RESUMO
Kupffer cells, the liver tissue resident macrophages, are critical in the detection and clearance of cancer cells. However, the molecular mechanisms underlying their detection and phagocytosis of cancer cells are still unclear. Using in vivo genome-wide CRISPR-Cas9 knockout screening, we found that the cell-surface transmembrane protein ERMAP expressed on various cancer cells signaled to activate phagocytosis in Kupffer cells and to control of liver metastasis. ERMAP interacted with ß-galactoside binding lectin galectin-9 expressed on the surface of Kupffer cells in a manner dependent on glycosylation. Galectin-9 formed a bridging complex with ERMAP and the transmembrane receptor dectin-2, expressed on Kupffer cells, to induce the detection and phagocytosis of cancer cells by Kupffer cells. Patients with low expression of ERMAP on tumors had more liver metastases. Thus, our study identified the ERMAP-galectin-9-dectin-2 axis as an 'eat me' signal for Kupffer cells.
Assuntos
Citofagocitose , Células de Kupffer , Humanos , Fagocitose/genética , Galectinas/genética , Galectinas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
Type 10 17ß-hydroxysteroid dehydrogenase (17ß-HSD10), a homo-tetrameric multifunctional protein with 1044 residues encoded by the HSD17B10 gene, is necessary for brain cognitive function. Missense mutations result in infantile neurodegeneration, an inborn error in isoleucine metabolism. A 5-methylcytosine hotspot underlying a 388-T transition leads to the HSD10 (p.R130C) mutant to be responsible for approximately half of all cases suffering with this mitochondrial disease. Fewer females suffer with this disease due to X-inactivation. The binding capability of this dehydrogenase to Aß-peptide may play a role in Alzheimer's disease, but it appears unrelated to infantile neurodegeneration. Research on this enzyme was complicated by reports of a purported Aß-peptide-binding alcohol dehydrogenase (ABAD), formerly referred to as endoplasmic-reticulum-associated Aß-binding protein (ERAB). Reports concerning both ABAD and ERAB in the literature reflect features inconsistent with the known functions of 17ß-HSD10. It is clarified here that ERAB is reportedly a longer subunit of 17ß-HSD10 (262 residues). 17ß-HSD10 exhibits L-3-hydroxyacyl-CoA dehydrogenase activity and is thus also referred to in the literature as short-chain 3-hydorxyacyl-CoA dehydrogenase or type II 3-hydorxyacyl-CoA dehydrogenase. However, 17ß-HSD10 is not involved in ketone body metabolism, as reported in the literature for ABAD. Reports in the literature referring to ABAD (i.e., 17ß-HSD10) as a generalized alcohol dehydrogenase, relying on data underlying ABAD's activities, were found to be unreproducible. Furthermore, the rediscovery of ABAD/ERAB's mitochondrial localization did not cite any published research on 17ß-HSD10. Clarification of the purported ABAD/ERAB function derived from these reports on ABAD/ERAB may invigorate this research field and encourage new approaches to the understanding and treatment of HSD17B10-gene-related disorders. We establish here that infantile neurodegeneration is caused by mutants of 17ß-HSD10 but not ABAD, and so we conclude that ABAD represents a misnomer employed in high-impact journals.
Assuntos
3-Hidroxiacil-CoA Desidrogenases , Álcool Desidrogenase , Doença de Alzheimer , Humanos , Álcool Desidrogenase/genética , Doença de Alzheimer/genética , Mutação de Sentido IncorretoRESUMO
Type 10 17ß-hydroxysteroid dehydrogenase (17ß-HSD10) is the HSD17B10 gene product playing an appreciable role in cognitive functions. It is the main hub of exercise-upregulated mitochondrial proteins and is involved in a variety of metabolic pathways including neurosteroid metabolism to regulate allopregnanolone homeostasis. Deacetylation of 17ß-HSD10 by sirtuins helps regulate its catalytic activities. 17ß-HSD10 may also play a critical role in the control of mitochondrial structure, morphology and dynamics by acting as a member of the Parkin/PINK1 pathway, and by binding to cyclophilin D to open mitochondrial permeability pore. 17ß-HSD10 also serves as a component of RNase P necessary for mitochondrial tRNA maturation. This dehydrogenase can bind with the Aß peptide thereby enhancing neurotoxicity to brain cells. Even in the absence of Aß, its quantitative and qualitative variations can result in neurodegeneration. Since elevated levels of 17ß-HSD10 were found in brain cells of Alzheimer's disease (AD) patients and mouse AD models, it is considered to be a key factor in AD pathogenesis. Since data underlying Aß-binding-alcohol dehydrogenase (ABAD) were not secured from reported experiments, ABAD appears to be a fabricated alternative term for the HSD17B10 gene product. Results of this study would encourage researchers to solve the question why elevated levels of 17ß-HSD10 are present in brains of AD patients and mouse AD models. Searching specific inhibitors of 17ß-HSD10 may find candidates to reduce senile neurodegeneration and open new approaches for the treatment of AD.
Assuntos
17-Hidroxiesteroide Desidrogenases , Doença de Alzheimer , Animais , Humanos , Camundongos , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , Álcool Desidrogenase/metabolismo , Doença de Alzheimer/metabolismo , Encéfalo/metabolismoRESUMO
MicroRNA (miRNA) is a posttranscriptional downregulator that plays a vital role in a wide variety of biological processes. In this study, we constructed five ovarian and testicular small RNA libraries using two somatic libraries as reference controls for the identification of sex-biased miRNAs and gonadal differentially expressed miRNAs (DEMs) of the Chinese mitten crab, Eriocheir sinensis. A total of 535 known and 243 novel miRNAs were identified, including 312 sex-biased miRNAs and 402 gonadal DEMs. KEGG pathway analysis showed that DEM target genes were statistically enriched in MAPK, Wnt, and GnRH signaling pathway, and so on. A number of the sex-biased miRNAs target genes associated with sex determination/differentiation, such as IAG, Dsx, Dmrt1, and Fem1, while others target the genes related to gonadal development, such as P450s, Wnt, Ef1, and Tra-2c. Dual-luciferase reporter assay in vitro further confirmed that miR-34 and let-7b can downregulate IAG expression, miR-9-5p, let-7d, let-7b, and miR-8915 can downregulate Dsx. Taken together, these data strongly suggest a potential role for the sex-biased miRNAs in sex determination/differentiation and gonadal development in the crab.
Assuntos
Braquiúros/genética , Regulação da Expressão Gênica , Gônadas/metabolismo , MicroRNAs/genética , Transcriptoma , Animais , Braquiúros/metabolismo , Feminino , Perfilação da Expressão Gênica , Gônadas/crescimento & desenvolvimento , Masculino , MicroRNAs/metabolismo , Caracteres Sexuais , Transdução de Sinais/genéticaRESUMO
Nuclear protein of the testis midline carcinoma (NMC) is a rare malignant tumor that is mostly located in the upper trachea,mediastinal midline,and paravertebral midline,and few literature has described the imaging features of NMC in the nasal cavity and paranasal sinuses. In this article we summarize the clinical,radiologic,and pathologic data of one case of pathologically confirmed NMC in the nasal cavity and paranasal sinus by focusing on its CT and magnetic resonance imaging features.
Assuntos
Neoplasias Nasais/diagnóstico por imagem , Neoplasias dos Seios Paranasais/diagnóstico por imagem , Humanos , Imageamento por Ressonância Magnética , Cavidade Nasal/patologia , Proteínas Nucleares , Seios Paranasais/patologia , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Hydroxysteroid (17beta) dehydrogenase X (HSD10) is a multifunctional protein encoded by the HSD17B10 gene at Xp11.2. In response to stress or hypoxia-ischemia its levels increase rapidly. Expression of this gene is also elevated significantly in colonic mucosa of the inactive ulcerative colitis patients. However, accurate information about its several transcripts is still lacking, and additional evidence for its escape from X-chromosome inactivation remains to be obtained in order to help settle a debate (He XY, Dobkin C, Yang SY: Does the HSD17B10 gene escape from X-inactivation? Eur J Hum Genet 2011, 19: 123-124). RESULTS: Two major HSD17B10 transcription start sites were identified by primer extension at -37 and -6 as well as a minor start site at -12 nucleotides from the initiation codon ATG. Epigenetic analysis of the 5'-flanking region of the HSD17B10 gene showed that there was little 5-methylcytosine (< 3%) in a normal male, and that none of CpG dinucleotides in the CpG island approached 50% methylation in females. CONCLUSION: The actual length of first exon of the HSD17B10 gene was found to be about a quarter larger than that originally reported. Its transcripts result from a slippery transcription complex. The hypomethylation of the CpG island provides additional evidence for the variable escape of the HSD17B10 gene from X-chromosome inactivation which could influence the range of severity of HSD10 deficiency, an inherited error in isoleucine metabolism, in heterozygous females.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Epigênese Genética , 3-Hidroxiacil-CoA Desidrogenases/genética , 5-Metilcitosina/química , 5-Metilcitosina/metabolismo , Sequência de Bases , Cromossomos Humanos X , Ilhas de CpG , Metilação de DNA , Éxons , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sítio de Iniciação de TranscriçãoRESUMO
Gonadotropin-releasing hormone (GnRH) plays a pivotal role in reproductive regulation in vertebrates. However, GnRH was rarely isolated and its function remains poorly characterized in invertebrates. The existence of GnRH in ecdysozoa has been controversial for a long. Here, we isolated and identified two GnRH-like peptides from brain tissues in Eriocheir sinensis. Immunolocalization showed that the presence of EsGnRH-like peptide in brain, ovary and hepatopancreas. Synthetic EsGnRH-like peptides can induce germinal vesicle breakdown (GVBD) of oocyte. Similar to vertebrates, ovarian transcriptomic analysis revealed a GnRH signaling pathway in the crab, in which most genes exhibited dramatically high expression at GVBD. RNAi knockdown of EsGnRHR suppressed the expression of most genes in the pathway. Co-transfection of the expression plasmid for EsGnRHR with reporter plasmid bearing CRE-luc or SRE-luc response element into 293T cells showed that EsGnRHR transduces its signal via cAMP and Ca2+ signaling transduction pathways. In vitro incubation of the crab oocyte with EsGnRH-like peptide confirmed the cAMP-PKA cascade and Ca2+ mobilization signaling cascade but lack of a PKC cascade. Our data present the first direct evidence of the existence of GnRH-like peptides in the crab and demonstrated its conserved role in the oocyte meiotic maturation as a primitive neurohormone.
Assuntos
Braquiúros , Hormônio Liberador de Gonadotropina , Animais , Feminino , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Oócitos/metabolismo , Ovário/metabolismo , Perfilação da Expressão Gênica , Transdução de Sinais , Braquiúros/genéticaRESUMO
Mutations in the HSD17B10 gene were identified in two previously described mentally retarded males. A point mutation c.776G>C was found from a survivor (SV), whereas a potent mutation, c.419C>T, was identified in another deceased case (SF) with undetectable hydroxysteroid (17beta) dehydrogenase 10 (HSD10) activity. Protein levels of mutant HSD10(R130C) in patient SF and HSD10(E249Q) in patient SV were about half that of HSD10 in normal controls. The E249Q mutation appears to affect HSD10 subunit interactions, resulting in an allosteric regulatory enzyme. For catalyzing the oxidation of allopregnanolone by NAD+ the Hill coefficient of the mutant enzyme is approximately 1.3. HSD10(E249Q) was unable to catalyze the dehydrogenation of 2-methyl-3-hydroxybutyryl-CoA and the oxidation of allopregnanolone, a positive modulator of the gamma-aminobutyric acid type A receptor, at low substrate concentrations. Neurosteroid homeostasis is critical for normal cognitive development, and there is increasing evidence that a blockade of isoleucine catabolism alone does not commonly cause developmental disabilities. The results support the theory that an imbalance in neurosteroid metabolism could be a major cause of the neurological handicap associated with hydroxysteroid (17beta) dehydrogenase 10 deficiency.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/genética , Deficiência Intelectual/enzimologia , Deficiência Intelectual/genética , Isoleucina/metabolismo , Mutação Puntual , Esteroides/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/química , 3-Hidroxiacil-CoA Desidrogenases/deficiência , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Biocatálise , Células Cultivadas , Fibroblastos/enzimologia , Humanos , Masculino , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Vasa gene encodes a protein member of DEAD-box superfamily of ATP-dependent RNA helicases, which plays a key role in germline development in metazoans. In present study, we identified a new germline-specific marker Mrvasa in the prawn Macrobrachium rosenbergii, whose genomic DNA sequence consists of 14 exons and 13 introns. A 2516 bp of full-length Mrvasa cDNA encodes a protein of 603 amino acids. It contains nine conserved motifs, a zinc-finger motif, and RGG repeats. RT-PCR indicated that Mrvasa mRNA was specifically expressed in gonads. QPCR analysis further revealed that the expression of Mrvasa mRNA is much higher in testis than in ovary. In testis, the relative expression level of Mrvasa mRNA in late developing stage is significantly higher than that in early-middle developing stage. During ovarian development, no significant difference in expression was found. In situ hybridization demonstrated that Mrvasa mRNA was localized in germline cells including spermatogonia, spermatocytes, and spermatozoa in testes, and previtellogenic and vitellogenic oocytes in ovary. We then isolated the Mrvasa promoter and determined the transcription core region of this promoter. This is the first report on identification of vasa core promoter in crustaceans. Our results will provide a useful germline-specific marker Mrvasa for tracing germline cell formation and development in M. rosenbergii.
Assuntos
Decápodes , Palaemonidae , Sequência de Aminoácidos , Animais , Feminino , Água Doce , Masculino , Palaemonidae/genética , Palaemonidae/metabolismo , EspermatogôniasRESUMO
BACKGROUND: Mitochondrial 17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10) is necessary for brain cognitive function, but its studies were confounded by reports of Aß-peptide binding alcohol dehydrogenase (ABAD), formerly endoplasmic reticulum-associated Aß-peptide binding protein (ERAB), for two decades so long as ABAD serves as the alternative term of 17ß-HSD10. OBJECTIVE: To determine whether those ABAD reports are true or false, even if they were published in prestigious journals. METHODS: 6xHis-tagged 17ß-HSD10 was prepared and characterized by well-established experimental procedures. RESULTS: The N-terminal 6xHis tag did not significantly interfere with the dehydrogenase activities of 17ß-HSD10, but the kinetic constants of its 3-hydroxyacyl-CoA dehydrogenase activity are drastically distinct from those of ABAD, and it was not involved in ketone body metabolism as previously reported for ABAD. Furthermore, it was impossible to measure its generalized alcohol dehydrogenase activities underlying the concept of ABAD because the experimental procedures described in ABAD reports violated basic chemical and/or biochemical principles. More incredibly, both authors and journals had not yet agreed to make any corrigenda of ABAD reports. CONCLUSION: Brain 17ß-HSD10 plays a key role in neurosteroid metabolism and further studies in this area may lead to potential treatments of neurodegeneration including AD.
Assuntos
3-Hidroxiacil-CoA Desidrogenases , Doença de Alzheimer , 17-Hidroxiesteroide Desidrogenases , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Álcool Desidrogenase , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Coenzima A , HumanosRESUMO
17ß-Hydroxysteroid dehydrogenases are indispensable for downstream enzyme steps of the neurosteroidogenesis. Neurosteroids are synthesized de novo in neurons and glia from cholesterol transported into mitochondria, or by conversion from proneurosteroids, e. g. dehydroepiandrosterone (DHEA) and pregnenolone, through the same metabolic pathway as revealed in the de novo neurosteroidogenesis. Hormonal steroids generated from endocrine glands are transported into brain from the circulation to exert neuronal activity via genomic pathway, whereas neurosteroids produced in brain cells without genomic targets identified could bind to cell surface targets, e.g., GABAA or NMDA receptors and elicit antidepressant, anxiolytic, anticonvulsant and anesthetic effects by regulating neuroexcitability. In a broad sense, neurosteroids include hormonal steroids in brain, and they are irrespective of origin playing important roles in brain development including neuroprotection, neurogenesis and neural plasticity. They are also a critical element in cognitive and memory functions. Mitochondrial 17ß-HSD10, encoded by the HSD17B10 gene mapping to Xp11.2, is found in various brain regions, essential for the maintenance of neurosteroid homeostasis. Mutations identified in this gene resulted in two distinct brain diseases, namely HSD10 deficiency and MRXS10, of which clinical presentations and pathogenetic mechanisms are quite different. Since elevated levels of 17ß-HSD10 was found in brains of Alzheimer's disease patients and AD mouse model, it may also act as an adverse factor in the AD pathogenesis due to an imbalance of neurosteroid metabolism.
Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Sistema Nervoso Central/metabolismo , Neuroesteroides/metabolismo , 17-Hidroxiesteroide Desidrogenases/genética , Animais , Homeostase , Humanos , Mutação de Sentido Incorreto/genéticaRESUMO
17ß-Hydroxysteroid dehydrogenase type 10 is a multifunctional, homotetrameric, mitochondrial protein encoded by the HSD17B10 gene at Xp 11.2. This protein, 17ß-HSD10, is overexpressed in brain cells of Alzheimer's disease (AD) patients. It was reported to be involved in AD pathogenesis as the endoplasmic reticulum-associated amyloid-ß binding protein (ERAB) and as amyloid-ß binding alcohol dehydrogenase (ABAD). However, the exaggerated catalytic efficiencies for ERAB/ABAD in these reports necessitated the re-characterization of the catalytic functions of this brain enzyme. In addition to isoleucine metabolism, 17ß-HSD10 is also responsible for the mitochondrial metabolism of neurosteroids such as 5α-androstane-3α,17ß-diol and 17ß-estradiol. These neurosteroids are inactivated by the oxidation catalyzed by 17ß-HSD10. Since neurosteroid homeostasis is presumably essential for cognitive function, analysis of the impact of 17ß-HSD10 and its inhibitor, amyloid-ß peptide (Aß), on the metabolism of neuroactive steroids offers a new approach to AD pathogenesis.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/metabolismo , Mitocôndrias/enzimologia , Neurotransmissores/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/genética , Doença de Alzheimer/genética , Encéfalo/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/genética , OxirreduçãoRESUMO
Human 17beta-hydroxysteroid dehydrogenase type 10 (17beta-HSD10) is a mitochondrial enzyme encoded by the SCHAD gene, which escapes chromosome X inactivation. 17Beta-HSD10/SCHAD mutations cause a spectrum of clinical conditions, from mild mental retardation to progressive infantile neurodegeneration. 17Beta-HSD10/SCHAD is essential for the metabolism of isoleucine and branched-chain fatty acids. It can inactivate 17beta-estradiol and steroid modulators of GABA(A) receptors, and convert 5alpha-androstanediol into 5alpha-dihydrotestosterone (DHT). Certain malignant prostatic epithelial cells contain high levels of 17beta-HSD10, generating 5alpha-DHT in the absence of testosterone. 17Beta-HSD10 has an affinity for amyloid-beta peptide, and might be linked to the mitochondrial dysfunction seen in Alzheimer's disease. This versatile enzyme might provide a new drug target for neuronal excitability control and for intervention in Alzheimer's disease and certain cancers.
Assuntos
17-Hidroxiesteroide Desidrogenases/fisiologia , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/genética , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Doença de Alzheimer/enzimologia , Doença de Alzheimer/etiologia , Animais , Hormônios Esteroides Gonadais/metabolismo , Humanos , Isoleucina/metabolismo , Neurotransmissores/metabolismo , Esteroides/metabolismoRESUMO
3-Hydroxyacyl-CoA dehydrogenase (HAD) functions in mitochondrial fatty acid beta-oxidation by catalyzing the oxidation of straight chain 3-hydroxyacyl-CoAs. HAD has a preference for medium chain substrates, whereas short chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) acts on a wide spectrum of substrates, including steroids, cholic acids, and fatty acids, with a preference for short chain methyl-branched acyl-CoAs. Therefore, HAD should not be referred to as SCHAD. SCHAD is not a member of the HAD family, but instead, belongs to the short chain dehydrogenase/reductase superfamily. Previously reported cases of SCHAD deficiency are due to an inherited HAD deficiency. SCHAD, also known as 17beta-hydroxysteroid dehydrogenase type 10, is important in brain development and aging. Abnormal levels of SCHAD in certain brain regions may contribute to the pathogenesis of some neural disorders. The human SCHAD gene and its protein product, SCHAD, are potential targets for intervention in conditions, such as Alzheimer's disease, Parkinson's disease, and an X-linked mental retardation, that may arise from the impaired degradation of branched chain fatty acid and isoleucine.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Doença de Alzheimer/enzimologia , Saúde , Deficiência Intelectual/enzimologia , Doença de Parkinson/enzimologia , 3-Hidroxiacil-CoA Desidrogenases/química , 3-Hidroxiacil-CoA Desidrogenases/deficiência , Doença de Alzheimer/metabolismo , Animais , Humanos , Deficiência Intelectual/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Doença de Parkinson/metabolismoRESUMO
The steroids allopregnanolone and allotetrahydrodeoxycorticosterone (3alpha,5alpha-THDOC) are positive allosteric modulators of GABA(A) receptors, generated by the reduction of 5alpha-dihydroprogesterone (5alpha-DHP) and 5alpha-DHDOC, respectively, under the catalysis of human type 3 3alpha-hydroxysteroid dehydrogenase (HSD). However, brain enzymes catalyzing the conversion of such tetrahydrosteroids back to the corresponding 5alpha-dihydrosteroids remain to be identified. Characterization of human type 10 17beta-HSD provides a new insight into its importance for the oxidation of steroid modulators of GABA(A) receptors. The apparent catalytic efficiency (k(cat)/K(m)) of this enzyme for the oxidation of allopregnanolone and 3alpha,5alpha-THDOC are 432 and 1381 min(-1) mM(-1), respectively. This enzyme has negligible 3-ketosteroid reductase activity for 5alpha-DHP and 5alpha-DHDOC even in an acidic environment. Immunoreactivity against 17beta-HSD10 was found in a number of neuronal populations. Taken together, evidence suggests that 17beta-HSD10 is the brain enzyme capable of catalyzing the oxidation of steroid modulators of GABA(A) receptors.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , 5-alfa-Di-Hidroprogesterona/metabolismo , Ansiolíticos/metabolismo , Encéfalo/enzimologia , Desoxicorticosterona/análogos & derivados , Desoxicorticosterona/metabolismo , Receptores de GABA-A/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Ansiolíticos/química , Catálise , Desoxicorticosterona/química , Feminino , Humanos , Hidroxiesteroide Desidrogenases/metabolismo , Masculino , Pessoa de Meia-Idade , OxirreduçãoRESUMO
Allopregnanolone is a positive allosteric modulator of GABAA receptors, generated by the reduction of 5alpha-dihydroprogesterone (5alpha-DHP) in astrocytes. This neuroactive steroid can be inactivated by its 3alpha-oxidation to yield 5alpha-DHP. It was found that 5alpha-DHP levels in HEK293 cells expressing type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSD10), but not its catalytic inactive mutant, increased significantly as allopregnanolone was added to culture media. The results demonstrate that mitochondrial 17beta-HSD10 effectively catalyzes the intracellular oxidation of allopregnanolone. Moreover, brain astrocytes contain a moderate level of 17beta-HSD10, which is elevated in activated astrocytes of brains with Alzheimer type pathology, including sporadic Alzheimer's disease (AD) and Down's syndrome with AD. The distribution of 17beta-HSD10 was found not to parallel that of 3alpha-HSD3. Cerebral cortex has the lowest level of 17beta-HSD10; whereas the hippocampus, hypothalamus, and amygdala possess relatively higher levels of this enzyme. The catalysis of 17beta-HSD10 appears to be essential for maintaining normal functions of GABAergic neurons. The elevated level of 17beta-HSD10 in activated astrocytes is a new feature found in brains of people with AD, and it may have important impact on AD pathogenesis.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Encéfalo/enzimologia , Líquido Intracelular/metabolismo , Pregnanolona/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , OxirreduçãoRESUMO
Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4+8+ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class I and class II molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.
Assuntos
Apoptose/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Comunicação Celular , Complexo Principal de Histocompatibilidade/imunologia , Células Estromais/imunologia , Animais , Comunicação Celular/imunologia , Linhagem Celular , Técnicas de Cocultura , Células Dendríticas/imunologia , Proteína Ligante Fas , Glicoproteínas de Membrana/imunologia , Camundongos , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Timo/citologia , Timo/imunologia , Receptor fas/imunologiaRESUMO
A full-length cDNA of mouse type 10 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD10) was cloned from brain, representing the accurate nucleotide sequence information that rendered possible an accurate deduction of the amino acid sequence of the wild-type enzyme. A comparison of sequences and three-dimensional models of this enzyme revealed that structures previously reported by other groups carry either a truncated or mutated amino-terminal sequence. Fusion of the first 11 residues of the wild-type enzyme to the green fluorescent protein directed the reporter protein into mitochondria. Thus, the N-terminus was identified as a mitochondrial targeting signal that accounts for the intracellular localization of the mouse enzyme. This enzyme is normally associated with mitochondria, not with the endoplasmic reticulum as suggested by its trivial name 'endoplasmic reticulum-associated amyloid-beta biding protein (ERAB)'. After its C-terminal region was used to raise rabbit anti-17 betaHSD10 antibodies, immunogold electron microscopy showed that an abundance of this enzyme could be found in hippocampal synaptic mitochondria of betaAPP transgenic mice, but not in normal controls. High levels of this enzyme may disrupt steroid hormone homeostasis in synapses and contribute to synapse loss in the hippocampus of the mouse Alzheimer's disease model.
Assuntos
17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/isolamento & purificação , 3-Hidroxiacil-CoA Desidrogenases , Doença de Alzheimer/enzimologia , Sequência de Aminoácidos/genética , Hipocampo/enzimologia , Mitocôndrias/enzimologia , Terminações Pré-Sinápticas/enzimologia , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Sequência de Bases/genética , Sítios de Ligação/genética , Clonagem Molecular , DNA Complementar/isolamento & purificação , Modelos Animais de Doenças , Hipocampo/patologia , Hipocampo/ultraestrutura , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , NAD/metabolismo , Terminações Pré-Sinápticas/patologia , Terminações Pré-Sinápticas/ultraestrutura , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/genética , Transdução de Sinais/genética , Esteroides/metabolismo , Triptofano/metabolismoRESUMO
In vitro enzyme assays have demonstrated that human type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSD10) catalyzes the oxidation of 5alpha-androstane-3alpha,17beta-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17beta-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17beta-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17beta-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17beta-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3alpha-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17beta-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.
Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , 3-Hidroxiacil-CoA Desidrogenases , 3-alfa-Hidroxiesteroide Desidrogenase (B-Específica)/metabolismo , 17-Hidroxiesteroide Desidrogenases/genética , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , Di-Hidrotestosterona/metabolismo , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Oxirredução , Próstata/citologia , Próstata/enzimologia , Próstata/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
17ß-Hydroxysteroid dehydrogenase type 10 (17ß-HSD10) is encoded by the HSD17B10 gene mapping at Xp11.2. This homotetrameric mitochondrial multifunctional enzyme catalyzes the oxidation of neuroactive steroids and the degradation of isoleucine. This enzyme is capable of binding to other peptides, such as estrogen receptor α, amyloid-ß, and tRNA methyltransferase 10C. Missense mutations of the HSD17B10 gene result in 17ß-HSD10 deficiency, an infantile neurodegeneration characterized by progressive psychomotor regression and alteration of mitochondria morphology. 17ß-HSD10 exhibits only a negligible alcohol dehydrogenase activity, and is not localized in the endoplasmic reticulum or plasma membrane. Its alternate name - Aß binding alcohol dehydrogenase (ABAD) - is a misnomer predicated on the mistaken belief that this enzyme is an alcohol dehydrogenase. Misconceptions about the localization and function of 17ß-HSD10 abound. 17ß-HSD10's proven location and function must be accurately identified to properly assess this enzyme's important role in brain metabolism, especially the metabolism of allopregnanolone. The brains of individuals with Alzheimer's disease (AD) and of animals in an AD mouse model exhibit abnormally elevated levels of 17ß-HSD10. Abnormal expression, as well as mutations of the HSD17B10 gene leads to impairment of the structure, function, and dynamics of mitochondria. This may underlie the pathogenesis of the synaptic and neuronal deficiency exhibited in 17ß-HSD10 related diseases, including 17ß-HSD10 deficiency and AD. Restoration of steroid homeostasis could be achieved by the supplementation of neuroactive steroids with a proper dosing and treatment regimen or by the adjustment of 17ß-HSD10 activity to protect neurons. The discovery of this enzyme's true function has opened a new therapeutic avenue for treating AD.