Farber disease (FD) is a severe inherited disorder of lipid metabolism characterized by deficient lysosomal acid ceramidase (ACDase) activity, resulting in ceramide accumulation. Ceramide and metabolites have roles in cell apoptosis and proliferation. We introduced a single-nucleotide mutation identified in human FD patients into the murine Asah1 gene to generate the first model of systemic ACDase deficiency. Homozygous Asah1P361R/P361R animals showed ACDase defects, accumulated ceramide, demonstrated FD manifestations and died within 7–13 weeks. Mechanistically, MCP-1 levels were increased and tissues were replete with lipid-laden macrophages. Treatment of neonates with a single injection of human ACDase-encoding lentivector diminished the severity of the disease as highlighted by enhanced growth, decreased ceramide, lessened cellular infiltrations and increased lifespans. This model of ACDase deficiency offers insights into the pathophysiology of FD and the roles of ACDase, ceramide and related sphingolipids in cell signaling and growth, as well as facilitates the development of therapy.

The utility of the urinary proteome in infectious diseases remains unclear. Here we analyzed the proteome and metabolome of urine and serum samples from patients with COVID-19 and healthy controls. Our data show that urinary proteins effectively classify COVID-19 by severity. We detect 197 cytokines and their receptors in urine but only 124 in serum using TMT-based proteomics. Decrease of urinary ESCRT complex proteins correlates with active SARS-CoV-2 replication. Downregulation of urinary CXCL14 in severe COVID-19 cases positively correlates with blood lymphocyte counts. Integrative multiomic analysis suggests that innate immune activation and inflammation triggered renal injuries in patients with COVID-19. COVID-19-associated modulation of the urinary proteome offers unique insights into the pathogenesis of this disease. This study demonstrates the added value of including the urinary proteome in a suite of multiomic analytes in evaluating the immune pathobiology and clinical course of COVID-19 and, potentially, other infectious diseases.

This work describes the application of the catch-and-release electrospray ionization-mass spectrometry (CaR-ESI-MS) assay, implemented using picodiscs (complexes comprised of saposin A and lipids, PDs), to screen mixtures of glycolipids (GLs) against water-soluble proteins to detect specific interactions. To demonstrate the reliability of the method, seven gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD2, and GT1b) were incorporated, either individually or as a mixture, into PDs and screened against two lectins: the B subunit homopentamer of cholera toxin (CTB5) and a subfragment of toxin A from Clostridium difficile (TcdA-A2). The CaR-ESI-MS results revealed that CTB5 binds to six of the gangliosides (GM1, GM2, GM3, GD1a, GD1b, and GT1b), while TcdA-A2 binds to five of them (GM1, GM2, GM3, GD1a, and GT1b). These findings are consistent with the measured binding specificities of these proteins for ganglioside oligosaccharides. Screening mixtures of lipids extracted from porcine brain and a human epithelial cell line against CTB5 revealed binding to multiple GM1 isoforms as well as to fucosyl-GM1, which is a known ligand. Finally, a comparison of the present results with data obtained with the CaR-ESI-MS assay implemented using nanodiscs (NDs) revealed that the PDs exhibited similar or superior performance to NDs for protein-GL binding measurements.

Protein interactions with glycolipids are implicated in diverse cellular processes. However, the study of protein-glycolipid complexes remains a significant experimental challenge. Here, we describe a powerful new assay that combines electrospray ionization mass spectrometry (ESI-MS) and picodiscs, which are composed of human sphingolipid activator protein saposin A and a small number of phospholipids, to display glycolipids in a lipid environment for protein-glycolipid interaction studies in aqueous solution. Time-resolved measurements of enzyme catalyzed hydrolysis of glycolipid substrates and the detection of low, moderate, and high affinity protein-glycolipid interactions serve to demonstrate the reliability and versatility of the assay.

EPDR1, a member of the ependymin-related protein family, is a relatively uncharacterized protein found in the lysosomes and secretomes of most vertebrates. Despite having roles in human disease and health, the molecular functions of EPDR1 remain unknown. Here, we present crystal structures of human EPDR1 and reveal that the protein adopts a fold previously seen only in bacterial proteins related to the LolA lipoprotein transporter. EPDR1 forms a homodimer with an overall shape resembling a half-shell with two non-overlapping hydrophobic grooves on the flat side of the hemisphere. EPDR1 can interact with membranes that contain negatively charged lipids, including BMP and GM1, and we suggest that EPDR1 may function as a lysosomal activator protein or a lipid transporter. A phylogenetic analysis reveals that the fold is more widely distributed than previously suspected, with representatives identified in all branches of cellular life.