A FRET sensor for the real-time detection of long chain acyl-CoAs and synthetic ABHD5 ligands.

Intracellular long-chain acyl-coenzyme As (LC-acyl-CoAs) are thought to be under tight spatial and temporal controls, yet the ability to image LC-acyl-CoAs in live cells is lacking. Here, we developed a fluorescence resonance energy transfer (FRET) sensor for LC-acyl-CoAs based on the allosterically regulated interaction between α/ß hydrolase domain-containing 5 (ABHD5) and Perilipin 5. The genetically encoded sensor rapidly detects intracellular LC-acyl-CoAs generated from exogenous and endogenous fatty acids (FAs), as well as synthetic ABHD5 ligands. Stimulation of lipolysis in brown adipocytes elevated intracellular LC-acyl-CoAs in a cyclic fashion, which was eliminated by inhibiting PNPLA2 (ATGL), the major triglyceride lipase. Interestingly, inhibition of LC-acyl-CoA transport into mitochondria elevated intracellular LC-acyl-CoAs and dampened their cycling. Together, these observations reveal an intimate feedback control between LC-acyl-CoA generation from lipolysis and utilization in mitochondria. We anticipate that this sensor will be an important tool to dissect intracellular LC-acyl-CoA dynamics as well to discover novel synthetic ABHD5 ligands.

Molecular Modeling of ABHD5 Structure and Ligand Recognition.

Alpha/beta hydrolase domain-containing 5 (ABHD5), also termed CGI-58, is the key upstream activator of adipose triglyceride lipase (ATGL), which plays an essential role in lipid metabolism and energy storage. Mutations in ABHD5 disrupt lipolysis and are known to cause the Chanarin-Dorfman syndrome. Despite its importance, the structure of ABHD5 remains unknown. In this work, we combine computational and experimental methods to build a 3D structure of ABHD5. Multiple comparative and machine learning-based homology modeling methods are used to obtain possible models of ABHD5. The results from Gaussian accelerated molecular dynamics and experimental data of the apo models and their mutants are used to select the most likely model. Moreover, ensemble docking is performed on representative conformations of ABHD5 to reveal the binding mechanism of ABHD5 and a series of synthetic ligands. Our study suggests that the ABHD5 models created by deep learning-based methods are the best candidate structures for the ABHD5 protein. The mutations of E41, R116, and G328 disturb the hydrogen bonding network with nearby residues and suppress membrane targeting or ATGL activation. The simulations also reveal that the hydrophobic interactions are responsible for binding sulfonyl piperazine ligands to ABHD5. Our work provides fundamental insight into the structure of ABHD5 and its ligand-binding mode, which can be further applied to develop ABHD5 as a therapeutic target for metabolic disease and cancer.

Leucine zipper transcription factor-like 1 (LZTFL1), an intraflagellar transporter protein 27 (IFT27) associated protein, is required for normal sperm function and male fertility.

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella, including mammalian sperm tails. Depletion of IFT27, a component of the IFT complex, in male germ cells results in infertility associated with disrupted sperm flagella structure and motility. Leucine zipper transcription factor-like 1 (LZTFL1) is an IFT27 associated protein. LZTFL1, also known as BBS17, is a Bardet-Biedl syndrome (BBS) associated protein. Patients carrying biallelic variants of LZTFL1 gene exhibit the common BBS phenotypes. The global Lztfl1 knockout mice showed abnormal growth rate and retinal degeneration, typical of BBS phenotype. However, it is not clear if Lztfl1 has a role in male fertility. The LZTFL1 protein is highly and predominantly expressed in mouse testis. During the first wave of spermatogenesis, the protein is only expressed during spermiogenesis phase from the round spermatid stage and displays a cytoplasmic localization with a vesicular distribution pattern. At the elongated spermatid stage, LZTFL1 is present in the developing flagella and appears also close to the manchette. Fertility of Lztfl1 knockout mice was significantly reduced and associated with low sperm motility and a high level of abnormal sperm (astheno-teratozoospermia). In vitro assessment of fertility revealed reduced fertilization and embryonic development when using sperm from homozygous mutant mice. In addition, we observed a significant decrease of the testicular IFT27 protein level in Lztfl1 mutant mice contrasting with a stable expression levels of other IFT proteins, including IFT20, IFT81, IFT88 and IFT140. Overall, our results support strongly the important role of LZTFL1 in mouse spermatogenesis and male fertility.

Dynamic interactions of ABHD5 with PNPLA3 regulate triacylglycerol metabolism in brown adipocytes.

Patatin-Like Phospholipase Domain Containing 2 (PNPLA2)/Adipose Triglyceride Lipase (ATGL) and PNPLA3/Adiponutrin are close paralogs that appear to have opposite functions on triacylglycerol (TAG) mobilization and storage. PNPLA2/ATGL is a major triglyceride lipase in adipose tissue and liver, whereas a common human variant of PNPLA3, I148M, greatly increases risk of hepatosteatosis. Nonetheless, the function of PNPLA3 and the mechanism by which the I148M variant promotes TAG accumulation are poorly understood. Here we demonstrate that PNPLA3 strongly interacts with α/ß hydrolase domain-containing 5 (ABHD5/CGI-58), an essential co-activator of PNPLA2/ATGL. Molecular imaging experiments demonstrate that PNPLA3 effectively competes with PNPLA2/ATGL for ABHD5, and that PNPLA3 I148M is more effective in this regard. Inducible overexpression of PNPLA3 I148M greatly suppressed PNPLA2/ATGL-dependent lipolysis and triggered massive TAG accumulation in brown adipocytes, and these effects were dependent on ABHD5. The interaction of PNPLA3 and ABHD5 can be regulated by fatty acid supplementation and synthetic ABHD5 ligands, raising the possibility that this interaction might be targeted for treatment of fatty liver disease.

Novel Pharmacological Probes Reveal ABHD5 as a Locus of Lipolysis Control in White and Brown Adipocytes.

Current knowledge regarding acute regulation of adipocyte lipolysis is largely based on receptor-mediated activation or inhibition of pathways that influence intracellular levels of cAMP, thereby affecting protein kinase A (PKA) activity. We recently identified synthetic ligands of α-ß-hydrolase domain containing 5 (ABHD5) that directly activate adipose triglyceride lipase (ATGL) by dissociating ABHD5 from its inhibitory regulator, perilipin-1 (PLIN1). In the current study, we used these novel ligands to determine the direct contribution of ABHD5 to various aspects of lipolysis control in white (3T3-L1) and brown adipocytes. ABHD5 ligands stimulated adipocyte lipolysis without affecting PKA-dependent phosphorylation on consensus sites of PLIN1 or hormone-sensitive lipase (HSL). Cotreatment of adipocytes with synthetic ABHD5 ligands did not alter the potency or maximal lipolysis efficacy of the ß-adrenergic receptor (ADRB) agonist isoproterenol (ISO), indicating that both target a common pool of ABHD5. Reducing ADRB/PKA signaling with insulin or desensitizing ADRB suppressed lipolysis responses to a subsequent challenge with ISO, but not to ABHD5 ligands. Lastly, despite strong treatment differences in PKA-dependent phosphorylation of HSL, we found that ligand-mediated activation of ABHD5 led to complete triglyceride hydrolysis, which predominantly involved ATGL, but also HSL. These results indicate that the overall pattern of lipolysis controlled by ABHD5 ligands is similar to that of isoproterenol, and that ABHD5 plays a central role in the regulation of adipocyte lipolysis. As lipolysis is critical for adaptive thermogenesis and in catabolic tissue remodeling, ABHD5 ligands may provide a means of activating these processes under conditions where receptor signaling is compromised.