Unmasking crucial residues in adipose triglyceride lipase for coactivation with comparative gene identification-58.

Lipolysis is an essential metabolic process that releases unesterified fatty acids from neutral lipid stores to maintain energy homeostasis in living organisms. Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis and can be coactivated upon interaction with the protein comparative gene identification-58 (CGI-58). The underlying molecular mechanism of ATGL stimulation by CGI-58 is incompletely understood. Based on analysis of evolutionary conservation, we used site directed mutagenesis to study a C-terminally truncated variant and full-length mouse ATGL providing insights in the protein coactivation on a per-residue level. We identified the region from residues N209-N215 in ATGL as essential for coactivation by CGI-58. ATGL variants with amino acids exchanges in this region were still able to hydrolyze triacylglycerol at the basal level and to interact with CGI-58, yet could not be activated by CGI-58. Our studies also demonstrate that full-length mouse ATGL showed higher tolerance to specific single amino acid exchanges in the N209-N215 region upon CGI-58 coactivation compared to C-terminally truncated ATGL variants. The region is either directly involved in protein-protein interaction or essential for conformational changes required in the coactivation process. Three-dimensional models of the ATGL/CGI-58 complex with the artificial intelligence software AlphaFold demonstrated that a large surface area is involved in the protein-protein interaction. Mapping important amino acids for coactivation of both proteins, ATGL and CGI-58, onto the 3D model of the complex locates these essential amino acids at the predicted ATGL/CGI-58 interface thus strongly corroborating the significance of these residues in CGI-58-mediated coactivation of ATGL.

DIP2 is a unique regulator of diacylglycerol lipid homeostasis in eukaryotes.

Chain-length-specific subsets of diacylglycerol (DAG) lipids are proposed to regulate differential physiological responses ranging from signal transduction to modulation of the membrane properties. However, the mechanism or molecular players regulating the subsets of DAG species remain unknown. Here, we uncover the role of a conserved eukaryotic protein family, DISCO-interacting protein 2 (DIP2) as a homeostatic regulator of a chemically distinct subset of DAGs using yeast, fly, and mouse models. Genetic and chemical screens along with lipidomics analysis in yeast reveal that DIP2 prevents the toxic accumulation of specific DAGs in the logarithmic growth phase, which otherwise leads to endoplasmic reticulum stress. We also show that the fatty acyl-AMP ligase-like domains of DIP2 are essential for the redirection of the flux of DAG subspecies to storage lipid, triacylglycerols. DIP2 is associated with vacuoles through mitochondria-vacuole contact sites and such modulation of selective DAG abundance by DIP2 is found to be crucial for optimal vacuole membrane fusion and consequently osmoadaptation in yeast. Thus, the study illuminates an unprecedented DAG metabolism route and provides new insights on how cell fine-tunes DAG subspecies for cellular homeostasis and environmental adaptation.

Lipids, such as fats and hormones, constitute one of the main building blocks of cells. There are thousands of different lipids each with distinctive chemical properties that allow them to carry out specific roles. For example, a group of lipids called diacylglycerols help cells perform a myriad of tasks, like sensing external signals, making membranes, and storing energy. The production and breakdown of diacylglycerols is therefore tightly regulated. However, very little is known about the molecules involved in this metabolic process. One possible candidate is the enzyme DIP2 which is comprised of a protein module known as FAAL (short for fatty acyl-AMP ligase). FAAL belongs to a family of enzymes that synthesize lipid-like molecules in bacteria. In 2021, a group of researchers tracked the evolutionary trajectory of these bacterial proteins and found that most of them were lost in eukaryotes, such as animals and fungi. FAAL-like proteins, however, had been retained through evolution and incorporated in to DIP2. Here, Mondal, Kinatukara et al. ­ including some of the researchers involved in the 2021 study ­ have used a combination of genetic and biochemical experiments to investigate whether and how DIP2 contributes to lipid metabolism in eukaryotes. They found that yeast cells without the gene for DIP2 had higher levels of diacylglycerols which hampered the shape and function of certain cellular compartments. The mutant cells were also unable to convert diacylglycerols in to another group of lipids which are involved in energy storage. This effect was observed in fruit flies and mice lacking DIP2, suggesting that this role for DIP2 is conserved across most eukaryotes. Further experiments in yeast cells revealed that unlike other enzymes that metabolize diacylglycerols, DIP2 only acted on a sub-population of diacylglycerols at specific locations and times. Furthermore, yeast cells lacking DIP2 could still grow under ideal conditions, but could not cope with high or low salt concentrations in their surroundings, suggesting that the enzyme helps cells deal with environmental stresses. Since DIP2 is found in most eukaryotes, understanding how it works could be useful for multiple branches of biology. For example, some pathogenic fungi that cause diseases in crop plants and humans also rely on DIP2. Further studies are needed to better understand the role that DIP2 plays in other eukaryotic species which may shed light on other processes the enzyme is involved in.

A universal pocket in fatty acyl-AMP ligases ensures redirection of fatty acid pool away from coenzyme A-based activation.

Fatty acyl-AMP ligases (FAALs) channelize fatty acids towards biosynthesis of virulent lipids in mycobacteria and other pharmaceutically or ecologically important polyketides and lipopeptides in other microbes. They do so by bypassing the ubiquitous coenzyme A-dependent activation and rely on the acyl carrier protein-tethered 4'-phosphopantetheine (holo-ACP). The molecular basis of how FAALs strictly reject chemically identical and abundant acceptors like coenzyme A (CoA) and accept holo-ACP unlike other members of the ANL superfamily remains elusive. We show that FAALs have plugged the promiscuous canonical CoA-binding pockets and utilize highly selective alternative binding sites. These alternative pockets can distinguish adenosine 3',5'-bisphosphate-containing CoA from holo-ACP and thus FAALs can distinguish between CoA and holo-ACP. These exclusive features helped identify the omnipresence of FAAL-like proteins and their emergence in plants, fungi, and animals with unconventional domain organizations. The universal distribution of FAALs suggests that they are parallelly evolved with FACLs for ensuring a CoA-independent activation and redirection of fatty acids towards lipidic metabolites.