Identification of diverse lipid-binding modes in the groove of zinc α2 glycoprotein reveals its functional versatility.

ZAG is a multifunctional glycoprotein with a class I MHC-like protein fold and an α1-α2 lipid-binding groove. The intrinsic ZAG ligand is unknown. Our previous studies showed that ZAG binds the dansylated C11 fatty acid, DAUDA, differently to the boron dipyrromethane C16 fatty acid, C16 -BODIPY. Here, the molecular basis for this difference was elucidated. Multi-wavelength analytical ultracentrifugation confirmed that DAUDA and C16 -BODIPY individually bind to ZAG and compete for the same binding site. Molecular docking of lipid-binding in the structurally related Cluster of differentiation 1 proteins predicted nine conserved ligand contact residues in ZAG. Twelve mutants were accordingly created by alanine scanning site directed mutagenesis for characterisation. Mutation of Y12 caused ZAG to misfold. Mutation of K147, R157 and A158 abrogated C16 -BODIPY but not DAUDA binding. L69 and T169 increased the fluorescence emission intensity of C16 -BODIPY but not of DAUDA compared to wild-type ZAG and showed that C16 -BODIPY binds close to T169 and L69. Distance measurements of the crystal structure revealed K147 forms a salt bridge with D83. A range of bioactive bulky lipids including phospholipids and sphingolipids displaced DAUDA from the ZAG binding site but unexpectedly did not displace C16 -BODIPY. We conclude that the ZAG α1-α2 groove contains separate but overlapping sites for DAUDA and C16 -BODIPY and is involved in binding to a bulkier and wider repertoire of lipids than previously reported. This work suggested that the in vivo activity of ZAG may be dictated by its lipid ligand.

High levels of modified ceramides are a defining feature of murine and human cancer cachexia.

BACKGROUND: Cancer cachexia (CCx) is a multifactorial energy-wasting syndrome reducing the efficiency of anti-cancer therapies, quality of life, and survival of cancer patients. In the past years, most studies focused on the identification of tumour and host-derived proteins contributing to CCx. However, there is still a lack of studies addressing the changes in bioactive lipids. The aim of this study was to identify specific lipid species as a hallmark of CCx by performing a broad range lipid analysis of plasma from well-established CCx mouse models as well as cachectic and weight stable cancer patients. METHODS: Plasma from non-cachectic (PBS-injected mice, NC26 tumour-bearing mice), pre-cachectic and cachectic mice (C26 and LLC tumour-bearing mice, ApcMin/+ mutant mice), and plasma from weight stable and cachectic patients with gastrointestinal cancer, were analysed using the Lipidyzer™ platform. In total, 13 lipid classes and more than 1100 lipid species, including sphingolipids, neutral and polar glycerolipids, were covered by the analysis. Correlation analysis between specific lipid species and readouts of CCx were performed. Lipidomics data were confirmed by gene expression analysis of metabolic organs to analyse enzymes involved in sphingolipid synthesis and degradation. RESULTS: A decrease in several lysophosphatidylcholine (LPC) species and an increase in numerous sphingolipids including sphingomyelins (SMs), ceramides (CERs), hexosyl-ceramides (HCERs) and lactosyl-ceramides (LCERs), were mutual features of CCx in both mice and cancer patients. Notably, sphingolipid levels gradually increased during cachexia development. Key enzymes involved in ceramide synthesis were elevated in liver but not in adipose, muscle, or tumour tissues, suggesting that ceramide turnover in the liver is a major contributor to elevated sphingolipid levels in CCx. LPC(16:1), LPC(20:3), SM(16:0), SM(24:1), CER(16:0), CER(24:1), HCER(16:0), and HCER(24:1) were the most consistently affected lipid species between mice and humans and correlated negatively (LPCs) or positively (SMs, CERs and HCERs) with the severity of body weight loss. CONCLUSIONS: High levels of sphingolipids, specifically ceramides and modified ceramides, are a defining feature of murine and human CCx and may contribute to tissue wasting and skeletal muscle atrophy through the inhibition of anabolic signals. The progressive increase in sphingolipids during cachexia development supports their potential as early biomarkers for CCx.

Emerging evidence for the modulation of exocytosis by signalling lipids.

Membrane fusion is a key event in exocytosis of neurotransmitters and hormones stored in intracellular vesicles. In this process, soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins are essential components of the exocytotic molecular machinery, while lipids have been seen traditionally as structural elements. However, the so-called signalling lipids, such as sphingosine and arachidonic acid, interact with SNAREs and directly modulate the frequency and mode of fusion events. Interestingly, recent work has proved that the sphingosine analogue FTY-720, used in the treatment of multiple sclerosis, mimics the effects of signalling lipids. In the present Review, we discuss recent investigations suggesting that endogenous signalling lipids and synthetic analogues can modulate important physiological aspects of secretion, such as quantal release, vesicle recruitment into active sites, vesicle transport and even organelle fusion in the cytosol. Therefore, these compounds are far from being merely structural components of cellular membranes.