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Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) (EC 3.1.4.3) and phosphatidylethanolamine (PE)-specific PLC (PE-PLC) (EC 3.1.4.62), which generate diacylglycerol (DG) and are tricyclodecan-9-yl-xanthogenate (D609)-sensitive, were detected in detergent-insoluble fractions of mammalian tissues approximately 70 and 35 years ago, respectively. However, the genes and proteins involved in PC-PLC and PE-PLC activities remain unknown. In a recent study, we observed that mammalian sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr) display PC-PLC and PE-PLC activities in vitro. In the present study, we showed that human SMS2, which is located in detergent-insoluble fractions of the plasma membrane, also possesses PC-PLC activity (approximately 41% of SMS activity), PE-PLC activity (approximately 4%), ceramide phosphoethanolamine synthase (CPES) activity (approximately 46%), and SMS activity in the presence of phospholipid-detergent mixed micelles. Moreover, purified SMS2 reconstituted in detergent-free proteoliposomes (near-native environments) showed PC-PLC, PE-PLC, and CPES activities. Notably, in the presence of approximately 2 mol% ceramide and 4 mol% PC (1:2 ratio), PC-PLC activity was almost equal to SMS activity. SMS2 as PC/PE-PLC showed substrate selectivity for saturated fatty acid- and/or monounsaturated fatty acid-containing PC and PE species. The PC-PLC/SMS inhibitor D609 inhibited all enzyme activities (SMS, PC-PLC, PE-PLC, and CPES) of SMS2. Moreover, Zn 2+ strongly inhibited all the enzymatic activities of SMS2. Interestingly, DG inhibited the SMS activity of SMS2 (feedback control). These results indicate that mammalian SMS2 has unique enzymatic properties and is a candidate for a long-sought mammalian PC/PE-PLC., Competing Interests: Conflict of interest The authors declare no conflicts of interest associated with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Sphingomyelin (SM) synthase 1 (SMS1), which is involved in lipodystrophy, deafness, and thrombasthenia, generates diacylglycerol (DG) and SM using phosphatidylcholine (PC) and ceramide as substrates. Here, we found that SMS1 possesses DG-generating activities via hydrolysis of PC and phosphatidylethanolamine (PE) in the absence of ceramide and ceramide phosphoethanolamine synthase (CPES) activity. In the presence of the same concentration (4.7 mol%) of PC and ceramide, the amounts of DG produced by SMS and PC-phospholipase C (PLC) activities of SMS1 were approximately 65% and 35% of total DG production, respectively. PC-PLC activity showed substrate selectivity for saturated and/or monounsaturated fatty acid-containing PC species. A PC-PLC/SMS inhibitor, D609, inhibited only SMS activity. Mn 2+ inhibited only PC-PLC activity. Intriguingly, DG attenuated SMS/CPES activities. Our study indicates that SMS1 is a unique enzyme with PC-PLC/PE-PLC/SMS/CPES activities., (© 2023 Federation of European Biochemical Societies.)

Chemogenetic methods enabling the rapid translocation of specific proteins to the plasma membrane (PM) in a single protein-single ligand manner are useful tools in cell biology. We recently developed a technique, in which proteins fused to an Escherichia coli dihydrofolate reductase (eDHFR) variant carrying N-terminal hexalysine residues are recruited from the cytoplasm to the PM using the synthetic myristoyl-d-Cys-tethered trimethoprim (m D cTMP) ligand. However, this system achieved PM-specific translocation only when the eDHFR tag was fused to the N terminus of proteins, thereby limiting its application. In this report, we engineered a universal PM-targeting tag for m D cTMP-induced protein translocation by grafting the hexalysine motif into an intra-loop region of eDHFR. We demonstrate the broad applicability of the new loop-engineered eDHFR tag and m D cTMP pair for conditional PM recruitment and activation of various tag-fused signaling proteins with different fusion configurations and for reversibly and repeatedly controlling protein localization to generate synthetic signal oscillations., Competing Interests: Declarations of interest S. Suzuki, A.N., T.Y., and S.T. are co-inventors of a patent application related to this work. The other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The extracellular ionic environment in neural tissue has the capacity to influence, and be influenced by, natural bouts of neural activity. We employed optogenetic approaches to control and investigate these interactions within and between cells, and across spatial scales. We began by developing a temporally precise means to study microdomain-scale interactions between extracellular protons and acid-sensing ion channels (ASICs). By coupling single-component proton-transporting optogenetic tools to ASICs to create two-component optogenetic constructs (TCOs), we found that acidification of the local extracellular membrane surface by a light-activated proton pump recruited a slow inward ASIC current, which required molecular proximity of the two components on the membrane. To elicit more global effects of activity modulation on 'bystander' neurons not under direct control, we used densely-expressed depolarizing (ChR2) or hyperpolarizing (eArch3.0, eNpHR3.0) tools to create a slow non-synaptic membrane current in bystander neurons, which matched the current direction seen in the directly modulated neurons. Extracellular protons played contributory role but were insufficient to explain the entire bystander effect, suggesting the recruitment of other mechanisms. Together, these findings present a new approach to the engineering of multicomponent optogenetic tools to manipulate ionic microdomains, and probe the complex neuronal-extracellular space interactions that regulate neural excitability.