Functional fat body proteomics and gene targeting reveal in vivo functions of Drosophila melanogaster α-Esterase-7

Carboxylesterases constitute a large enzyme family in insects, which is involved in diverse functions such as xenobiotic detoxification, lipid metabolism and reproduction. Phylogenetically, many insect carboxylesterases are represented by multienzyme clades, which are encoded by evolutionarily ancient gene clusters such as the α-Esterase cluster. Much in contrast to the vital importance attributed to carboxylesterases in general, the in vivo function of individual α-Esterase genes is largely unknown. This study employs a functional proteomics approach to identify esterolytic enzymes of the vinegar fly Drosophila melanogaster fat body. One of the fat body carboxylesterases, α-Esterase-7, was selected for mutational analysis by gene targeting to generate a deletion mutant fly. Phenotypic characterization of α-Esterase-7 null mutants and transgenic flies, which overexpress a chimeric α-Esterase-7:EGFP gene, reveals important functions of α-Esterase-7 in insecticide tolerance, lipid metabolism and lifespan control. The presented first deletion mutant of any α-Esterase in the model insect D. melanogaster generated by gene targeting not only provides experimental evidence for the endogenous functions of this gene family. It also offers an entry point for in vivo structure-function analyses of α-Esterase-7, which is of central importance for naturally occurring insecticide resistance in wild populations of various dipteran insect species.     

Physical and genetic structure of the glpK-cpxA interval of the Escherichia coli K-12 chromosome

Summary Mutations at the cpxA locus of Escherichia coli K-12 affect cellular processes that are not otherwise related. We have now determined the physical and genetic structure of the E. coli chromosome in the region of cpxA (87.5 min). Our results indicate that cpxA is a single gene. Previous studies showed cpxA to be linked to tpiA. We therefore isolated two tpiA ⁺ recombinant plasmids, pRA200 and pRA300, from EcoRI and BamHI digests of F133, respectively. By genetic complementation or enzyme overproduction, the 9.5 kb EcoRI fragment in pRA200 was shown to include glpK, tpiA and cdh. The 13.6 kb BamHI fragment of pRA300 lacks glpK, but includes tpiA, pfkA and cpxA. Neither fragment complemented a deletion of the rha operon. These data indicate the chromosomal gene order: 87 min-rha-cpxA-pfkA-cdh-tpiA-glpK-88 min. The EcoRI and BamHI fragments overlap in an interval corresponding to about 8.2 kb of DNA. The total region of the E. coli K12 chromosome covered by the two fragments is about 15 kb. A terminal 2 kb EcoRI-BamHI fragment from pRA300 complemented the chromosomal cpxA2[Ts] allele with respect to isoleucine and valine synthesis, RNA bacteriophage sensitivity and surface exclusion in Hfr strains, and envelope protein composition. Complementation occurred when the fragment was subcloned in pBR325 but not when it was subcloned in pBR322, suggesting that the 2 kb fragment lacks expression sequences that are supplied by cat (chloramphenicol acetyltransferase gene) expression sequences of pBR325. The cpxA locus on the E. coli chromosome was established with respect to two chromosomal Tn10 insertions by a combination of genetic and physical analyses. The locus established by those analyses was consistent with the location of the 2 kb EcoRI-BamHI fragment in the physical map of the region. Physical analyses of (rha-pfkA) and (rha-tpiA) deletion strains showed that they lack cpxA and surrounding genes. Since these strains were viable, cpxA is not essential under all growth conditions.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Three-way interactions between mosquito population,viral strain and temperature underlying chikungunya virus transmission potential

Interactions between pathogens and their insect vectors in nature are under the control of both genetic and non-genetic factors, yet most studies on mosquito vector competence for human pathogens are conducted in laboratory systems that do not consider genetic and/or environmental variability. Evaluating the risk of emergence of arthropod-borne viruses (arboviruses) of public health importance such as chikungunya virus (CHIKV) requires a more realistic appraisal of genetic and environmental contributions to vector competence. In particular, sources of variation do not necessarily act independently and may combine in the form of interactions. Here, we measured CHIKV transmission potential by the mosquito Aedes albopictus in all combinations of six worldwide vector populations, two virus strains and two ambient temperatures (20°C and 28°C). Overall, CHIKV transmission potential by Ae. albopictus strongly depended on the three-way combination of mosquito population, virus strain and temperature. Such genotype-by-genotype-by-environment (G × G × E) interactions question the relevance of vector competence studies conducted with a simpler set of conditions. Our results highlight the need to account for the complex interplay between vectors, pathogens and environmental factors to accurately assess the potential of vector-borne diseases to emerge.     

The lipolytic proteome of mouse adipose tissue

Hydrolysis of triacylglycerols and cholesteryl esters is a key event in energy homeostasis of animals. However, many lipolytic activities still await their molecular identification. Here we report on a novel tool for concomitant analysis of lipases in complex proteomes. Fluorescent activity tags mimicking lipid substrates were used to label the proteome of mouse adipose tissue. Analysis by two-dimensional gel electrophoresis and LC-MS/MS led to the identification of all known intracellular lipases as well as a number of novel candidates. One of them was recently shown to be involved in triacylglycerol mobilization in adipocytes and therefore named adipose triglyceride lipase. Functional characterization of expressed enzymes demonstrated that lipolytic and esterolytic activities could be well discriminated. Thus our results show the first map of the lipolytic proteome of mouse adipose tissue and demonstrate the general applicability of our method for rapid profiling and identification of lipolytic activities in complex biological samples.     

Adipose Triglyceride Lipase (ATGL) and Hormone-Sensitive Lipase (HSL) Deficiencies Affect Expression of Lipolytic Activities in Mouse Adipose Tissues

Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are key enzymes involved in intracellular degradation of triacylglycerols. It was the aim of this study to elucidate how the deficiency in one of these proteins affects the residual lipolytic proteome in adipose tissue. For this purpose, we compared the lipase patters of brown and white adipose tissue from ATGL (−/−) and HSL (−/−) mice using differential activity-based gel electrophoresis. This method is based on activity-recognition probes possessing the same substrate analogous structure but carrying different fluorophores for specific detection of the enzyme patterns of two different tissues in one electrophoresis gel. We found that ATGL-deficiency in brown adipose tissue had a profound effect on the expression levels of other lipolytic and esterolytic enzymes in this tissue, whereas HSL-deficiency hardly showed any effect in brown adipose tissue. Neither ATGL- nor HSL-deficiency greatly influenced the lipase patterns in white adipose tissue. Enzyme activities of mouse tissues on acylglycerol substrates were analyzed as well, showing that ATGL-and HSL-deficiencies can be compensated for at least in part by other enzymes. The proteins that responded to ATGL-deficiency in brown adipose tissue were overexpressed and their activities on acylglycerols were analyzed. Among these enzymes, Es1, Es10, and Es31-like represent lipase candidates as they catalyze the hydrolysis of long-chain acylglycerols.Excess lipids are stored as intracellular triacylglycerol and steryl ester deposits in animals, plant seeds, and fungi. In mammals adipose tissue is the body''s largest storage organ for triacylglycerols (TAG)¹ as the primary source of energy during periods of starvation and increased energy demand. Two types of adipose tissue, namely brown (BAT) and white (WAT) adipose tissue exist in mammals, localizing to anatomically distinct areas. BAT and WAT differ in almost all their structural and functional features. Whereas BAT develops prenatally, WAT is subject to maturation postnatally. The different appearance of brown and white adipose tissue is caused by differences in lipid content and the abundance of mitochondria in the constituent adipocytes. Brown fat cells contain several small multilocular lipid droplets and a high number of large mitochondria with numerous cristae. In addition, BAT is highly vascularized and highly innervated by the sympathetic nervous system. In contrast, white adipocytes, usually contain one major unilocular lipid droplet that fills most of the cytoplasm leaving space for only few mitochondria (1–3). WAT accumulates excess energy as triacylglycerols, whereas BAT dissipates energy through adaptive thermogenesis. The thermogenic activity of BAT is caused by the expression of one protein unique in brown adipocytes, the uncoupling protein 1 (UCP1). This polypeptide is a facultative proton transporter and localizes to the inner mitochondrial membrane. It generates heat instead of ATP by uncoupling oxidation in the respiratory chain (3).Lipolysis in WAT is the catabolic process responsible for the release of free fatty acids from triacylglycerol (4, 5). The balance of lipid storage and mobilization is tightly regulated to ensure whole body energy homeostasis. The mobilization of triacylglycerol stores by activation of lipolytic enzymes is specifically stimulated by hormones and chemical agents. In addition, a number of specific physiological conditions owing to exercise, aging, and nutritional status (feeding, fasting) also regulate degradation of TAGs (6). Impairment of lipolysis in adipocytes may be associated with clinical symptoms including obesity, insulin resistance, diabetes mellitus, and dyslipidaemia. All these conditions seem to have a common substrate called lipotoxicity (7–10).The sequential hydrolysis of triacylglycerols in adipocytes producing FFAs is catalyzed by a cascade of lipolytic enzymes, with different substrate preferences (11). The committed enzyme catalyzing the first step of TAG hydrolysis is ATGL, which was identified in three different laboratories in 2004 (12–14). Its activity appears to be largely dependent on association with CGI-58 (14, 15). HSL exhibits a much broader substrate spectrum, with preference for diacylglycerols as well as cholesteryl and retinyl esters (16, 17). In the final step of lipolysis, monoacylglycerol lipase (MGL) degrades MAG thereby generating free fatty acid and glycerol (18). ATGL is the major TAG lipase in adipose tissue. Expression in other tissues is rather low. Currently it cannot be excluded, that other lipases also exist that are important for lipid catabolism (19). Recent functional proteomic screens in various mouse tissues led to the identification of enzyme candidates that are currently subject to functional characterization (unpublished data).The intracellular degradation of triacylglycerols is catalyzed by a cascade of lipolytic enzymes. There appears to be an overlap of substrate preferences as well as a redundancy of lipases to ensure a proper function of this important catabolic process if individual lipase activities are reduced or entirely absent. This study aimed at identifying the effects of ATGL and HSL-deficiency on the expression of other lipolytic enzymes in adipose tissue. For this purpose, we compared the lipolytic proteomes of BAT and WAT from ATGL (−/−) and HSL (−/−) mice with the enzyme patterns of wt tissues using differential activity-based gel electrophoresis (DABGE) (20). This method is based on activity-recognition probes containing same substrate analogous structures but carrying different fluorophores for specific detection of the individual enzyme patterns of two different tissues. These inhibitors react with the nucleophilic serine in the active center of lipolytic enzymes thereby generating covalent bound lipid-protein complexes, which can be separated by gel electrophoresis. We found, that ATGL-deficiency in BAT had a profound effect on the expression levels of other lipolytic and esterolytic enzymes in this tissue, whereas HSL-deficiency hardly showed any effect in BAT. Neither ATGL- nor HSL-deficiency greatly influenced the lipase patterns in WAT. ATGL-deficiency led to a significant but not total reduction in the TAG hydrolyzing activity of adipose tissues. Obviously, there must be (an)other enzyme(s) compensating for the hydrolytic capacity of ATGL. Three proteins that responded to ATGL-deficiency in BAT were overexpressed and their activities on acylglycerols were analyzed. Among these proteins, Es1, Es10, and Es31-like emerged as novel lipase candidates in these studies.     

The role of serotonin in sleep disordered breathing associated with Parkinson disease: a correlative [11C]DASB PET imaging study

Sleep dysfunction and excessive daytime sleepiness are common in Parkinson disease (PD). Several studies suggest that PD patients exhibit high prevalence of sleep-disordered breathing (SDB). PD has a complex profile of neurochemical deficits in which abnormalities of different neurotransmitter systems may play significant and differing roles in the development of non-motor features. In the present study, we investigated whether SDB in PD is related to serotoninergic neuron degeneration. We used a cross-sectional design to assess the correlation between SDB and measures of caudal brainstem serotonin neuron integrity. Fifty one PD participants with mean disease duration of 6.0 (SD 3.7) years and mean age of 63.9 (SD 6.2) years were studied. We measured caudal brainstem serotoninergic innervation with [(11)C]DASB positron emission tomography (PET) imaging and striatal dopaminergic innervation with [(11)C]DTBZ PET imaging. SDB was assessed with polysomnography (PSG) and sleepiness with multiple sleep latency tests. Greater than half of participants exhibited PSG evidence of significant SDB; 12 participants had normal PSGs, 6 had mild SDB, 20 had moderate SDB, and 13 had severe SDB. We found no association between severity of SDB and caudal brainstem serotoninergic innervation in PD participants. Striatal dopaminergic denervation did not correlate with severity of SDB. We did find significant correlations between measures of motor function impairment and sleep quantity and quality in PD. Neither serotoninergic nor dopaminergic neuron degeneration is likely to play a major role in SDB observed in PD patients.     

Na+ and K+ ion imbalances in Alzheimer's disease

Alzheimer's disease (AD) is associated with impaired glutamate clearance and depressed Na(+)/K(+) ATPase levels in AD brain that might lead to a cellular ion imbalance. To test this hypothesis, [Na(+)] and [K(+)] were analyzed in postmortem brain samples of 12 normal and 16 AD individuals, and in cerebrospinal fluid (CSF) from AD patients and matched controls. Statistically significant increases in [Na(+)] in frontal (25%) and parietal cortex (20%) and in cerebellar [K(+)] (15%) were observed in AD samples compared to controls. CSF from AD patients and matched controls exhibited no differences, suggesting that tissue ion imbalances reflected changes in the intracellular compartment. Differences in cation concentrations between normal and AD brain samples were modeled by a 2-fold increase in intracellular [Na(+)] and an 8-15% increase in intracellular [K(+)]. Since amyloid beta peptide (Aβ) is an important contributor to AD brain pathology, we assessed how Aβ affects ion homeostasis in primary murine astrocytes, the most abundant cells in brain tissue. We demonstrate that treatment of astrocytes with the Aβ 25-35 peptide increases intracellular levels of Na(+) (~2-3-fold) and K(+) (~1.5-fold), which were associated with reduced levels of Na(+)/K(+) ATPase and the Na(+)-dependent glutamate transporters, GLAST and GLT-1. Similar increases in astrocytic Na(+) and K(+) levels were also caused by Aβ 1-40, but not by Aβ 1-42 treatment. Our study suggests a previously unrecognized impairment in AD brain cell ion homeostasis that might be triggered by Aβ and could significantly affect electrophysiological activity of brain cells, contributing to the pathophysiology of AD.