OxPhos biogenesis and, more broadly, demonstrate the power of transomics for defining genuine targets of RBPs.HIGHLIGHTSThe RNA binding protein (RBP) Puf3p regulates coenzyme Q (CoQ) biosynthesisTransomic analysis of RNAs, proteins, lipids, and metabolites defines RBP targetsPuf3p regulates the potentially toxic CoQ biosynthesis enzyme Coq5pPuf3p couples regulation of CoQ with a broader program for controlling mitochondria

scholarly journals Lipoid congenital adrenal hyperplasia due to STAR mutations in a Caucasian patient

2016 ◽  
Vol 2016 ◽  
Author(s):  
Jasmeet Kaur ◽  
Luis Casas ◽  
Himangshu S Bose
Keyword(s):  
Congenital Adrenal Hyperplasia ◽  
De Novo ◽  
Medical Center ◽  
Regulatory Protein ◽  
Background Level ◽  
Severe Form ◽  
List Type ◽  
Adrenal Hyperplasia ◽  
University Of North Dakota ◽  
Finger Printing

Summary Lipoid congenital adrenal hyperplasia (lipoid CAH), the most severe form of CAH, is most commonly caused by mutations in steroidogenic acute regulatory protein (STAR), which is required for the movement of cholesterol from the outer to the inner mitochondrial membranes to synthesize pregnenolone. This study was performed to evaluate whether the salt-losing crisis and the adrenal inactivity experienced by a Scandinavian infant is due to a de novo STAR mutation. The study was conducted at the University of North Dakota, the Mercer University School of Medicine and the Memorial University Medical Center to identify the cause of this disease. The patient was admitted to a pediatric endocrinologist at the Sanford Health Center for salt-losing crisis and possible adrenal failure. Lipoid CAH is an autosomal recessive disease, we identified two de novo heterozygous mutations (STAR c.444C>A (STAR p.N148K) and STAR c.557C>T (STAR p.R193X)) in the STAR gene, causing lipoid CAH. New onset lipoid CAH can occur through de novo mutations and is not restricted to any specific region of the world. This Scandinavian family was of Norwegian descent and had lipoid CAH due to a mutation in S TAR exons 4 and 5. Overexpression of the STAR p.N148K mutant in nonsteroidogenic COS-1 cells supplemented with an electron transport system showed activity similar to the background level, which was ∼10% of that observed with wild-type (WT) STAR. Protein-folding analysis showed that the finger printing of the STAR p.N148K mutant is also different from the WT protein. Inherited STAR mutations may be more prevalent in some geographical areas but not necessarily restricted to those regions. Learning points STAR mutations cause lipoid CAH. This is a pure population from a caucasian family. Mutation ablated STAR activity. The mutation resulted in loosely folded conformation of STAR.

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