Exploring the Therapeutic Landscape of Sphingomyelinases.

Sphingosine, ceramide, sphingosine-1-phosphate, and other related sphingolipids have emerged as important bioactive molecules involved in a variety of key cellular processes such as cell growth, differentiation, apoptosis, exosome release, and inter- and intracellular cell communication, making the pathways of sphingolipid metabolism a key domain in maintaining cell homeostasis (Hannun and Obeid, Trends Biochem Sci 20:73-77, 1995; Hannun and Obeid, Nat Rev Mol Cell Biol 9:139-150, 2008; Kosaka et al., J Biol Chem 288:10849-10859, 2013). Various studies have determined that these pathways play a central role in regulating intracellular production of ceramide and the other bioactive sphingolipids and hence are an important component of signaling in various diseases such as cancer, diabetes, and neurodegenerative and cardiovascular diseases (Chaube et al., Biochim Biophys Acta 1821:313-323, 2012; Clarke et al., Adv Enzyme Regul 51:51-58, 2011b; Horres and Hannun, Neurochem Res 37:1137-1149, 2012). In this chapter, we discuss one of the major enzyme classes in producing ceramide, sphingomyelinases (SMases), from a biochemical and structural perspective with an emphasis on their applicability as therapeutic targets.

The juxtamembrane linker in neutral sphingomyelinase-2 functions as an intramolecular allosteric switch that activates the enzyme.

Neutral sphingomyelinase 2 (nSMase2) produces the bioactive lipid ceramide and has important roles in neurodegeneration, cancer, and exosome formation. Although nSMase2 has low basal activity, it is fully activated by phosphatidylserine (PS). Previous work showed that interdomain interactions within nSMase2 are needed for PS activation. Here, we use multiple approaches, including small angle X-ray scattering, hydrogen-deuterium exchange-MS, circular dichroism and thermal shift assays, and membrane yeast two-hybrid assays, to define the mechanism mediating this interdomain interactions within nSMase2. In contrast to what we previously assumed, we demonstrate that PS binding at the N-terminal and juxtamembrane regions of nSMase2 rather acts as a conformational switch leading to interdomain interactions that are critical to enzyme activation. Our work assigns a unique function for a class of linkers of lipid-activated, membrane-associated proteins. It indicates that the linker actively participates in the activation mechanism via intramolecular interactions, unlike the canonical linkers that typically aid protein dimerization or localization.

Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation.

Neutral sphingomyelinase 2 (nSMase2, product of the SMPD3 gene) is a key enzyme for ceramide generation that is involved in regulating cellular stress responses and exosome-mediated intercellular communication. nSMase2 is activated by diverse stimuli, including the anionic phospholipid phosphatidylserine. Phosphatidylserine binds to an integral-membrane N-terminal domain (NTD); however, how the NTD activates the C-terminal catalytic domain is unclear. Here, we identify the complete catalytic domain of nSMase2, which was misannotated because of a large insertion. We find the soluble catalytic domain interacts directly with the membrane-associated NTD, which serves as both a membrane anchor and an allosteric activator. The juxtamembrane region, which links the NTD and the catalytic domain, is necessary and sufficient for activation. Furthermore, we provide a mechanistic basis for this phenomenon using the crystal structure of the human nSMase2 catalytic domain determined at 1.85-Å resolution. The structure reveals a DNase-I-type fold with a hydrophobic track leading to the active site that is blocked by an evolutionarily conserved motif which we term the "DK switch." Structural analysis of nSMase2 and the extended N-SMase family shows that the DK switch can adopt different conformations to reposition a universally conserved Asp (D) residue involved in catalysis. Mutation of this Asp residue in nSMase2 disrupts catalysis, allosteric activation, stimulation by phosphatidylserine, and pharmacological inhibition by the lipid-competitive inhibitor GW4869. Taken together, these results demonstrate that the DK switch regulates ceramide generation by nSMase2 and is governed by an allosteric interdomain interaction at the membrane interface.

Alternate splicing of dysferlin C2A confers Ca²âº-dependent and Ca²âº-independent binding for membrane repair.

Dysferlin plays a critical role in the Ca²âº-dependent repair of microlesions that occur in the muscle sarcolemma. Of the seven C2 domains in dysferlin, only C2A is reported to bind both Ca²âº and phospholipid, thus acting as a key sensor in membrane repair. Dysferlin C2A exists as two isoforms, the "canonical" C2A and C2A variant 1 (C2Av1). Interestingly, these isoforms have markedly different responses to Ca²âº and phospholipid. Structural and thermodynamic analyses are consistent with the canonical C2A domain as a Ca²âº-dependent, phospholipid-binding domain, whereas C2Av1 would likely be Ca²âº-independent under physiological conditions. Additionally, both isoforms display remarkably low free energies of stability, indicative of a highly flexible structure. The inverted ligand preference and flexibility for both C2A isoforms suggest the capability for both constitutive and Ca²âº-regulated effector interactions, an activity that would be essential in its role as a mediator of membrane repair.