Voltage-gated sodium channels (Na v ) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Na v ␣ subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Na v s at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Na v s.Voltage-gated sodium channels (Na v ) 3 produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. These channels are heteromeric membrane proteins composed of an ␣ subunit, which is sufficient for channel gating, and one or more auxiliary ␤ subunits, which tune voltage dependence and kinetics of channel gating (for review, see Ref.

We have previously shown that glycosaminoglycan (GAG) storage in animal models of the mucopolysaccharidoses (MPS) leads to inflammation and apoptosis within cartilage. We have now extended these findings to synovial tissue and further explored the mechanism underlying GAG-mediated disease. Analysis of MPS rats, cats, and/or dogs revealed that MPS synovial fibroblasts and fluid displayed elevated expression of numerous inflammatory molecules, including several proteins important for lipopolysaccharide signaling (eg, Toll-like receptor 4 and lipoprotein-binding protein). The expression of tumor necrosis factor, in particular, was elevated up to 50-fold, leading to up-regulation of the osteoclast survival factor, receptor activator of nuclear factor-B ligand, and the appearance of multinucleated osteoclast-like cells in the MPS bone marrow. Treatment of normal synovial fibroblasts with GAGs also led to production of the prosurvival lipid sphingosine-1-phosphate, resulting in enhanced cell proliferation, consistent with the hyperplastic synovial tissue observed in MPS patients. In contrast, GAG treatment of normal chondrocytes led to production of the proapoptotic lipid ceramide, confirming the enhanced cell death we had previously observed in MPS cartilage. These findings have important implications for the pathogenesis and treatment of MPS and have further defined the mechanism of GAG-stimulated disease. (Am J Pathol

Recent studies suggest that the lipid, ceramide, induces the default apoptosis process in eggs. Yet, it is obscure how newly formed embryos overcome this fate. Acid ceramidase (AC) is a key regulatory enzyme involved in ceramide metabolism, and mutations in the AC gene (Asah1) result in Farber Lipogranulomatosis, a fatal human genetic disorder. Our previous studies revealed that AC knockout (Asah1-/-) mice had a lethal phenotype, and herein we reveal the mechanism underlying this observation. A single-cell, polymerase chain reaction (PCR) genotyping method was developed to analyze individual embryos from Asah1 +/- intercrosses. Combined with Annexin V staining, this genotype analysis demonstrated that Asah1-/- embryos could not survive beyond the 2-cell stage, and underwent apoptotic death. Notably, sphingosine-1-phosphate (S1P) treatment of early 2-cell embryos from the Asah1 +/- intercrosses rescued Asah1-/- embryos, and enabled their progression from the 2-cell to 4-8-cell stage. Quantitative PCR also revealed that expression of the Asah1 gene in healthy embryos was initiated at the 2-cell stage, coincident with embryonic genome activation (EGA). AC activity and Western blot analyses further demonstrated high expression and activity of the enzyme in normal, unfertilized eggs, which likely provide the protein to newly formed embryos prior to EGA. Based on these observations, we suggest that AC is an essential factor required for embryo survival that functions by removing ceramide from the newly formed embryos, thus inhibiting the default apoptosis pathway.

Herein we report the mechanism of human acid ceramidase (AC; N-acylsphingosine deacylase) cleavage and activation. A highly purified, recombinant human AC precursor underwent self-cleavage into ␣ and ␤ subunits, similar to other members of the N-terminal nucleophile hydrolase superfamily. This reaction proceeded with first order kinetics, characteristic of selfcleavage. AC self-cleavage occurred most rapidly at acidic pH, but also at neutral pH. Site-directed mutagenesis and expression studies demonstrated that Cys-143 was an essential nucleophile that was required at the cleavage site. Other amino acids participating in AC cleavage included Arg-159 and Asp-162. Mutations at these three amino acids prevented AC cleavage and activity, the latter assessed using BODIPY-conjugated ceramide. We propose the following mechanism for AC self-cleavage and activation. Asp-162 likely forms a hydrogen bond with Cys-143, initiating a conformational change that allows Arg-159 to act as a proton acceptor. This, in turn, facilitates an intermediate thioether bond between Cys-143 and Ile-142, the site of AC cleavage. Hydrolysis of this bond is catalyzed by water. Treatment of recombinant AC with the cysteine protease inhibitor, methyl methanethiosulfonate, inhibited both cleavage and enzymatic activity, further indicating that cysteine-mediated self-cleavage is required for ceramide hydrolysis. Human acid ceramidase (AC;3 N-acylsphingosine deacylase; EC 3.5.1.23) hydrolyzes the sphingolipid, ceramide, into sphingosine and free fatty acid. AC is considered a lysosomal enzyme since it has optimal in vitro activity at acidic pH, and most of the lipid storage in Farber disease patients (the genetic disorder resulting from the deficiency of this enzyme) occurs within late endosomes and/or lysosomes (1). It is therefore thought that the main function of AC is to participate in lysosomal membrane turnover. A low level, secreted form of AC also has been described (2-4), although its biological function remains unknown.In addition to its important housekeeping function in sphingolipid metabolism, AC participates in signal transduction pathways that regulate various physiological and pathological processes. Recently, it was shown that the AC gene (Asah1) is among the first genes expressed in newly formed mouse embryos, and its deficiency causes embryo death at the two-cell stage (5). In addition, many studies have reported the involvement of AC in complex diseases. For example, AC is overexpressed in several types of human cancer (prostate, head and neck squamous cell, etc.) (6, 7), and cancer therapy based on the inhibition of AC activity has recently been proposed (8, 9). Moreover, impaired ceramide metabolism has been implicated in the pathogenesis of diabetes, Alzheimer disease, atherosclerosis, thrombosis, and cardiomyocyte apoptosis (10 -14). Although the precise role of AC in these common diseases is unknown, the enzyme is likely to act as a rheostat controlling the levels of ceramide, sphingosine, and sphingosine-1-phosphate in cel...