Computational design and molecular dynamics simulations suggest the mode of substrate binding in ceramide synthases.

Until now, membrane-protein stabilization has relied on iterations of mutations and screening. We now validate a one-step algorithm, mPROSS, for stabilizing membrane proteins directly from an AlphaFold2 model structure. Applied to the lipid-generating enzyme, ceramide synthase, 37 designed mutations lead to a more stable form of human CerS2. Together with molecular dynamics simulations, we propose a pathway by which substrates might be delivered to the ceramide synthases.

Rheumatoid arthritis and Hailey-Hailey disease treated with methotrexate.

We report a rare case of long-standing Hailey-Hailey disease in a Caucasian Portuguese 69-year-old woman, recently diagnosed with rheumatoid arthritis. The patient's skin lesions remained active and exudative despite topical and oral treatments with corticosteroids, tetracyclines, antifungals, and oral treatment with azathioprine. After introduction of methotrexate for rheumatoid arthritis treatment, the skin lesions regressed, with significant impact on the patient's quality of life. This case report supports the clinical evidence of methotrexate's potential role in Hailey-Hailey disease treatment.

Fatty acid transport protein 2 interacts with ceramide synthase 2 to promote ceramide synthesis.

Dihydroceramide is a lipid molecule generated via the action of (dihydro)ceramide synthases (CerSs), which use two substrates, namely sphinganine and fatty acyl-CoAs. Sphinganine is generated via the sequential activity of two integral membrane proteins located in the endoplasmic reticulum. Less is known about the source of the fatty acyl-CoAs, although a number of cytosolic proteins in the pathways of acyl-CoA generation modulate ceramide synthesis via direct or indirect interaction with the CerSs. In this study, we demonstrate, by proteomic analysis of immunoprecipitated proteins, that fatty acid transporter protein 2 (FATP2) (also known as very long-chain acyl-CoA synthetase) directly interacts with CerS2 in mouse liver. Studies in cultured cells demonstrated that other members of the FATP family can also interact with CerS2, with the interaction dependent on both proteins being catalytically active. In addition, transfection of cells with FATP1, FATP2, or FATP4 increased ceramide levels although only FATP2 and 4 increased dihydroceramide levels, consistent with their known intracellular locations. Finally, we show that lipofermata, an FATP2 inhibitor which is believed to directly impact tumor cell growth via modulation of FATP2, decreased de novo dihydroceramide synthesis, suggesting that some of the proposed therapeutic effects of lipofermata may be mediated via (dihydro)ceramide rather than directly via acyl-CoA generation. In summary, our study reinforces the idea that manipulating the pathway of fatty acyl-CoA generation will impact a wide variety of down-stream lipids, not least the sphingolipids, which utilize two acyl-CoA moieties in the initial steps of their synthesis.

Ceramide synthases: Reflections on the impact of Dr. Lina M. Obeid.

Sphingolipids are a family of lipids that are critical to cell function and survival. Much of the recent work done on sphingolipids has been performed by a closely-knit family of sphingolipid researchers, which including our colleague, Dr. Lina Obeid, who recently passed away. We now briefly review where the sphingolipid field stands today, focusing in particular on areas of sphingolipid research to which Dr. Obeid made valued contributions. These include the 'many-worlds' view of ceramides and the role of a key enzyme in the sphingolipid biosynthetic pathway, namely the ceramide synthases (CerS). The CerS contain a number of functional domains and also interact with a number of other proteins in lipid metabolic pathways, fulfilling Dr. Obeid's prophecy that ceramides, and the enzymes that generate ceramides, form the critical hub of the sphingolipid metabolic pathway.

Biophysical Analysis of Lipid Domains in Mammalian and Yeast Membranes by Fluorescence Spectroscopy.

The use of steady-state and time-resolved fluorescence spectroscopy to study sterol and sphingolipid-enriched lipid domains as diverse as the ones found in mammalian and fungal membranes is herein described. We first address how to prepare liposomes that mimic raft-containing membranes of mammalian cells and how to use fluorescence spectroscopy to characterize the biophysical properties of these membrane model systems. We further illustrate the application of Förster resonance energy transfer (FRET) to study nanodomain reorganization upon interaction with small bioactive molecules, phenolic acids, an important group of phytochemical compounds. This methodology overcomes the resolution limits of conventional fluorescence microscopy allowing for the identification and characterization of lipid domains at the nanoscale.We continue by showing how to use fluorescence spectroscopy in the biophysical analysis of more complex biological systems, namely the plasma membrane of Saccharomyces cerevisiae yeast cells and the necessary adaptations to the filamentous fungus Neurospora crassa , evaluating the global order of the membrane, sphingolipid-enriched domains rigidity and abundance, and ergosterol-dependent properties.

Ceramide Domains in Health and Disease: A Biophysical Perspective.

Ceramides are the central molecules in sphingolipid metabolism. In addition, they are recognized as important modulators of cell function, playing key roles in several cellular processes that range from cell proliferation to cell death. Moreover, ceramides were implicated in multiple diseases, including cancer, neurodegenerative and metabolic diseases, and also in infection by different pathogens. The mechanisms underlying the diverse biological and pathological actions of ceramides are yet to be fully elucidated. Several lines of evidence suggest that the structural features of ceramides, namely their high hydrophobicity and ability to establish strong H-bond network, are responsible for changes in the biophysical properties of biological membranes that can affect the activity of proteins and activate signaling pathways. Ceramide-induced alterations in membrane biophysical properties might also influence the internalization, trafficking and sorting of lipids, proteins, drugs and even pathogens contributing to cell pathophysiology. In this chapter, we critically discuss the ability of ceramides to form lipid domains with atypical biophysical properties and how these domains can be involved in those processes.