AKR1C3 (type 5 17ß-hydroxysteroid dehydrogenase/prostaglandin F synthase): Roles in malignancy and endocrine disorders.

Aldo-Keto-Reductase 1C3 (type 5 17ß-hydroxysteroid dehydrogenase (HSD)/prostaglandin (PG) F2α synthase) is the only 17ß-HSD that is not a short-chain dehydrogenase/reductase. By acting as a 17-ketosteroid reductase, AKR1C3 produces potent androgens in peripheral tissues which activate the androgen receptor (AR) or act as substrates for aromatase. AKR1C3 is implicated in the production of androgens in castration-resistant prostate cancer (CRPC) and polycystic ovarian syndrome; and is implicated in the production of aromatase substrates in breast cancer. By acting as an 11-ketoprostaglandin reductase, AKR1C3 generates 11ß-PGF2α to activate the FP receptor and deprives peroxisome proliferator activator receptorγ of its putative PGJ2 ligands. These growth stimulatory signals implicate AKR1C3 in non-hormonal dependent malignancies e.g. acute myeloid leukemia (AML). AKR1C3 moonlights by acting as a co-activator of the AR and stabilizes ubiquitin ligases. AKR1C3 inhibitors have been used clinically for CRPC and AML and can be used to probe its pluripotency.

The impact of the clinical CYP11B2 mutation V386A strongly depends on the enzyme's genetic background.

Human cytochrome P450 11B2 (CYP11B2) is an essential enzyme in the steroid hormone biosynthesis, which catalyzes the last three reaction steps of the aldosterone synthesis. These reactions comprise a hydroxylation at position C11 of the steroid intermediate deoxycorticosterone yielding corticosterone, followed by a hydroxylation at position C18 yielding 18-hydroxy-corticosterone and a subsequent oxidation of the hydroxyl group at C18, which results in the formation of aldosterone. Alterations in the amino acid sequence of CYP11B2 often cause severe disease patterns. We previously described a procedure for expression and purification of human CYP11B2 employing recombinant E. coli, which allows the rapid characterization of CYP11B2 mutants on a molecular level. This system was now utilized for the examination of the influence of the polymorphism at position 173 in combination with the mutation V386A on the activity of CYP11B2. Our in vitro findings show that the combination of the V386A mutation with the variant CYP11B2 173Arg only slightly reduces the 18-hydroxylase and 18-oxidase activity, whereas the V386A mutation with the CYP11B2 173Lys variant almost abolishes the 18-hydroxylation and 18-oxidation. In both cases the 11-hydroxylase activity is not affected. These findings highlight the importance of the genetic background of an enzyme when regarding the effect of clinical mutations.

The proprotein convertases, 20 years later.

The proprotein convertases (PCs) are secretory mammalian serine proteinases related to bacterial subtilisin-like enzymes. The family of PCs comprises nine members, PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, SKI-1/S1P, and PCSK9 (Fig. 3.1). While the first seven PCs cleave after single or paired basic residues, the last two cleave at non-basic residues and the last one PCSK9 only cleaves one substrate, itself, for its activation. The targets and substrates of these convertases are very varied covering many aspects of cellular biology and communication. While it took more than 22 years to begin to identify the first member in 1989-1990, in less than 14 years they were all characterized. So where are we 20 years later in 2011? We have now reached a level of maturity needed to begin to unravel the mechanisms behind the complex physiological functions of these PCs both in health and disease states. We are still far away from comprehensively understanding the various ramifications of their roles and to identify their physiological substrates unequivocally. How do these enzymes function in vivo? Are there other partners to be identified that would modulate their activity and/or cellular localization? Would non-toxic inhibitors/silencers of some PCs provide alternative therapies to control some pathologies and improve human health? Are there human SNPs or mutations in these PCs that correlate with disease, and can these help define the finesses of their functions and/or cellular sorting? The more we know about a given field, the more questions will arise, until we are convinced that we have cornered the important angles. And yet the future may well reserve for us many surprises that may allow new leaps in our understanding of the fascinating biology of these phylogenetically ancient eukaryotic proteases (Fig. 3.2) implicated in health and disease, which traffic through the cells via multiple sorting pathways (Fig. 3.3).

Inhibitor screening of proprotein convertases using positional scanning libraries.

Proprotein convertases represent an important class of biosynthetic enzymes that are increasingly viewed as targets for therapeutic approaches to infection, cancer, and potentially endocrine disorders. The identification of potent inhibitors can be accomplished by screening synthetic combinatorial libraries containing thousands of small molecules to millions of peptides. In this chapter, the screening of positional scanning libraries is described for the identification of PC1/3 and PC2 inhibitors.

Aspartyl, arginyl and alanyl aminopeptidase activities in the hippocampus and hypothalamus of streptozotocin-induced diabetic rats.

Acid (aspartyl), basic (arginyl) and neutral (alanyl) aminopeptidases degrade angiotensins, vasopressin, oxytocin, bradykinin and enkephalins. These peptides regulate memory, energy homeostasis, water-salt balance and blood pressure, functions that are mainly exerted in the hippocampus and hypothalamus, and that can be affected by diabetes mellitus. To evaluate the relationship between the diabetes mellitus and processing and inactivation roles of these representative aminopeptidases, we measured their activities in both brain structures of control and streptozotocin-diabetic rats. Hypothalamic soluble aspartyl and arginyl aminopeptidases presented significant decreased activity levels in diabetic rats, which were mitigated by insulin therapy. In addition to membrane-bound puromycin sensitive and insensitive alanyl aminopeptidases, its soluble puromycin sensitive form did not differ between diabetic and control rats in both brain structures. Glucose and/or insulin did not seem to alter in vitro the hypothalamic activities of soluble aspartyl and arginyl aminopeptidases. The implied hypothalamic control of regulatory peptide activity by aspartyl and arginyl aminopeptidases supports the hypothesis that the hydrolytic ability of these enzyme types could be a common link for the disruptions of water-salt balance, blood pressure and energy homeostasis in diabetes mellitus.

17beta-hydroxysteroid dehydrogenase-3 deficiency: a rare endocrine cause of male-to-female sex reversal.

Deficiency of 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3), due to mutations in the gene encoding the enzyme, results in a rare autosomal recessive form of male-to-female sex reversal. Mutated genes encode an abnormal enzyme with absent or reduced ability to convert Delta4-androstenedione to testosterone in the testis. Affected individuals are genetically males who developed internal male Wolffian structures but female external genitalia. Such individuals are usually raised as females and diagnosis is made at puberty, when they show virilization. Correct diagnosis is mandatory to optimize treatment and follow-up. In the present paper we report the clinical history, endocrine evaluation and molecular genetics of a prepubertal girl affected by 17beta-HSD3 deficiency, in whom an erroneous diagnosis of androgen insensitivity syndrome was made. The clinical, endocrine and genetic features of 17beta-HSD3 deficiency are also reviewed.

[Leptin--a new hormone in endocrinology]. / Leptin--novyi gormon v éndokrinologii.

Research of recent years has fundamentally revised modern endocrinology. Many organs and tissue that have never before been treated as endocrinal or involved in production of various hormones, became such. In particular, adipose tissue secreting to blood an important hormone--leptin--became the study object of particular interest.

Minireview: 11beta-hydroxysteroid dehydrogenase type 1- a tissue-specific amplifier of glucocorticoid action.

11beta-hydroxysteroid dehydrogenases (11beta-HSDs) catalyze the interconversion of active glucocorticoids (cortisol, corticosterone) and inert 11-keto forms (cortisone, 11-dehydrocorticosterone). 11beta-HSD type 2 has a well recognized function as a potent dehydrogenase that rapidly inactivates glucocorticoids, thus allowing aldosterone selective access to otherwise nonselective mineralocorticoid receptors in the distal nephron. In contrast, the function of 11beta-HSD type 1 has, until recently, been little understood. 11beta-HSD1 is an ostensibly reversible oxidoreductase in vitro, which is expressed in liver, adipose tissue, brain, lung, and other glucocorticoid target tissues. However, increasing data suggest that 11beta-HSD1 acts as a predominant 11beta-reductase in many intact cells, whole organs, and in vivo. This reaction direction locally regenerates active glucocorticoids within expressing cells, exploiting the substantial circulating levels of inert 11-keto steroids. While the biochemical determinants of the reaction direction are not fully understood, insights to its biological importance have been afforded by use of inhibitors in vivo, including in humans, and the generation of knockout mice. Such studies suggest 11beta-HSD1 effectively amplifies glucocorticoid action at least in the liver, adipose tissue, and the brain. Inhibition of 11beta-HSD1 represents a potential target for therapy of disorders that might be ameliorated by local reduction of glucocorticoid action, including type 2 diabetes, obesity, and age-related cognitive dysfunction.

Receptores de membrana y sus mecanismos enzimáticos de acción: patologías endocrinas asociadas / Membrane receptors, their enzymatic mechanisms of action and associated endocrine diseases

The cell, as a functional unit of a living individual, has the capacity to recognize signals from the extracellular compartment and to respond to these signals in a specific, precise and characteristic way. This review analyzes some membrane receptor mediated processes, characterized by a complex chain of events from the external signal to the induction of specific genes. Additionally, some endocrine diseases associated with molecular defects in some of these stages are analyzed

[Membrane receptors and their enzymatic mechanisms of action. Associated endocrine diseases]. / Receptores de membrana y sus mecanismos enzimáticos de acción. Patologías endocrinas asociadas.

The cell, as a functional unit of a living individual, has the capacity to recognize signals from the extracellular compartment and to respond to these signals in a specific, precise and characteristic way. This review analyzes some membrane receptor mediated processes, characterized by a complex chain of events from the external signal to the induction of specific genes. Additionally, some endocrine diseases associated with molecular defects in some of these stages are analyzed.

[Glutathione peroxidases: value of their determination in clinical biology]. / Les glutathion peroxydases: intérêt de leur dosage en biologie clinique.

The role of glutathione peroxidase in the oxidative metabolism and recent advances in the demonstration of the consequences of the desequilibrium in the proxidant/antioxidant balance on biological molecules oxidation, intracellular signals transduction, apoptosis and necrosis, have led to new approach in the knowledge of many pathological processes. Methods for determining antioxidant capacity have been developed. The measurement of glutathione peroxidase activity is a key step in the study of oxidative stress. Its determination in clinical biology needs optimal conditions for standardised assays which will be used for epidemiological studies aimed to evaluate the role of nutritional factors involved in the pathogeny of diseases caused or accompanied by oxidative stress.

Endocrine disorders associated with inappropriately high aromatase expression.

Aromatase P450 (P450arom) is responsible for conversion of C19 steroids to estrogens in a number of human tissues, such as the placenta, gonads, adipose tissue, skin and the brain. Aromatase expression in human tissues is regulated by use of alternative promoters in the placenta (promoter I.1), adipose tissue (promoters I.4, I.3 and II) and gonads (promoter II). Aromatase expression is absent in the disease-free adult liver, adrenal and uterine tissues. Excessive or inappropriate aromatase expression in adipose fibroblasts and endometriosis-derived stromal cells, as well as in testicular, hepatic, adrenal and uterine tumors, is associated with abnormally high circulating estrogen levels and/or with increased local estrogen concentrations in these tissues. Whether systemically delivered or locally produced, elevated estrogen levels will in turn promote the growth of hormone-responsive tissues. We recently studied aromatase expression in testicular tumor and adipose tissue samples from prepubertal boys with gynecomastia, in hepatocellular cancer and adrenocortical tumor samples from adult men with gynecomastia, in breast adipose tissue samples proximal to breast tumors, and in endometrial cancer, leiomyoma and endometriosis tissues. Excessive aromatase activity and P450arom transcript levels were found in these tissue samples or in cultured cells derived from these tissues. In these neoplastic or non-neoplastic tissues or cells, the regulation of aromatase expression was studied in terms of alternative promoter use, both in vivo and in response to various hormonal stimuli. Our results were suggestive of a common metabolic abnormality associated with activation of a cyclic AMP-dependent signalling pathway that gives rise to transcriptional transactivation of aromatase expression via promoters I.3 and II in all of the above tissues. This article describes the common pathophysiological and molecular features of excessive aromatase expression in these disease states.

Carboxypeptidase E is a regulated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in Cpe(fat) mice.

A proposed mechanism for sorting secretory proteins into granules for release via the regulated secretory pathway in endocrine-neuroendocrine cells involves binding the proteins to a sorting receptor at the trans-Golgi network, followed by budding and granule formation. We have identified such a sorting receptor as membrane-associated carboxypeptidase E (CPE) in pituitary Golgi-enriched and secretory granule membranes. CPE specifically bound regulated secretory pathway proteins, including prohormones, but not constitutively secreted proteins. We show that in the Cpe(fat) mutant mouse lacking CPE, the pituitary prohormone, pro-opiomelanocortin, was missorted to the constitutive pathway and secreted in an unregulated manner. Thus, obliteration of CPE, the sorting receptor, leads to multiple endocrine disorders in these genetically defective mice, including hyperproinsulinemia and infertility.

Growth failure, encephalopathy, and endocrine dysfunctions in two siblings, one with 5-oxoprolinase deficiency.

UNLABELLED: Two female siblings, born to consanguineous parents, presented with a similar phenotype characterized by severe growth and developmental failure, dysmorphic features, thyroid and gonadal dysfunction, autistic traits and hand stereotypes resembling Rett syndrome. In the elder patient, analysis of urinary organic acids disclosed a very high excretion of 5-oxoproline (4.2 to 8.1 mol/mol creatinine) and enzyme assays of leucocyte extracts revealed a profound deficiency of 5-oxoprolinase. However, normal urinary organic acid profiles were found in the younger child. In view of their distinct dysmorphic features and severe growth deficiency, these siblings cannot be considered as Rett Syndrome variants. The Dubowitz and carbohydrate-deficient glycoprotein syndromes were also excluded clinically and biochemically respectively. We conclude that these patients suffer from a hitherto undescribed autosomal recessive disorder, unrelated to the 5-oxoprolinase deficiency of the elder sib. CONCLUSION: The present findings give evidence that 5-oxoprolinase deficiency is not associated with a distinct morbid phenotype.

Structure-function relationships and molecular genetics of the 3 beta-hydroxysteroid dehydrogenase gene family.

The isoenzymes of the 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene-isomerase (3 beta-HSD) gene family catalyse the transformation of all 5-ene-3 beta-hydroxysteroids into the corresponding 4-ene-3-keto-steroids and are responsible for the interconversion of 3 beta-hydroxy- and 3-keto-5 alpha-androstane steroids. The two human 3 beta-HSD genes and the three related pseudogenes are located on the chromosome 1p13.1 region, close to the centromeric marker D1Z5. The 3 beta-HSD isoenzymes prefer NAD+ to NADP+ as cofactor with the exception of the rat liver type III and mouse kidney type IV, which both prefer NADPH as cofactor for their specific 3-ketosteroid reductase activity due to the presence of Tyr36 in the rat type III and of Phe36 in mouse type IV enzymes instead of Asp36 found in other 3 beta-HSD isoenzymes. The rat types I and IV, bovine and guinea pig 3 beta-HSD proteins possess an intrinsic 17 beta-HSD activity specific to 5 alpha-androstane 17 beta-ol steroids, thus suggesting that such "secondary" activity is specifically responsible for controlling the bioavailability of the active androgen DHT. To elucidate the molecular basis of classical form of 3 beta-HSD deficiency, the structures of the types I and II 3 beta-HSD genes in 12 male pseudohermaphrodite 3 beta-HSD deficient patients as well as in four female patients were analyzed. The 14 different point mutations characterized were all detected in the type II 3 beta-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3 beta-HSD gene predominantly expressed in the placenta and peripheral tissues. The mutant type II 3 beta-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact transfected cells, at the exception of L108W and P186L proteins, which have some residual activity (approximately 1%). Mutations found in nonsalt-loser patients have some residual activity ranging from approximately 1 to approximately 10% compared to the wild-type enzyme. Characterization of mutant proteins provides unique information on the structure-function relationships of the 3 beta-HSD superfamily.

[Diagnostic value of glutamate dehydrogenase determination in the dog]. / Diagnostischer Wert der Glutamat-dehydrogenase-Bestimmung beim Hund.

Overproportional GLDH-increase was found to be the most frequently appearing pathological enzyme pattern in canine practice. Thus it could be shown that GLDH deviates, in spite of its mitochondrial localization and greater molecular weight, more frequently and to a higher degree from its reference value than the parameters ALT, AST, AP, GGT and Bilirubin. The results of the study suggest that the liberation of the enzyme is less determined by the intensity than by the intralobular target of the liver insult. Therefore an increase in GLDH-activity should no longer be interpreted as the result of severe liver damage. On the contrary, the enzyme appeared to be the most sensitive indicator for the diagnosis of primary and secondary hepatopathies. The phenomenon of isolated GLDH-increase could be interpreted in almost every disease group as an appearance of the over-proportional increase and can therefore be understood as a serological expression of a slight, perivenous liver affection. Only with effusion patients the enzyme pattern should be regarded as an independent finding, because it has extrahepatic reasons. The induction of the enzyme in cases of diabetes mellitus is discussed.

Twin genes and endocrine disease: CYP21 and CYP11B genes.

CYP21 and CYP11B genes have a common feature: they are "twin" genes. It seems as if doubling and subsequent drifting apart rendered these genes particularly sensitive to defect acquisition by mechanisms involving recombinatorial events. This creates specific difficulties in the molecular diagnosis of defects.

Small adrenocortical tumors without apparent clinical endocrine abnormalities. Immunolocalization of steroidogenic enzymes.

Immunohistochemical analysis of steroidogenic enzymes (P-450 side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase, P-450 C21-hydroxylase, P-450 17 alpha-hydroxylase and P-450 11 beta-hydroxylase) was performed on fifteen cases of small adrenocortical adenomas, which were detected incidentally in hormonally asymptomatic patients (non-functioning adrenocortical tumor), in order to study steroidogenesis in these tumors. Immunolocalization revealed that all cases examined expressed all the enzymes in the adrenocortical steroidogenic pathway to various degrees, and in twelve cases abnormalities of precursor hormones and steroid metabolites were clinically observed. Attached non-neoplastic adrenals were present in twelve cases. Among these twelve cases, eight showed cortical atrophy, especially in the zona fasciculata. These atrophied adrenals expressed little immunoreactivity of the enzymes examined. These results strongly indicate that most of small non-functioning adrenocortical tumors have the capacity to produce biologically active steroids including cortisol, although not necessarily associated with hypercorticism. Especially in the cases with cortical atrophy in attached non-neoplastic adrenals, it is considered that autonomous neoplastic production and secretion of cortisol may be insufficient to cause clinical and routine laboratory abnormalities but sufficient to subtly alter the hypothalamic-pituitary-adrenal axis by suppressing ACTH and/or CRF secretion and to result in adrenocortical atrophy. It should be recognized that corticosteroidogenesis does take place in the majority of clinically small non-functioning adrenocortical tumors incidentally detected in hormonally asymptomatic patients when managing these patients.