Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro.

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA-independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.

Twenty-three-year demographic history of the Affenberg Japanese macaques (Macaca fuscata), a translocated semi-free-ranging group in southern Austria.

Demographic studies on translocated primate groups provide a unique opportunity to study population dynamics, social strategies, and reproductive parameters of a species adapting to new environments. In 1996, 38 Japanese macaques (Macaca fuscata) of the Minoo-H group (Osaka Prefecture, Japan) were translocated to Affenberg Landskron, a four-hectare naturally forested park in southern Austria. By January 2020, the population had increased to 160 individuals, and a total of 223 births were recorded. Births peaked in late April to late May, and the timing was influenced by neither offspring sex nor parity status of the mother. Infant mortality was low (8.97%), mostly involving primiparous females, and the average interbirth intervals were shorter following the death of an infant (1.10 years) than a surviving infant (1.77 years). Females rarely had offspring with the same males repeatedly, and the reproductive success among males declined with increasing years of presence in the group. The main aspects of reproduction, mortality, and mate choice are consistent with published data on natural and provisioned populations in Japan and those translocated to other countries. The life expectancy for females, however, was relatively high (11.72% chance of reaching the age of 20), whereas birth control prevented them from using their lifetime reproductive potential. By January 2020, the number of old individuals (> 18 years; 17.5%) was close to that of juveniles (< 4 years; 22.5%). The specific group composition, along with the inability of males to emigrate out of their natal group, may affect the social dynamics of the population, which merits further attention in future studies.

Genes Activated by Vibrio cholerae upon Exposure to Caenorhabditis elegans Reveal the Mannose-Sensitive Hemagglutinin To Be Essential for Colonization.

During its life cycle, the facultative human pathogen Vibrio cholerae, which is the causative agent of the diarrheal disease cholera, needs to adapt to a variety of different conditions, such as the human host or the aquatic environment. Importantly, cholera infections originate from the aquatic reservoir where V. cholerae persists between the outbreaks. In the aquatic environment, bacteria are constantly threatened by predatory protozoa and nematodes, but our knowledge of the response pathways and adaptation strategies of V. cholerae to such stressors is limited. Using a temporally controlled reporter system of transcription, we identified more than 100 genes of V. cholerae induced upon exposure to the nematode Caenorhabditis elegans, which emerged recently as a valuable model for environmental predation during the aquatic lifestyle of V. cholerae Besides others, we identified and validated the genes encoding the mannose-sensitive hemagglutinin (MSHA) type IV pilus to be significantly induced upon exposure to the nematode. Subsequent analyses demonstrated that the mannose-sensitive hemagglutinin is crucial for attachment of V. cholerae in the pharynx of the worm and initiation of colonization, which results in growth retardation and developmental delay of C. elegans Thus, the surface adhesion factor MSHA could be linked to a fitness advantage of V. cholerae upon contact with bacterium-grazing nematodes.IMPORTANCE The waterborne diarrheal disease cholera is caused by the bacterium Vibrio cholerae The facultative human pathogen persists as a natural inhabitant in the aquatic ecosystem between outbreaks. In contrast to the human host, V. cholerae requires a different set of genes to survive in this hostile environment. For example, predatory micrograzers are commonly found in the aquatic environment and use bacteria as a nutrient source, but knowledge of the interaction between bacterivorous grazers and V. cholerae is limited. In this study, we successfully adapted a genetic reporter technology and identified more than 100 genes activated by V. cholerae upon exposure to the bacterium-grazing nematode Caenorhabditis elegans This screen provides a first glimpse into responses and adaptational strategies of the bacterial pathogen against such natural predators. Subsequent phenotypic characterization revealed the mannose-sensitive hemagglutinin to be crucial for colonization of the worm, which causes developmental delay and growth retardation.

Hypoxia-inducible lipid droplet-associated protein inhibits adipose triglyceride lipase.

Elaborate control mechanisms of intracellular triacylglycerol (TAG) breakdown are critically involved in the maintenance of energy homeostasis. Hypoxia-inducible lipid droplet-associated protein (HILPDA)/hypoxia-inducible gene-2 (Hig-2) has been shown to affect intracellular TAG levels, yet, the underlying molecular mechanisms are unclear. Here, we show that HILPDA inhibits adipose triglyceride lipase (ATGL), the enzyme catalyzing the first step of intracellular TAG hydrolysis. HILPDA shares structural similarity with G0/G1 switch gene 2 (G0S2), an established inhibitor of ATGL. HILPDA inhibits ATGL activity in a dose-dependent manner with an IC50 value of ∼2 µM. ATGL inhibition depends on the direct physical interaction of both proteins and involves the N-terminal hydrophobic region of HILPDA and the N-terminal patatin domain-containing segment of ATGL. Finally, confocal microscopy combined with Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicated that HILPDA and ATGL colocalize and physically interact intracellularly. These findings provide a rational biochemical explanation for the tissue-specific increased TAG accumulation in HILPDA-overexpressing transgenic mouse models.