Analysis of the psychrotolerant property of hormone-sensitive lipase through site-directed mutagenesis

Mammalian hormone-sensitive lipase (HSL) has given its name to a family of primarily prokaryotic proteins which are structurally related to type B carboxylesterases. In many of these alpha/beta hydrolases, a conserved HG-dipeptide flanks the catalytic pocket. In HSL this dipeptide is followed by two additional glycine residues. Through site-directed mutagenesis, we have investigated the importance of this motif for enzyme activity. Since the presence of multiple glycine residues in a critical region could contribute to cold adaptation by providing local flexibility, we studied the effect of mutating these residues on the psychrotolerant property of HSL. Any double mutation rendered the enzyme completely inactive, without any major effect on the enzyme stability. The partially active single mutants retained the same proportion of activity at reduced temperatures as the wild-type enzyme. These results do not support a role for the HGGG motif in catalysis at low temperatures, but provide further validation of the current three-dimensional model of HSL. Rat HSL was found to be relatively more active than human HSL at low temperatures. This difference was, however, not due to the 12 amino acids which are present in the regulatory module of the rat enzyme but absent in human HSL.     

Hop,an active Mutator-like element in the genome of the fungus Fusarium oxysporum

A new type of active DNA transposon has been identified in the genome of Fusarium oxysporum by its transposition into the niaD target gene. Two insertions within the final exon, in opposite orientations at the same nucleotide site, have been characterized. These elements, called Hop, are 3,299 bp long, with perfect terminal inverted repeats (TIRs) of 99 bp. The sequencing of genomic copies reveals a 9-bp target site duplication and no apparent sequence specificity at the insertion sites. The sequencing of a cDNA indicates that Hop does not contain an intron and encodes a putative transposase of 836 amino acids. The structural features (length, TIRs size, and 9-bp duplication), together with the presence of conserved domains in the transposase, strongly suggest that Hop is a Mutator-like element (MULE). Hop is thus the first active member of this family found beyond plants. The high rate of excision observed indicates that Hop is very active and thus represents a promising efficient tagging system for the isolation of fungal genes. The distribution of Hop elements within the Fusarium genus revealed that they are present in different species, suggesting that related elements could be present in other fungal genomes. In fact, Hop-related sequences have been identified in the survey of the entire genome sequence of three other ascomycetes, Magnaporthe grisea, Neurospora crassa, and Aspergillus fumigatus.     

Evolution of the Fot1 transposons in the genus Fusarium: discontinuous distribution and epigenetic inactivation

To understand the evolution of Fot1, a member of the pogo family widely dispersed in ascomycetes, we have performed a phylogenetic survey across the genus Fusarium divided into six sections. The taxonomic distribution of Fot1 is not homogeneous but patchy; it is prevalent in the Fusarium oxysporum complex, absent in closely related sections, and found in five species from the most distant section Martiella. Multiple copies of Fot1 were sequenced from each strain in which the element occurs. In three species, the Fot1 nucleotide sequence is 98% identical to that from F. oxysporum (Fox), whereas nucleotide divergence for host genes is markedly higher: 11% for partial nuclear 28S rDNA and up to 30% for the gene encoding nitrate reductase (nia). In two species, sequence divergence of Fot1-related elements relative to Fox ranged from 7% to 23% (16% average). Most of the sequence differences (82%) were C-to-T and G-to-A transitions. These mutations are distributed throughout the Fot1 sequences, although they tend to be concentrated in the middle portion of the elements. Analysis of the local sequence context of transitions revealed a hierarchy of site preferences. These characteristics are typical of the repeat-induced point mutation process, first discovered in Neurospora crassa. The spotty distribution of Fot1 elements among species together with the high degree of similarity between Fot1 sequences present in distant species strongly suggests a case of horizontal transfer.     

The plant nitrogen mobilization promoted by Colletotrichum lindemuthianum in Phaseolus leaves depends on fungus pathogenicity

Nitrogen plays an essential role in the nutrient relationship between plants and pathogens. Some studies report that the nitrogen-mobilizing plant metabolism that occurs during abiotic and biotic stress could be a 'slash-and-burn' defence strategy. In order to study nitrogen recycling and mobilization in host plants during pathogen attack and invasion, the Colletotrichum lindemuthianum/Phaseolus vulgaris interaction was used as a model. C. lindemuthianum is a hemibiotroph that causes anthracnose disease on P. vulgaris. Non-pathogenic mutants and the pathogenic wild-type strain were used to compare their effects on plant metabolism. The deleterious effects of infection were monitored by measuring changes in chlorophyll, protein, and amino acid concentrations. It was shown that amino acid composition changed depending on the plant-fungus interaction and that glutamine accumulated mainly in the leaves infected by the pathogenic strain. Glutamine accumulation correlated with the accumulation of cytosolic glutamine synthetase (GS1 alpha) mRNA. The most striking result was that the GS1 alpha gene was induced in all the fungus-infected leaves, independent of the strain used for inoculation, and that GS1 alpha expression paralleled the PAL3 and CHS defence gene expression. It is concluded that a role of GS1 alpha in plant defence has to be considered.     

Adipose gene expression prior to weight loss can differentiate and weakly predict dietary responders

Background

The ability to identify obese individuals who will successfully lose weight in response to dietary intervention will revolutionize disease management. Therefore, we asked whether it is possible to identify subjects who will lose weight during dietary intervention using only a single gene expression snapshot.

Methodology/Principal Findings

The present study involved 54 female subjects from the Nutrient-Gene Interactions in Human Obesity-Implications for Dietary Guidelines (NUGENOB) trial to determine whether subcutaneous adipose tissue gene expression could be used to predict weight loss prior to the 10-week consumption of a low-fat hypocaloric diet. Using several statistical tests revealed that the gene expression profiles of responders (8–12 kgs weight loss) could always be differentiated from non-responders (<4 kgs weight loss). We also assessed whether this differentiation was sufficient for prediction. Using a bottom-up (i.e. black-box) approach, standard class prediction algorithms were able to predict dietary responders with up to 61.1%±8.1% accuracy. Using a top-down approach (i.e. using differentially expressed genes to build a classifier) improved prediction accuracy to 80.9%±2.2%.

Conclusion

Adipose gene expression profiling prior to the consumption of a low-fat diet is able to differentiate responders from non-responders as well as serve as a weak predictor of subjects destined to lose weight. While the degree of prediction accuracy currently achieved with a gene expression snapshot is perhaps insufficient for clinical use, this work reveals that the comprehensive molecular signature of adipose tissue paves the way for the future of personalized nutrition.     

Profiling of adipokines secreted from human subcutaneous adipose tissue in response to PPAR agonists

The role of PPARs in the regulation of human adipose tissue secretome has received little attention despite its potential importance in the therapeutic actions of PPAR agonists. Here, we have investigated the effect of selective PPARgamma, PPARalpha, and PPARbeta/delta agonists on the production of adipokines by human subcutaneous adipose tissue. Antibody arrays were used to measure secreted factors in media from cultured adipose tissue explants. Sixteen proteins were produced in significant amounts. Activation of PPARs regulated the production of five proteins. Treatments with the three PPAR agonists decreased the secretion of leptin and interleukin-6. PPARalpha and beta/delta agonists markedly enhanced hepatocyte growth factor secretion whereas PPARbeta/delta down-regulated angiogenin and up-regulated TIMP-1 release. Hepatocyte growth factor, interleukin-6, and TIMP-1 are chiefly expressed in cells from the stromal vascular fraction whereas angiogenin is expressed in both adipocytes and cells from the stromal vascular fraction. Our data show that PPAR agonists modulate secretion of bioactive molecules from the different cell types composing human adipose tissue.     

Lipolysis and lipid mobilization in human adipose tissue

Triacylglycerol (TAG) stored in adipose tissue (AT) can be rapidly mobilized by the hydrolytic action of the three main lipases of the adipocyte. The non-esterified fatty acids (NEFA) released are used by other tissues during times of energy deprivation. Until recently hormone-sensitive lipase (HSL) was considered to be the key rate-limiting enzyme responsible for regulating TAG mobilization. A novel lipase named adipose triglyceride lipase/desnutrin (ATGL) has been identified as playing an important role in the control of fat cell lipolysis. Additionally perilipin and other proteins of the surface of the lipid droplets protecting or exposing the TAG core of the droplets to lipases are also potent regulators of lipolysis. Considerable progress has been made in understanding the mechanisms of activation of the various lipases. Lipolysis is under tight hormonal regulation. The best understood hormonal effects on AT lipolysis concern the opposing regulation by insulin and catecholamines. Heart-derived natriuretic peptides (i.e., stored in granules in the atrial and ventricle cardiomyocytes and exerting stimulating effects on diuresis and natriuresis) and numerous autocrine/paracrine factors originating from adipocytes and other cells of the stroma-vascular fraction may also participate in the regulation of lipolysis. Endocrine and autocrine/paracrine factors cooperate and lead to a fine regulation of lipolysis in adipocytes. Age, anatomical site, sex, genotype and species differences all play a part in the regulation of lipolysis. The manipulation of lipolysis has therapeutic potential in the metabolic disorders frequently associated with obesity and probably in several inborn errors of metabolism.     

Decreased expression and function of adipocyte hormone-sensitive lipase in subcutaneous fat cells of obese subjects.

Decreased lipolytic effect of catecholamines in adipose tissue has repeatedly been demonstrated in obesity and may be a cause of excess accumulation of body fat. However, the mechanisms behind this lipolysis defect are unclear. The role of hormone-sensitive lipase was examined using abdominal subcutaneous adipocytes from 34 obese drug-free and otherwise healthy males or females and 14 non-obese control subjects. The enzyme catalyzes the rate-limiting step of the lipolysis pathway. The maximum lipolytic capacity of fat cells was significantly decreased in obesity when measured using either a non-selective beta-adrenergic receptor agonist (isoprenaline) or a phosphodiesterase resistant cyclic AMP analogue (dibutyryl cyclic AMP). Likewise, enzyme activity, protein expression, and mRNA of hormone-sensitive lipase were significantly decreased in adipocytes of obese subjects. The findings were not influenced by age or gender. The data suggest that a decreased expression of hormone-sensitive lipase in subcutaneous fat cells, which in turn causes decreased enzyme function and impaired lipolytic capacity of adipocytes, is present in obesity. Impaired expression of the hormone-sensitive lipase gene might at least in part explain the enzyme defect.