Comparison of predicted and experimental subcellular localization of two putative rat steroid dehydrogenases from the short-chain dehydrogenase/reductase protein superfamily.

In the characterization of newly identified proteins, subcellular localization studies can provide important hints to the proteins' metabolic functions. Depending on the biochemical task of an enzyme, certain subcellular environmental conditions as pH or availability of cofactors and substrates have to be fulfilled. Consequently, misdirected proteins often cannot conduct the proper chemical reaction. This study is aimed at detecting differences in bioinformatic and wet lab experiments and presenting ways for reliable analysis of subcellular localization. On a set of ten enzymes from the short-chain dehydrogenase/reductase (SDR) superfamily, we have performed predictions and experimental analyses of subcellular localization. Exemplarily, we show the localization studies on rat short-chain dehydrogenases/reductases dhrs7b and dhrs8. We demonstrate in particular that all of the prediction algorithms tested failed to assign the SDR enzymes to an experimentally verified subcellular compartment.

Role of short-chain hydroxyacyl CoA dehydrogenases in SCHAD deficiency.

Short-chain hydroxyacyl CoA dehydrogenase deficiency is an ill-defined, severe pediatric disorder of mitochondrial fatty acid beta-oxidation of short-chain hydroxyacyl CoAs. To understand the relative contributions of the two known short-chain hydroxyacyl CoA dehydrogenases (HADH) tissue biopsies of six distinct family individuals were analyzed and kinetic parameters were compared. Steady-state kinetic constants for HADH 1 and HADH 2 suggest that type 1 is the major enzyme involved in mitochondrial beta-oxidation of short-chain hydroxyacyl-CoAs. Two patients are heterozygous carriers of a HADH 1 polymorphism, whereas no mutation is detected in the HADH 2 gene of all patients. The data suggest that protein interactions rather than HADH mutations are responsible for the disease phenotype. Pull-down experiments of recombinant HADH 1 and 2 with human mitochondrial extracts reveal two proteins interacting with HADH 1, one of which was identified as glutamate dehydrogenase. This association provides a possible link between fatty acid metabolism and the hyperinsulinism/hyperammonia syndrome.

Structural and biochemical characterization of human orphan DHRS10 reveals a novel cytosolic enzyme with steroid dehydrogenase activity.

To this day, a significant proportion of the human genome remains devoid of functional characterization. In this study, we present evidence that the previously functionally uncharacterized product of the human DHRS10 gene is endowed with 17beta-HSD (17beta-hydroxysteroid dehydrogenase) activity. 17beta-HSD enzymes are primarily involved in the metabolism of steroids at the C-17 position and also of other substrates such as fatty acids, prostaglandins and xenobiotics. In vitro, DHRS10 converts NAD+ into NADH in the presence of oestradiol, testosterone and 5-androstene-3beta,17beta-diol. Furthermore, the product of oestradiol oxidation, oestrone, was identified in intact cells transfected with a construct plasmid encoding the DHRS10 protein. In situ fluorescence hybridization studies have revealed the cytoplasmic localization of DHRS10. Along with tissue expression data, this suggests a role for DHRS10 in the local inactivation of steroids in the central nervous system and placenta. The crystal structure of the DHRS10 apoenzyme exhibits secondary structure of the SDR (short-chain dehydrogenase/reductase) family: a Rossmann-fold with variable loops surrounding the active site. It also reveals a broad and deep active site cleft into which NAD+ and oestradiol can be docked in a catalytically competent orientation.

RDH12, a retinol dehydrogenase causing Leber's congenital amaurosis, is also involved in steroid metabolism.

Three retinol dehydrogenases (RDHs) were tested for steroid converting abilities: human and murine RDH 12 and human RDH13. RDH12 is involved in retinal degeneration in Leber's congenital amaurosis (LCA). We show that murine Rdh12 and human RDH13 do not reveal activity towards the checked steroids, but that human type 12 RDH reduces dihydrotestosterone to androstanediol, and is thus also involved in steroid metabolism. Furthermore, we analyzed both expression and subcellular localization of these enzymes.

Transcriptional regulation of human and murine 17beta-hydroxysteroid dehydrogenase type-7 confers its participation in cholesterol biosynthesis.

In both humans and mice, 17beta-hydroxysteroid dehydrogenase type-7 (HSD17B7) was described as possessing dual enzymatic functionality. The enzyme was first shown to be able to convert estrone to estradiol in vitro. Later involvement of this enzyme in postsqualene cholesterol biosynthesis was postulated (conversion of zymosterone to zymosterol) and could be proven in vitro. In this work, we performed a detailed analysis of the transcriptional regulation of both the human and murine genes. Despite relatively low sequence similarity, both promoters contain similar contexts of transcription factor-binding sites. The participation of these sites in transcriptional regulation of HSD17B7 was proven by electro-mobility shift assay and site-directed mutagenesis of the corresponding binding sites. We describe novel involvement of vitamin D receptor/retinoid X receptor and provide new information on the regulation of HSD17B7 expression by sterol regulatory element-binding protein and hepatocyte nuclear factor 4, the latter known from other genes of cholesterogenic enzymes. The results of our study provide unequivocal evidence for a role of HSD17B7 in cholesterol biosynthesis.

Speech prosody, voice quality and personality.

The 'individuation' of oral language is still largely an unknown territory, especially with respect to the individual use of speech prosody (rhythm, intonation and intensity). An initial study (Zellner Keller, 2004) has suggested an interesting relationship between speakers' prosodic styles and their personality profiles, as perceived by listeners. In this study, it is shown for 34 male speakers that distributions of F0 (for the intonation) and dB (for the intensity) give interesting information. F0 distributions among speakers are not similar, whether the speech task is the same or not, but intensity distributions are nearly perfectly superposed on each other. Individual intonational styles in relationship with personality styles are then examined in two tasks: reading texts and spontaneous speech.

Mindfulness-Based Cognitive Approach for Seniors (MBCAS): Program Development and Implementation.

A number of cognitive interventions have been developed to enhance cognitive functioning in the growing population of the elderly. We describe the Mindfulness-Based Cognitive Approach for Seniors (MBCAS), a new training program designed especially for seniors. It was conceived in the context of self-development for seniors who wish to enhance their relationship with their inner and outer selves in order to navigate their aging process more easily and fluently. Physical and psychosocial problems related to aging, as well as some temporal issues, were taken into account in developing this program. Unlike clinically oriented mindfulness-based programs, which are generally delivered during an 8-week period, the MBCAS training program is presented over a period of 8 months. The main objectives of this program are to teach seniors to observe current experiences with nonjudgmental awareness, to identify automatic behaviors or reactions to current experiences that are potentially nonadaptive, and to enhance and reinforce positive coping with typical difficulties that they face in their daily lives. Details of the program development and initial implementation are presented, with suggestions for evaluating the program's effectiveness.